Molecular biology of papovaviruses.

ANNUAL REVIEWS

Further

Quick links to online content Ann. Rev. Biochem. 1977. 46:471-522

Annu. Rev. Biochem. 1977.46:471-522. Downloaded from www.annualreviews.org by WIB6242 - Universitaets- und Landesbibliothek Duesseldorf on 12/07/13. For personal use only.

Copyright

©

1977 by Annual Reviews Inc. All rights reserved

MOLECULAR BIOLOGY OF PAPOVAVIRUSES George C Fareed Molecular Biology Institute and Department of Microbiology and Immunology, University of California, Los Angeles, California 90024

Dana Davoli Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115

CONTENTS PERSPECTIVES AND SUMMARY ............................................................................

472

POLYOMA VIRUSES....................................................................................................

475

PROTEINS OF POLYOMA VIRUS AND SV40........................................................

476

GENOMES OF PAPOVAVIRUSES ............................................................................

477

Properties of SV40 and Polyoma DNAs ............................................................

477

Physical Mapping of SV40 and Polyoma DNAs.............................................. Comparison of Virus Strains ........... ................................ .. .................................

481

REPLICATION OF SV40 AND POLYOMA DNAs ................................................

482

Initiation ..................................................................................................................

483

.

.

481

Polynucleotide Chain Propagation and Unwinding of Template Strands..

484

Termination..............................................................................................................

484

VIRAL NUCLEOPROTEIN COMPLEXES................................................................

485

TRANSCRIPTION OF SV40 AND POLyOMA........................................................

485

Early Transcription ................................................................................................ Late Transcription ..................................................................................................

486

Comparison of SV40 and Polyoma ....................................................................

487

...............................

489

HUMAN PAPOVAVIRUSES..............................................

486

DAR Virus................................................................................................................

490

JC and BK Viruses .................................... ............................................................

490

471


472

FAREED & DAVOLI

MUTANTS OF POLYOMA VIRUSES. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ . . . . . . . . . ......

492

Temperature-Sensitive Mutants............................................................................ Host-Range Mutants of Polyoma ...................... ....... .......... .................................

493

Location of Mutations............................................................................................ Adeno-SV40 Hybrids..............................................................................................

494

492 493

Mutants Arising During Serial Passage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

497

CONSTRUCTION OF MUTANTS IN VITRO . . . . . . . . . . . . . . . ........................ . . . . . . . ............

502

EVIDENCE FOR VIRUS-CODED PROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . ... . . . . . . . . . . . . . . . . . . .

504

PROPERTIES OF SV40 T ANTIGEN .... ....... . . . . . ................. . ......................................

505

SEQUENCING OF SV40 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......

506

TRANSFORMATION BY POLYOMA VIRUSES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

507

Transcription in Transformed Cells.................................................................... Transformation by DNA Fragments . . ..................... . .... . ..... . .. ............ ........ . . . . . . . . .

511

PAPOVAVIRUSES AS VEHI CLES FOR PROPAGATING FOREIGN DNA I N EUKARYOTIC CELLS . . . . . . . . . . . ....... . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .

511

PAPILLOMA VIRUSES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

514

510

PERSPECTIVES AND SUMMARY

Simian virus 40 (SV40) and polyoma virus are two members of the papovavirus group that have been intensively investigated during the past ten years. Polyoma Virus and SV40 were isolated from cells of their natural host species-mice or rhesus monkeys, respectively-and human papovavi ruses have been detected in the brain cells of patients suffering from progres sive multifocal leukoencephalopathy, a demyelinating neurological disease, and in the urine of patients receiving immunosuppressive therapy. When papovaviruses, such as SV40 and polyoma, infect cells of their natural hosts (permissive cells), the viral genomes are replicated and viral proteins are made, which results in release of progeny virions and cell lysis. These viruses can also infect cells of other species, termed nonpermissive cells, which do not support the efficient replication of the viral genome. Instead, infection of these cells results occasionally in their transformation to a malignant phenotype by a process that requires the expression of at least one viral gene. The result of this transformation event in vivo is the forma tion of tumors. The viral particles of SV40 and polyoma virus are composed of individual circular duplex DNA molecules (- 3.3 X 1 06 mol wt) enclosed in a 4S-nm icosahedron of class T7 with protein capsids and no membrane coats. These viruses, due to their small size, can code for only four to five proteins of average molecular weight (5 X 1 04) and thus rely heavily on the host cell functions for replication and transcription during the lytic cycle. For this reason, the interaction between these viruses and their permissive host cells has been used as a model system to elucidate the mechanism and regulation


MOLECULAR BIOLOGY OF PAPOVAVIRUSES

473

of RNA and DNA synthesis. The small size of their DNA has made analysis of the structure and function of the viral genome feasible and has facilitated the study of the processes leading to transformation and growth in permissive cells. When viral DNA is extracted from purified virions or from cells infected by these viruses, two species are found. The majority of the viral DNA is in the form of a covalently closed, circular duplex DNA molecule called DNA I, which for SV40 contains about 26 negative superhelical turns. A smaller proportion of the viral DNA is in the form of an open circular duplex DNA (DNA II), which is generated from DNA I molecules by breaking a single phosphodiester bond in either strand. Bacterial restriction endonucleases, which recognize specific nucleotide sequences in duplex DNA and cleave both strands at these sites, have been used to construct physical maps of papovavirus genomes. This was accomplished by cleavage of the viral genome with various restriction enzymes, separation of the resulting fragments by gel electrophoresis, and determination of the size and order of these fragments. The technique was first applied to map the SV40 genome by Nathans and co-workers, who used the restriction enzymes from Haemophilus injluenzae (endo R· Hin dII/I1I) and Haemophilus para injluenzae (endo R· Hpal), which cleave SV40 DNA into 1 3 and 3 frag ments, respectively. The endo R· HindII/II1 and R· Hpal sites were also mapped relative to the cleavage site of endo R . EeoRI, a restriction enzyme that introduces one specific cleavage into the SV40 genome to generate a unit-length linear molecule; the EeoRI site has been chosen arbitrarily as zero map units on the genome. Many other restriction enzymes have been isolated subsequently and used to produce detailed maps of the SV40, polyoma, human papovavirus BK, and human papilloma virus DNAs. Recently, more refined restriction-endonuclease mapping procedures have been applied to analyze the orientation and site specificity for viral DNA integration in transformed cells. The early steps in the productive infection of permissive cells by papovaviruses, which begins with adsorption and penetration of the cell by the virus, followed by uncoating of the virus in the cell nucleus, remains largely obscure and awaits the application of biochemical and genetic tech niques for better clarification. The lytic cycle that follows has been divided into two phases: early and late. During the early phase of SV40 or polyoma virus infection, which lasts until the beginning of synthesis of viral DNA, virus-specific early RNA is made, as well as the virus-specific neoantigens. Substantial evidence now exists to indicate that one of these neoantigens, the nuclear T antigen, is coded for in the early-gene region. In the late phase, viral DNA is replicated and the late viral mRNA and structural proteins


474

FAREED & DAVOLI

of the virion (VP 1 , VP2, and VP3) are synthesized. The lytic cycle termi nates in the assembly, maturation, and release of progeny virions, phenom ena that are still poorly understood. When SV40 infects permissive cells, the snythesis of neither host RNA nor proteins is inhibited. Therefore, virus-specific RNA represents a small proportion of the total RNA synthesis of infected cells and can be studied only by molecular-hybridization techniques. The size of the SV40 early RNA as well as the position on the viral genome from which it is transcribed has been determined. Early mRNA has an s value of 1 9 and a molecular weight of 0.7 to 0.8 X 106, or about half the size of one strand of viral DNA. The region of the SV40 genome from which this RNA is transcribed was determined by hybridization of early viral mRNA to separated strands of the Hind fragments of SV40. The early 1 9S mRNA is transcribed from the early or E strand of Hind fragments B, I, H, and A. This region from which early mRNA is transcribed is termed the early-gene region. During the late phase of the lytic cycle, the 1 9S early mRNA is made, as well as late mRNA. The late mRNA consists of two species which sediment at 1 9S and 1 6S. The late 19S species is complementary to one half of the late or L strand and is transcribed from the L strand of SV40 Hind fragments C, D, E, K, F, J, and G. The 1 6 S RNA is identical to the 3'-terminal half of the 1 9S L-strand RNA; therefore, the 1 9S and 1 6S RNAs copied from the L strand are overlapping. The region of the genome from which late mRNAs are transcribed is termed the late-gene region. Analysis of transcripts of the viral genome in transformed cells has shown the early mRNA to be formed; no functional late mRNA has been detected. Of current interest are the regulation of viral transcription and the location and structure of early and late promoters using in vitro assays. The basic steps in replication of viral DNA have been elucidated for SV40 and polyoma, and progress has been made in duplicating the steps after initiation of replication with in vitro systems. Since the early-gene function (gene A) of SV40 and polyoma is involved in the initiation reaction, much effort continues to be directed toward developing an assay for this reaction that could be used for the clarification of the biological function of the gene-A product in both the lytic infection and transformation. From a genetic standpoint SV40 and polyoma virus are probably the most thoroughly characterized DNA-containing animal viruses. Both viral genomes have been nearly completely covered with genetic and physical markers from both temperature-sensitive (Is) conditional lethal mutants and deletion mutants. With the aid of constructed deletion mutants and complementary findings from cell-free translation of both normal in vivo transcripts of viral DNA and the transcripts produced from specific frag ments of viral DNA, maps of the location of SV40 and polyoma viral genes


475

on their DNAs have been constructed. These data are being refined further with the completion of nucleotide sequence analysis of major regions of these genomes.


POLYOMA VIRUSES

The papovaviruses can be divided into two groups on the basis of mor phology: papilloma viruses and polyoma viruses ( 1). The papilloma virus group includes human papilloma (wart) virus, Shope rabbit papilloma, and various other species-specific papilloma viruses. Among the polyoma viruses are polyoma virus, SV40, rabbit kidney vacuolating virus, stump tailed macaque virus, and the human viruses DAR, Ie, and BK. The virus particles of all of these viruses are composed of a circular duplex DNA molecule enclosed in a spherical protein capsid with icosahedral symmetry. Polyoma virus particles have a diameter of 450 A and contain approxi mately 3 X 106 daltons of DNA. Papilloma viruses are larger (550 A) and contain about 5 X 106 daltons of DNA. All of the papovaviruses are capable of initiating a lytic infection in permissive cells. However, there is no known tissue culture system in which papilloma viruses can be propagated. Since in vitro systems have been developed to support the growth of the polyoma viruses, much of the molecular biology has been elucidated with members of this group, espe cially polyoma and SV40. Permissive cells support the complete replication of polyoma viruses, which leads to the release of progeny virus and cell death. The productive infection by polyoma (in mouse cells) or SV40 (in African green monkey cells) begins with adsorption and penetration of the cell by the virus, fol lowed by uncoating in the cell nucleus. The ensuing lytic cycle has been divided into two phases: early and late. The early phase lasts until the beginning of viral DNA synthesis (about 16-1 8 hr after infection). During this phase, virus-specific early RNA is made, as well as the nuclear T and U antigens and the tumor-specific transplantation cell-surface antigen (TrAg). The late phase is marked by the onset of replication of the viral DNA and includes the synthesis of late viral mRNA and the structural proteins of the virion (V or capsid antigen). The cycle terminates in the assembly, maturation, and release of progeny. virions. Polyoma viruses can cause tumors when injected into susceptible ani mals. Transformation of nonpermissive cells growing in vitro by these viruses is believed to be an analogous process to the induction of tumors in animals because cells transformed in vitro acquire a set of properties characteristic of tumor cells. The transformation of susceptible cells by SV40 and polyoma requires expression of a virus-specific gene(s). While


476

FAREED & DAVOLI

no viral DNA synthesis or viral capsid proteins are detected, both early virus-specific RNA and the T, U, and TrAg antigens are made. The DNA of the polyoma viruses is about 3 X 106 daltons. Therefore, they can code for only four to five proteins of average molecular weight and must rely heavily on the host-cell functions for replication and transcription during the lytic cycle. For this reason, SV40 and polyoma virus have been used as model systems to try to understand more about the regulation and mechanism of RNA and DNA synthesis in eukaryotic cells. Their small size also makes it feasible to attempt to map the viral gene products and to determine their function, especially the viral gene(s) involved in transforma tion of nonpermissive cells. PROTEINS OF POLYOMA VIRUS AND SV40

The T antigen, a nonstructural virus-induced protein, is found in the nu cleus of cells lytically infected with polyoma viruses (2-8), as well as in the nucleus of cells transformed by these viruses (2, 6, 9). Recent evidence (pre sented later) has shown that it is coded for by the early-gene region of polyoma and SV40 and has an estimated molecular weight of 80,000 to 1 00,000 ( 10-1 2). SV40 T antigen does not cross-react immunologically with polyoma T antigen; however, SV40 T antigen does share immunological properties with T antigen induced by human papovavirus BK ( 1 3). The function of T antigen is unknown, but it may be important for the initiation of viral DNA synthesis in the lytic cycle and for the establishment and maintenance of transformation of nonpermissive cells. The U antigen, which is localized in the nuclear membrane ( 1 4), and TrAg, found at the cell surface ( 1 5-2 1), are induced early in the lytic cycle and are present in transformed cells. The function of U antigen is unknown; TrAg may be involved in the tumor rejection mechanism in vivo. Late in the lytic cycle, a new intranuclear antigen (V antigen) is detected (22-24). This antigen most likely represents the viral coat protein(s) and (as is shown later) is coded for by the late-gene region of SV40 and polyoma. The viral coat proteins have been characterized by gel electrophoresis (254 1). The major virus-coded polypeptide of these viruses (VP 1 ) has a molecu lar weight of 45,000-48,000. Minor polypeptides (VP2 and VP3) of polyoma have a molecular weight of 36,000 and 23,000 and those of SV40 are 35,000 and 25,000. The two minor polypeptides of polyoma share common sequences but are unrelated to the major polypeptide (37, 38, 40). SV40 VP l and VP3 are also structurally unrelated (39). In addition to the virus-specific proteins VPI-VP3, the SV40 and polyoma virions con tain the four cellular histones H2A, H2B, H3, and H4 (32, 35, 37, 38, 4 1 ).


477


GENOMES OF PAPOVAVIRUSES

In this group of oncogenic viruses, the genomes of SV40 and polyoma virus have been most extensively analyzed. In addition to these two viruses, structural information has been obtained for the genomes of human JC and BK papovaviruses, human papilloma (wart) virus, and papilloma viruses of various other species. When the viral DNA is extracted from purified papovavirus particles, two and occasionally three different DNA species are found (I, 42, 43). The majority of the viral DNA is in the form of DNA I, which contains negative superhelical turns. Smaller proportions of the viral DNA extracted from virions are in the form of DNA II or of a unit-length linear molecule (DNA III). The molecular weights of papovavi ral DNAs have been determined by electron microscopic contour-length measurements of DNA II molecules, agarose gel electrophoresis, and velocity-gradient centrifugation. The molecular weight of SV40 is 3.3 X 1 06 (44, 45), and of polyoma, 3. 1 X 106 (46, 47). The size of BK DNA was determined to be 3.4 X 1 06, based on an assumed size of 3.6 X 106 for SV40 (48). Although no plaque-purified virus preparations of JC have been obtained, its genome has been estimated at 3.0 X 106 daltons relative to that of SV40 (49, 50). In many preparations of polyoma viral DNA and in some from SV40, host-cell fragments have been encapsulated into virions during the process of viral maturation and give rise to DNA III forms (5 1-56). Both DNA I and II are infectious, and certain preparations of linear duplex viral DNA molecules that can form circles in vivo without deleting a functional genetic site are also infectious in permissive cells (57, 58), al though the host-cell DNA fragments are not. Transformation of nonper missive cells by circular and linear viral DNA preparations has also been demonstrated (59-62). The general properties including base composition, conformation, sedi mentation characteristics, and nearest-neighbor base frequencies of papovavirus DNAs have been reviewed by Levine (4 1). The present descrip tion focuses primarily on more recently obtained physical and genetic findings. Properties of SV40 and Polyoma DNAs

The continuous circular polynucleotide strands of SV40 or polyoma DNA I have been shown to be topologically wound around each other through base pairing in the normal Watson-Crick double helix (63) and cannot be separated by denaturation procedures (alkaline pH or high temperatures) unless one or more single-strand breaks are introduced (64, 65). In addition, the DNA I molecules contain tertiary turns in which the duplex strands are twisted on themselves to impart a more compact shape (64) and lower


478

FAREED & DAVOLI

intrinsic viscosity (66) to the molecules. The presence of negative superheli cal turns in all naturally occurring, closed, circular duplex DNAs is a consequence of the molecules being partially unwound at the time of closure of the final phosphodiester bond. The recent studies of SV40 and polyoma minichromatin has provided the first plausible explanation for the existence of these tertiary turns or superhelical structure (67, 68). In these nucleo protein particles, obtained from virions or the nuclei of infected cells, the viral DNA is associated with histones that are derived from their host cells (69-73) and these complexes have structural features similar to those of cellular chromatin. By electron microscopic analysis, these particles appear as circular molecules that possess 2 1 DNA-protein beads or nucleosomes (68, 74). Procedures that remove the histones result in the superhelical configuration only for the closed, circular DNA I molecules (67). The accurate determination of superhelical turns in DNA might shed light on both its physical and biological significance and help to understand the organization of DNA in chromatin. Six methods have been used to determine superhelix density (or the number of superhelical turns per 10 base pairs) of SV40 or polyoma DNAs. These methods include four based on the binding of ethidium bromide (75): titrations of the supercoiled DNA monitored by sedimentation velocity, by buoyant density, by viscometry, and by buoyant separation in high concentrations of ethidium bromide or propidium diiodide. Covalently closed, circular DNAs bind lower amounts of intercalating dyes, such as ethidium bromide, than do relaxed circular molecules (76, 77). Thus, the DNA I and II species can be effectively separated in intercalative dye-CsCI density gradients where DNA I has a higher density (78). The fifth method is alkaline buoyant-density titration (75), and the most recently developed method estimates the number of superhelical turns in a given DNA by counting the resolved bands present after agarose gel electrophoresis of the DNA, which had been partially relaxed by the nicking-closing or DNA-relaxing protein from mammalian cells (79-82). The initial results from the latter method suggest that 24 ± 2 negative superhelical turns existed in SV40 DNA I and, when correlated to the buoyant shift of completely relaxed SV40 DNA in a CsCI-propidium diiodide gradient, a helix unwinding angle of 26-28° for intercalation of one molecule of ethidium bromide was calculated (83). These values were subse quently made more accurate by a method of overlapping the results ob tained after agarose gel electrophoresis under two different sets of conditions (82). It was determined that virion SV40 DNA contains 26 ± 0. 5 superhelical turns, and the mean value for unwinding the double helix by ethidium bromide is 23 ± 3°. Since there are 2 1 ± 1 nucleosomes per SV40 chromatin, 1 .25 ± 0.09 was calculated to correspond to the average number of superhelical turns within a nucleosome. If the regions of internu-



479

cleosomal DNA are relaxed, then 1 .25 refers to the average number of superhelical turns accommodated within a nucleosome. A mathematical clarification of supercoiling in SV40 chromatin structure has recently been described (84) in which the writhing number (a property of the overall structure such as its axis) is equal to the linking number (L) minus the twist (1) of the structure. The number of superhelical turns (L) is the difference between the mean L for the minichromosome and the mean L for the completely relaxed form of SV40 DNA without supercoils. The units of twist (1) are metrical and T N sin a, where N is the sum of turns up plus the turns down the double helix and a is the angle of inclination of the helix. The stress of superhelical structure in SV40 or polyoma DNA has been shown to induce localized interruptions in base pairing primarily at regions rich in adenine and thymine (A-T-rich). This has been demonstrated by cleavage with single-strand-specific endonucleases, with binding of T4 gene-32 protein or Escherichia coli unwinding protein, and by chemical modification of the unpaired regions. The single-strand-specific nuclease from Aspergillus oryzae, S1> was shown to cleave both strands of covalently closed, circular superhelical SV40 (85) and polyoma (86) DNAs to generate unit-length linear duplex molecules with intact single strands. The sites of cleavage were mapped within regions that were readily denaturable in a topologically constrained superhelical molecule. At moderate salt concen trations (75 mM), SV40 DNA was cleaved once eitl:!er between 0. 1 5 and 0.25 or between 0.45 and 0.55 SV40 map units (Figure 2). In higher ionic strength (250 mM), cleavage occurred preferentially within the 0.45 to 0.55 region of the map. In accord with the S, nuclease cleavage results, the bacteriophage T4 gene-32 protein, which binds to single-stranded but not to duplex DNA, forms a specifically located denaturation loop in SV40 DNA I at 0.46 (87). More recently, the E. coli unwinding protein was shown to bind to SV40 DNA I at either of two preferred sites: 0.46 or 0.9 map units (88). It has been found that in polyoma DNA I both S, endonu clease (89) and T4 gene-32 protein (90) may bind to three to four different A-T-rich regions of the genome. Superhelical SV40 DNA I can be chemically modified with N-cyclohex yl-N-,8-(4-methylmorpholinium)-ethylcarbodiimide (CMC) (9 1), which reacts preferentially with the imino sites of unpaired thymine or guanine residues to form a stable covalent product in the neutral pH range. The locations on the SV40 genome that were reactive with CMC also contained the sites susceptible to S, endonuclease action (92). These findings, using both chemical and enzymatic probes of superhelical SV40 and polyoma DNAs, support the view that regions of localized interrupted secondary structure exist that may be capable of forming intrastrand hairpin struc=

480

tures. Superhelical SV40 DNA is asymmetrically transcribed in vitro by coli RNA polymerase (93) and the strand copied corresponds to that copied early in the lytic infection of monkey kidney cells (94). The preferen tial initiation sites for E coli RNA polymerase have been mapped at or near the SI nuclease cleavage regions (95). In vitro transcription studies of SV40 DNA I relative to the linear form by calf thymus and rat liver RNA polymerases (96-98) have revealed an enhanced symmetrical transcription for SV40 DNA I. The enhanced rate has been explained by an increased binding of RNA polymerase at unpaired sites (96). Studies of the iodination of SV40 DNA I have recently raised the possibility that some of these weakly hydroden-bonded sites are generated by certain denaturing agents or other conditions. Although DNA I reacted more rapidly than DNA II with iodine, the iodination occurred uniformly throughout the DNA (99). When SV40 or polyoma DNA is extracted from infected cells, rather than from virions, oligomeric forms of viral DNA are found in addition to DNA I and II molecules of unit length ( 1 00-106). These oligomeric forms are infectious and include dimers, trimers, tetramers, pentamers, and hex amers of SV40 and polyoma. Both catenated (interlocked circular mono mers) and concatenated (connected series of monomers) oligomers have been found. Mouse 3T3 cells transformed by polyoma tsA mutants do not contain free viral DNA at the nonpermissive temperature (38.5°C), but do after a temperature shift from 38.5 to 3 1 °C ( 1 07). The oligomeric polyoma viral DNAs produced at the permissive temperature (31 DC) by two related sublines of Py tsA -transformed mouse 3T3 cells have been analyzed (108). The major oligomeric components were head-to-tail dimers from which a segment of different length was deleted in the different sub lines. These could have resulted from excision of tandemly integrated genomes followed by specific deletion events. Recent reports have shown that during productive infection of permissive cells by both SV40 and polyoma, many copies of the viral genome become integrated into the cellular DNA (109-1 1 1 ). Holzel & Sokol developed a procedure to separate cellular DNA of SV40-infected monkey cells from free viral DNA. Using hybridization techniques it was shown that late after infection, when replication of viral DNA was at a maximum, cellular DNA contained as much as 2% integrated viral DNA. This corresponds to more than 20,000 integrated virus genome equivalents per cell. An alternative basis for these findings has been suggested by Martin and his co-workers ( 1 1 2), who have identified large quantities of high-molecular-weight oli gomers of SV40 during the productive infection. These nonintegrated circu lar viral genomes of high molecular weight could have easily been trapped among cellular DNA fragments on extraction and, since they sediment in E


FAREED & DAVOLI


48 1

regions of alkaline sucrose gradients characteristic of high-molecular weight cellular DNA, falsely high estimates for integrated genome equiva lents could be obtained.


Physical Mapping of SV40 an d Polyoma DNAs

The type-II bacterial restriction endonucleases ( 1 1 3), which recognize spe cific nucleotide sequences in unmodified duplex DNA and cleave both strands at these sites, have been used to construct physical maps of papovavirus genomes. This was done by cleaving the viral genome with various restriction enzymes, separating the resulting fragments on gels, and then determining the size and order of these fragments (see 1 14). The technique was first applied to map the SV40 genome by Danna & Nathans ( 1 14, 1 1 5) using the restriction enzymes from H. i njl uenzae (Hi n d II and -III), which cleaves SV40 into eleven major fragments, and from H. parainjluenzae (Hpa I and -II), which cleaves SV40 into four fragments. The Hpa and Hi n d sites were also mapped relative to the cleavage site of the restriction enzyme EcoRI. EcoRI has been shown to make one specific

cleavage in the SV40 genome ( 1 1 6, 1 1 7) and this site has arbitrarily been chosen as zero map units on the genome. Since this earlier work with Hpa, Hi n d, and EcoRI, other restriction enzymes such as EcoRII, Haelll, HhaI, Hi nf, Al uI , and many others have been isolated and used to produce a very detailed map of the SV40 genome ( 1 1 8- 1 26) (see Figure 1). More recently, the same technology has been used to construct a cleavage map of the polyoma genome ( 1 27-1 33). Once a cleavage map has been con structed, it is then possible to use this knowledge to compare related DNAs and to localize certain genome functions. Com parison of Virus Strains

Nathans & Danna ( 1 34) compared the Hin d digest pattern of small-plaque, large-plaque, and minute-plaque SV40 viral DNA. Cleavage of small plaque SV40 DNA with the restriction enzyme Hi n d produces eleven major fragments, lettered A through K in order of decreasing size. The cleavage of large-plaque SV40 DNA also yielded eleven fragments; however, one of these fragments, Hi n d-F, had an altered mobility, which suggests a deletion of 25 base pairs relative to the Hi n d-F of small-plaque SV40 DNA. The cleavage pattern of minute-plaque SV40 was also altered relative to that of small-plaque SV40 due to the deletion of 1 50 base pairs in Hi n d-C. There fore, the difference in these three strains is due to one small deletion in different parts ofthe genome. Huang et al ( 1 35) used the restriction enzyme from Haemophil us aegypti us (Hae) to compare small- and large-plaque SV40 strains and again the difference in cleavage patterns could be ex-

482

FAREED

& DAVOLI A

Hho I Hinf

8 .730

B

I

E

J

C

8

Haem

.105

F

C

H GI

.308.351

.211

D .21

0

.497 .535

N

E .59

.99

K Flo M Fib (I,LlH .725

.66


F2

G J 8

.795 .85 .905 .95

Hoerr

.835

F

A

Eca Rn

H KL

M

C

F

J .06 .105

G

I

B . 175

B

I

.435

.150 .225.27 .30

Hind (lI+m)

G

N�

H

C

A . 6 55

.325 .375 .430

0

0

. 595 .635 .70 .735

Hpall Hpo

!

A .645

A

.28

A .827

E

. 845

D

l.M .760

Jz A . 935

E

K F

.860 .9 45.98�

.730

C

A

I

B

.1 75

o

Eca RI Site

0.1

0.2

A .760

.375

0. 3

0.4

0.5

FRACTIONAL MAP

0.6

0.7

0. 8

0.9

LENGTH

1.0

Eca RI Site

Figure 1

Coordinates on SV40 DNA for cleavage sites by different bacterial restnctlon endonucleases. This diagram was kindly provided by Dr. K. Sub ramanian. The genome ofSV40 is shown as a linear structure in this diagram opened at the EcoRI cleavage site (1.010.0 map units).

plained by a small deletion in one strain relative to the other . Variations in the cleavage pattern between various polyoma strains have also been de tected ( 1 36, 1 37). REPLICATION OF SV40 AND POLYOMA DNAs

Rirt first provided evidence that the replication of polyoma occurs in a semiconservative fashion ( 1 3 8). Since then, replicating molecules of SV40 and polyoma have been isolated from lytically infected cells. These mole cules appear in the electron microscope as Cairns-type structures with two branch points, three branches, and no free ends ( 1 39-142). From length measurements, the two branches, which are of equal length, correspond to the replicating part of the molecules. The unreplicated part of the molecule contains superhelical turns because the parental strands remain covalently closed during replication.


483

The replication of SV40 and polyoma can be divided into three stages: 1 . initiation, 2. polynucleotide chain propagation, and 3. termination (see 4 1 , 44, 1 43).


In itiation

Various experimental approaches have been used to demonstrate that DNA synthesis is initiated at a specific site on SV40 and polyoma. These methods depend on the cleavage of SV40- or polyoma-replicating molecules or newly synthesized molecules by restriction enzymes. Danna & Nathans ( 1 44) determined the pattern of labeling of the Hin d fragments in newly repli cated SV40 DNA I after cleavage of these molecules with this enzyme. If there is a preferred site for initiation of replication, then molecules com pleted during a pulse time shorter than the time needed for complete replication of one molecule will be most highly labeled in those regions synthesized last (near the terminus of replication). With a pulse time longer than the replication time, all regions of newly completed molecules will contain label, but there will be a gradient of labeling that reflects the temporal order of synthesis of the different parts of the genome. After determining the amount of pulse-label present in individual Hin d frag ments, Danna and Nathans concluded that the origin of replication was at 0.67 map units in Hin d-C near its junction with Hin d-A. It was also possible to conclude that replication proceeded equally in both directions from the origin and terminated 1 800 from it in fragment Hin d-G. Fareed et al ( 145) cleaved replicating molecules of SV40 with the restric tion enzyme EcoRI. This cleavage converts the circular replicating mole cules into linear structures and introduces a reference point (the end of the molecule) for determining the position of the replication fork relative to the cleavage site. The linear structures produced after cleavage of SV40replicating molecules contained one bubble at a specific site in the molecule. The length of the linear segments decreased at the same rate as replication proceeded, which proved replication to be bidirectional. The measurements also allowed one to conclude that replication always begins 33% from the EcoRI cleavage site and that both replicating forks move at the same rate. Similar experiments have been carried out with various strains of polyoma virus. As with SV40, in the majority of the molecules, replication was initiated at a unique site in Hpa II-5 near the Hpa II-5-3 junction ( 1 46). This replication proceeded bidirectionally from the origin, terminating 1 800 from it in HpalI-6 (136). However, about 1 0% of the replicating molecules initiated at another origin, which is near the EcoRI cleavage site ( 1 47). Replication appears to proceed unidirectionally from this origin.

484

FAREED & DAVOLI


Polynucleotide Chain Propagation and Un win ding of Tem plate Stran ds

The mechanism of chain growth during replication of 8V40 and polyoma DNA has been studied both in vivo ( 148-1 52) and in vitro ( 1 53- 1 67). When replicating intermediates are labeled for a brief time in such systems, two populations of newly synthesized strands are found. One population con sists of growing viral DNA chains ranging from 68 to almost 1 68 (one complete strand), while the second consists of 48 fragments (about 1 50 nucleotides). These 48 fragments can be chased into the growing strands (6-1 68) and are therefore the likely intermediate in DNA chain growth. Although RNA primers have not been detected from in vivo studies, ribonucleotides are incorporated in vitro at the 5'-terminus of the 48 chains of polyoma and 8V40 ( 1 56, 1 6 1 , 1 62, 1 65). The results from both in vivo and in vitro experiments suggest the following series of events in the replica tion of SV40 and polyoma: 1 . Once DNA replication is initiated at the origin, replication proceeds bidirectionally with an equal rate of chain growth at each of the replica tion forks. 2. Unwinding of template strands occurs at or proximal to the replication forks ( 1 68). The template strands are covalently closed and supercoiled in the isolated replicative intermediates ( 1 4 1 , 1 42). The involvement of a mammalian DNA untwisting protein (79-8 1) in this process appears to be required, although no direct evidence for this has been obtained. 3. Chain growth occurs by a discontinuous mechanism in which 48 DNA fragments are first synthesized. All DNA polymerases can only add onto the 3'-end of an oligonucleotide primer. Therefore, the short stretches of RNA found at the 5'-end of each 4 S fragment may fulfill the primer requirement. 4. After removal of the RNA primer, the gaps between the 4S fragments and the longer growing chain are filled. 5. The newly synthesized fragments are then j oined to the growing chain by a covalent link that is probably formed by DNA ligase. Term ination

Polynucleotide chain propagation proceeds until the two replicating forks meet at a position 1 800 from the origin. The two interlocked replicating circles then separate, generating a DNA II molecule ( 1 69). This DNA II molecule contains all of the newly synthesized DNA in the discontinuous strand. In addition, the interruption in the discontinuous strand has been shown to be a gap located in Hin d-G, 1 800 away from the initiating site ( 1 69, 1 70). The size of this specific gap has been found to be about 22 to


485


73 nucleotides (171). These DNA II molecules are converted to SV40 DNA I, possibly by the action of a polymerase, a nuclease, DNA ligase, and DNA gyrase (not yet identified in mammalian cells). From analysis of replicating intermediates of deletion mutants of SV40, Nathans and co-workers (172, 173) have concluded that termination of replication occurs whenever the two growing forks meet and that it does not involve a specific nucleotide (signal) sequence. VIRAL NUCLEOPROTEIN COMPLEXES

The DNA of eukaryotic cells and some small DNA viruses is associated with histones in compact structures called nucleosomes or nu bodies. These are about 100 A in diameter and contain about 200 base pairs of DNA and eight histone molecules. Viral nucleoprotein complexes have been isolated from infected cells treated with nonionic detergents (174). These mini chromatin complexes, which sediment at 44-55 S, may in part be precursors in the encapsulation process. The analysis of both polyoma and SV40 minichromatin with bacterial restriction endonucleases has shown the posi tioning ofnucleosomes to be random with respect to DNA sequences (175). Recently, a 90S complex has been extracted at low ionic strength from the nuclei of SV40-infected monkey kidney cells (164). This complex represents a soluble material that supports the later steps in SV40 DNA replication when supplemented with cytoplasmic factors and the deoxynucleotide tri phosphates. A salt-stable protein-DNA complex in closed circular SV40 DNA was extracted from viruses at pH 10.5 (1 76). After nicking with X-rays and cleavage with restriction endonucleases, the "knob" representing the pro tein-DNA complex mapped at or near the origin of viral DNA replication. When viruses were subjected to more stringent lysis using sodium dodecyl sulfate in the presence of dithiothreitol, protein has been found to be cova lently linked at a phosphodiester-bond cleavage site also near the origin or 0.67 SV40 map units ( 1 77). After alkaline denaturation of the intact com plex, the protein remains attached to one end of unit-length linear single strands of DNA. Characterization of the denatured linear strands from the nicked circular molecules has suggested that the nicks are introduced in a staggered fashion and are separated by about 200 nucleotides on the two complementary strands. TRANSCRIPTION OF SV40 AND POLYOMA

As previously mentioned, the lytic cycle of polyoma and SV40 can be divided into two phases. During the early phase, which lasts until replica-

486

FAREED & DAVOLI


tion begins, there is synthesis of early mRNA and the viral antigens T, U, and TrAg. In the late phase, both early and late viral RNA are made, as well as the capsid protein(s). Because these viruses do not inhibit cellular RNA synthesis, studies of viral RNA metabolism rely on hybridization methods to detect virus-specific RNAs (44). Currently, soluble nucleo protein complexes ( 1 78) are being used to explore transcriptional control mechanisms. Early Transcription

Early virus-specific RNA represents about 0.01 % of the total RNA synthe sis of the cell ( 1 79, 1 80). The size of early viral RNA, as well as the position of the viral genome from which it is transcribed, have been determined for both the viral primary transcript and the viral mRNA found in the cyto plasm. Pulse-labeled SV40- or polyoma-specific RNA, which represents the primary transcription product, sediments at 1 9S in sucrose gradients ( 1 8 1 , 1 82). This corresponds to a molecular weight of 0.7--0.8 X 106, or half the size of one strand of viral DNA. To determine the sequence composition of the primary transcript of polyoma, unlabeled nuclear RNA was hybri dized with small amounts of radioactively labeled, separated viral DNA strands ( 1 83). The nuclear RNA hybridized with only 40-50% of the sequences of one strand of the viral DNA (called the early or E strand). There was no hybridization with the complementary strand (late or L strand). Early viral mRNA found in the cytoplasm appears to be identical in size and in sequence to the early primary transcript. It has an s value of 1 9S ( 1 84, 1 85) and hybridizes to only 40-50% of the E strand (94, 1 86- 1 89). From summation experiments, it was shown that both early cytoplasmic mRNA and nuclear RNA contain the same sequences ( 1 83). Since there is no change in size or sequences of early RNA between its transcription in the nucleus and its appearance in the cytoplasm as mRNA, the primary transcript is assumed to be the early mRNA. The early mRNA of both SV40 and polyoma, which is polyadenylated ( 1 90), has been located on the physical map of the viral genome by hybridization of the RNA to separated strands of various restriction enzymes. The early mRNA of SV40 is tran scribed from the E strand of SV40 Hin d-B, -I, -H, and -A (counterclockwise from 0.65--0. 1 7), whereas early mRNA of polyoma is transcribed from the E strand of the polyoma Hpa l l fragments 5, 4, 8, 7, 2, and part of 6 or clockwise from 72 to 25 map units (1 88, 1 89, 1 9 1- 193). Late Transcription

The late primary transcript of virus-specific RNA represents up to 1 % of the RNA labeled in infected cells ( 1 8 1 , 1 9 1 , 1 94). In both polyoma- and SV40-infected cells, the pulse-labeled late virus-specific RNA sediments as



487

an RNA species as large as or larger than genome size ( 1 8 1 , 1 84, 1 94). The nature of the sequences in these late primary transcripts was first elucidated by experiments done by Aloni (1 95), who found that sequences from both viral DNA strands are transcribed in the same region of the genome, giving rise to self-complementary RNA. By hybridizing unlabeled nuclear RNA to the separated strands of labeled viral DNA or to separated strands of restriction fragments of viral DNA (94, 1 69, 1 88, 1 89), it was possible to show that all L-strand sequences are represented in late nuclear RNA. Most or all of the E-strand sequences are also present, but these are in considera bly lower concentration than the L-strand transcript (1 88, 196). This pre dominance of L-strand transcript is due to more efficient transcription of the L strand in vivo (1 97). Late virus-specific mRNA found in the cytoplasm sediments as two peaks with s values of 1 6S and 1 9S (1 84, 1 98). Hybridization of this mRNA with separated strands of viral DNA demonstrated that late mRNA is comple mentary to the same one half of the E strand from which early mRNA is copied, as well as being complementary to one half of the L strand (94, 1 86- 1 89, 1 99). Thus late mRNA contains the same sequences as early mRNA plus additional sequences copied from the L strand. The region of the genome from which the 1 9S and 1 6S species are transcribed has been determined by hybridization of the RNA to separated strands of various restriction-enzyme fragments of the genome. There are two 19S species ( 1 90, 200, 20 1). The minor 1 9S component, which is complementary to one half of the E strand, is identical to early 1 98 mRNA (Figures 2 and 3). The major 1 9S species is complementary to one half of the L strand and contains most or all of the L-strand sequences represented in late mRNA. This 1 9S species is transcribed from the L strand of the SV40 Hi n d fragments C, D, E, K, F, J, and G, and from the L strand of the polyoma Hpa II fragments 1 , 3, and part of 6. The most abundant late viral mRNA is the 1 6S species, which is identical to the 3'-terminal half of the 1 9S L-strand RNA. There fore, the 1 6S and the 1 9S copied from the L strand are overlapping. All of these late mRNA species are polyadenylated at their 3'-end ( 1 90) and methylated and capped at their 5'-end (202, 203). Com parison of S V40 an d Polyoma

The previous results have shown that there is a great deal of similarity between polyoma and SV40 in both the structure and organization of the virion proteins and the viral DNA within the virions. The work from the transcriptional mapping demonstrates that this similarity extends to the number and the size of viral mRNAs and their disposition on the physical map of the viral DNA. In both viral DNAs, the early and late regions each constitute about half of the molecule, and the early and late RNAs are


488

FAREED & DAVOLI

/

ORIGIN OF REPLICATION

Figure 2

Functional map of SV40 DNA. The map coordinates are identical to those in Figure I and are oriented clockwise from the EcoRI cleavage site at l .0/0.0. The "stable" polyadenylated cytoplasmic mRNAs are demarcated by the inner arcs. The shaded areas correspond to regions where viable deletions have been located. The outer arcs provide approximate locations for virus-coded proteins and genetic complementation groups.

transcribed from opposite DNA strands. Also, in both, the 5'-ends of the mRNAs map very near the origin of viral DNA synthesis, whereas their 3'-ends are near the replication terminus. Although SV40 and polyoma are very similar in organization, they share only a small region of DNA homology. Ferguson & Davis (204), using an electron microscopic method that enabled them to detect small amounts of homology, detected a 1 5 % region o f weak sequence homology between SV40 and polyoma. This homology, mapped relative to the EcoRI site in each genome, is in similar portions of the map of SV40 and polyoma (from 0.83 to 0.99 map units in SV40 for a region of weak homology and from 0.93 to 0.98 SV40 map units for a region of strong homology) in the late-gene region. With monospecific antisera against SV40 VP 1, it was shown that SV40 and polyoma share a common VP 1 antigenic determinant (205). The structural gene coding for SV40 VP 1 has been located on the SV40 physical map between positions



�

70\ �� 3

489

LATE REGION 50

VP-2

7 ..... VP-3 --....___

. Figure 3

Functional map of polyoma viral DNA. The inner concentric circle containing fragments numbered 1 to 8 is the restriction-nuclease Hpa II cleavage map and the outer circle with fragments numbered 1 to 4 is the EcoRI plus HhaI map. The unique site of cleavage by EcoRI is located at 0 map units at the top. Orientations of the three polyadenylated cytoplasmic RNAs and virus-coded proteins are indicated by the outer arcs.

0.95 (N-terminus) and 0. 1 8 (C-terminus) (206, 207). Based on the nucleo tide sequence-homology results, it is possible that the antigenic determinant common to SV40 and polyoma may lie at the N-terminus of VP l . The immunologic cross-reactivity observed between SV40 and polyoma viruses extends to other members of the SV40-polyoma subgroup of papovaviruses (BK virus, rabbit vacuolating virus, and stump-tailed macaque virus), which suggests that all members of this subgroup share a common antigenic determinant located in their major capsid polypeptides (205). HUMAN PAPOVAVIRUSES

The human papovaviruses have been isolated from immunosuppressed indi viduals. They fall into three classes: DAR, JC, and BK. DAR virus was

490

FAREED & DAVOLI


only occasionally obtained from brain tissue of patients with progressive muItifocal leukoencephalopathy (PML), a rare demyelinating disease of the brain (208, 209). The most frequent isolate from PML is JC virus (2 10), which is distinguishable genetically from DAR. BK virus was isolated from urine specimens of kidney-transplant patients who were immunosuppressed (2 1 1 ) or from the urine of patients with the Wiskott-Aldrich syndrome of combined immunodeficiency (2 1 2). DAR Virus

The virus particles of DAR (also referred to as PML-2) resemble SV40 both morphologically and antigenically (209). DAR differs from SV40 in that DAR can grow efficiently in both monkey cells and human cells, whereas SV40 grows efficiently only in monkey cells. Sack et al (2 1 3) purified the nucleic acid from one isolate of DAR and compared its genome to that of SV40 DNA. DAR DNA is a covalently closed, circular duplex with about the same molecular weight of SV40 DNA. It was shown to have extensive homology with SV40 DNA both by hybridization of the two DNAs and by comparison of their Hin d cleavage patterns. When DAR DNA is digested with Hin d, eleven major fragments are produced. Nine of these fragments coincide in mobility with nine of the Hin d fragments of SV40. Two SV40 fragments, Hin d-C and -F, are not present in the DAR digest but are replaced by two smaller fragments, which suggests that there are two small deletions in the DAR genome relative to the genome of SV40, one in Hi n d-C and the other in Hin d-F. Since these same types of variation are seen with stable SV40 plaque-morphology variants, DAR is considered to be a variant of SV40. Je and BK Viruses

JC and BK viruses differ both biologically and genetically from polyoma and SV40. Serological studies have shown JC and BK to be ubiquitous in the human population, and both viral DNAs have distinct restriction cleavage patterns from that of SV40 and polyoma (2 14-2 1 6). They are similar to SV40 and polyoma with respect to size and morphology of both the virion and the DNA. JC and BK also induce in hamster cells an identical or closely related T antigen to that induced by SV40 and show some minor cross-reaction with the SV40 capsid proteins ( 1 3 , 2 1 1 , 2 1 7). However, in certain human cells transformed or acutely infected by JC virus, the viral T antigen is immunologically distinct from that of SV40 or BK virus (2 1 8). Osborn et al (2 1 6) purified BKV and JC viral DNAs and showed that they are circular DNA duplexes with a molecular weight of about 3 X 106• Comparison of restriction-enzyme cleavage patterns of JC, BK, and SV40 DNA showed that they all differed, in both number and size of the frag-



49 1

ments. More recently, several groups (2 1 9-223) have further compared the genomes of BK, JC, and SV40. From reassociation kinetics, it was con cluded that there is about 1 1-12% sequence homology between BK and SV40 DNA. Under less stringent conditions that involved formation of heteroduplexes between the two genomes, 50% homology was found. The region of SV40 that is homologous to BK DNA was determined from the heteroduplex analysis and from DNA-DNA hybridization studies employ ing Hin d fragments of SV40. The common sequences are located princi pally in the late-gene region of SV40 DNA, corresponding to portions of SV40 Hin d fragments C, D, G, J, and K, and to a lesser extent to Hin d-E and -F. Little or no homology was detected in reassociation experiments and in electron microscopic heteroduplex analysis with the early-gene re gions of SV40. These results are consistent with the minor cross-reaction between BK and SV40 virion antigens, since the homology between the two viruses lies in the late-gene region of SV40 that codes for V antigens. They do not explain the strong immunological cross-reaction of SV40 and BK T antigens. SV40 T antigen is coded for by the early-gene region of SV40. A more sensitive test for homology between two related DNAs utilizes the method of Ferguson & Davis (204), in which the two DNAs are joined in vitro at a single restriction-endonuclease cleavage site common to both. When such a recombinant dimer was formed between SV40 and BK DNAs linked together via their EeoRI sites, extensive homology was detected in the early-gene region as well as parts of the late-gene region (224). Studies of the kinetics of reassociation of JC DNA in the presence of a large excess of SV40 or BK DNAs revealed polynucleotide sequence homology of 1 1 % between the genomes of JC and SV40, and 25% between the genomes of JC and BK viruses (2 19). In addition, the DNA sequences shared by JC and SV40 were shown to be a subset of the sequences shared by JC and BK. A physical map of the BK viral genome has been constructed (222). Since the areas of homology in SV40 and BK have been mapped relative to their EeoRI site, it is possible to orient the two genomes around this site. BK Hin d, EeoRI-A l , and EeoRI-A2 are homologous with those SV40 se quences expressed late in the lytic cycle (SV40 Hin d-C, -D, -G, -J, and -K) and should therefore contain sequences expressed late in cells lytically infected by BK. The regions in BK corresponding to Hin d-B, -D, and -C are probably transcribed early in infection. Two other isolates of BK have been mapped and the results suggest that the region extending from 0.52 to 0.73 map units in the BK genome can vary among different isolates. DNA sequences from BK virus have recently been detected by reassocia tion kinetic analysis in DNA preparations from certain human tumor tis sues and human tumor cell lines (225). Whether this association causes the tumor state or is a trivial association has not yet been resolved.

492

FAREED & DAVOLI


MUTANTS OF POLYOMAVIRUSES

Since the genetics of papovavirus mutants have been described in two cogent reviews (226, 328), this section is brief and primarily concerned with newer findings. The naturally occurring mutants include temperature-sensi tive (ts) mutants, host-range mutants, adeno-SV4O hybrid DNAs, and mu tants arising during serial passage of polyomaviruses. Recently, by use of various biochemical techniques, mutants have been constructed in vitro that contain deletions or insertions in the viral DNA. Tem perature-Sensitive Mutants

Four or five distinguishable classes of ts mutants of polyoma (226-229) and SV40 (230-243) have been isolated on the basis of complementation tests. tsA OF SV40 AND POLYOMA This complementation group is defective in an early viral function. At the nonpermissive temperature, these mutants can infect permissive cells and synthesize early viral RNA (244), but the T antigen made appears to be abnormal (245-25 1). The gene product of this complementation group is required for the initiation of SV40 and polyoma DNA synthesis (236, 243, 252) and for the initiation of synthesis of late viral mRNA (244). At the nonpermissive temperature, these mutants are unable to transform cells (226-228, 236, 242). The SV40 tsA function is also required for maintenance of some aspects of transformation (253-257). This gene function has also been shown to be dominant over mutations in late genes (B, C, and BC) of SV40 from studies of mutants carrying two ts mutations (258). LATE MUTANTS OF SV40 AND POLYOMA These mutants are defective in a late-gene function(s) and are classified as tsB, C, and BC for SV40 and ts1260, tslO, tsC, MP208, and ts59 for polyoma. Although no infectious virions are formed at the nonpermissive temperature, the early viral func tions, such as viral DNA synthesis, appear normal. Many of the virions from mutants of this class are thermally unstable and induce the synthesis of an altered capsid protein (V antigen) (227, 228, 23 1 -233, 24 1-243). tsD OF SV40 AND POLYOMA ts3 The mutant virions from this class appear to be defective in several early functions during the lytic cycle at the nonpermissive temperature (234, 235, 238, 239, 242, 259), since no early viral mRNA is synthesized. However, infection with ts viral DNA leads to a normal lytic cycle and production of ts progeny virions (242, 259). It seems most likely that these mutants are blocked at the nonpermissive temperature in some stage of uncoating.


493


Host-Rang e Mutan ts of Polyoma

A host-range mutant of polyoma virus, NG 1 8, was originally selected for its ability to grow on polyoma-transformed, but not on normal, mouse 3T3 cells (260). This mutant was also defective in transformation of rat and hamster cells. More recent work (26 1) on NG 1 8 and additional host-range mutants of this type has shown that these mutants can grow on a variety of untransformed cell types; therefore, the permissive state does not require a cell-associated polyoma genome as the basis for complementation. It has been suggested that these mutants are unable to induce a certain cellular function necessary for viral growth in 3T3 cells. Recently, a deficiency in histone H3 and H4 acetylation has been attributed to this mutation (262). The host-range mutants have been shown to complement tsA mutants, which may indicate the presence of two functional segments in the early region of polyoma. Location of Mutations

Two techniques have recently been developed to determine the location of mutations of the genome. These are especially useful in mapping ts mutants and other point mutations or small deletions that cannot be detected by restriction-enzyme analysis or electron microscopic heteroduplex analysis. One such technique, marker rescue, relies on the ability of a fragment of wild-type DNA, which has no infectivity by itself, to produce wild-type virus when annealed to full-length mutant DNA and introduced into a cell. This technique was first applied to SV40 by Lai & Nathans (263, 264) to map ts mutants. Heteroduplex molecules were formed between mutant single-stranded circular DNA and a wild-type fragment produced by cleav age of the SV40 genome with Hin d or Hpa . These partial heteroduplexes were then tested for infectivity by infecting cells at the nonpermissive temperature and counting the number of plaques formed, with the assump tion that cellular enzymes would convert the partial heteroduplexes into double-stranded circular DNA. It was found that for each ts mutant, only one Hpa and one Hin d fragment were the most efficient in correcting the ts mutation, thus locating the mutation to that segment of SV40 DNA from which the active fragment was derived. With this approach it was possible to map 41 ts mutants of SV40 from complementation groups A, B, e, Be, and D. The tsA mutants, known to be defective in an early function, map in the early region of the genome as defined by transcriptional mapping. This gene region includes Hin -I and at least parts of Hin -H and Hin -B. Mutants from the B and e complementation groups map in Hin -K, -F, -J, and -G, respectively, which are in the late-gene region. These results suggest that B and C mutations are in the same gene and probably complement


494

FAREED & DAVOLI

each other at the protein level. The tsBC mutants map in Hin -J and Hin -G and appear to be part of the gene for the B/C protein. The muta tional site of complementation group D has been localized to Hin -E, which demonstrates that the D-gene product is a late-gene protein. Mantel et al (265) have reached the same conclusion using this technique. This type of study has been used to map ts mutants of polyoma (266) and to map NG 1 8 and other host-range mutants of polyoma (267). A comparison of these maps again leads to the conclusion that the genetic organization of SV40 and polyoma are very similar. This includes a distinct clustering effect of the ts mutants, in that no ts mutations have been mapped in approximately '50% of the genome, which comprises roughly half of the early-gene region and half of the late-gene region surrounding the origin of DNA replication. Another technique that is useful for mapping ts mutants and other types of mutations was developed by Shenk et al (268). This involves mapping alterations in DNA with the nuclease S I. This nuclease, from Aspergillus oryzae, degrades single- but not double-stranded DNA and is capable of cleaving an intact DNA strand opposite a nick. This latter property of S I makes it useful for determining the location of small deletions, insertions, or any difference in base sequence between homologous DNAs. The single strand-specific nucleases from Neurospora crassa have also been shown to be useful for these analyses (269). For example, heteroduplex DNA mole cules formed between complementary strands of a deletion mutant and wild-type SV40 DNA contain a single-stranded loop at the position corre sponding to the deletion. S 1 will break this single�stranded loop and subse quently cleave the intact strand at the resulting nick (or gap). If the molecules used in formation of the heteroduplex were first cleaved with EcoRI, S l cleavage of the heteroduplex will produce fragments whose length corresponds to the position of the deletion loop from the Eco RI site. S 1 can also cleave some heteroduplexes between a ts mutant and wild-type DNA, making it possible to map the ts mutants of SV40. Shenk et al mapped mutants of the complementation groups A and D and their results agree with those of the marker-rescue technique. Aden o-S V40 Hybrids

Human adenoviruses are propagated in human cells. They do not grow in monkey cells unless these cells are coinfected with SV40 (270, 271), which suggests that SV40 infection induces a "helper factor" required by the adeno (Ad) genome (272-277). Some of the adenovirus stocks that were grown in monkey kidney cells in the presence of SV40 were found to be able to productively infect monkey cells in the absence of SV40 virions. This is now known to be the result of the formation of Ad-SV40 hybrid virions, which consist of recombinant DNA containing all or part of the SV40


495


genome and all or part of the Ad genome enclosed in Ad capsids. When these hybrid viruses infect monkey cells, a part of the SV40 genome is expressed since the SV40 antigens T, U, and TrAg are induced by some hybrids. By correlating the function expressed with the segment of the SV40 genome retained in the hybrid, it has been possible to map these early-gene functions on the SV40 genome. Ad7-SV40 HYBRID E46+ This hybrid is a defective hybrid between SV40 and Ad7 DNA (278-2 8 1 ) and requires coinfection with a normal Ad7 genome for infection (282). Upon infection of monkey cells, this hybrid virus induces SV40 T antigen, TrAg, and U antigen (279-28 1) and synthe sizes SV40-specific early-RNA sequences (283). The structure of this hybrid has been studied in detail by electron microscopic heteroduplex analysis and hybridization methods. The summary of these results shows that in this hybrid, about 16% of the Ad7 genome is deleted and the deleted DNA is replaced by a single substitution of SV40 DNA (284). This SV40 DNA includes the region between 0. 1 1 and 0.66 SV40 map units, with the region , between 0.50 and 0.66 being repeated in tandem (285). Therefore, the SV40 DNA present in this hybrid virus contains all of the SV40 early-gene region. Since U, T, and TrAg are all synthesized by this hybrid, these antigens must be induced by the early-gene region. Ad2-SV40 HYBRIDS These hybrid viruses fall into two main groups. The Ad2HEY and Ad2LEY are mixed virus populations containing Ad2-SV40 hybrid particles and Ad2 particles (286, 287), and thus resemble E46+. However, they differ from E46+ in that Ad2LEY and -HEY can yield infectious SV40 virions on infection of monkey cells (288). Mapping of Ad2HEY and -LEY by heteroduplex analysis showed that both populations were heterogeneous (289). Ad2HEY contains three hybrids-HEY I, HEYII, and HEYIII-whose hybrid genomes differ only in their content of SV40 DNA (0.45, 1 .43, and 2 . 3 9 SV40 genomes, respectively). Ad2LEY contains two hybrids, LEYI and LEYII, which contain 0.03 and 1 .05 SV40 genomes, respectively. In the hybrids containing more than one com plete SV40 genome, the excess DNA is organized as tandem repeats. The production of infectious virions from these hybrids is thought to occur when a unit-length SV40 molecule is excised from the adeno-SV40 hybrid, then replicated and encapsulated. NONDEFECTIVE ADENO-SV40 HYBRIDS Another group of Ad2-SV40 hybrids are the nondefective hybrids Ad2+ND l , Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5 (286, 290). These can replicate in human cells and monkey cells without a helper Ad2 virus. The structure of these hybrids


496

FAREED & DAVOLI

and the SV4O-specific RNA and antigens they induce have been studied in detail (29 1 -298). It was found that 1. each nondefective hybrid contains a single substitution (SV40 DNA inserted, Ad2 DNA deleted); 2. in all of the hybrids, the SV40 DNA sequences begin 14% from one end of the Ad DNA molecule; 3. the lengths of the SV40 DNAs inserted are completely overlap ping, with a common endpoint at 0. 1 1 SV40 map units. By correlating the segment of SV40 DNA retained in each hybrid with the SV40 antigen(s) induced by that hybrid, it has been possible to map the genes cOding for these antigens. Ad2+ND l , which contains 1 8 % of the SV40 genome, codes for U antigen, whereas Ad2+ND2 contains 32% of the genome and codes for both U and TrAg. Ad2+ND4, which contains the largest SV40 segment (48%), codes for U, TrAg, and T antigens. This localizes that segment coding for U antigen at the common end of the hybrids between 0. 1 1 and 0.28 SV40 map units. TrAg and T antigen would be associated with 0.28--0.39 and 0.39--0.59 units, respectively. An alternative model, sug gested by the experiments that have placed the molecular weight of T antigen between 60,000 and 100,000, would be that the entire region from 0. 1 1 to 0.59 map units is needed to code for T antigen; thus U antigen and TrAg might be cleavage products of T antigen. The mapping data are consistent with the studies of SV4O-specific RNA induced by these hybrids (294, 295). The SV40 RNA species induced by a given hybrid are com pletely represented in the RNA species induced by all those hybrids con taining more SV40 DNA. Also, the SV40 RNA sequences specified by Ad2+ND4 include all the RNA sequences transcribed early in the infec tion of monkey kidney cells with wild-type SV40. Two of the hybrids, Ad2+ND3 and Ad2+ND5, induce no known antigens. The SV40 se quences in Ad2+ND3 (7% of SV40) may be too small to code for any protein. In Ad2+ND5, which contains almost as much SV40 DNA as Ad2+ND2, the SV40 sequences are transcribed. The lack of antigen forma tion may be due to some defect in translation of the mRNA or due to the synthesis of an unstable protein that never accumulates in quantities sufficient to be detectable by immunological techniques (29 1 , 296). New nondefective adenovirus-SV40 hybrids have been isolated from cells coinfected with SV40 and H7 1-a host-range mutant of Ad2+ND l that fails to grow in monkey cells (299). Structural studies of these hybrids have suggested that they arise by unequal crossover during recombination. A series of novel hybrids has also been generated using restriction endonu cleases to fragment a new defective Ad2-SV40 hybrid (300). The resulting DNA fragments have been used to complement in vivo DNA of a tsA SV40 mutant. In this way a series of novel hybrids was selected whose circular genomes consisted of the early region of SV40 covalently attached to seg ments of adenovirus DNA.


497


Grodzicker, Lewis & Anderson (299) have used Ad2+ND l to isolate host-range mutants that are defective in growth on monkey cells but not for growth on human cells and appear to carry point mutations rather than deletions. A 30,OOO-dalton protein unique to Ad2+ND 1 -infected cells is not seen in monkey cells infected with certain of those host-range mutants, and translation in vitro of SV40-specific mRNA from these cells produces new unique polypeptides, instead of the 30,000-dalton protein. Mutan ts A rising during Serial Passage

When polyomaviruses are serially passaged at high mUltiplicities of infec tion in permissive cells, virus particles containing defective genomes accu mulate. Early studies demonstrated that these defective particles, which were in many cases the predominant viral species, contained covalently closed, circular DNA molecules smaller than wild-type DNA. Many types of rearrangements had occurred (301-306), including deletions, inversions, reiterations, and insertions at many locations in the viral genome (307-309). Some of these variants also contained cellular DNA sequences covalently linked to viral DNA (3 10-32 1). More recent data has suggested that, in general, after a few high mUltiplicity passages, most of the progeny virions contain simple deletions and rearrangements involving only viral DNA (3 1 3-323). After prolonged serial passage, however, viruses with more extensively altered genomes arise, consisting predominantly of cellular DNA ( 1 72, 3 2 1 , 324, 325). To study the structure and function of a defective molecule, it is most useful to purify the defective from the helper wild-type genomes in the viral stock. This can be done in many ways, including separation of the mutant from the wild-type genome on the basis of size or on the basis of sensitivity to a particular restriction enzyme. Since defective particles arising during serial passage contain shortened genomes, these defectives can be partially separated from wild-type-sized particles in equilibrium gradients. Alterna tively, the shortened DNA can be separated from the full-length DNA on sucrose gradients or agarose gels (3 13, 320). A third selective procedure relies on the fact that during serial passage of papovaviruses, deletion mutants arise with altered sensitivity to restriction endonucleases. Nor mally, SV40 and polyoma are cleaved once by EcoRI. During serial pas sage, however, a large percentage of the viral DNA becomes resistant to cleavage by this and other restriction endonucleases, presumably due to a deletion of the restriction site (308, 309, 3 1 4, 3 1 5, 326). Cleavage of the serially passaged DNA permits separation of the endonuclease-resistant molecules from wild-type genomes or other classes of defectives (3 1 5, 3 1 8320, 322). A specific class of deletion mutants, termed reiteration mutants, consist of tandem or head-to-tail repeats of a small segment of the wild-type


498

FAREED & DAVOLI

genome ( 1 72, 3 1 4, 3 1 5, 3 1 7, 322, 323, 326, 327). If the monomer segment of a reiteration mutant preserves a particular restriction-endonuclease site, then cleavage of the reiteration mutant with this enzyme will yield less than unit-length segments, which can be separated from the linears of the wild type genome (3 1 4, 3 1 7, 323, 327). Since serially passaged DNA contains many different classes of defectives in addition to the helper virus, defectives separated from the wild-type genome by the techniques just discussed will usually consist of a mixed population of defective molecules. Therefore, additional techniques have been developed to isolate populations enriched for one type of defective. In one such technique, infectious-center plaquing, serially passaged stocks of virus are used to infect permissive cells at a low virus-to-cell ratio to permit infection of individual cells by only one defective particle and one nondefec tive particle (3 1 3 , 3 1 6, 3 1 8). Single plaques from such an infection, which contain the progeny from a single cell, are grown to prepare a viral lysate enriched in individual distinct classes of defective molecules. Additional selective procedures can be used in conjunction with this infectious-center technique to enrich for clones of SV40 variants, which yield virus with specifically altered genomes. For example, Brockman & Nathans (3 1 3) used centrifugation to partially separate defective particles containing shortened genomes from wild-type particles of a serially passaged stock. These light defective virions were then used to infect cells in the presence of ts mutants at a nonpermissive temperature. Infection with either a ts mutant virus alone or the shortened defective virus alone resulted in few plaques, whereas coinfection with both a ts mutant and the defective virions resulted in many plaques. Only shortened defectives expressing those functions that comple ment the defect in the particular ts mutants used in the coinfection will be found in the viral lysates prepared from such plaques. For example, defec tives that still express early-gene functions will complement tsA mutants, while defectives expressing late-gene functions will complement tsB mu tants. Mertz et al (308) isolated clones of defective SV40 deletion mutants lacking the EcoRI or Hpa ll restriction-endonuclease cleavage site by first preparing endonuclease-resistant DNA from a serially passaged viral stock. This Hpa II- or EcoRI-resistant DNA was then used in a complementation plaque assay to isolate individual clones of resistant molecules. Using the techniques described, various groups have isolated specific mutant DNAs from early passage stocks of SV40 and polyoma and have studied their structure. Using restriction-enzyme analysis and heteroduplex analysis, Mertz et al (308, 3 1 8, 3 1 9) determined the structure of defective deletion mutants lacking the Eco RI and/or the Hpall restriction site. Most of the defective molecules studied had a large deletion encompassing a unique region of the SV40 genome, but the exact location of the deletion



499

varied from one cloned defective to another. The deleted region always included the EcoRI and/or Hpa ll site, which explains the resistance to cleavage by these enzymes. In addition, most of the mutants also had a unique duplication of SV40 DNA that included the site of initiation of DNA replication. Brockman et al ( 1 72, 307, 3 1 3) also studied the structure of early-passage evolutionary variants of SV40, which had been selected on the basis of their shortened genome length and their ability to complement specific ts mutants. These variants contained a limited deletion in either the late- or early-gene region of the genome in addition to a unique duplication of that portion of the SV40 genome containing the origin of replication. Cloned deletion mutants lacking portions of both late genes (BIC and D), but with an intact early genomic segment, were able to induce T antigen in infected cells, replicate their DNA in the absence of helper virus, stimu late thymidine incorporation into cellular DNA, and transform mouse and hamster cells (3 10). Mertz & Berg (3 I S) also found that some of the Hpa II-resistant deletion mutants isolated were viable. Plaques produced by this mutant DNA in the absence of any helper virus displayed a small-plaque phenotype characteris tic of a slower-than-normal growth cycle. The DNA isolated from these plaques was Hpa ll resistant, as expected. Cleavage of the DNA with Hin d showed that for each mutant, only the fragment Hin d-C had an altered mobility compared to wild-type SV40 DNA. This was due to a deletion of from so to 190 base pairs in this fragment at the position of the Hpa II restriction-endonuclease site. These results suggested that at least one re gion of the SV40 genome, the viral DNA segment around the Hpall cleavage site, was dispensable. Reiteration mutants, which contain tandem or head-to-tail repeats of small segments of the wild-type genome, have also been isolated from early serially passaged stocks of papovavirus DNA. The presence of such reiter ation mutants can be detected by reassociation-kinetic analysis and by their sensitivity to various restriction enzymes. The renaturation of DNA is inversely proportional to its physical complexity. Therefore, if a reiteration mutant contained, for example, a triplication of a specific one third of the viral genome, it would reassociate three times more rapidly than the wild type genome. This type of analysis has been used to detect reiteration mutants in serially passaged stocks of polyoma, SV40, and DAR virus. Fried and co-workers (320, 323) isolated a reiteration mutant of polyoma virus using the infectious-center technique in conjunction with the resis tance of this mutant to EcoRI. The results from structural studies were consistent with the fact that the defective DNA is composed of tandemly repeating units, each of which is 1 7 % of the wild-type genome and includes the viral sequences from the Hpa II-5 fragment and parts of Hpa-3 and


500

FAREED & DAVOLI

Hpa-4. Since this mutant is half the size of polyoma, it must contain three tandem repeats of a segment of the genome that includes the origin for DNA replication. Folk & Fishel (322) also isolated reiteration mutants of polyoma containing tandem repeats of the origin of replication. Other groups have studied the structure of reiteration mutants from SV40 and DAR viral stocks. Fareed et al (3 14) determined the structure of a DAR triplication mutant in which one third of the DAR genome had been preserved and tandemly triplicated to produce a molecule the size of the wild-type genome. The EcoRI site was retained in each monomer segment of this mutant; therefore, Eco RI cleavage generated fragments one third the size of the DAR genome. Structural studies of this monomer fragment led to the conclusion that the one third of the DAR genome preserved in this triplication mutant included sequences from two noncontiguous regions of the genome (31 7). Several additional reiteration mutants have been isolated that are tetramers, pentamers, and hexamers of specific regions of the DAR or SV40 genome (327). In each mutant the monomer fragment has always retained the initiation site for DNA replication; therefore, each reiterant contains multiple origins for replication.

Prolonged serial passage of papovaviruses re sults in production of viruses with more extensively altered genomes con taining both viral and cellular DNA. Tai et al (306) showed that upon random nicking and self-renaturation of DNA from such stocks, the heteroduplexes that formed were a result of both deletions and substitutions in the SV40 genome. Extensive rearrangements were also shown by the significant alterations in the Hi nd digest patterns of these mutants com pared to that of wild-type DNA (307, 308). Winocour's group found host DNA in both SV40 and polyoma DNA I from serially passaged stocks using filter hy.bridization, which suggested that the viral DNA was substi tuted with cellular sequences (3 1 1). From reassociation kinetic analyses, both reiterated and nonreiterated host-cell DNA were found in the viral genomes, with the bulk predominantly of the nonreiterated type ( 1 72, 307, 326, 329-332). The results also suggested that, although populations of defectives arising during different sets of serial passages contained different host sequences, these host sequences did not represent a random selection of the total sequences present in the host genome (330-332). The mecha nism by which cellular DNA becomes incorporated into viral DNA is unknown, but it may be a result of integration of the viral DNA into the host-cell genome during the lytic cycle, followed by excision of molecules containing both cellular and viral D NA ( 1 72, 32 1 , 330, 33 1 ). Measurements of heteroduplexes formed from DNA of serially passaged stocks cleaved with EcoRI (309, 33 1) indicate that substitutions of cellular DNA can LATE-PASSAGE STOCKS



501

occur in many different parts of the viral genome. However, there may be a few preferred sites on SV40 DNA where these substitutions occur most often, which suggests some specific integration of SV40 into the host-cell DNA during the lytic infection. Lee et al (32 1 ) and Davoli et al (324) have isolated specific defectives from late-passage stocks of SV40 and DAR, respectively, that contain host-cell DNA. Structural studies showed that in the variants analyzed, the genomes are made up of small tandemly repeated segments of DNA consisting of cellular DNA and that portion of the viral genome containing the initiation site for DNA replication. One such SV40 defective, called ev 1 103, is made up of a repeating unit that is 8.8% the length of SV40 DNA. Since these genomes are about 78% the length of SV40 DNA, they contain nine tandem repeats of the 8.8% units. Each unit has a segment of SV40 derived from the junction around the Hin -A-C junction, which makes up about one fourth to one third of the repeating unit. The rest of the DNA is derived from cell DNA, including reiterated and nonreiterated sequences. The structural studies on the variants isolated from both early and late serial passages of papovaviruses suggest that active recombination occurs between and within viral genomes and between viral DNA and cellular DNA. Since the rearrangements have been shown to occur at many differ ent sites on the viral genome, extensive base-sequence homology may not be required for recombination in virus-infected cells. While many different types of defectives are probably generated during serial passage, only those with a selective advantage would become the predominant DNA species in the population. Many of the variants isolated from early-passage stocks contain a duplication of the region containing the origin of DNA replica tion. The mechanism by which such duplications of the origin occur is not known, but it has been suggested that they are generated by specific defects in replication (3 1 5, 320, 327) or, alternatively, by recombination within dimer molecules (325). Molecules containing a duplication of the origin might be expected to have a selective advantage in replication if a protein or cell site required for initiation is limiting. Therefore, these molecules would emerge as the predominant part of the population. Defective mole cules may also have a selective advantage in replication because of their shortened length. In addition, only DNA molecules of the appropriate size will be encapsulated and propagated. The reiteration mutants that arise during serial passage contain multiple initiation sites for DNA replication and this is probably the basis for their selective replication in vivo. Early-passage reiteration mutants contain only viral DNA, while late-passage mutants contain both cellular and viral DNA. It has been suggested that formation of reiteration mutants involves recombination events within the viral genome (3 1 7) or between viral and


502

FAREED & DAVOLI

cellular DNA ( 1 72) that generate the monomer segments. This is followed by amplification of the small monomer segment by a reiteration process to generate DNA genomes that can be encapsulated. The existence of such a reiteration process has been shown by studies in which the short monomer segments isolated from DAR reiteration mutants in vitro were amplified in vivo through an arithmetic series of oligomers to generate the original reiteration mutant and mutant oligomers larger than wild-type DNA (327, 333, 334). Griffin & Fried (323) and Shenk & Berg (335) have also demon strated this reiteration process with segments of polyoma and SV40, respec tively. Studies of mutants arising during serial passage of viral genomes have been useful in analyzing the organization and expression of the SV40 genome. For example, it has been possible to conclude that the iniJiation signal is probably the only cis function required for DNA replication ( 1 72, 308, 324, 327). Structural studies of a late-passage reiteration mutant showed that the only segment of the SV40 genome preserved is a region of about 1 50 base pairs containing the origin for DNA replication. However, analysis of replicating molecules led to the conclusion that most or perhaps all of the molecules with multiple initiation sites begin replication at only one of the possible sites (1 72, 3 1 4). Also, it was found that termination of replication is always 1 800 from the initiation site and, therefore, termination does not involve a specific nucleotide sequence ( 1 72, 321). CONSTRUCTION OF MUTANTS IN VITRO

Deletion mutants of viral DNA should be useful in mapping structural genes and in identifying viral gene products. The evolutionary variants just described contain specific deletions, but many also contain other alterations such as duplications and insertions of cellular DNA. Therefore, to obtain simple deletions in various parts of the genome, biochemical techniques have been employed. One method involves construction of deletion mutants in vitro from wild-type DNA by using the restriction enzymes Hin dIII or EcoRII to excise specific segments of DNA from the viral genome. The resulting deleted DNA is then cloned by complementation with ts mutants as previ ously described. Lai & Nathans (336) cleaved SV40 DNA I with limiting concentrations of the restriction enzyme Hin dIII, which cleaves SV40 into six fragments. This restriction enzyme makes a staggered break at its cleav age site, generating fragments with cohesive termini. Such fragments will form covalently closed circles in infected cells, as previously shown by Mertz & Davis (337) for full-length SV40 DNA linears with cohesive termini. After digestion of SV40 with Hin dUI, the partial digest products



503

were fractionated according to length by electrophoresis in agarose gels. Fragments of a size appropriate for encapsulation (65 % to nearly 100% the length of SV40) were isolated and used to infect monkey kidney cells, which were then coinfected with an early or late ts mutant. Infected cells were plated at the nonpermissive temperature and the plaques formed were tested for the presence of virus with short genomes. The cloned deletion mutants, isolated by electrophoresis, were mapped by heteroduplex analysis and by restriction-enzyme analysis. Both "excisional" and "extended" deletions were found. In the excisional deletion mutants, as expected, the limits of the deleted segment corresponded to the Hin dIII cleavage site. In the extended deletion mutants, however, the deletion extended beyond the Hin dIll cleavage sites. These were probably generated by intramolecular recombination near the ends of the linear Hin dIII fragments. Of the nine deletion mutants analyzed, two had deletions in the early region of the SV40 genome, while six had deletions in the late-gene regions and one had a deletion spanning both regions. Although genomes lacking the Hin dIII-C segment (Hin d-C-D) might be expected, none were found, probably be cause such a mutant would lack the initiation site for DNA replication present in Hin d-C. Mertz et al (308) used the enzyme EcaRII, which has 1 6 cleavage sites in SV40 and generates cohesive termini, to construct deletion mutants. Mutants of SV40 with constructed deletions in Hin fragments K, F, J, and G were used to define precisely the BIC gene in the late region of the genome (206). Complementation tests indicated that the BIC deletion mu tants were in a cistron distinct from tsA and tsD mutations and that the junction between the BIC and D genes was within Hin -K. Furthermore, analysis of new proteins in cells infected with these mutants revealed altered VP l polypeptides. These results indicated that the BIC gene (1 200 nucleo tide pairs) codes for VP l . The ability of cells to cyclize linear fragments of SV40 has made it possible to generate deletion mutants in another fashion. Berg and co workers have cleaved SV40 DNA I with EeaRI (338), Hpa ll (338), or DNase I in the presence of MnH (339) to generate unit-length linear molecules. EeaRI and Hpal l make one specific break in SV40, whereas cleavage of the genome with DNase (MnH) yields randomly cleaved linear DNA. The linear DNA produced by cleavage with these enzymes was, in some cases, further treated with A-51-exonuclease to remove a limited (2530) number of nucleotides from the 51-ends. These linears were then used to infect monkey cells in the presence or absence of ts mutants. Covalently closed circular deletion mutants were formed in vivo. Defective mutants, which lacked the EeaRI site, were isolated using this approach and could only be grown when complemented by WT SV40 or a tsA mutant at the


504

FAREED & DAVOLI

nonpermissive temperature. Viable mutants that lack the Hpa II site were also found. These viable mutants produced plaques with the small-plaque morphology found in mutants arising during serial passage lacking the Hpa ll site. With DNase I used in the presence of MnH to generate linears, it has been possible to isolate both viable and defective deletion mutants. The viable deletion mutants have been mapped by 8 1 nuclease and restric tion-endonuclease digestion, and the location and size of the deletion deter mined. Deletions found in at least three regions of the SV40 chromosome have slight or no effect on the rate of viral multiplication or on the yield of virus in permissive cells. The ability of these mutants to transform cells is indistinguishable from wild-type 8V40. The regions deleted are located at 0. 1 7 to 0. 1 8, 0.54 to 0.59, and 0.68 to 0.74 on the SV40 genome (339). These results are consistent with those from mapping ts mutants of 8V40 and polyoma, which suggests that certain regions of the viral genome may be nonessential (Figure 2). An alternative method for obtaining 8V40 mutants defective in a small region of the viral genome is to insert short segments of extraneous DNA covalently into the viral DNA at specific sites. For example, Carbon et al (340) inserted a poly(dA-dT) segment (about 50 base pairs) at the Hpall site in SV40. These molecules were used to infect monkey cells and were found to be viable. In addition, the plaques formed were similar in growth rate to those formed by the naturally arising mutants lacking the Hpa II site and those Hpa ll-resistant mutants that arose after infection of cells with Hpa ll linears treated with A-exonuclease. EVIDENCE FOR VIRUS-CODED PROTEINS

The previous mapping experiments have suggested that T, U, and TrAg antigens are viral gene products coded by the early-gene region, while the viral capsid proteins are coded by the late-gene region. Direct evidence that the viral genome codes for some of these proteins has come from two different types of experiments. The first type of experiment utilizes cell-free systems to translate 8V40 and polyoma RNA isolated from infected cells or viral RNA made in vitro with E. coli RNA polymerase (341-346). As previously discussed, in vivo late polyoma and 8V40 mRNA consist of a 1 98 and a 1 68 RNA. The 1 9S is complementary to one half of the L strand and contains most or all of the L-strand sequences represented in late mRNA. The 1 6S species is identical to the 3'-terminal half of the 1 9S species. This 1 68 viral RNA can be translated in a cell-free system into a polypeptide that migrates together with the major capsid protein (VP 1 ) in SDS-polyacrylamide gels (34 1 , 342, 345, 346). Comparison of the polypeptide with the major capsid protein



505

isolated from virions by peptide mapping substantiates this conclusion. Therefore, the region coding for this 1 6S RNA (see Figures 2 and 3) must specify the major capsid protein. Since the SV40 ts mutants B, C, and BC and the polyoma late ts mutants have been localized to the same segment of the genome, these mutants must contain a ts defect in this protein. Cell-free translation of the late SV40 1 9S RNA results in the production of a polypeptide (X) that may be a precursor to VP3 (342). The precise localization of the region of SV40 DNA coding for viral structural proteins VP 1 , VP2, and VP3 was determined with a linked transcription-translation cell-free system (343, 344). Three SV40 DNA fragments derived from the late-gene region were purified. These fragments were Hpa I-A (0.76-0. 1 75 map units), BglI-EcoRI-B (0.672-0.0), and Hpa II-EcoRI-B (0.735-0.0). From analysis of polypeptides synthesized in vitro, it was concluded that the region of SV40 DNA encoding the informa tion for VP l was between 0.835 and 0. 175, VP2 was between 0.76 and 0.0, and VP3 was between 0.835 and 0.0 map units. Thus, VP2 and VP3 are coded for by overlapping DNA sequences. Graessmann et al have shown that "early" SV40-specific RNA contains information for the formation of T antigen (347). When SV40 DNA I is transcribed in vitro with E coli RNA polymerase, RNA complementary to the early (E) strand is the only RNA made. Following purification of the RNA, it was injected into cells of confluent primary mouse-cell cultures and was found to induce T-antigen formation in up to 80% of the cells. Concen trations of actinomycin D sufficient to block cellular-RNA synthesis did not prevent the induction of T antigen. These results support the hypothesis that SV40-specific T antigen is a virus-coded protein and that the early virus-specific RNA contains this information. Similar experiments have been made by using cRNA transcribed from polyoma DNA in vitro (348). The polyoma RNA made, which is transcribed from both the E and the L strands, can induce V and T antigens. PROPERTIES OF SV40 T ANTIGEN

During lytic infection, transcription of gene A of SV40 leads to the forma tion of early 19S cytoplasmic mRNA, which is translated into A protein, synonymous with T antigen (347, 349). The A protein is synthesized contin uously during the course of infection and accumulates in the nucleus (247). The A protein may also be modified in some fashion to allow for insertion into the nuclear and plasma membranes (350, 3 5 1 ). Several observations have suggested that the SV40 A protein regulates its own synthesis by controlling the transcription of gene A. The synthesis of A protein is stimulated when cells infected by ts mutants in gene A (tsA mutants) are


506

FAREED & DAVOLI

shifted to a restrictive temperature (352). Both the rate of synthesis and the intracellular amount of early mRNA are higher in cells infected with tsA mutant than in cells infected with wild-type virus (353). At 32°C cells infected by tsA mutants synthesize early RNA approximately twice as fast as cells infected with wild-type virus. However, after a shift to 4 1 °C, the rate of synthesis in the tsA infection has been found to increase to 1 5 times the rate in the wild-type infection. These findings are consistent with an autoregulatory role for the A protein in its own expression. Immunoprecipitation with antisera from hamsters bearing SV40-induced tumors has been employed to demonstrate that the gene-A protein is about 1 00,000 daltons and is an SV40 gene product (354). A constructed deletion mutant, DI- 1 00 1 , that lacks 20% of the early-gene region, directed the synthesis of a 33,OOO-dalton polypeptide immunoreactive with anti-T serum and not detected in cells infected with wild-type virus. Maps of the tryptic peptides from the 1 00,OOO-dalton protein and the 33,000-dalton deletion fragment showed common peptides. Also, the phosphorylation site (350) and a DNA-binding site were within this segment of the A protein (354). The same anti-T serum that precipitates the l OO,OOO-dalton SV40 A protein has been shown to immunoprecipitate l OO,OOO-dalton protein(s) from BK infected cells (355). Peptide maps of these A proteins from BK- and SV40infected cells are remarkably similar, but not identical. Purified preparations of SV40 T antigen bind to DNA (248, 356-359). The antigen binds a variety of circular viral DNA molecules and appears to have some specificity with respect to binding sites on SV40 DNA. Elec tron microscopic studies indicated a preferred binding site at the origin of viral DNA replication (358) and nitrocellulose filter assays have shown the antigen to have affinity for several sites in the early-gene region (359). SEQUENCING OF SV40 DNA

Weissman and his collaborators (360-363), Fiers et al ( 1 20, 1 23, 1 25), and others (364) have begun to analyze the nucleotide sequences at certain biologically important sites in SV40 DNA. Their approach has been to isolate and sequence virus-specific RNA obtained from transcripts of DNA fragments in vitro or from cellular RNA that was purified by annealing with DNA fragments. More recently, direct chemical sequencing of short DNA fragments has been employed. Weissman's group has located and determined the nucleotide sequence of the regions of SV40 DNA spanning the 3'- and 5'-ends of "early" and "late" 1 9S mRNA and the region in which DNA replication begins. The 3'-ends of SV40 early and late mRNA lie very close to the Hi n -G-B junction, while the 5'-ends are close to the Hi n -A-C junction. Cytoplasmic



507

mRNA was isolated from monkey cells infected with SV40 and annealed to SV40 DNA fragments produced by cleavage with EeoRII endonuclease. The viral RNA that was resistant to RNase attack was sequenced (365). The 5'-ends of early and late species of SV40 1 9S mRNA, which were transcribed from opposite strands, were found to overlap for a region of 60 to 1 00 nucleotides. This region of overlap included a portion of the segment of DNA in which the origin of DNA replication was located. Several stretches of six or more deoxy-adenylic acids are found in the DNA and appear to be preferentially located near the 5'- and 3'-ends on the strand whose transcript is not represented in stable mRNA. Such stretches are similar to sequences in prokaryotic DNA that function to initiate or termi nate transcription. Interpretation of the results obtained from sequencing stable mRNA are limited, however, by the possibility that the sites of initiation and termination of transcription on viral DNA may not corre spond to the ends of the cytoplasmic RNA because of nucleolytic processing of precursor RNA. By analysis of those SV40 sequences retained in naturally occurring or biochemically constructed deletion mutants of SV40 that are capable of replicating in the presence of helper virus, it has been concluded that most or all of the sequences essential for DNA replication lie in the Hin -C fragment of SV40 DNA ( 1 72) within 1 50 to 1 75 base units of the Hin -A fragment. At or near this initiation site for replication, symmetrical se quences are found, as well as some prominent palindromic arrangements of nucleotides. One of these was a stretch of 27 nucleotides located at about 0.67 map units, which was rich in G and C residues and possessed a perfect twofold axis of symmetry (363). A tandem repeat of 2 1 nucleotides was located near the presumed origin of DNA replication and a stretch of 1 5 nucleotides having twofold symmetry was located at the Hin d(II+III)-A C junction. Between the Hin -A-C junction and 0.67 map position was an additional palindromic sequence of 17 nucleotides in length. Such sequences may be necessary for recognition by proteins (such as T antigen) required for initiation of replication. TRANSFORMATION BY POLYOMA VIRUSES

Polyoma virus and SV40 can cause tumors when injected into susceptible animals ( 1). Transformation of cells growing in vitro by these viruses is believed to be a process analogous to the induction of tumors in animals; therefore, in vitro transformation provides a model system to study events leading to the development of human cancer. The transformation of susceptible cells by SV40 and polyoma requires the expression of a virus-specific gene(s). While no viral DNA is synthesized


508

FAREED & DAVOLI

(366) or viral capsid protein (V antigen) detected, both virus-specific RNA ( 1 78- 1 8 1 , 1 83, 1 87, 1 88, 1 9 1 , 1 99, 366-377) and the antigens U ( 1 4,303), T (2, 9, 6), and TrAg ( 1 5-29) are made. Also, as mentioned, the tsA gene product is necessary for the initiation of transformation by both polyoma and SV40. In addition, the tsA function has also been shown to be involved in some aspects of maintenance of transformation in SV40-transformed cells, but not in polyoma-transformed cells. Nonpermissive cells transformed by SV40 and polyoma are free of detect able virus and nO viral DNA is synthesized. However, it is known that at least one complete viral genome is present in many cells that are trans formed because SV40, and less often polyoma, can be recovered from the transformed cells by one of two methods. The first method involves coculti vation of transformed cells with permissive cells (378-388), and the second involves infection of permissive cells with DNA extracted from virus-trans formed cells (389, 390). Some transformed cells from which virus cannot be rescued may contain defective copies of the viral genome, i. e. less than one complete viral copy. Direct physical evidence of the presence of the viral genome has been hard to obtain since one molecule of viral DNA is only about 10-6 the size of transformed-cell DNA. It was first shown by Sambrook et al (39 1 ) that SV40 DNA is covalently integrated into the cellular DNA. Many types of experiments were then done to determine 1 . the structure of the integrated viral genome and 2 . the site of integration of the viral DNA. The structure of the integrated viral DNA has been studied by hybridization methods. Early work, involving hybridization of virus-specific RNA made in vitro to DNA from transformed cells, showed that there were multiple copies (2-60 copies) of viral DNA in each trans formed cell (392-396). However, the conclusion from these types of experi ments were overestimates due to a systematic error in the method (397). The second method, DNA-DNA hybridization, was first developed by Gelb et al (398). It took advantage of the fact that the rate of reannealing in solution of any sequence of denatured DNA is proportional to its concen tration. It is therefore possible to determine directly the amount of viral DNA in the genome of transformed cells by following the rate of reanneal ing of small amounts of labeled viral DNA in the presence of DNA from transformed cells. Gelb et al allowed highly radioactive, denatured SV40 DNA to anneal in the presence of denatured DNA from a variety of transformed and untransformed cell lines. Since the rate of annealing of the labeled viral DNA was faster in the presence of transformed-cell DNA than in the presence of untransformed-cell DNA, they were able to calculate that the SV40-transformed cell lines contained between one and three copies of SV40 DNA. Ozanne et al (375) and Kamen et al ( 1 88) arrived at a similar conclusion using the same approach with SV40- and polyoma-transformed



509

cell lines, respectively. However, in these experiments it was assumed that all sequences of the SV40 genome were present at equal frequencies in the genome of transformed cells. This assumption is not justified, as shown by experiments done later by Botchan et al (399). They studied the kinetics of renaturation of SV40 Hpa I-EcoRI fragments in the presence of DNA from the SV40-transformed, mouse cell line SVT2. It was found that these cells contain about six copies of a segment of DNA that includes the early-gene region and about one copy of the late viral sequences. Therefore, trans formed cells can contain combinations of complete and partial genomes. An alternative procedure for analyzing the structure of the viral genome within transformed cells is to first rescue this viral DNA by fusion with permissive cells. The viral particle produced can then be studied. Botchan et al (400) used restriction-enzyme analysis to analyze the viral genomes rescued from different clones of SVT2 that had been transformed by the same SV40 stock. Three different types of rescued DNAs were found. One was identical in its cleavage pattern to the wild-type SV40 virus that had been used to transform the cells, while the other two types of viral DNAs rescued had small deletions in Hin d-F or Hin d-C. Nathans & Danna (1 34) also showed that a virus rescued from an SV40-transformed cell line had been modified to give deletions in Hin d-F and -Co Defective SV40 genomes have also been rescued from transformed ceJl lines (384, 385, 387, 388). The site of integration of SV40 into the cellular genome has been studied by two different approaches. One approach involves the formation of somat ic-cell hybrids between mouse cells and SV40-transformed human lines (40 1). In such a hybrid, the human chromosomes are shed preferentially (402, 403). Results from Croce et al (402, 404) and Khoury & Croce (405) suggest that SV40 is integrated into human chromosome #7. This comes from results that showed a concordant segregation of SV40 T antigen with human chromosome #7 and that SV40 DNA is present only in those hybrid clones containing human chromosome #7 and expressing SV40 T antigen. Recent work (406) has shown human chromosome # 1 7 to be another possible location for integration. In the second approach, SV40-transformed cell DNA was cleaved with the restriction enzyme Eco RI (407). If the entire SV40 genome were inte grated into the cellular genome, two fragments containing viral sequences would be produced after EcoRI cleavage since one EcoRI site would be within the integrated viral genome and one would be on either side in the cellular genome. Following cleavage of the transformed-cell DNA, the resulting fragments were fractionated by agarose-gel electrophoresis. DNA was extracted from each fraction of the gel and hybridized with radioactive RNA complementary to SV40 DNA. All of the SV40 DNA on the gel was detected in two peaks, corresponding to fragments having molecular


5 10

FAREED & DAVOLI

weights of 3. 1 X 106 and 1 . 8 X 1 06• These bands contained 70% and 30% , respectively, of the SV40 sequences. If this result arises from integration of one SV40 genome at a single site in the transformed-cell DNA, it implies that the integration site lies 30% away from the EcoRI site. This also shows that the viral DNA is inserted at a limited number of chromosomal sites. Ketner & Kelly (408) have used a similar approach to examine five indepen dent SV40-transformed mouse cell lines. After digestion of the transformed mouse-cell DNA by Hpa ll or Bam I and transfer of denatured DNA fragments to nitrocellulose filters, each line yielded, by hybridization analy sis, a simple pattern of fragments containing SV40 DNA. In addition, each of the lines yielded a different fragment pattern; therefore, the structure of the integrated SV40 DNA and/or its location in the host-cell DNA must be different in the various transformed lines. This suggests that integration is not absolutely site-specific. Although there may be specificity with respect to the recombination site on either the viral genome or the host genome, there does not seem to be specificity with respect to both sites simulta neously. However, such an interpretation rests on the assumption that rearrangements of the viral DNA do not commonly occur prior to or subsequent to the primary integration event. Similar conclusions were reached in a separate study of 1 1 different SV40-transformed rat cell lines (409). Transcription in Transformed Cells

The pattern of transcription in SV40- and polyoma-transformed cells is quite different from that observed in lytic cells. The RNA from transformed cells can be isolated and the virus-specific RNA studied by using various hybridization techniques. Virus-specific RNA sequences occur in both heterogeneous nuclear and stable cytoplasmic RNA. In the nucleus, the viral RNA is present in large molecules of RNA that are longer than a single strand of SV40 DNA ( 1 8 1 , 370, 37 1 , 373, 374) and the RNA contains covalently linked host and viral sequences (37 1 ). The cytoplasmic species contain only viral sequences. Early experiments using competition hybridization ( 1 79, 1 80) suggested that the viral mRNA made in transformed cells included those RNA spe cies transcribed early in the lytic cycle. More recent experiments using hybridization of transformed-cell RNA against separated strands of intact SV40 or polyoma DNA ( 1 83, 1 87, 1 88, 1 99, 375, 376) or restriction endonuclease fragments ( 1 88, 377) have shown that in different transformed cell lines, different percentages of the viral genome are expressed. In the SV40 cell lines examined (376, 377), from 30% to 80% of the early strand was transcribed. The majority of the RNA hybridized to the E strand of Hin d-A, -H, -I, and -B; therefore, the SV40 sequences expressed in these



511

transformed cell lines are similar if not identical to those expressed during the early lytic cycle. Less abundant RNA species are also found that hybri dize to the regions of the minus-DNA strand contiguous with the early-gene region. In some transformed lines, RNA complementary to the L strand was also found, but in very low concentrations. Kamen et al ( 1 88) reached similar conclusions from studies of the RNA found in polyoma-transformed cells. As with SV40-transformed cells, a different percentage of the E viral strand (between 50% and 80%) is transcribed in different cell lines. This includes transcription from Hpa II-5 through the initial two thirds of the early region and ending somewhere in Hpa l I-2. These results suggest that expression of the transformed phenotype re quires the presence of RNA coded for by the early-gene region of SV40 and polyoma DNA. The results are consistent with the expression of the early antigens T, U, and TrAg in transformed cells and with the experiments showing the need for the tsA -gene protein to maintain transformation. Transformation by DNA Fragments

Abrahams & van der Eb (61 ) have shown that rat and mouse cells can be transformed by SV40 DNA by the calcium technique. More recently, linear SV40 DNA molecules of genome length and DNA fragments smaller than genome length were prepared with restriction enzymes and tested for trans forming activity on rat cells (62). The linear molecules of unit length, prepared with EcoRI, Bam I, or HpaII, could all transform cells with the same efficiency of SV40 DNA I. A 74% fragment produced from cleavage of the genome with EcoRI and Hpall (or Hpa I) included that region of the genome between 0.0 and 0.74 map units and could transform cells. A 59% fragment extending from 0. 1 5 to 0.74 map units (Bam I!Hpa I) could also transform cells. The fragments produced by Hpa I have no transform ing capability, probably since one of the cleavage sites is in the early-gene region. Since the DNA fragments able to transform cells all contained the early-gene region intact, this further substantiates the need for expression of this region in transformation. PAPOVAVIRUSES AS VEHICLES FOR PROPAGATING FOREIGN DNA IN EUKARYOTIC CELLS

Specific segments of eukaryotic and prokaryotic DNA have been isolated by methods used for cloning DNA in E. coli. These methods involve the in vitro construction of recombinant DNA molecules between a specific eukaryotic or prokaryotic DNA segment and a replicon from E. coli such as a plasmid DNA of a bacteriophage A genome (4 10). When the hybrid


512

FAREED & DAVOLI

molecules are introduced into bacterial cells, their replication is mediated by the E. cali replicon portion of the hybrid. In this way it has been possible to prepare large quantities of defined segments of DNA from both prokar yotic and eukaryotic sources in E coli. This permits study of the structure of such DNA in detail and allows for the genetic manipulation of these sequences in vitro, for example, by introduction of mutations into specific regions. Methods analogous to those employed for cloning DNA in E. coli have been developed to propagate foreign DNA in eukaryotic cells. Because the initiation site of SV40 is the only cis-required function for DNA replication, a segment of viral DNA containing the origin of replication will be able to replicate in the presence of a helper SV40 DNA molecule, which provides the necessary trans function(s). Any foreign DNA that is covalently joined to the viral segment containing the origin should also be replicated in monkey cells. The propagation of such hybrid molecules in monkey cells should make it possible to study the requirements for the expression of genes in mammalian cells such as from eukaryotic DNAs that have been previously cloned in E. coli. In addition, the ability of SV40 to integrate into the chromosomal DNA may allow the expression of the genes it contains. The first hybrid recombinant DNA containing SV40 DNA was constructed by Jackson, Symons & Berg (4 1 1 ). This hybrid contained Adv- Gal linked at the EcaR! cleavage site of SV40 DNA. Subsequently, Ganem et al (4 1 2) and Nussbaum et al (4 13) constructed and propagated SV40 segments carrying well-defined sequences of bacterio phage A DNA. The SV40 DNA used as the vectors in these studies was derived from reiteration mutants of SV40 DNA. As previously shown, the monomer fragments of such reiteration mutants contain the initiation site for viral DNA replication and can be isolated in vitro by restriction nuclease cleavage of the parent reiterant. For example, a hybrid SV40-A. DNA molecule was constructed between a segment of A. DNA and a segment of the monomer fragment of a triplication mutant of SV40. The A segment that was used contained the leftward operator-promoter region of A and was isolated by cleavage of the A genome with restriction enzymes followed by purification of the desired A segment by gel electrophoresis. The A. fragment used was 2400 base pairs long and contained one Bam I and one Hin dlIl cohesive termini. The SV40 segment, which was 940 base pairs long and contained the origin of replication but no SV40 genes, was isolated after cleavage of the triplication mutant with Bam I and Eco R I The A segment and the SV40 segment were then joined in vitro through their Bam cohesive termini with ligase and used to infect monkey kidney cells in the presence of a wild-type SV40 helper genome. Circularization and replica tion of the SV40-A. hybrid DNA molecule was observed, as well as its .



513

encapsulation into progeny SV40 virions. After partial cloning of this hy brid by infectious-center plaquing, the structure of the hybrid was studied in detail (4 13, 4 1 4). The presence of A DNA sequences in the hybrid DNA was demonstrated by its specific affinity when denatured to anneal to single stranded A DNA immobilized on filters, and by its specific affinity for A repressor. Studies with bacterial restriction endonucleases and electron mi croscopic heteroduplex analysis showed that the A sequences in the hybrid were not substantially rearranged. A second A-SV40 hybrid, constructed using an SV40 vector from a different reiteration mutant and a different segment of A DNA, was also propagated in monkey cells (4 1 2). Structural studies of this hybrid detected no rearrangements of the A DNA segment. These results, then, demonstrated that defective SV40 replicons could be used to propagate foreign DNA in mammalian cells. Hamer et al (4 1 5) have used similar methods to construct an SV40 segment carrying a bacterial suppressor gene. The SV40 vehicle used was a 3700 base-pair fragment of wild-type SV40 DNA produced by cleavage of the genome with EeoRI and Hpa ll and included the region from 0.0 to 0.74 SV40 map units. This part of the genome contains all of the sequences included in the early-gene region of SV40 in addition to the origin of replication. It lacks the sequences necessary for the expression of the late genes and, therefore, a recombinant made with this virus is defective. It can be propagated as virus only if bite genes are supplied by a helper genome. This fragment of SV40 was joined in vitro to a fragment of E. eoli DNA carrying the bacterial suppressor gene su+III, which specifies the tRNA that translates the amber codon VAG as tyrosine. After coinfection of monkey kidney cells with the SV40-su+III molecules and a tsA mutant of SV40 at the nonpermissive temperature, which supplies the late-gene func tion necessary for virus production, the recombinant DNA was replicated and encapsulated into virions. Structural studies of the hybrid showed that the E. coli DNA suffered no observable sequence alterations. In addition, monkey cells infected with the SV40-su+III virus transcribed the bacterial DNA fragment, but did not produce functional suppressor tRNA (4 1 5). Since the SV40 vehicle used in the formation of this hybrid contains all the information necessary for transformation of nonpermissive cells, it has been possible to obtain SV40-transformed rat cell lines carrying the bacterial DNA linked to the SV40 vehicle (4 1 6). Goff & Berg (41 7) have constructed an SV40 recombinant in which a fragment of bacteriophage A DNA is inserted, by poly(dA-dT) joints into the viral late-gene region. The A se quences replaced the SV40 segment between 0.74 and 0. 1 5 of the late-gene region. In contrast to findings with the SV40-su+III hybrid, no detectable levels of A-specific mRNA were present in monkey cells infected with this SV40-A hybrid.

5 14

FAREED & DAVOLI


PAPILLOMA VIRUSES

Papilloma viruses have been isolated from many different species (human, rabbit, bovine, hamster, canine) and usually cause benign skin papillomas or warts in their natural host ( 1). The papilloma viruses from different species are different in that there is no detectable sereological cross-reaction between virus particles, the base compositions of the DNAs are different, and there is no cross-hybridization between DNAs. The papilloma viruses are larger (55 mm) than the viruses of the polyoma group. Their DNAs have a molecular weight of about 5 X 106 and are in the form of a covalently closed, circular molecule. Since there is no satisfactory tissue-culture system for the analysis of either productive infection or of transformation by papil loma virus, little is known about the molecular biology of these viruses apart from their structure (41 8, 4 1 9). Recently, two groups (420, 42 1 ) have physically mapped human papil loma viral DNA using bacterial restriction endonucleases. The size of the genome by electron microscopy and by gel electrophoresis is 4.8-4.9 X 106 daltons. Gissmann & Zur Hausen (42 1) found that there is some heterogeneity within virus purified from histologically similar tissue as shown by the presence of additional restriction-enzyme cleavage sites in different viral preparations. Furthermore, differences in the epidemiological pattern of specific warts and nucleic acid hybridization tests with papilloma viral DNA from plantar wart and condyloma acuminata or laryngeal papil lomas (422) are consistent with the existence of various types of human papilloma virus. In accord with this prediction, these investigators have identified a new human papilloma virus that predominates in warts with rather low particle production (423). ACKNOWLEDGMENTS

The research work cited from the authors' laboratories was supported in part by Grants 5ROl CA- 1 4885 and 7RO l CA-20794 from the US Public Health Service. G. C. F. was the recipient of USPHS Research Career Program Award K04 CA-00057, and D. D. was a USPHS predoctoral trainee.


515


Literature Cited 1 . Tooze, J. 1 973. In The Molecular Biology o/ Tumor Viruses, ed. J. Tooze, p. 269. New York: Cold Spring Harbor Laboratory 2. Black, P. H., Rowe, W. P., Turner, H. C., Huebner, R. J. 1 963. Proc. Natl. Acad. Sci. USA 50: 1 148 3. Pope, J. H., Rowe, W. P. 1 964. J. Exp. Med. 1 20: 1 2 1 4 . Rapp, F., Kitahara, T., Butel, J. S., Melnick, J. L. 1 964. Proc. Natl. A cad. Sci. USA 52: 1 1 3 8 5. Gilden, R V . , Carp, R. I., Taguchi, F., Defendi, V. 1 965. Proc. Natl. Acad. Sci. USA 53 :684 6. Habel, K. 1 965. Virology 25:55 7. Takemoto, K. K., Habel, K. 1 966. Virology 30:20 8. Hoggan, M. D. , Rowe, W. P., Black, P. H., Huebner, R. J. 1 965. Proc. Natl. Acad. Sci. USA 53: 1 2 9. Fogel, M., Gilden, R , Defendi, V. 1 967. Proc. Soc. Exp. BioI. Med. 24: 1 047

10. Del Villano, B. c., Defendi, V. 1 973. Virology 5 1 :34 1 1 . Tegtmeyer, P. 1 975. Cold Spring Har bor Symp. Quant. Bioi. 39:9 12. Ahmad-Zadeh, C., Allet, B., Green blatt, J., Weil, R. 1 976. Proc. Natl. Acad. Sci. USA 73: 1097 1 3 . Takemoto, K. K . • Mullarkey, M . F. 1 973. J. Virol. 1 2:625 14. Lewis, A. M. Jr., Rowe, W. P. 1 97 1 . J. Virol. 7: 1 89 1 5. Habel, K. 1 96 1 . Proc. Soc. Exp. Bioi. Med. 1 06:722 1 6. Sjogen, H. 0., Hellstrom, I., Klein, G. 1 9 6 1 . Cancer Res. 2 1 :329 17. Defendi, V. 1 963. Proc. Soc. Exp. Bioi. Med. 1 1 3 : 1 2 1 8. Habel, K . , Eddy, B. E . 1 965. Proc. Soc. Exp. Bioi. Med. 1 1 3 : I 1 9 . Khera, K. S., Ashkenazi, A., Rapp, F., Melnick, J. L. 1 963. J. Immunol. 9 1 :604 20. Koch, M. A., Sobin, A. B. 1 963. Proc. Soc. Exp. Bioi. Med. 1 1 3:4 2 1 . Girardi, A. I., Defendi, V. 1 970. Virology 42:688 22. Mayor, H. D., Stinebaugh, S. E., Jami son, R .M., Jordan, L. E., Melnick, J. L. 1 962. Exp. Mol. Pathol. 1 :397 23. Melnick, J. L., Stinebaugh, S. E., Rapp, F. 1 964. J. Exp. Med. 1 1 9:3 1 3 24. Ozer, H . L., Tegtmeyer, P . 1 972. J.

Virol. 9:52

25. Anderer, F. A., Schlumberger, H. D., Koch, M. A., Frank, H., Eggers, H. I. 1 967. Virology 32:5 1 1 26. Fine, R., Mass, M., Murakami, W. T. 1 968. J. Mol. Bioi. 3 6 : 1 67

27. Girard, M., Marty, L., Suarez, F. 1 970. Biochem. Biophys. Res. Commun, 40:97 28. Barban, S., Goor, R. S. 1 97 1 . J. Virol. 7 : 1 98 29. Estes, M. K., Huang, E., Pagano, J. 1 97 1 . J. Virol. 7:635 30. Hirt, B., Gesteland, R. F. 1 97 1 . Lepetit Colloq. BioI. Med. 2:98 3 1 . Roblin, R., Harle, E., Dulbecco, R. 1 9 7 1 . Virology 45:555 32. Frearson, P. M., Crawford, L. V. 1 972. J. Gen. Virol. 14: 1 4 1 3 3 . Friedmann, T., David, D. 1 972. J. Virol. 10:776 34. Huang, E. S., Nonoyama, M., Pagano, J. S. 1 972. J. Virol. 9:930 35. Lake, R. S., Barban, S., Salzman, N. P. 1 973. Biochem. Biophys. Res. Commun, 54:640 36. Fey, G., Hirt, B. 1 975. Cold Spring Harbor Symp. Quant. Bioi. 39:235 37. Gibson, W. 1 974. Virology 62:3 1 9 38. Gibson, W . 1 975. Virology 68:539 39. Hewick, R. M., Fried, M., Waterfield, M. D. 1 975. Virology 66:408 40. Pett, D. M., Estes, M. K., Pagano, J. S. 1 975. J. Virol. 1 5 :379 4 1 . Levine, A. J. 1 974. Progr. Med. Virol. 17:1 42. Dulbecco, R., Vogt, M . 1 963. Proc. Natl. Acad. Sci. USA 50:236 43. Bauer, W., Vinograd, J. 1 968. J. Mol. BioI. 3 3 : 1 4 1 44 . Salzman, N. P . , Khoury, G. 1 974. In Comprehensive Virology, ed. H. Fraen kel-Conrad, R. R. Wagner, 3:63. New York: Plenum 45. Dugaiczyk, A., Boyer, H. W., Good man, H. M. 1975. J. Mol. Bioi. 96:7 1 46. Weil, R., Vinograd, J. 1963. Proc. Natl. Acad. Sci. USA 50:730 47. Chen, M. C. Y., Chang, K. S. S., Salz man, N. P. 1 975. J. ViroL 1 5 : 1 9 1 48. Howley, P . M., Khoury, G., Bryne, J. c., Takemoto, K. K., Martin, M. A. 1 975. J. Virol. 1 6:959 49. Osborn, J. E., Robertson, S. M., Pad gett, B. L., ZuRhein, G. M., Walker, D. L., Weisblum, B. 1 974. J. Virol. 1 3 :6 1 4 50. Howley, P . M . , Khoury, G., Takemoto, K. K., Martin, M. A. 1 976. Virology 73:303 5 1 . Michel, M. R., Hirt, B., Weil, R 1 967. Proc. Natl. Acad. Sci. USA 58: 1 3 8 1 52. Winocour, E . 1 968. Virology 34:571 53. Ritzi, E., Levine, A. J. 1 969. J. Virol. 5:686 54. Levine, A. J., Teresky, A. K. 1 970. J. Virol. 5:45 1 55. Trilling, D. M., Axelrod, D. 1 972. Virology 47:360


516

FAREED & DAVOLI

56. Kashmiri, S. V. S., Aposhian, H. V. 1 974. Proc. Natl. Acad. Sci. USA 7 1 ;3834 57. McCutchan, J., Pagano, J. S. 1 968. J. Natl. Cancer Inst. 4 1 :35 1 58. Mertz, J. E., Davis, R. W. 1 972. Proc. Natl. Acad. Sci. USA 69:3370 59. Aaronson, S. A., Todaro, G. J. 1 969. Science 1 66:390 60. Aaronson, S. A., Martin, M. A. 1 970. Virology 42:848 6 1 . Abrahams, P. J., van der Eb, A. J. 1 975. J. Virol. 1 6:206 62. Abrahams, P. J., Mulder, C., Van de Voorde, A., Warnaar, S. 0., van der Eb, A. J. 1 975. 1. Virol. 1 6: 8 1 8 63. Vinograd, J., Lebowitz, J . 1 966. J. Gen. Physiol. 49:103 64. Vinograd, J., Lebowitz, J., Radloff, R., Watson, R., Laipis, P. 1 965. Proc. Natl. Acad. Sci. USA 5 3 : 1 104 65. Vinograd, J., Lebowitz, 1., Watson, R. 1 968. J. Mol. Bioi. 33: 1 73 66. Opschoor, A., Pouwels, P. H., Knijnen burg, C. M., Aten, J. B. T. 1 968. 1. Mol. Bioi. 37: 1 3 67. Germond, J . E., Hirt, B . , Oudet, P., Gross-Bellard, M., Cham bon, P. 1 975. Proc. Natl. Acad. Sci. USA 72: 1 843 68. Griffith, J. 1 975. Science 1 87: 1 202 69. Fearson, P. M., Crawford, L. V. 1 972. 1. Gen. Virol. 1 4 : 1 4 1 70. Lake, R. S . , Barban, S . , Salzman, N. P. 1 973. Biochem. Biophys. Res. Commun. 54:640 7 1 . Fey, G., Hirt, B. 1 975. Cold Spring Har bor Symp. Quant. Bioi. 39:235 72. Louie, A. J. 1 975. Cold Spring Harbor Symp. Quant. Bioi. 39:259 73. Pelt, D. M., Estes, M. K., Pagano, J. S. 1 975. J. Virol. 1 5 :379 74. Oudet, P., Gross-Bellard, M., Cham bon, P. 1 975. Cell 4:2 8 1 75. Bauer, W . , Vinograd, J. 1974. In Basic Principles in Nucleic Acid Chemistry, ed. P. O. P. T'so, 2:262. New York, London: Academic 76. Crawford, L. V., Waring, M. J. 1 967. J. Mol. Bio!. 25:23 77. Bauer, W., Vinograd, J. 1 968. J. Mol. Bioi. 3 3 : 1 4 1 78. Radloff, R . , Bauer, W . , Vinograd, J . 1 967. Proc. Natl. Acad. SCI: USA 57: 1 5 1 4 79. Champoux, 1. J., Dulbecco, R. 1 972. Proc. Natl. Acad. Sci. USA 69: 1 43 80. Vosberg, H. P., Grossman, L. I., Vino grad, J. 1 974. Fed. Proc. 3 3 : 1 356 (Abstr. 7 5 1 ) 8 1 . Keller, W. 1 975. Proc. Natl. Acad. Sci. USA 72:2550

82. Shure, M., Vinograd, 1. 1 976. Cell 8: 215 83. Keller, W. 1 975. Proc. Nat!' Acad. Sci. USA 72:4876 84. Crick, F. H. C. 1 976. Proc. Nat!. Acad. SCI: USA 73:2639 85. Beard, P., Morrow, J. F., Berg, P. 1 973. J. Virol. 1 2 : 1 303 86. Germond, J. E., Vogt, V. M., Hirt, B. 1 974. Eur. J. Biochem. 43:59 1 87. Morrow, J. F., Berg, P. 1 973. 1. Virol. 1 2 : 1 63 1 88. Reed, S. I., Ferguson, J., Davis, R. W., Stark, G. R. 1 975. Proc. Natl. Acad. Sci. USA 72: 1 605 89. Parodi, A., Rouge!, P., Croissant, 0., Blangy, D., Cuzin, F. 1 975. Cold Spring Harbor Symp. Quant. Bioi. 39:247 90. Yaniv, M., Croissant, 0., Cuzin, F. 1 974. Biochem. Biophys. Res. Commun. 57: 1074 9 1 . Lebowitz, J., Garon, C. F., Chen, M., Salzman, N. P. 1 976. J. Virol. 1 8:205 92. Chen, M., Lebowitz, J., Salzman, N. P. 1 976. 1. Virol. 1 8:2 1 1 93. Westphal, H . 1 970. 1. Mol. Bioi. 50:407 94. Khoury, G., Howley, P. M., Nathans, D., Martin, M. 1 975. J. Virol. 1 5:433 95. Lebowitz, P., Bloodgood, R. 1 975. J. Mol. Bioi. 94: 1 83 96. Hossenlopp, P., Oudet, P., Chambon, P. 1 974. Eur. J. Biochem. 41 :397 97. Mandel, J. L., Chambon, P. 1 974. Eur. J. Biochem. 4 1 :379 98. Mandel, J. L., Chambon, P. 1 974. Eur. J. Biochem. 4 1 :367 99. Anderson, D. M., Folk, W. R. 1 976. Biochemistry 1 5 : 1022 1 00. TUrler, H . 1 975. J. Virol. 1 5 : 1 1 58 1 0 1 . Jaenisch, R., Levine, A. J. 1 97 1 . Virology 44:480 102. Jaenisch, R., Levine, A. 1. 1 97 1 . J Mol. Bioi. 6 1 :735 103. Rush, M. G., Eason, R., Vinograd, J. 1 97 1 . Biochim. Biophys. Acta 228:585 104. Jaenisch, R., Levine, A. J. 1 972. Virology 48:373 105. Bourgaux, P. 1 973. J. Mol. Bioi. 77: 1 97 106. Jaenisch, R., Levine, A. J. 1 973. 1. Mol. Bioi. 73:99 1 07. Cuzin, F., Vogt, M., Dieckmann, M., Berg, P. 1 970. J. Mol. Bio!. 47:3 1 7 108. Vogt, M . , Bacheler, L . T., Boice, L. 1 976. 1. Viral 1 7: 1 009 109. Hirai, K., Defendi, V. 1 972. J. Viral. 9:705 1 1 0. Ralph, R. K., Colter, J. S. 1 972. Virology 48:49 I l l . Holze!, F., Sokol, F. 1 974. J. Mol. Bio!. 84:423 1 1 2. Martin, M. A., Howley, P. M., Byrne, J. C., Garon, C. F. 1 976. Virology 7 1 :2 8


MOLECULAR BIOLOGY OF PAPOVAVIRUSES 1 1 3. Nathans, D., Smith, H . O. 1975. Ann. Rev. Biochem. 44:273 1 14. Danna, K. J., Sack, G. H. Jr., Nathans, D. 1973. J. Mol. BioI. 78:363 1 1 5. Danna, K., Nathans, D. 1 97 1 . Proc. Natl. Acad. Sci. USA 68:29 1 3 1 1 6. Morrow, J . F., Berg, P . 1 972. Proc. Natl. Acad. Sci. USA 69:3365 1 1 7 . Mulder, C, De1ius, H. 1 972. Proc. Natl. Acad. Sci. USA 69:3 2 1 5 1 1 8. Subramanian, K . , Zain, B. S . , Roberts, R. J., Weissman, S. M. 1977. J. Mol. Bioi. In press 1 1 9. Sharp, P. A., Sugden, B., Sambrook, J. 1973. Biochemistry 1 2 : 3055 1 20. Fiers, W., Danna, K. J., Rogiers, R., Van de Voorde, A., van Herreweghe, J., van Heuverswyn, H., Vo1ckaert, G., Yang, R. 1 975. Cold Spring Harbor Symp. Quant. Bioi. 39: 1 79 1 2 1 . Roberts, R. J., Breitmeyer, J. B., Ta bachnik, N. F., Myers, P. A. 1 875. J. Mol. Bioi. 9 1 : 1 2 1 1 22. Dhar, R . , Subramanian, K., Zain, B . S., Pan, 1., Weissman, S. M. 1 975. Cold Spring Harbor Symp. Quant. Bioi. , 39 : 1 53 1 23. Yang, R., Danna, K. J., Van de Voorde, A., Fiers, W. 1 975. Virology 68:260 1 24. Subramanian, K. N., Pan, 1., Zain, S., Weissman, S. M. 1 974. Nucl. Acid. Res. 1 :727 1 25. Van de Voorde, A., Contreras, R., Rogiers, R., Fiers, W. 1 976. Cell 9: 1 1 7 1 26. Lebowitz, P., Siegel, W., Sklar, J. 1 974. 1. Mol. Bioi. 8 8 : 1 05 127. Griffin, B. E., Fried, M., Cowie, A. 1974. Proc. Natl. Acad. Sci. USA 7 1 : 2077 1 28. Griffin, B. E., Fried, M. 1977. Adv. Can cer Res. In press 1 29. Chen, M., Chang, K., Salzman, N. P. 1975. J. Virol. 1 5 : 1 9 1 1 30. Folk, W. R . , Fishel, B. R . , Anderson, D. M. 1975. Virology 64:277 1 3 1 . Griffin, B. E., Fried, M . 1975. Nature 256: 1 7 5 1 32. Summers, J. 1975. J. Virol. 1 5 :946 1 33. Crawford, L. V., Robbins, A. K. 1 976. 1. Gen. Virol. 3 1 :3 1 5 1 34. Nathans, D . , Danna, K . J . 1972. J. Mol. BioI. 64: 5 1 5 1 35. Huang, E., Newbold, J. E . , Pagano, J . S . 1973. J. Virol. 1 1 :508 1 36. Crawford, L. V., Robbins, A. K., Nic klin, P. M. 1 974. J. Gen. Virol. 25: 1 33 1 37 . Fried, M., Griffin, B. E., Lund, E., Rob berson, D. L. 1975. Cold Spring Harbor Symp. Quant. Bioi. 39:45 1 38. Hirt, B. 1 966. Proc. Natl. Acad. Sci. USA 55:997 1 39. Hirt, B. 1 969. J. Mol. Bioi. 40: 1 4 1

517

140. Levine, A. J . , Kang, H. S . , Billheimer, F. E. 1 970. 1. Mol. Bioi. 50:549 1 4 1 . Jaenisch, R., Mayer, A., Levine, A. 1 9 7 1 . Nature New Bioi. 233:72 142. Sebring, E. D., Kelly, T. J. Jr., Thoren, M. M., Salzman, N. P. 1 97 1 . J. Virol. 8:478 143. Kasamatsu, H., Vinograd, J. 1 974. Ann. Rev. Biochem. 43 :695 144. Danna, K. J., Nathans, D. 1 972. Proc. Natl. Acad. Sci. USA 69:3097 145. Fareed, G. C, Garon, C. F., Salzman, N. P. 1972. J. Viral. 10:484 146. Crawford, L. V., Syrett, C, Wilde, A. 1973. 1. Gen. Viral. 2 1 :5 1 5 147. Robberson, D . L. , Crawford, L . V., Sy rett, C., James, A. W. 1975. J. Gen. Viral. 26:59 148. Fareed, G . C, Salzman, N, P. 1972. Na ture New BioI. 238:274 149. Fareed, G. C, Khoury, G., Salzman, N. P. 1973. J. Mol. Bioi. 77:457 1 50. Magnusson, G. 1 973. J. Viral. 1 2:600 l S I . Salzman, N. P., Thoren, M. M. 1 973. 1. Viral. 1 1 :721 1 52. Laipis, P., Levine, A. J. 1974. Virology 56:580 1 5 3. Magnusson, G., Winnacker, E. L., Eliasson, R., Reichard, P. 1972. 1. Mol. BioI. 72:539 1 54. Winnacker, E. L., Magnusson, G., Reichard, P. 1972. 1. Mol. Bioi. 72:523 I S S. Magnusson, G. 1973. J. Viral. 1 2 :609 1 56. Magnusson, G., Pigiet, V., Winnacker, E. L., Abrams, R., Reichard, P. 1973. Proc. Natl. Acad. Sci. USA 70:4 1 2 1 57. Pigiet, V . , Winnacker, E. L . , Eliasson, R., Reichard, P. 1 973. Nature New Bioi. 245:203 1 5 8. Eliasson, R., Martin, R., Reichard, P. 1974. Biochim. Biophys. Acta 59:307 1 59. Francke, B., Hunter, T. 1 974. J. Mol. Bioi. 83:99 1 60. Hunter, T., Francke, B. 1 974. 1. Viral. 1 3 : 1 25 1 6 1 . Hunter, T., Francke, B. 1974. J. Mol. Bioi. 83: 1 23 1 62. Reichard, P., Eliasson, R. Soderman, G. 1 974. Proc. Natl. Acad. Sci. USA 7 1 :490 1 1 63. DePamphilis, M. L., Berg, P. 1 975. 1. Bioi. Chem. 250:4348 1 64. Su, R., DePamphilis, M. L. 1 976. Proc. Natl. Acad. Set: USA 73:3466 1 65. Kaufman, G., Anderson, S., DePam philis, M. L. 1977. In preparation 1 66. Francke, B., Hunter, T. 1975. 1. Viral. 1 5 :759

1 67. Otto, B., Reichard, P. 1 975. 1. Viral. 1 5 :259 1 68. Sebring, E. D., Garon, C. F., Salzman, N. P. 1 974. J. Mol. Bioi. 90:371


518

FAREED & DAVOLI

1 69. Fareed, G. c., McKerlie, 1., Salzman, N. P. 1 973. J. Mol Biol 74:95 1 70. Lai pis, P., Sen, A., Levine, A. J., Mul der, C. 1 975. Virology 69: 1 1 5 1 7 1 . Chen, M. C. Y., Birkenmeier, E., Salz man, N. P. 1 976. J. Virol. 1 7 : 6 1 4 1 72. Brockman, W. W . , Gutai, M. W., Na thans, D. 1 975. Virology 66:36 1 73. Lai, c.-J., Nathans, D. 1 975. J. Mol. BioI. 97: 1 1 3 1 74. Green, M. H., Miller, H. I., Hendler, S. 1 97 1 . Proc. Natl Acad. Sci. USA 68 : 1032 1 75. Polisky, B., McCarthy, B. J. 1 975. Proc. Natl Acad. Sci. USA 72:2895 1 76. Griffith, J., Dieckmann, M., Berg, P. 1 975. J. Virol. 1 5 : 1 67 1 77. Kasamatsu, H., Wu, M. 1 976. Proc. Natl Acad. Sci. USA 73 : 1 945 1 78. Gariglio, P., Mousset, S. 1 975. FEES Lett. 56: 1 49 1 79. Aloni, Y., Winocour, E., Sachs, 1. 1 968. J. Mol. Biol 3 1 :4 1 5 1 80. Oda, K., Dulbecco, R . 1 968. Proc. Natl. Acad. Sci. USA 60:525 1 8 1 . Tonegawa, S., Walter, G., Bernardini, A., Dulbecco, R. 1 970. Cold Spring Harbor Symp. Quant. BioI. 3 5 : 823 1 82. Weil, R., Salomon, c., May, E., May, P. 1 974. In Viruses, Evolution and Can cer, ed. E. Kurstak, K. Marmorosch, p. 455. New York: Academic 1 83. Beard, P., Acheson, N. H., Maxwell, I. H. 1 976. J. Virol. 1 7:20 1 84. Weinberg, R. A., Warnaar, S. 0., Wino cour, E. 1 972. J. Virol 10: 1 93 1 85. Weil, R., Salomon, C., May, E., May, P. 1 974. Cold Spring Harbor Symp. Quant. BioI. 39:381 1 86. Lindstrom, D. M., Dulbecco, R. 1 972. Proc. Natl. Acad. Sci. USA 69: 1 5 1 7 1 87. Sambrook, J., Sharp, P . A., Keller, W. 1 972. J. Mol. Bioi. 70:57 1 88. Kamen, R., Lindstrom, D. M., Shure, H., Old, R. W. 1 975. Cold Spring Har bor Symp. Quant. BioI. 39: 1 87 1 89. Khoury, G., Howley, P. M., Brown, M . , Martin, M. A. 1975. Cold Spring Harbor Symp. Quant. Bioi. 39: 147 1 90. Weinberg, R. A., Ben-Ishai, Z., New bold, J. E. 1 974. J. Virol. 1 3 : 1 263 1 9 1 . Khoury, G., Martin, M. A., Lee, T. N. H., Danna, K. J., Nathans, D. 1 973. J. Mol. BioI. 78:377 1 92. Sambrook, J., Sugden, B., Keller, W., Sharp, P. A. 1 973. Proc. Natl Acad. Sci. USA 70:37 1 1 1 9 3 . Kamen, R., Shure, H. 1 976. Cell 7:361 1 94. Acheson, N. H., Buetti, E., Scherrer, K., Wei1, R. 1 9 7 1 . Proc. Natl. Acad. Sci. USA 68:223 1

1 95. Aloni, Y. 1 972. Proc. Natl Acad. Sci. USA 69:2404 1 96. Aloni, Y. 1 975. Cold Spring Harbor Symp. Quant. Bioi. 39: 1 65 1 97. Laub, 0. , Aloni, Y. 1 975. J. Viral. 1 6: 1 1 7 1 1 98. Buetti, E . 1 974. J. Virol. 14:249 1 99. Khoury, G., Byrne, J. c., Martin, M. A. 1 972. Proc. Natl. Acad. Sci. USA 69: 1 925 200. Khoury, G., Carter, B. J., Ferdinand, F. J., Howley, P. M . , Brown, M., Martin, M. A. 1 976. J. Virol 1 7 :832 20 1 . May, E., Kopecka, H., May, P. 1 975. Nucl Acid Res. 2: 1 995 202. Lavi, S., Shatkin, A. 1 975. Proc. Natl. Acad. Sci. USA 72:20 1 2 203. Aloni, Y. 1 975. FEBS Lett. 54:363 204. Ferguson, J., Davis, R. W. 1 975. J. Mol. BioI. 94: 1 35 205. Shah, K. Y., Ozer, H. 1., Ghazey, H. N., Kelly, T. J. Jr. 1 977. J. Viral. 2 1 : 1 79 206. Lai, C.-J., Nathans, D. 1 976. Virology 75:335 207. Prives, ' C. 1., Aviv, H., Gilboa, E., Revel, M . , Winocour, E. 1 975. Cold Spring Harbor Symp. Quant. BioI. 39:309 208. Weiner, 1. P., Herndon, R. M . , Narayan, 0., Johnson, R. T . , Shaw, K . , Rubinstein, 1. J., Preziosi, T . J., Con ley, F. K. 1972. N. Engl. J. Med. 286:385 209. Weiner, 1. P., Herndon, R. M., Narayan, 0. , Johnson, R. T. 1 972. J. Virol. 10: 147 2 10. Padgett, B. 1., Walker, D. 1., ZuRhein, G. M., Eckroade, R. 1., Dessel, B. H. 1 97 1 . Lancet 1 : 1 257 2 1 1 . Gardner, S. D., Field, A. M . , Coleman, D. Y., Hulme, B. 1 97 1 . Lancet 1 : 1 253 2 1 2. Takemoto, K. K., Rabson, A. S., Mul larkey, M. F., Blease, R. M., Garon, C. F., Nelson, D. 1 974. J. Natl Cal/cer II/st. 53: 1 205 2 1 3 . Sack, G. H. Jr., Narayan, 0., Danna, K. J., Weiner, 1. P., Nathans, D. 1 973. Virology 51 :345 2 14. Gardner, S. D. 1 973. Br. Med. J. 1 :77 2 1 5. Padgett, B. 1., Walker, D. 1. 1 973. J. Infect. Dis. 1 27:467 2 1 6. Osborn, J. E., Robertson, S. M., Pad gett, B. 1., ZuRhein, G. M., Walker, D. 1., Weisblum, B. 1 974. J. Virol. 1 3 :614 2 1 7. Penney, J. B., Narayan, O. 1 973. II/feet. Immun. 8:299 2 1 8. Fareed, G. C., Takemoto, K. K., Gim brone, M. A. 1 977. In preparation 2 1 9. Howley, P. M., Khoury, G., Takemoto, K. K., Martin, M. A. 1 976. Virology 73:303


MOLECULAR BIOLOGY OF PAPOVAVIRUSES 220. Osborn, J. E., Robertson, S. M., Pad gett, B. 1., Walker, D. 1., Weisblum, B. 1 976. J. Viral. 1 9:675 2 2 1 . Howley, P. M., Mullarkey, M . F., Takemoto, K. K., Martin, M. A. 1 975. J. Virol. 1 5 : 1 73 222. Howley, P. M., Khoury, G., Bryne, J. C., Takemoto, K. K., Martin, M. A. 1 975. J. Virol. 16:959 223. Khoury, G., Howley, P. M., Garon, C., Mullarkey, M. F., Takemoto, K. K., Martin, M. A. 1 975. Proc. Natl. Acad. Sci. USA 72:2563 224. Newell, N., Kelly T. J. Jr. 1 977. In preparation 225. Fiori, M., DiMayorca, G. 1 976. Proc. Natl. Acad. Sci. USA 73 :4662 226. Eckhart, W. 1 974. Ann. Rev. Genet. 8:30 1 - 1 7 227. DiMayorca, G . , Callender, J . , Marin, G., Giordano, R. 1 969. Virology 38: 1 26 228. Eckhart, W. 1 969. Virology 38: 1 20 229. Fried, M. 1 970. Virology 40:605 230. Kit, S., Tokuno, K., Nakajima, K., Trkula, D., Dubbs, D. R. 1 970. J. Viral. 6:286 23 1 . Tegtmeyer, P., Dohan, C. Jr., Rez nikoff, C 1 970. Proc. Nat!. Acad. Sci. USA 66:745 232. Tegtmeyer, P., Ozer, H. 1. 1 9 7 1 . J. Virol. 8 : 5 1 6 2 3 3 . Kimura, G . , Dulbecco, R. 1 972. Vir ology 49:394 234. Robb, J. A., Martin, R. G. 1 972. J. Virol. 9:956 235. Robb, 1. A., Smith, H. S., Scher, C D. 1 972. J. Virol. 9:969 236. Tegtmeyer, P. 1 972. J. Virol. 10:591 237. Kimura, G., Dulbecco, R. 1 973. Virology 52:529 238. Robb, J. A. 1 973. J. Virol. 1 2: 1 1 87 239. Chou, J. Y., Martin, R. G. 1 974. J. Virol. 1 3 : 1 1 0 1 240. Dubbs, D . R . , Rachmeier, M., Kit, S . 1 974. Virology 5 7 : 1 6 1 24 1 . Robb, J . A . , Tegtmeyer, P . , Ishikawa, A., Ozer, H. 1. 1 974. J. Virol. 1 3 :662 242. Chou, J. Y., Martin, R. G. 1 975. J. Viral. 1 5 : 1 2 7 243. Chou, J. Y., Avila, J., Martin, R. G. 1 974. J. Virol. 1 4: 1 1 6 244. Cowan, K., Tegtmeyer, P., Anthony, D. D. 1 973. Proc. Natl. Acad. Sci. USA 70: 1 927 245. Oxman, M. N., Takemoto, K. K . , Eck hart, W. 1 972. Virology 49:675 246. Osborn, M. J., Weber, K. 1 975. Cold Spring Harbor Symp. Quant. Bioi. 39:267 247. Alwine, J. C., Reed, S. I., Ferguson, J., Stark, G. R. 1 975. Cell 6:529

519

248. Tenen, D. G., Baygell, P., Livingston, D. M. 1 975. Proc. Natl. A cad. Sci. USA 72:43 5 1 249. Kuchino, T . , Yamaguchi, N. 1 975. J. Virol. 1 5 : 1 302 250. Paulin, D., Cuzin, F. 1975. J. Virol. 1 5 :393 25 1 . Dulbecco, R., Eckhart, W. 1 970. Proc. Natl. Acad. Sci. USA 67: 1 775 252. Francke, B., Eckhart, W. 1 973. Virology 55: 1 27 253. Brugge, J. S., Butel, J. S. 1 975. J. Virol. 1 5 :6 1 9 254. Tegtmeyer, P. 1 975. J. Virol. 1 5 : 6 1 3 255. Osborn, M . J . , Weber, K . 1 975. J. Virol. 1 5 :636 256. Martin, R. G., Chou, J. Y. 1975. J. Virol. 1 5 :599 257. Kimura, G., Itagaki, A. 1 975. Proc. Natl. Acad. Sci. USA 72:673 258. Chou, J. Y. 1 976. J. Virol. 20:39 259. Eckhart, W., Du1becco, R. 1 974. Virology 60:359 260. Benjamin, T. 1. 1 970. Proc. Nat!. Acad. Sd USA 67:394 26 1 . Goldman, E., Benjamin, T. L. 1 975. Virology 66:372 262. Schaffhausen, B. S., Benjamin, T. 1. 1 976. Proc. Natl. Acad. Sci. USA 7 3 : 1 092 263. Lai, C, Nathans, D. 1 974. Virology 60:466 264. Lai, C., Nathans, D. 1 975. Virology 66:70 265. Mantel, N., Boyer, H . W., Goodman, H. M. 1 975. J. Virol. 1 6:754 266. Miller, L. K., Fried, M. 1 976. J. Virol. 1 8 :824 267. Feunteun, J., Sompayrac, 1., Fluck, M . , Benjamin, T . 1. 1 976. Proc. Natl. Acad. Sci. USA 73:4 1 69 268. Shenk, T. E., Rhodes, C D. Jr., Rigby, P. W. J., Berg, P. 1 975. Proc. Natl. Acad. Sci. USA 72:989 269. Bartok, K., Fraser, M. 1., Fareed, G. C. 1 974. Biochem. Biophys. Res. Commun. 60:507 270. O'Conor, G. T., Rabson, A. S., Malmgren, R. A., Berezesky, I. K., Paul, F. J. 1 965. J. Natl. Cancer Inst. 34:679 27 1 . Friedman, M. P., Lyons, M. J., Gins berg, H. S. 1 970. J. Viral. 5:586 272. Hashimoto, K., Nakajima, K., Oda, K., Shimojo, H. 1 973. J. Mol. Bioi. 81 :207 273. Jerkofsky, M., Rapp, F. 1 973. Virology 5 1 :466 274. Nakajima, K., Ishitsuka, H., Oda, K. 1 974. Nature 252:649 275. Eron, L., Westphal, H., Khoury, G. 1 975. J. Viral. 1 5 : 1 256


520

FAREED & DAVOLI

276. Jerkofsky, M . , Rapp, F. 1 975. J. Viral. 1 5 :253 277. Nakajima, K., Oda, K. 1 975. Virology 67:85 278. Hartley, J. W., Huebner, R. J., Rowe, W. P. 1 956. Proc. Soc. Exp. Biol Med. 92:667 279. Huebner, R. J. 1964. Proc. Natl. Acad. Sci. USA 52: 1 333 280. Rapp, F., Melnick, J. L., Butel, J. S., Kitahara, T. 1 964. Proc. Natl. Acad. Sci. USA 5 2 : 1 348 28 1 . Rowe, W. P., Baum, S. G. 1 964. Proc. Natl. Acad. Sci. USA 52: 1 340 282. Rowe, W. P., Baum, S. G. 1 965. J. Exp. Med. 1 22:955 283. Lebowitz, P., Khoury, G. 1 974. J. Viral. 15:1214 284. Kelly, T . J . Jr., Rose, J. A . 1 97 1 . Proc. Natl. Acad. Sci. USA 68: 1 037 285. Kelly, T. J. Jr. 1 975. J. Viral. 1 5 : 1 267 286. Lewis, A. M. Jr., Levin, M. J., Wiese, W. H . , Crumpacker, C. S., Henry, P. H. 1 969. Proc. Natl. Acad. Sci. USA 63: 1 1 2 8 287. Crumpacker, C . S . , Levin, M. J., Wiese, W. H., Lewis, A. M. Jr., Rowe, W. P. 1 9 70. J. Viral 6:788 288. Lewis, A. M. Jr., Rowe, W. P. 1 970. J. Viral 5:4 1 3 289. Kelly, T . J. Jr., Lewis, A . M . Jr., Le vine, A. S., Siegel, S. 1 974. J. Mol. BioI. 89: 1 1 3 290. Lewis, A. M . Jr., Levine, A. S., Crum packer, C. S., Levin, M. J., Samaha, R. J., Henry, P. H. 1 973. J. Viral. 1 1 :655 29 1 . Walter, G., Martin, H. 1976. J. Viral 1 6 : 1 236 292. Henry, P. H., Schnipper, L. E., Samaha, R. J., Crumpacker, C. S., Lewis, A. M. Jr., Levine, A. S. 1 973. Virology 1 1 :665 293. Kelly, T. J. Jr., Lewis, A. M. 1 97 3 . J. Viral. 1 2:643 294. Khoury, G., Lewis, A. M. Jr., Oxman, M. N., Levine, A. S. 1973. Nature New BioI. 246:202 295. Levine, A. S., Levin, M. J., Oxman, M. N., Lewis, A. M. Jr. 1 973. J. Viral 1 1 :672 296. Deppert, W., Walter, G. 1 976. Proc. Natl Acad. Sci. USA 73:2505 297. Morrow, J. F., Berg, P., Kelly, T. J. Jr., Lewis, A. M. Jr. 1973. J. Viral 1 2 :653 298. Lebowitz, P., Kelly, T. J. Jr., Nathans, D . , Lee, T. N. H., Lewis, A. M. Jr. 1 974. Proc. Natl Acad. Sci USA 7 1 :441 299. Grodzicker, T., Lewis, J. B., Anderson, C. W. 1 976. J. Viral. 1 9:559 300. Sambrook, J., Hassell, J., Zain, S., Lukanidin, E. 1 977. In preparation 30 1 . Uchida, S., Watanabe, S., Kato, M. 1 966. Virology 27: 1 35

302. Thorne, H. V., Evans, J., Warden, D. 1 968. Nature 2 1 9:728 303. Uchida, S., Yoshiike, K., Watanabe, S., Furuno, A. 1968. Virology 34: I 304. Yoshiike, K. 1968. Virology 34:391 305. Blackstein, M. E., Stanners, C. P., Far milo, A. J. 1969. J. Mol. BioI. 42:301 306. Tai, H. T., Smith, C. A., Sharp, P. A., Vinograd, J. 1 972. J. Viral. 9:3 1 7 307. Brockman, W. W., Lee, T. N . H., Na thans, D. 1 973. Virology 54:384 308. Mertz, J. E., Carbon, J., Herzberg, M . , Davis, R. W., Berg, P. 1 975. Cold Spring Harbor Symp. Quant. BioI. 39:69 309. Risser, R., Mulder, C. 1 974. Virology 58 :424 3 1 0. Scott, W. A., Brockman, W. W., Na thans, D. 1 976. Virology 75:3 1 9 3 1 1 . Lavi, S . , Winocour, E. 1 972. J. Viral. 9:309 3 1 2. Lavi, S., Winocour, E. 1 974. Virology 57:296 3 1 3. Brockman, W. W., Nathans, D. 1 974. Proc. Natl. A cad. Sci. USA 71 :942 3 1 4. Fareed, G. c., Bryne, J. c., Martin, M. A. 1974. J. Mol Bioi. 87:275 3 1 5. Folk, W. R., Wang, H. E. 1 974. Virology 6 1 : 140 3 1 6. Fried, M. 1 974. J. Viral. 1 3:939 3 1 7. Khoury, G., Fareed, G. c., Berry, K., Martin, M. A., Lee, T. N. H., Nathans, D. 1 974. J. Mol. BioI. 87:289 3 1 8. Mertz, J. E., Berg, P. 1 974. Proc. Natl. Acad. Sci. USA 7 1 :4879 3 1 9. Mertz, J. E., Berg, P. 1 974. Virology 62: 1 1 2 320. Robberson, D. L., Fried, M. 1974. Proc. Natl. Acad. Sci. USA 7 1 :3497 32 1 . Lee, T. N. H., Brockman, W. W., Na thans, D. 1975. Virology 66:53 322. Folk, W. R., Fishel, B. R. 1 975. Virology 64:447 323. Griffin, B. E., Fried, M. 1 975. Nature 256: 1 75 324. Davoli, D., Ganem, D., Nussbaum, A. L., Fareed, G. c., Howley, P. M., Khoury, G., Martin, M. A. 1 977. Virology Vol. 77. In press 325. Rozenblatt, S., Lavi, S., Singer, M. F., Winocour, E. 1 973. J. Viral. 1 2:501 326. Martin, M . A., Ge1b, L. D., Fareed, G. c., Milstein, J. B. 1 973. J. Viral. 1 2:748 327. Ganem, D., Nussbaum, A., Davoli, D., Fareed, G. C. 1 976. J. Mol. BioI. 1 0 1 :57 328. Fried, M., Griffin, B. E. 1 977. Adv. Can cer Res. 24:67 329. Frenkel, N., Lavi, S., Winocour, E. 1 974. Virology 60:9 330. Oren, M., Kuff, E. L., Winocour, E. 1 976. Virology 7 3 :4 1 9 3 3 L Chow, L . T., Boyer, H . W., Tischer, E.


332. 333. 334.


335. 336. 337. 338. 339. 340. 34 1 . 342.

343.

344. 345. 346. 347. 348. 349. 350. 351. 352. 353. 354.

G., Goodman, H. M. 1 975. Cold Spring Harbor Symp. Quant. Bioi. 39: 109 Segal, S., Garner, M., Singer, M. F., Rosenberg, M. 1 976. Cell 9:247 Davoli, D., Fareed, G. C. 1 974. Nature 25 1 : 1 53 Davoli, D., Fareed, G. C. 1 975. Cold Spring Harbor Symp. Quant. BioL 39: 1 2 7 Shenk, T . E . , Berg, P. 1 976. Proc. Nat!. Acad. Sci. USA 73 : 1 5 1 3 Lai, c.-J., Nathans, D . 1 974. 1. Mol. Bioi. 89: 1 79 Mertz, J. E., Davis, R. W. 1 972. Proc. Natl. Acad. Sci. USA 69:3370 Carbon, J., Shenk, T. E., Berg, P. 1 975. Proc. Natl. Acad. Sci. USA 72: 1 392 Shenk, T. E., Carbon, J., Berg, P. 1 976. 1. Viral. 1 8:664 Carbon, J., Shenk, T. E., Berg, P. 1 975. 1. Mol. Bioi. 98: 1 Lodish, H. F., Weinberg, R. A., Ozer, H. L. 1 974. 1. ViraL 1 3:590 Prives, C. L., Aviv, H., Paterson, B. M., Roberts, B. E., Rozenblatt, S., Revel, M., Winocour, E. 1 974. Proc. Natl Acad. ScL USA 7 1 :302 Roberts, B. E., Gorecki, M., Mulligan, R. c., Danna, K. J., Rozenblatt, S., Rich, A. 1 975. Proc. Natl. Acad. Sci. USA 72: 1 922 Rozenblatt, S., Mulligan, R. C., Go recki, M., Roberts, B. E., Rich, A. 1 976. Proc. Nat!. Acad. Sci. USA 73:2747 Wheeler, T., Bayley, S. T., Harvey, R., Crawford, L. V., Smith, A. E. 1 977. 1. Virol. 2 1 :2 1 5 Smith, A. E., Bayley, S. T., Mangel, W. F., Shure, T., Wheeler, T., Kamen, R. I. 1 975. Proc. 10th FEBS Symp. 39; 1 5 1 Graessmann, M., Graessmann, A. 1 976. Proc. Nat!. Acad. Sci. USA 7 3 :366 Graessmann, M., Graessmann, A., Nie bel, J., Koch, H., Fogel, M., Mueller, C. 1975. Nature 258:756 Prives, C., Gilboa, E., Revel, M., Wino cour, E. 1 977. Proc. Nat!. Acad. Sci. USA 74:457 Tegtmeyer, P., Rundell, K., Collins, J. K. 1 977. 1. Viral. 2 1 :647 Anderson, J. L., Martin, R. F., Chang, C., Mora, P. T., Livingston, D. M. 1 977. Virology 76:420 Tegtmeyer, P., Schwartz, M., Collins, J. K., Rundell, K. 1 975. 1. Virol. 1 6; 1 68 Reed, S. I., Stark, G. R., Alwine, J. C. 1 976. Proc. Natl. Acad. Sci. USA In press 73:3083 Rundell, K., Collins, J. K., Tegtmeyer, P., Ozer, H. L., Lai, C. J., Nathans, D. 1 977. 1. Virol. 2 1 :636

52 1

355. Rundell, K., Tegtmeyer, P., Wright, P., DiMayorca, G. 1 977. In preparation 356. Jessel, D., Hudson, J., Landau, T., Tenen, D., Livingston, D. M . 1 975. Proc. Natl. Acad. Sci. USA 72: 1 960 357. Carroll, R. B., Hager, L., Dulbecco, R. 1 974. Proc. Natl. Acad. Sci. USA 7 1 :3754 358. Reed, S. I., Ferguson, J., Davis, R. W., Stark, G. R. 1 975. Proc. Nat!. Acad. Sci. USA 72: 1 605 359. Jessel, D., Landau, T., Hudson, J., Lalor, T., Tenen, D., Livingston, D. M. 1 976. Cell 8:535 360. Dhar, R., Zain, S., Weissman, S. M., Pan, J., Subramanian, K. 1 973. Proc. Natl. Acad. Sci. USA 7 1 :3 7 1 36 1 . Zain, B. S . , Weissman, S . M., Dhar, R., Pan, J. 1 974. Nucleic Acids Res. 1 :577 362. Zain, B. S., Dhar, R., Weissman, S. M., Lebowitz, P., Lewis, A. M. Jr. 1 973. 1. Virol. 1 1 ;682 363. Subramanian, K. N., Dhar, R., Weiss man, S. M. 1 977. 1. Bioi. Chem. 252: 333 364. Jay, E., Roychoudhury, R., Wu, R. 1 976. Biochem. Biophys. Res. Commun. 69:678 365. Dhar, R., Subramanian, K., Pan, J., Weissman, S. M. 1 977. 1. Bioi. Chem. 252:368 366. Du1becco, R., Vogt, M. 1 960. Proc. Natl Acad. Sci. USA 46: 1 6 1 7 367. Martin, M. A., Axelrod, D . 1 969. Science 1 64:68 368. Sauer, G., Kidwal, J. R. 1 968. Proc. Natl. Acad. Sci. USA 6 1 : 1 256 369. Martin, M. A., Axelrod, D. 1 969. Proc. Natl. Acad. Sci. USA 64: 1 203 370. Darnell, J. E. Jr., Pagoulatos, G. N., Lindberg, U., Balint, R. 1 970. Cold Spring Harbor Symp. Quant. Bioi. 35:555 3 7 1 . Wall, R., Darnell, J. E. 1 97 1 . Nature New Bioi. 232:73 372. Sauer, G. 1 97 1 . Nature New Bioi. 2 3 1 : 1 35 373. Martin, M. A. 1 970. Cold Spring Har bor Symp. Quant. Bioi. 3 5 :833 374. Lindberg, U., Darnell, J. E. 1 970. Proc. Natl. Acad. Sci. USA 65 : 1 089 375. Ozanne, B., Sharp, P. A., Sambrook, J. 1 973. 1. Virol 1 2:90 376. Khoury, G., Byrne, J. c., Takemoto, K. K., Martin, M. A. 1 973. 1. Virol. 1 1 :54 377. Khoury, G., Martin, M. A., Lee, T. N. H., Nathans, D. 1 975. Virology 63:263 378. Melnick, J. L., Khera, K. S., Rapp, F. 1 964. Virology 23:430 379. Gerber, P. 1 966. Virology 28:501 380. Koprowski, H., Jensen, F. c., Steplew-

522

38 1 . 382.

383.


384. 385. 386. 387. 388. 389.

390. 391. 392. 393. 394.

395. 396. 397. 398. 399. 400.

40 1 . 402.

FAREED & DAVOLI ski, Z. 1 967. Proc. Natl. Acad. Sci. USA 58:127 Watkins, J. F . , Dulbecco, R. 1 967. Proc. Natl. Acad. Sci. USA 58: 1 396 Fogel, M., Sachs, L. 1969. Virology 37:327 Summers, J., Vogt, M. 1 97 1 . Lepetit Colloq. Bioi. Med. 2:306 Croce, C. M., Huebner, K., Girardi, A. J., Koprowski, H. 1 974. Virology 60:276 Huebner, K., Croce, C. M., Koprowski, H. 1 974. Virology 59:570 Poste, G., Schaeffer, B., Reeve, P., Al exander, D. J. 1974. Virology 60:85 Yoshiike, K., Furuno, A., Uchida, S. 1 974. Virology 60:342 Huebner, K., Santoli, D., Croce, C. M., Koprowski, H. 1 975. Virology 63 : 5 1 2 Boyd, V. A . L., Butel, J . S. 1 972. J. Viral. 10:399 Shani, M., Huberman, E., Aloni, Y., Sachs, L. 1 974. Virology 6 1 :303 Sambrook, J., Westphal, H., Srinivasan, P. R., Dulbecco, R. 1 968. Proc. Natl. Acad. Sci. USA 60: 1 288 Westphal, H., Dulbecco, R. 1968. Proc. Natl. Acad. Sci. USA 59: 1 1 5 8 Tai, H. T., O'Brien, R . L . 1 969. Virology 38 :698 Levine, A. S., Oxman, M. N., Henry, P. H., Levin, M. J., Diamandopoulos, G. T., Enders, J. F. 1 970. J. Viral. 6 : 1 99 Westphal, H., Kiehn, E. D. 1 970. Cold Spring Harbor Symp. Quant. Bioi. 35:819 Hirai, K . , Defendi, V . 1 97 1 . Biochem. Biophys. Res. Commun. 42:7 1 4 Haas, M . , Vogt, M . , Dulbecco, R. 1 972. Proc. Natl. Acad. Sci. USA 69:2 1 60 Gelb, L. D., Kohne, D. E., Martin, M. A. 1 97 1 . J. Mol. Bioi. 57: 1 29 Ilotchan, M. R., Ozanne, B., Sugden, B., Sharp, P. A., Sambrook, J. 1 974. Proc. Natl. Acad. Sci. USA 7 1 :4 1 83 Botchan, M. R., Ozanne, B., Sambrook, J. 1 975. Cold Spring Harbor Symp. Quant. Bioi. 39:95 Weiss, M. C. 1 970. Proc. Natl. Acad. Sci. USA 66:79 Croce, C. M., Girardi, A. J., Koprow-

ski, H. 1 973. Proc. Nat!. Acad. Sci. USA 70:3 6 1 7 . 403. Croce, C. M., Aden, D., Koprowski, H . 1 975. Proc. Natl. Acad. Sci. USA 72: 1 397

404. Croce, C. M., Koprowski, H . 1 975. Proc. Natl. Acad. Sci. USA 72: 1 658 405. Khoury, G., Croce, C. M. 1975. Cell 6:535 406. Croce, C. M. 1 977. Proc. Natl. Acad. Sci. USA 74:3 1 5 407. Botchan, M . R., McKenna, G., Sharp, P. A. 1 974. Cold Spring Harbor Symp. Quant. Bioi. 38:383 408. Ketner, G., Kelly, T. J. Jr. 1 976. Proc. Natl. A cad. Sci. USA 7 3 : 1 102 409. BOlchan, M., Topp, W., Sambrook, J. 1976. Cell 9:269 4 10. Sinsheimer, R. L. 1 977. Ann. Rev. Bio chem. 46:415-38 4 1 1 . Jackson, D. A., Symons, R. H., Berg, P. 1 972. Proc. Natl. Acad. Sci. USA 69:2904 4 1 2. Ganem, D., Nussbaum, A. L., Davoli, D., Fareed, G. C. 1 976. Cell 7:349 4 1 3. Nussbaum, A. L., Davoli, D., Ganem, D., Fareed, G. C. 1 976. Pmc. Nat!. Acad. Sci. USA 7 3 : 1 068 4 1 4. Davoli, D., Nussbaum, A. L., Fareed, G. C. 1 976. J. Virol. 1 9 : 1 100 4 1 5. Hamer, D. H., Davoli, D., Thomas, C. A. Jr., Fareed, G. C. 1 977. J. Mol. BioI. In press 4 1 6. Upcroft, P., U pc roft , J. A., Baker, R. F, Fareed, G. C., Solomon, D., Khoury, G., Hamer, D. H. 1 977. In preparation 4 1 7. Goff, S., Berg, P. 1 977. Cell In press 4 1 8. Crawford, L. V., Crawford, E. M. 1 963. Virology 21 :258 4 1 9. Crawford, L. V. 1 969. Adv. Virus Res. 14:89 420. Favre, M., Orth, G., Croissant, 0., Yaniv, M. 1 976. Proc. Natl. Acad. Sci. USA 72:48 1 0 42 1 . Gissmann, L . , Z u r Hausen, H. 1 976. Proc. Nat!. A cad. Sci. USA 73: 1 3 10 422. Zur Hausen, H., Meinhof, W., Scheiber, W., Bornkamm, G. W. 1974. Int. J. Cancer 1 3 :650 423. Gissmann, L., Pfister, H., Zur Hausen, H. 1 977. Virology 76:569

Molecular biology of cytomegalovirus.

Molecular biology of retroelements.

Funding of molecular biology.

Molecular biology of selenoproteins.

Molecular biology of methanogens.

Computational systems biology methods in molecular biology, chemistry biology, molecular biomedicine, and biopharmacy.

Molecular biology: Marked progress.

Molecular biology in nutrition.

Molecular biology. Chlamydomonas surrenders.

Molecular biology of neurological diseases.

The Molecular Biology of Pestiviruses.

Molecular biology of gynecological cancer.

Molecular biology of lung cancer.

Molecular biology of adrenergic receptors.

The molecular biology of poliovaccines.

Molecular biology of bacterial bioluminescence.

Molecular biology of the Bunyaviridae.

Molecular biology of cellulose degradation.

Molecular biology of dopamine receptors.

Molecular biology of serotonin receptors.

Molecular biology in medicine.

Molecular biology: an overview.

Molecular biology. Inflammatory activities.

The molecular biology of selenocysteine.