Proc. Natl. Acad. Sci. USA Vol. 74, No. 10, pp. 4195-4199, October 1977

Biochemistry

Synthesis of qX174 viral DNA in vitro depends on qX replicative form DNA (DNA replication/E. coli dna gene products)

CHIKAKO SUMIDA-YASUMOTO AND JERARD HURWITZ Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461

Contributed by Jerard Hurwitz, July 11, 1977 A cell-free system that catalyzes 4X174 repliABSTRACT cative form I (supercoiled circular duplex, RFI)dependent 4X174 DNA synthesis has been isolated from Escherichia coli infected with 4X174 phage. The products formed with such preparations are viral strands as judged by hybridization to poly(U,G) followed by equilibrium centrifugation in CsCl. This 4X174 DNA-synthesizing involves formation of DNAprotein complexes that sediment in neutral sucrose with S values of 50, 60-70, and higher. The S0S complex contained a rolling-circle replicative intermediate DNA with an extended tail of singlestranded viral DNA. The DNA contained in the 60-70S region was a mixture of circular and linear single-stranded DNA, RFI, and RFII with an extended single-stranded tail. Such complexes have been isolated during in vivo progeny 4X174 DNA synthesis [Fujisawa, H. & Hayashi, M. (1976) J. Virol. 19, 409]. In vitro, maximal 4X174 DNA synthesis was shown to require the genetically defined proteins E. coli dna B, dna C, dna G, dna Z, rep, OX174 gene A product, and other kX174 coded proteins. The synthesis of OX174 DNA is ATP-dependent and is inhibited by nalidixic acid and novobiocin but is resistant to rifampicin.

Previous in vitro studies have shown that bacteriophage 4X174 (kX174) single-stranded (ss) circular DNA was converted to double-stranded circular replicative form of 4X174 DNA containing at least one discontinuity (RFII) in the presence of at least 11 distinct host proteins (1, 2), including several known to be involved in Escherichia coli DNA replication. In addition to the above host proteins, replication of kX174 replicative forms I (RFI) requires the OX174 A gene product (which specifically introduces a discontinuity in the A cistron of the viral strand of supertwisted 4X174 RFI) (3, 4) and the E. coli rep protein (5, 6). Furthermore, replication of the double-stranded circular replicative form of kX174 DNA (RF) is inhibited by nalidixic acid and novobiocin (6). In vitro, the last stage in the replication of qX174 DNA viral progeny synthesis is also complex. In addition to being sensitive to nalidixic acid and novobiocin, this system depends on gene products coded by the OX 174 genome. In this communication we describe the isolation of soluble fractions from kX174-infected E. coli that catalyze RFI-dependent synthesis of viral circular ss DNA. In accord with in vivo observations of Fujisawa and Hayashi (7), ss DNA synthesis occurs via formation of protein-nucleic acid inter-

mediates.

MATERIALS AND METHODS Bacterial and Phage Strains. Bacterial and phage strains were as described (6) except for E. coli strain AX727 (dna zts, kX174r) and OX am H57 (F gene) which were obtained from J. R. Walker and M. Hayashi, respectively.

Table 1. Requirements for RF and ss DNA synthesis in vitro

Additions

Complete Omit 4X174 RFI Omit receptor ammonium sulfate fraction Omit fraction II Omit ATP or omit Mg2+ or omit dATP, dGTP, dCTP Complete + N-ethylmaleimide (4 mM) Complete + rifampicin (10 Mg/ml) Complete + nalidixic acid (240 Mug/ml) Complete + novobiocin (240 jg/ml)

dTMP incorporated, pmol/40 min RF ss DNA 218 64 3 14 2 4

2 4

2-4 3 230 18 22

2-4 4

67 6 16

Reaction mixtures (0.05 ml) contained, in order of addition: 20 mM Tris-HCl (7.5), 10 mM MgCl2, 4 mM dithiothreitol, 1 mM ATP, 0.2 mM NAD+, 40MAM each of dATP, dGTP, dCTP, and [a-32P]dTTP (200-700 cpm/pmol), 0.1 mM each of CTP, UTP, and GTP, 1 nmol of kX174 [3H]RFI (1-6 cpm/pmol), 0.2 mg of ammonium sulfate fraction from E. coli strain BT1029, 11 Mg of fraction II for RF replication or 23 Mg of fraction II for ss DNA synthesis, and 0.1 unit of dna C. Reaction mixtures were incubated as indicated and acid-insoluble radioactivity was measured (6). In the case of the ss DNA system, reactions were stopped by addition of 10 mM EDTA (final) and 0.2% sodium dodecyl sulfate (final) and heated at 650 for 15 min followed by a 4 hr incubation at 370 with autodigested proteinase K (1 mg/ml). The mixtures were dialyzed against 50 mM Tris-HCl, pH 8.0/5 mM EDTA for 12 hr. Viral strand formation was measured after hybridization to poly(U,G) followed by CsCl isopycnic centrifugation for 70 hr as described by Baas et al. (9). The amount of viral strand that banded at the expected density is the value reported above. Under these conditions, >90% of the incorporated 32p was recovered in this fraction. Reference markers of OX174 [14C]DNA, [3H]RFI, and

[3H]RFII were run in parallel gradients.

Preparation of DNA. qX174 [14C]DNA was prepared by the procedure of Francke and Ray (8); [3H]RFI and [3H]RFII were prepared from E. coli HF 4704 infected with OX am3 phage in the presence of chloramphenicol (30,ug/ml) and purified by two phenol extractions followed by pancreatic RNase treatment (1 hr at 370 with 2 ,ug per A260 unit); the DNA was further purified by isopycnic banding in propidium diiodide/ CsCI followed by neutral sucrose gradient centrifugation. Preparation of Protein Fractions. Unless indicated, protein fractions were as described (6), and growth of E. coli and X174 infection were carried out at 300. Ammonium sulfate fractions from uninfected E. coli were as described (6) except that the Abbreviations: 4X174, bacteriophage OX174; ss, single-stranded; RFII, double-stranded circular replicative form of OX174 DNA containing at least one discontinuity; RF, replicative form of 4X174 DNA, a double-stranded circular DNA; RFI, supercoiled circular duplex

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RF.

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Biochemistry: Sumida-Yasumoto and Hurwitz

Table 2. Gene products required for RF replication and s DNA synthesis in vitro

dTMP incorporated, Source of extract pmol/40 min and phage used for fraction II RF ss DNA 1. BT1029 (dna B ts) (OX am3) 43 21 2. 1 + dna B protein 210 68 3. LD332 (dna C ts) (,X am3) 1 5 4. 3 + dna C protein 222 251 5. NY73 4Xs (dna G ts) (+X am3) 55 35 6. 5 + dna G protein 218 109 7. AX729(dnaZts) 4 11 8. 7 +dna Zprotein 55 33 9. D92 (rep-) (,tX am3) 3 4 10. 9+ rep protein 248 102 11. BT1029 (OX amH90) 3 4 12. 11+,X174Aprotein 153 99 13. BT1029 (,kX amH57) 117 0 14. 11 + 13 0 118 Reaction mixtures (0.05 ml) were as described in Table 1 with the exception that the RF system contained ammonium sulfate fractions from the E. coli strains indicated, 0.3 unit of purified X174 A protein (4) except in reaction 11, and 0.2 unit of dna C protein except in reaction 3; fraction II was replaced by purified OX174 A protein. In the case of the ss DNA system, ammonium sulfate fractions used for the RF system supplemented with 0.2 unit of dna C protein were added. Fraction II (for ss DNA synthesis) was added as described in Table 1. This fraction was prepared from the necessary E. coli mutants infected with X as indicated. In reaction 7, fraction II was the same as that used in reaction 1. In reactions 1, 2, 5, and 6, fraction II (for ss DNA synthesis) and uninfected ammonium sulfate fractions were heated as described (6). Fractions from LD332 and AX729 did not require heat treatment. Purified preparations of dna gene products (0.1-0.3 unit) were added where indicated. In reactions 13 and 14, extracts were prepared from cells infected for 50 min with OX amH57.

ammonium sulfate precipitation was repeated twice. Fraction II for the RF system was prepared as described (6) except that E. coli BT1029 (dna B ts) was infected with wild-type 4X174 for 25 min. Fraction II, which catalyzed ss DNA synthesis, was prepared from E. coli BT1029 infected with OX am3 for 50 min (6) except for the following modification: the lysis mixture was incubated at 00 for 45 min followed by centrifugation at 50,000 rpm (in a Beckman 65 rotor) for 90 min at 20 (the latter step removed mature OXX174 from the supernatant fraction).

RESULTS Requirements of the RF and ss DNA Systems. In all experiments, OX 174-infected E. coli was used as a source of viral coded proteins (fraction II) whereas ammonium sulfate fractions from uninfected E. coli were used as a source of E. coli coded proteins. Both fractions were deficient in dna C and all reaction mixtures were supplemented with this protein. Fraction II preparations that catalyzed RF replication of ss DNA synthesis differed; the former system was isolated from cells infected with wild-type 4X174 for 25 min* which permitted formation of the kX174 A protein but limited accumulation of other viral coded proteins (OX A, OX B, OX C, OX D, OX F, OX G, and OX H). *

In the case of the RF system, the addition of purified 4X174 A protein satisfied the requirement for phage-coded protein and completely replaced fraction II.

10

10

20

30 40 50 FRACTION

60

70

80

FIG. 1. Poly(U,G)/CsCl gradients of DNA products formed in RF (A) and ss DNA (B) synthesizing systems in vitro. Conditions were as described in Table 1 except that reaction mixtures were incubated at 300 for 20 min. C strand, complementary strand; V strand, viral strand. ss DNA synthesis depended on the presence of RFI, the two protein fractions, ATP, Mg2+ and the four dNTPs and was in-

hibited by N-ethylmaleimide, nalidixic acid, and novobiocin but was unaffected by rifampicin (Table 1); these requirements were the same for the RF system. A comparison of E. coli gene products required for RF and for ss DNA synthesis is given in Table 2. RF replication was measured by using ammonium sulfate fractions from uninfected E. coil dna mutants supplemented with purified dna C (as indicated) and OX174 A proteins. Fraction II used to catalyze ss DNA synthesis was prepared as described above from each of the dna mutants except the dna Z mutant. Where necessary, fractions were heated at nonpermissive temperatures and assayed in the presence or absence of purified gene products at 300. dna B, dna C, dna G, dna Z, rep, and OX174 A proteins were required for maximal RF replication as well as ss DNA synthesis. In experiments with the dna Z protein, fraction II was derived from dna B infected extracts whereas uninfected ammonium sulfate fractions were derived from the dna Z ts mutant of E. coil (AX 727); this mutant is an E. coli K-12 strain and resistant to OX 174 infection. Fraction II derived from cells infected with 4X amH57 (F gene mutant) did not

Proc. Nati. Acad. Scf. USA 74 (1977)

Biochemistry: Sumida-Yasumoto and Hurwitz

90

7T N

4197

x

E

UL

N

so x

E 0.

E

0

0.

a:

I-

0

0

I-

.0

60

x

x

30 FRACTION

FIG. 2. Neutral sucrose gradient analyses of protein-DNA complexes formed in vitro during ss DNA synthesis. Conditions were as described in Table 1, except that reactions were incubated for 15 min (A), 60 min (B), and 140 min (C and D) with 0.6 nmol of [3HJRFI. A different preparation of fraction II for ss DNA synthesis (25 gg of protein) was used in the reaction shown in (D). Reactions were stopped by addition of 20 mM EDTA (final) at 00; reaction mixtures were immediately centrifuged through a 10 ml 5-30% sucrose gradient in buffer containing 20 mM Tris-HCl (pH 7.5), 5 mM EDTA, and 0.1 M NaCl with 0.5 ml of 55% CsCl solution as a cushion at the bottom of the tube. Centrifugation was carried out in a SW 41 rotor (at 39,000 rpm) for 150 min at 4°. 4C-Labeled XX am3 phage was run in a parallel gradient as a marker (114 S). Fractions 1-4 represent the CsCl cushion.

support ss DNA synthesis as in vlvo (10); in contrast, it did support RF replication. The combination of fraction II from OX A- infected cells with fraction II from OX F- infected cells catalyzed ss DNA synthesis preferentially. DNA Products Formed from RFI. The products formed in reactions primed with RFI depended upon the OX infected extract (Fig. 1). Fraction II prepared from cells infected for 25 min produced a mixture of RFII and RFI as determined by neutral sucrose gradient centrifugation (data not shown). When the mixture of RFII and RFI was annealed to the poly(U,G) after denaturation and subjected to isopycnic banding, three labeled products were detected (Fig. 1A)-i.e., viral strand, complementary strand, and RFI. Viral and complementary strands coming from RFII were equally labeled. When RFI was converted to RFII after nicking with pancreatic DNase and analyzed as above, both strands were also found to be equally labeled. Thus, both viral and complementary strands are equally labeled during RF replication. Products formed during a 20-min incubation with fraction II isolated from E. colt infected for 50 min with OX am3 were

E

E

C

UL

& 9 FRACTION

FIG. 3. Neutral sucrose gradient analysis of DNA from the 50S (A) and 60-70S (B) complexes and alkaline sucrose gradient of DNA from the 50S complex (C). Conditions were as described in Table 1. Reactions were carried out for 40 min with the same fraction II (25 ,gg of protein) used in Fig. 2D with [a-32P]dTTP (2000 cpm/pmol) and 0.6 nmol of RFI; reactions were stopped with 20 mM EDTA (final) addition and were centrifuged through a sucrose gradient as described in the legend of Fig. 2 (data not shown). Fractions 36-39 and 28-35, which correspond to the same fractions as indicated in Fig. 2, were pooled to obtain the 505 complex and 60-70S complexes, respectively. DNA was isolated from these complexes as described in Table 1. DNA samples were layered onto a 5-ml linear 5-20% neutral or alkaline sucrose gradient and sedimented as described (6), except that the alkaline sucrose gradient lacked NaCl and was run for 15 hr at 25,000 rpm at 40 with OX174 [14CJDNA (16 S) as a marker. Mixtures of 4X174 [14C]DNA, [3H]RFI, and [3H]RFII were run in parallel neutral sucrose gradients as markers.

shown by neutral sucrose gradient centrifugation to be a mixture consisting of 19% RFI, 61% RFII forms, and 20% ss DNA. The RFII included a structure containing an extended ss viral tail (a RFII). Approximately 75, 10, and 15% of the total label incorporated was recovered as viral strand, complementary strand, and RFI, respectively, after poly(U,G) annealing and CsCI isopycnic banding (Fig. 1B). The label present in RFI was due to the selective synthesis of the viral strand (data not shown). With the ss DNA system, the following was observed: (i) the rate of synthesis was linear for 20-25 min and then abruptly plateaued; (ii) the extent of synthesis varied [in some experiments, net synthesis was achieved (Table 2)]; (iii) prolonged incubation after viral DNA synthesis stopped resulted in formation of RFI from RFII but only the viral strand of both RF

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Biochemistry: Sumida-Yasumoto and Hurwitz -PProc. Nad. Acad. Scf. USA 74 (1977)

RFI

(e) RF I[

(C)

Phage 132S

COMPLEX

OX A Protein / RF I *-A COMPLEX

sk.

60 - 80S COMPLEX

(h) (d) in FIG. 4. Pathway of RF replication and ss circular DNA synthesis maturation of OX174 phage. The protein mixture required for the reactions included E. coli coded proteins dna B, dna C, dna G, dna Z., rep, nal A, DNA gyrase, E. coli binding protein, replication factors X, Y, and Z, elongation factors I and III, and dna E (pol III). Requirement of the last seven proteins is inferred from previous studies (2, 4-6) but has not been demonstrated. RFI (a) is attacked by the OX174 A protein which acts at the A cistron of the viral strand, yielding a RFII structure (b) containing the 4X174 A protein linked to the 5'-end. This intermediate is replicated by fractions devoid of DNA and 4X174 A protein activity (4). In the presence of the proteins listed above (and the 4X174 A protein, the only essential phage function), RFII is replicated by formation of a a RFII structure containing double-stranded tails which have been observed by electron microscopy (data not presented); both viral and complementary strands in these structures are synthesized de novo as small pieces (c) which can be chased into RFII and RFI (unpublished results). Synthesis continues until the A cistron of the viral strand appears as a ss region; this site will be cleaved by the OX174 A protein-generating structures d and e. It islpossiblethatanRFIIIL-4X174 A protein (e) is an intermediate which, upon circularization, yields d. We have observed RFIII forms (full-size linears forms) by electron microscopy but their role in RF replication is unknown. It is possible that the 4X174 A protein itself catalyzes circularization of RFIII structures and is removed from the complex to yield RFII. After removal of the OX174 A protein, RFII (d) can be converted in vitro by DNA ligase and DNA polymerase I to an RFIV form (relaxed RF, f) which can be converted to RFI by DNA gyrase (11, 12). The replication of RF is blocked by 4X174-coded proteins essential for phage maturation. This is detected by a cessation of complementary strand formation. In this way, upon conversion to RFII-4sX174 A protein complex, RFI can only support ss DNA synthesis. This pathway yields a RFII forms containing ss tails complexed to phage proteins in which only the viral strand is labeled (g). Viral strand DNA synthesis occurs by formation of short discontinuous fragments that are rapidly chased into large fragments (unpublished data) as in the case of RF replication. The 505 complex after action of the 4X174 A protein regenerates the RFII-A complex (d) and leads to formation of 60-80S complexes (h). These complexes vary in size due to changes in viral proteins associated with the DNA and formation of various DNA structures. Eventually, these intermediates are converted to 132 S (i) and then to phage [114 S, (j)].

forms was labeled; (iv) no net formation of RFII or RFI occurred. These results suggest that fractions that catalyzed ss DNA synthesis did not catalyze RF replication. Furthermore, RF replication was blocked by addition of fraction II which catalyzed ss DNA synthesis. Under these conditions, DNA synthesis was restricted to viral strand formation. Protein-DNA Complexes Formed in OX174 DNA Synthesis from RFI. In vivo, kX174 viral DNA synthesis is coupled to 4X174 maturation. Fujisawa and Hayashi (7) studied maturation of 4X174 in vivo and demonstrated the formation of protein-DNA complexes that sedimented with an S value of 50. This 50S complex contained a RFII molecules. We have detected similar complexes during in vitro ss DNA formation. Under the conditions used (Table 1), although ss DNA synthesis stopped after 25 min, the formation of protein-nucleic acid complexes continued over a period of 140 min (Fig. 2). After incubation at 300 for various times, reaction mixtures were

subjected to neutral (nondenaturing) sucrose gradients above a dense CsCl shelf. In these experiments, both 3H-labeled RFI template and newly 32P-labeled DNA rapidly formed protein complexes that distributed, with time, between a large 50S peak and material with an S value of 132 (Fig. 2 A, B, and C). In a separate experiment (Fig. 2D), a 32P-labeled product peak was detected in fraction 6, corresponding to 132 S; this fraction contained 3 X 108 plaque-forming units/ml whereas fraction 15 of the same gradient contained 108 plague-forming units/ ml.t When fraction II (as used in Fig. 2D) was increased 2-fold, radioactivity from the [3H]RFI template was recovered in fractions 1-10; 10, 20, and 35% of the input 3H was recovered sedimentation experiments were performed with reaction mixtures lacking RFI or fraction II; in these experiments, 4 X 104 or 0 plaque-forming units/ml, respectively, was detected in fractions near the CsCl shelf.

t Similar

Biochemistry: Sumida-Yasumoto and Hurwitz in the 114-132S region after 20, 40, and 140 min of incubation, respectively. Kinetic studies suggested that 30-50S DNA complexes were formed first and then were converted to 60-705 complexes and eventually to 132S and 114S complexes. The rate and extent of complex formation were maximal at 300 and decreased at 370 and were unaffected by rifampicin (20,gg/ ml). Once the reaction halted, addition of more fraction II was without effect. These large protein-DNA complexes (>50S) were not observed with the RF system. Structure of DNA in 50S and 60-70S Protein-DNA Complexes. After centrifugation (as in Fig. 2), material in the 50S region was pooled and deproteinized and the DNA was characterized by neutral sucrose gradient centrifugation (Fig. 3A). The DNA present in this complex was a mixture of RFI and RFII forms; the latter sedimented faster than RFII and contained RFII structures with extended ss tails (a forms). The DNA in the 60-70S complex, analyzed the same way, contained a mixture of RFI, a RFII, and circular and linear ss DNA (consisting of DNA one unit to less than 1 unit in length). The nature of these DNA structures was also verified by electron microscopy (data not shown). After deproteinization, the DNA in the 50S complex was analyzed by alkaline sucrose gradient centrifugation (Fig. 3C); DNA products longer than 1 unit length were detected. These results resemble those obtained in vivo and suggest that a RFII forms are intermediates in )X174 ss viral DNA formation.

DISCUSSION The results presented above define some of the requirements essential for in vitro ss viral DNA synthesis from RFI. A number of genetically defined proteins (dna B., dna C, dna G, rep, dna Z, and OX174 A protein) are essential for maximal RF replication as well as for ss DNA formation; these requirements mirror those found in vivo (10). In the presence of kX174 gene proteins and host proteins essential for morphogenesis of OX174, RFII rapidly complexes with OX174 coded proteins in vivo to form a 50S complex; in the process, a RFII structures are formed (7). In vitro, we observed similar intermediates (Fig. 2) that were converted, with time, to 60-70S and larger protein complexes. Analysis of the DNA in these complexes revealed the a RFII form and RFI (in 50S), whereas the 60-70 complexes contained a RFII as well as ss circular and linear viral DNA (Fig. 3). Eventually, one can detect large complexes (114-132 S) with infective biological activity. Extracts prepared from kX174-infected cells early in the replication cycle catalyzed RF replication. In contrast, extracts prepared from infected cells later in the replicative cycle only catalyzed ss DNA formation. This suggests that the temporal switch in RF replication to ss DNA synthesis is due to viral proteins that complex RF structures. The dominance of ss DNA

Proc. Natl. Acad. Sci. USA 74 (1977)

4199

synthesis over RF replication was observed when the above extracts were combined. Infection of E. coil with OX F- mutant (harvested at a late stage) yielded extracts the catalyzed only

RF replication. However, the addition of extracts from E. coli infected with OX A- mutants (harvested at a late stage) to OX F- extract blocked RF replication and resulted in ss DNA synthesis. A simplified scheme summarizing the two systems is presented in Fig. 4.

RF replication and ss DNA synthesis both are sensitive to nalidixic acid and novobiocin. They require a number of similar proteins essential for E. coli DNA replication. It is particularly striking that the dna C protein (a markedly labile component) is absolutely required for each pathway. In the ss DNA system, the initial round of DNA synthesis yields viral DNA containing no label from added dNTPs. However, there is a rapid conversion of the label from input RFI to large molecular weight forms. It is only after the first round of ss DNA synthesis that labeled dNTPs are incorporated into ss DNA. This is not true in the case of RF replication, in which the complementary strand is labeled during the first round of DNA synthesis. The striking feature of the ss DNA system in the rolling circle model is the catalytic role of the complementary strand. The results presented above are in accord with this model. Recently, Mukai and Hayashi (13) described the in tvtro incorporation of dNTPs into phage-specific DNA catalyzed by extracts from OX174-infected cells. Their results and our results are in accord: ss DNA synthesis involves the formation of DNA-protein

complexes.

This work was supported by the National Institutes of Health, the National Science Foundation, and The American Cancer Society. 1. Scheckman, R., Weiner, J. J. & Kornberg, A. (1974) Science 186, 987-993. 2. Wickner, S. & Hurwitz, J. (1975) Proc. Natl. Acad. Sci. USA 71, 1549-1553. 3. Henry, T. J. & Knippers, R. (1974) Proc. Natl. Acad. Sci. USA 71, 1549-1553. 4. Ikeda, J., Yudelevich, A. & Hurwitz, J. (1976) Proc. Natl. Acad. Sci. USA 73,2669-2673. 5. Eisenberg, S., Scott, J. F. & Kornberg, A. (1976) Proc. Natl. Acad. Sci. USA 73, 1594-1597. 6. Sumida-Yasumoto, C., Yudelevich, A. & Hurwitz, J. (1976) Proc. Natl. Acad. Sci. USA 73,1887-1891. 7. Fujisawa, H. & Hayashi, M. (1977) J. Virol. 19, 409-415. 8. Francke, B. & Ray, D. S. (1972) Proc. Natl. Acad. Sci. USA 69, 475-479. 9. Baas, P. O., Kansz, H. S. & Sinsheimer, R. L. (1976) J. Mol. Biol.

102,633-656.

10. Denhardt, D. T. (1975) CRC Crit. Rev. Microbiol. 161-224. 11. Gellert, M., Mizuchi, K., O'Dea, M. H. & Nash, H. A. (1976) Proc. Natl. Acad. Sci. USA 73,3872-3876. 12. Marians, J. K., Ikeda, J., Schlagman, S. & Hurwitz, J. (1977) Proc. Natl. Acad. Sci. USA 74,1965-1968. 13. Mukai, R. & Hayashi, M. (1977) J. Virol. 22, 619-625.