ANNUAL REVIEWS
Annu. Rev. Biochem. 1990.59:661-688. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 10/12/13. For personal use only.
Annu. Rev. Biochem.
Further
Quick links to online content
1990. 59:661-88
REGlJLATION OF VACCINIA VIRUS TRAr�SCRIPTIONl Bernard Moss Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda Maryland 20892
KEY WORDS :
DNA-dependent RNA polymerase , promoter, mRNA, transcription termina tion signal, posttranscriptional modification.
CONTENTS PERSPECTIVES AND SUMMARY . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
662
BIOLOGICAL CONSIDERATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
662 662 663 663
Po xvirus Family. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . Re pli ca ti on Cy cle .. . . . .. . . . . . . . ... . . . . . . . . . . . . . . . ... . . ..... . . . . . . . . ......... . . . . . . . . . . . . . . . . . . . . . . Vi rus P a"ti cle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
EARLY TRANS CRIPTION. . . . . . . . . . . . . . . . . . . . . . .... .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
mRNA . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
P ro mo te rs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Te rmina tio n S ig na l . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . In Vi tro Transcri ptio n Sy ste ms. .. . . . . . . ... . . . ... . . . ..... . . . . . ...... . . . . . . . . . . . . . . . . . .. . . . . . . . . . Enz y me s a nd Fa cto rs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LATE TRANSCRIPTION . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .
664 664 665 666 667 669
La te Ge ne s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P ro mo ters . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vi tro Transcriptio n S y ste m . . . .... . . . . . . ..... ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . .. . . . . . Inte rmedia te Ge ne s . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of DNA R e plica tio n. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . .
674 674 674 676 678 680 680
POSTTRANSCRIPTIONAL R EGULATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .
681
INTERACTIONS WITH THE HOST TRANSCRIPTION APPARATUS . . . . . . . . . . . . . . . . .
682
GENE REGULATION CYCLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
682
.
.
IThe US Government has the right to retain a nonexclusive royalty-free license in and to any copyright covering this paper.
661
662
MOSS
Annu. Rev. Biochem. 1990.59:661-688. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 10/12/13. For personal use only.
PERSPECTIVES AND SUMMARY Poxviruses, of which vaccinia virus is the prototype, differ from other eu karyotic DNA viruses in that they replicate within the cytoplasmic compart m ent of the cell rather than in the nucleus. As a necessary part of their life-style, these viruses encode a eukaryoticlike multisubunit RNA polymerase as well as many-perhaps all---o f the additional enzymes and factors needed to synthesize capped, methylated, and polyadenylated mRNA. Remarkably, all of the proteins necessary for transcription of the early class of genes are packaged within the virus particle. The transcription system is activated upon infection, and a cascade of events, involving an assortment of cis- and trans-acting factors , leads to the successive expression of several different classes of genes. Viral DNA replication plays a crucial role in controlling these events. Pox viruses thus provide an unusual opportunity to combine biochemical and genetic approaches for the investigation of transcriptional and posttran scriptional regulatory mechanisms. Such studies also may shed light on the origin of these largest and most self sufficient of all DNA viruses and their relationship to eukaryotic cells. In addition , vaccinia virus has become a popular expression vector, and a deeper understanding of its transcriptional m echanisms is crucial for maximal exploitation of the system.
BIOLOGICAL CONSIDERATIONS Poxvirus Family The poxviruses form a diverse family of complex DNA v iruses that infect both vertebrate and invertebrate hosts (1). Nevertheless, all members share the characteristic features of cytoplasmic replication and several use the same transcriptional regulatory signals. The vertebrate poxviruses have been grouped into six genera. Of these, we are most familiar with two members of the Orthopoxvirus genus that no longer exist in nature: variola virus and vaccinia virus (2). Variola virus, the causative agent of smallpox, has been eradicated. The origin of vaccinia virus, which was used until recently as a live vaccine against smallpox, is not precisely known; it may have existed in animals as an independent species or arisen as a hybrid strain during its long vaccine passage history (3). Advances in genetic engineering and molecular biology have led to a new interest in vaccinia virus as an expression vector, and recombinant vaccinia viruses are being considered as live vaccines against important diseases including AIDS (4). Vaccinia virus is relatively safe to work with and is easily grown in a wide variety of eukaryotic cells, contributing to its value as an experimental system. Indeed, vaccinia virus was the first animal virus to be seen microscopically, grown in tissue culture, accurately titered, physically purified, and chemically analyzed.
VACCINIA VIRUS TRANSCRIPTION
663
Annu. Rev. Biochem. 1990.59:661-688. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 10/12/13. For personal use only.
Replication Cycle The principal events in the reproductive cycle of poxviruses include virus entry, regulated gene expression, DNA replication , virion asembly, and virus dissemination (1) . Under suitable conditions, a rise in infectious virus is detected in 4 to 6 hours and a maximum yield of about 1 0,000 particles is obtained by 24 hours. Entry of vaccinia virus into the cytoplasm involves fusion with cell mem branes and subsequent virus internalization (5 , 6). Concomitantly, release of phospholipid and about 50% of the virion protein occurs (7). The latter process , known as the first stage of uncoating, is not blocked by inhibitors of either RN A or protein synthesis and results in the formation of structures resembling cores (8 , 9). The second stage of uncoating, measured by the susceptibility of the genome to DNase (which is not necessarily synonymous with release of naked DNA from the core structure) , begins after a lag period of 0.5 to 2 hours, depending on the multiplicity of infection, and requires 1 . 25 to 2.5 hours for 50% of the DNA to become DNase sensitive. The extent of second stage uncoating varies from 50 to 70% and is never complete. In contrast to first stage uncoating, the second stage requires RNA and protein synthesis and is prevented by ultraviolet irradiation of the virus (10). In tuitively, it has been thought that the second stage of uncoating might be necessary to provide free DNA for initiation of replication as well as for transcription of a delayed early class of genes, but such a requirement has never been demonstrated directly (11). Although a putative viral uncoating protein wiith trypsinl ike activity has been partially purified from extracts of infected cells, genetic studies are needed to establish its role (12) . DNA replication begins within a few hours of infection, requires the synthesis of virus-encoded enzymes including DNA polymerase , and involves long concatemeric DNA intermediates. Replication marks the end of the early phase of gene expression and the beginning of the late phase. V irion assem bly, a complex process involving the cleavage of structural proteins from higher-molecular-weight precursors, is poorly understood. Virus Particle particle, or virion, is composed of a lipoprotein bilayer surroundilllg a biconcave core with a structure of unknown function, called a lateral body, in each concavity . The genome , located within the core, consists of a l inear duplex DNA molecule of about 185 kilobase pairs (kbp). The ends of the poxvirus genome are hairpin loops that connect the two DNA strands into one covalently continuous molecule (13). Although the genome is com
The infectious virus
posed largely of unique sequences, there is a lO-kbp inverted terminal repeti
tion (14, 1 5) within which are blocks of short tandem repeats as well as several genes (16, 17). The majority of essential genes map within the central
Annu. Rev. Biochem. 1990.59:661-688. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 10/12/13. For personal use only.
664
MOSS
region of the genome, which is highly conserved among poxviruses; the genes that are not essential for replication in tissue culture and those involved in host range are nearer the ends ( 1 8). The apparent absence of introns, the short promoter sequences (to be discussed), and the relatively small sizes of many open-reading-frames (ORPs), account for the packing of an estimated 150 to 200 genes into the 185-kbp DNA molecule. B oth strands of the DNA are transcribed, yet extensive overlapping of ORFs is uncommon. ORFs fre quently occur in head-to-tail tandem arrays with some clusters containing predominantly early genes and others late genes. The virion contains a large number of polypeptides; more than 1 00 have b een resolved by two-dimensional polyacrylamide gel electrophoresis, but some of this complexity may result from posttranslational modifications (19). Although all of the enzymes involved in transcription are l ocated in the core, four structural proteins of 74, 62, 25, and 1 1 kd account for about 70% of the mass (20-23). In place of histones, several viral proteins are strongly bound to the DNA and apparently help to maintain it in a folded, supercoiled state (24, 25). B oth the l l -kd and the 25-kd proteins have a strong affinity for DNA (26, 27).
EARLY TRANSCRIPTION mRNA Immediately after infection, vaccinia virus cores are released into the cyto plasm, initiate transcription (28-30), and produce capped (3 1 ) and polyade nylated mRNA (32). The cap structure is required for binding of vaccinia virus mRNA to ribosomes (33) and probably for mRNA stability. Transcrip tion is not prevented by inhibitors of protein or DNA synthesis. On the c ontrary, viral RNA synthesis is enhanced and prolonged when uncoating of the core is prevented by protein synthesis inhibitors (34). In the presence of inhibitors of DNA replication, the initial rate of RNA synthesis is proportional to the size of the virus inoculum, but uncoating does occur and under these conditions early RNA synthesis graduall y decreases. Thus, the activation and decline of early transcription may correspond to the first and second stages of uncoating, respectively. Sequestration of the RNA pol ymerase complex with in the residual core structure could explain the low efficiency of transcription of transfected plasmids containing early promoters (35). RNA:DNA hybridization studies revealed that about one half of the genome is transcribed prior to DNA replication, and that there are several abundance classes of early RNA (36-39). Although there is evidence for two temporal classes of early genes-immediate early and delayed early, which are expressed b efore and after viral protein synthesis, respectively (29) recent data have not supported such a division.
Annu. Rev. Biochem. 1990.59:661-688. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 10/12/13. For personal use only.
VACCINIA VIRUS TRANSCRIPTION
665
Early mRNAs typically have short untranslated leaders, since DNA se quences corresponding to the start sites, as determined by nuclease digestion of RNA-DNA hybrids andlor by reverse transcriptase extension of DNA primers, are usually located not far upstream of the ORFs (40--44). The predominant early viral mRNAs are of discrete length ( 1 7 , 45), and no indication of splicing has been obtained. Evidence against functional polycistronic precursors was provided by ultraviolet target-size de terminations in vivo (46) and in vitro (see later section). The stability of early mRNA has been measured following the addition of actinomycin D to block further transcription; half-life values of several hours were obtained (38, 47). Promoters The DNA segments upstream of the ORFs of early genes are extremely rich in A + T residues. Evidence for promoter function was obtained by fusing such DNA segments to a reporter gene encoding an easily assayable enzyme, and the chimeric DNA was then recombined into the vaccinia genome. Studies with the recombinant vaccinia viruses indicated that the promoters for several early genes extend only about 30 bp upstream of the RNA start sites (48-53). There is no evidence for specific regulatory sequences further upstream. A fine structure analysis, including every possible single nucleotide muta tion (Figun! 1 ) and many multiple mutations, was made for one early promot er (54). The effects of these mutations were measured by expression of the ,a-galactosidase reporter gene as well as by in vivo and in vitro transcription. On the basis of these results, the promoter was divided into three regions relative to the RNA start site at + 1 : a 1 5-bp A-rich critical region (-1 3 to 28 ) in which most single-nucleotide substitutions have a major effect, separated by 1 1 bp of a less critical T-rich sequence from a 7-bp region within which initiation with a purine occurs. The fine structure of the critical region indicated that A residues are essential at several l ocations and optimal at all positions except -14, - 1 5 , -2 1 , -22, and -23. A G residue is needed at -2 1 and Ts are important at -22 or 23 Like the TATA box of higher eukaryotic RNA polymerase II promoters, the critical region specifies the location of the downstream site of transcription initiation. mRNA does not ordinarily start with a pyrimidine, and the precise point of initiation shifted a few nucleotides upstream or downstream when purine-to-pyrimidine sub stitutions were made. When the location of the critical region is fixed, the range of flexibility for initiation appears to be about 7 bp (54). An analysis of multiple substitutions within the critical region indicated that most behaved independently, although some of the up mutations in Figure 1 could partially compensate for potentially detrimental nucleotides at other positions (54). For the latter reason, there is more individual nucleotide variability in the critical region of early promoters than might have been -
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.
666
Be
MOSS
.G fiilIA
300
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Annu. Rev. Biochem. 1990.59:661-688. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 10/12/13. For personal use only.
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Annu. Rev. Biochem. 1990.59:661-688. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 10/12/13. For personal use only.
Annu. Rev. Biochem.
Further
Quick links to online content
1990. 59:661-88
REGlJLATION OF VACCINIA VIRUS TRAr�SCRIPTIONl Bernard Moss Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda Maryland 20892
KEY WORDS :
DNA-dependent RNA polymerase , promoter, mRNA, transcription termina tion signal, posttranscriptional modification.
CONTENTS PERSPECTIVES AND SUMMARY . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
662
BIOLOGICAL CONSIDERATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
662 662 663 663
Po xvirus Family. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . Re pli ca ti on Cy cle .. . . . .. . . . . . . . ... . . . . . . . . . . . . . . . ... . . ..... . . . . . . . . ......... . . . . . . . . . . . . . . . . . . . . . . Vi rus P a"ti cle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
EARLY TRANS CRIPTION. . . . . . . . . . . . . . . . . . . . . . .... .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
mRNA . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
P ro mo te rs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Te rmina tio n S ig na l . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . In Vi tro Transcri ptio n Sy ste ms. .. . . . . . . ... . . . ... . . . ..... . . . . . ...... . . . . . . . . . . . . . . . . . .. . . . . . . . . . Enz y me s a nd Fa cto rs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LATE TRANSCRIPTION . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .
664 664 665 666 667 669
La te Ge ne s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P ro mo ters . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vi tro Transcriptio n S y ste m . . . .... . . . . . . ..... ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . .. . . . . . Inte rmedia te Ge ne s . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of DNA R e plica tio n. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . .
674 674 674 676 678 680 680
POSTTRANSCRIPTIONAL R EGULATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .
681
INTERACTIONS WITH THE HOST TRANSCRIPTION APPARATUS . . . . . . . . . . . . . . . . .
682
GENE REGULATION CYCLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
682
.
.
IThe US Government has the right to retain a nonexclusive royalty-free license in and to any copyright covering this paper.
661
662
MOSS
Annu. Rev. Biochem. 1990.59:661-688. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 10/12/13. For personal use only.
PERSPECTIVES AND SUMMARY Poxviruses, of which vaccinia virus is the prototype, differ from other eu karyotic DNA viruses in that they replicate within the cytoplasmic compart m ent of the cell rather than in the nucleus. As a necessary part of their life-style, these viruses encode a eukaryoticlike multisubunit RNA polymerase as well as many-perhaps all---o f the additional enzymes and factors needed to synthesize capped, methylated, and polyadenylated mRNA. Remarkably, all of the proteins necessary for transcription of the early class of genes are packaged within the virus particle. The transcription system is activated upon infection, and a cascade of events, involving an assortment of cis- and trans-acting factors , leads to the successive expression of several different classes of genes. Viral DNA replication plays a crucial role in controlling these events. Pox viruses thus provide an unusual opportunity to combine biochemical and genetic approaches for the investigation of transcriptional and posttran scriptional regulatory mechanisms. Such studies also may shed light on the origin of these largest and most self sufficient of all DNA viruses and their relationship to eukaryotic cells. In addition , vaccinia virus has become a popular expression vector, and a deeper understanding of its transcriptional m echanisms is crucial for maximal exploitation of the system.
BIOLOGICAL CONSIDERATIONS Poxvirus Family The poxviruses form a diverse family of complex DNA v iruses that infect both vertebrate and invertebrate hosts (1). Nevertheless, all members share the characteristic features of cytoplasmic replication and several use the same transcriptional regulatory signals. The vertebrate poxviruses have been grouped into six genera. Of these, we are most familiar with two members of the Orthopoxvirus genus that no longer exist in nature: variola virus and vaccinia virus (2). Variola virus, the causative agent of smallpox, has been eradicated. The origin of vaccinia virus, which was used until recently as a live vaccine against smallpox, is not precisely known; it may have existed in animals as an independent species or arisen as a hybrid strain during its long vaccine passage history (3). Advances in genetic engineering and molecular biology have led to a new interest in vaccinia virus as an expression vector, and recombinant vaccinia viruses are being considered as live vaccines against important diseases including AIDS (4). Vaccinia virus is relatively safe to work with and is easily grown in a wide variety of eukaryotic cells, contributing to its value as an experimental system. Indeed, vaccinia virus was the first animal virus to be seen microscopically, grown in tissue culture, accurately titered, physically purified, and chemically analyzed.
VACCINIA VIRUS TRANSCRIPTION
663
Annu. Rev. Biochem. 1990.59:661-688. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 10/12/13. For personal use only.
Replication Cycle The principal events in the reproductive cycle of poxviruses include virus entry, regulated gene expression, DNA replication , virion asembly, and virus dissemination (1) . Under suitable conditions, a rise in infectious virus is detected in 4 to 6 hours and a maximum yield of about 1 0,000 particles is obtained by 24 hours. Entry of vaccinia virus into the cytoplasm involves fusion with cell mem branes and subsequent virus internalization (5 , 6). Concomitantly, release of phospholipid and about 50% of the virion protein occurs (7). The latter process , known as the first stage of uncoating, is not blocked by inhibitors of either RN A or protein synthesis and results in the formation of structures resembling cores (8 , 9). The second stage of uncoating, measured by the susceptibility of the genome to DNase (which is not necessarily synonymous with release of naked DNA from the core structure) , begins after a lag period of 0.5 to 2 hours, depending on the multiplicity of infection, and requires 1 . 25 to 2.5 hours for 50% of the DNA to become DNase sensitive. The extent of second stage uncoating varies from 50 to 70% and is never complete. In contrast to first stage uncoating, the second stage requires RNA and protein synthesis and is prevented by ultraviolet irradiation of the virus (10). In tuitively, it has been thought that the second stage of uncoating might be necessary to provide free DNA for initiation of replication as well as for transcription of a delayed early class of genes, but such a requirement has never been demonstrated directly (11). Although a putative viral uncoating protein wiith trypsinl ike activity has been partially purified from extracts of infected cells, genetic studies are needed to establish its role (12) . DNA replication begins within a few hours of infection, requires the synthesis of virus-encoded enzymes including DNA polymerase , and involves long concatemeric DNA intermediates. Replication marks the end of the early phase of gene expression and the beginning of the late phase. V irion assem bly, a complex process involving the cleavage of structural proteins from higher-molecular-weight precursors, is poorly understood. Virus Particle particle, or virion, is composed of a lipoprotein bilayer surroundilllg a biconcave core with a structure of unknown function, called a lateral body, in each concavity . The genome , located within the core, consists of a l inear duplex DNA molecule of about 185 kilobase pairs (kbp). The ends of the poxvirus genome are hairpin loops that connect the two DNA strands into one covalently continuous molecule (13). Although the genome is com
The infectious virus
posed largely of unique sequences, there is a lO-kbp inverted terminal repeti
tion (14, 1 5) within which are blocks of short tandem repeats as well as several genes (16, 17). The majority of essential genes map within the central
Annu. Rev. Biochem. 1990.59:661-688. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 10/12/13. For personal use only.
664
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region of the genome, which is highly conserved among poxviruses; the genes that are not essential for replication in tissue culture and those involved in host range are nearer the ends ( 1 8). The apparent absence of introns, the short promoter sequences (to be discussed), and the relatively small sizes of many open-reading-frames (ORPs), account for the packing of an estimated 150 to 200 genes into the 185-kbp DNA molecule. B oth strands of the DNA are transcribed, yet extensive overlapping of ORFs is uncommon. ORFs fre quently occur in head-to-tail tandem arrays with some clusters containing predominantly early genes and others late genes. The virion contains a large number of polypeptides; more than 1 00 have b een resolved by two-dimensional polyacrylamide gel electrophoresis, but some of this complexity may result from posttranslational modifications (19). Although all of the enzymes involved in transcription are l ocated in the core, four structural proteins of 74, 62, 25, and 1 1 kd account for about 70% of the mass (20-23). In place of histones, several viral proteins are strongly bound to the DNA and apparently help to maintain it in a folded, supercoiled state (24, 25). B oth the l l -kd and the 25-kd proteins have a strong affinity for DNA (26, 27).
EARLY TRANSCRIPTION mRNA Immediately after infection, vaccinia virus cores are released into the cyto plasm, initiate transcription (28-30), and produce capped (3 1 ) and polyade nylated mRNA (32). The cap structure is required for binding of vaccinia virus mRNA to ribosomes (33) and probably for mRNA stability. Transcrip tion is not prevented by inhibitors of protein or DNA synthesis. On the c ontrary, viral RNA synthesis is enhanced and prolonged when uncoating of the core is prevented by protein synthesis inhibitors (34). In the presence of inhibitors of DNA replication, the initial rate of RNA synthesis is proportional to the size of the virus inoculum, but uncoating does occur and under these conditions early RNA synthesis graduall y decreases. Thus, the activation and decline of early transcription may correspond to the first and second stages of uncoating, respectively. Sequestration of the RNA pol ymerase complex with in the residual core structure could explain the low efficiency of transcription of transfected plasmids containing early promoters (35). RNA:DNA hybridization studies revealed that about one half of the genome is transcribed prior to DNA replication, and that there are several abundance classes of early RNA (36-39). Although there is evidence for two temporal classes of early genes-immediate early and delayed early, which are expressed b efore and after viral protein synthesis, respectively (29) recent data have not supported such a division.
Annu. Rev. Biochem. 1990.59:661-688. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 10/12/13. For personal use only.
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Early mRNAs typically have short untranslated leaders, since DNA se quences corresponding to the start sites, as determined by nuclease digestion of RNA-DNA hybrids andlor by reverse transcriptase extension of DNA primers, are usually located not far upstream of the ORFs (40--44). The predominant early viral mRNAs are of discrete length ( 1 7 , 45), and no indication of splicing has been obtained. Evidence against functional polycistronic precursors was provided by ultraviolet target-size de terminations in vivo (46) and in vitro (see later section). The stability of early mRNA has been measured following the addition of actinomycin D to block further transcription; half-life values of several hours were obtained (38, 47). Promoters The DNA segments upstream of the ORFs of early genes are extremely rich in A + T residues. Evidence for promoter function was obtained by fusing such DNA segments to a reporter gene encoding an easily assayable enzyme, and the chimeric DNA was then recombined into the vaccinia genome. Studies with the recombinant vaccinia viruses indicated that the promoters for several early genes extend only about 30 bp upstream of the RNA start sites (48-53). There is no evidence for specific regulatory sequences further upstream. A fine structure analysis, including every possible single nucleotide muta tion (Figun! 1 ) and many multiple mutations, was made for one early promot er (54). The effects of these mutations were measured by expression of the ,a-galactosidase reporter gene as well as by in vivo and in vitro transcription. On the basis of these results, the promoter was divided into three regions relative to the RNA start site at + 1 : a 1 5-bp A-rich critical region (-1 3 to 28 ) in which most single-nucleotide substitutions have a major effect, separated by 1 1 bp of a less critical T-rich sequence from a 7-bp region within which initiation with a purine occurs. The fine structure of the critical region indicated that A residues are essential at several l ocations and optimal at all positions except -14, - 1 5 , -2 1 , -22, and -23. A G residue is needed at -2 1 and Ts are important at -22 or 23 Like the TATA box of higher eukaryotic RNA polymerase II promoters, the critical region specifies the location of the downstream site of transcription initiation. mRNA does not ordinarily start with a pyrimidine, and the precise point of initiation shifted a few nucleotides upstream or downstream when purine-to-pyrimidine sub stitutions were made. When the location of the critical region is fixed, the range of flexibility for initiation appears to be about 7 bp (54). An analysis of multiple substitutions within the critical region indicated that most behaved independently, although some of the up mutations in Figure 1 could partially compensate for potentially detrimental nucleotides at other positions (54). For the latter reason, there is more individual nucleotide variability in the critical region of early promoters than might have been -
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Annu. Rev. Biochem. 1990.59:661-688. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 10/12/13. For personal use only.
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