Replication of vaccinia DNA in mouse L cells III. Intracellular forms of viral DNA

VIROLOGY

82, 308-322 (1977)

Replication

of Vaccinia

III. Intracellular

MARIANO Department

of Microbiology,

ESTEBAN

DNA in Mouse

L Cells

Forms of Viral DNA AND

JOHN A. HOLOWCZAK’

College of Medicine and Dentistry of New Jersey, Rutgers Piscataway, New Jersey 08854

Medical

School,

Accepted June 20,1977

Parental and replicating vaccinia DNA molecules, labeled with [3H]thymidine or [i4Clthymidine, present in infected L cells were analyzed by sedimentation in neutral and alkaline sucrose gradients. Alkaline sucrose gradient sedimentation analysis of labeled parental genomes present in infected cells showed that: (a) Such genomes were not degraded to acid-soluble products during the infection cycle; (b) cell-associated, cross-linked parental molecules (102 and 90-92 S) were “nicked”; parental DNA molecules sedimenting at 70-72 S were detected, but further nicking or degradation did not occur; and (c) molecules sedimenting at 90-92 S appeared to accumulate in the cytoplasm of infected cells and could serve as the templates for semiconservative DNA replication. When analyzed in neutral sucrose gradients, about 90% of viral DNA molecules labeled from 1 to 2 hr postinfection were associated with large aggregates or complexes which pelleted under the conditions of analysis used in these studies. With time (2-3 hr) after infection, viral DNA molecules could be dissociated from such aggregates and resolved by sedimentation in neutral sucrose gradients. The dissociation of labeled viral DNA molecules from complexes required continuous protein synthesis. Viral DNA could be released from complexes by treatment with alkali or digestion with S-l nuclease, but not with RNase or Pronase. The results suggest that single-stranded (ss) DNA regions per se or proteins having affinity for ssDNA may bind the replicating vaccinia DNA molecules together in complexes. INTRODUCTION

Intracellular vaccinia DNA has been shown to be associated with characteristic structures in infected cells called “factories.” Initial studies utilizing autoradiographic techniques (Cairns, 1960), staining with fluorescent antibody specific for viral proteins (Kato et al., 1959,1964), and electron microscopy (Dales and Siminovitch, 1961; Harford et al., 1966) showed that the viral DNA replicated in association with viral proteins in such factories in the cytoplasm of infected cells. Joklik and Becker (1964) demonstrated that both parental viral DNA and newly synthesized viral DNA existed in the cytoplasm of infected cells associated with large aggregates or complexes which were heteroge-

nous in size and required Mg2+ ions for their stability. During the early stages of the infection cycle, practically all viral DNA was associated with such complexes; after 3 to 4 hr, progressively increasing amounts of viral DNA became dissociated from them (Joklik and Becker, 1964). Dahl and Kates (1970a, b) were able to isolate rapidly sedimenting structures containing newly replicated viral DNA in the form of a DNA-protein complex within which transcription of “early” and “late” viral mRNA synthesis could be demonstrated in vitro. The protein components of the complex were shown to consist mainly of two virus-specific DNA-binding proteins with molecular weights of about 28,000 and 33,000 (Polisky and Kates, 1972, 1975; Sarov and Joklik, 1973). Another protein, an arginine-rich basic polypeptide, of molecular weight ll,OOO-12,000 was shown to be

i Author to whom reprint requests should be addressed. 308 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0042-6822

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associated with vaccinia DNA in the cytoplasmic factories (Pogo et al., 1975). Treatment of the complex with Pronase, ionic detergents, or high concentrations of salts abolished the rapid sedimentation properties of the complex-associated DNA, leading to the suggestion that associated proteins played a structural role in noncovalently complexing the viral DNA molecules (Polisky and Kates, 1972). Further studies have shown that the proteins isolated from the aggregates would form complexes not only with vaccinia DNA but also with DNA from other sources (Polisky and Kates, 1976). Recently, involvement of the host cell nucleus in the replication of poxvirus DNA has been suggested on the basis of autoradiographic studies (Walen, 1971) and molecular DNA-DNA hybridization (Bolden et al., 1975; Gafford and Randall, 1976; LaColla and Weissbach, 1975). The exact interaction of cytoplasm and nucleus in the replication of poxvirus DNA remains to be determined. Here we report our characterization of the intracellular forms of vaccinia DNA present in intact infected mouse L cells, under conditions of analysis which preserved the structure of viral DNA. Cellassociated parental DNA labeled with [3H]thymidine and newly synthesized viral DNA labeled with [14Clthymidine were analyzed from the time when viral DNA synthesis begins in infected cells to the time of virion formation. The techniques employed permitted separation of viral DNA from host DNA and in addition completely removed proteins bound to the viral DNA. It will be shown that, initially, replicating vaccinia DNA existed in the form of large complexes in the cytoplasm of infected cells from which a pool of replicating intermediate strands and mature viral DNA molecules could be dissociated. The experiments will also show that continuous protein synthesis is required to obtain an orderly dissociation of newly replicated DNA molecules from the complexes under the conditions used in the analyses to be described. It will be demonstrated that digestion with S-l nuclease, but not with pancreatic RNase or Pronase,

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309

resulted in the release of DNA molecules from complexes. The results indicate that replicating vaccinia virus DNA molecules are held together in complexes by alkalilabile material and that ssDNA species may link, or in some way facilitate the linking together of, DNA molecules in the complexes. 2 MATERIALS

AND

METHODS

Virus and cells. Growth of L cells in suspension culture and the purification of vaccinia virus strain WR from mouse L cells have been described previously (Holowczak and Joklik, 1967; Holowczak and Diamond, 1976). Virion pools were used immediately after purification. Special care was taken to avoid damage of parental DNA since introduction of nicks occurred during the manipulation of purified virus (Holowczak, 1976). This phenomenon has been related to the action of the virion associated ssDNase (Pogo and Dales, 1974a, b) in cutting ssDNA regions (unwinding) that can be seen by electron microscopy in the structure of virion DNA (Esteban, Flores, and Holowczak, submitted). In any given purified virus preparation (six preparations labeled with either [3H]thymidine or 13*Plorthophosphoric acid) the relative proportion of viral DNA molecules, Type III (102-106 S, with two cross-links) to Type II (92-94 S, with one cross-link), was variable. Only virions containing a relatively high proportion of molecules with two cross-links (Type III, 102106 S) were used for these experiments (Geshelin and Berns, 1974; Holowczak, 1976; Holowczak and Diamond, 1976). The EB/PFU ratio was about 5O:l when titrated in primary chick embryo fibroblast cell cultures. Virions labeled in their DNA were prepared by labeling infected L cells (1 x lo6 cells/ml) with 2 &i/ml of 13Hlthymidine (50.8 Ci/mmol). The isotope was added 2 hr 2 Abbreviations used: EB, elementary body; PFU, plaque-forming unit; BSA, bovine serum albumin; ssDNA, single-stranded DNA, dsDNA, doublestranded DNA; TCA, trichloroacetic acid; m.o.i., multiplicity of infection; Sp act, specific activity; p.i., postinfection; HU, hydroxyurea; BME; p-mercaptoethanol.

310

ESTEBAN

AND

after virus infection, and the virus particles were purified 24-28 hr later. The specific activity of the purified virion preparation labeled with 13Hlthymidine was 4 x lo4 cpm/pg of DNA. Adenovirus type 2 DNA labeled with [3Hlthymidine was purified as described previously (Burlingham and Doerfler, 1971) and had a specific activity of 3 x lo4 cpm/pg of DNA. Infection of cells. Mouse L cells grown in suspension and concentrated to 1 x 10’ cells/ml in Puck’s saline A plus 20 n&f Mg2+ and 1% fetal calf serum were infected with vaccinia virus (m.o.i. = 1000 EB/cell) for 15 min at 37” (Becker and Joklik, 1964; Esteban and Metz, 1973). At the end of the adsorption period (designated zero time) the cells were diluted to 1 x lo6 cells/ml in Eagle’s MEM containing 5% calf serum and 0.5% lactoalbumin hydrolysate. Labeling of infected cells and preparation of cell lysates. Infected and mock-infected cells were labeled with [14Clthymidine (2-3 PCilml) or 13Hlthymidine (lo-20 &i/ml) for the times indicated in the legends of the appropriate figures. At the end of the labeling time, 1 x lo6 cells were removed from the cultures, poured into 3.0 ml of ice-cold buffered saline solution (50 miV Tris-HCl buffer, pH 7.6; 150 miV NaCl; 5 miV EDTA) and collected by centrifugation at 1000 rpm for 2 min. The cells were washed once with 4.0 ml of buffered saline, harvested by centrifugation, and resuspended in 1.0 ml of buffered saline. An aliquot of the cell suspension (2-3 x lo5 cells) was loaded onto either a neutral or an alkaline sucrose gradient which had been previously overlaid with 0.8 ml of lysis solution (see below). Cells were allowed to complete lysis for 16-20 hr at 4” in the dark (Esteban and Holowczak, 1977). It has been demonstrated that when a maximum of 2-3 x lo5 cells is analyzed under these conditions, 90% of the host cell DNA molecules pellet after centrifugation in alkaline sucrose gradients, while viral DNA molecules, free of protein, can be resolved in the gradients (Esteban and Holowczak, 1977). In neutral sucrose gradients both host and viral DNA molecules pelleted when samples were analyzed

HOLOWCZAK

early (O-2.5 hr) after infection. By our delinition, viral molecules which pellet in neutral sucrose gradients under the conditions of analysis described are part of large DNA complexes. In order to distinguish between viral and host cell molecules early after infection, viral DNA molecules labeled after hydroxyurea reversal were studied as described in a subsequent section. For labeling proteins, 1 x lo6 cells were removed from the cultures at 1.5, 2, and 2.5 hr p.i. and collected by centrifugation at 1000 rpm for 2 min. The cells were then resuspended in 1.0 ml of Eagle’s medium minus methionine buffered with 20 mM Tris, pH 7.8, and labeled with 10 $i of [35S]methionine for 20 min with continuous shaking at 37”. Aliquots (2 x lo5 cells) of the labeled culture were then processed as described above. Use of hydroxyurea and enzymatic digestion of vaccinia DNA complexes. Mouse L fibroblasts (1 x lo7 cells) were infected as described above, and at the end of the adsorption period the infected cells were diluted into medium containing 5 mM hydroxyurea (HU). At 4 hr p.i., the HU was removed by washing, as described previously (Pogo and Dales, 1971; Fil et al., 1974), and resuspended in fresh medium at a concentration of 1 x lo6 cells/ml and 1 x lo6 cells/ml resuspended in fresh medium. During the period of HU treatment, host functions, including DNA synthesis, are shut off with kinetics similar to those observed in infected cells not treated with HU (Jungwirth and Launer, 1968; Pogo and Dales, 1971; Fil et al., 1974). After HU reversal, more than 90% of [3H]thymidine incorporated by infected cells is present in viral DNA (Pogo and Dales, 19711. Using this technique, one can follow the fate of [3Hlthymidine-labeled viral DNA associated with rapidly sedimenting complexes (pellet fraction), since host DNA, which also pellets under our conditions of analysis, is not significantly labeled after HU reversal of infected cells. To learn more about the nature of the material which holds the complexes together, lysates prepared from infected cells containing complexes were digested

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with Pronase (1 mg/ml), pancreatic ribonuclease (100 pg/ml), or S-l nuclease (10, 100, or 1000 U/ml). The amount of viral DNA released from complexes by these enzymatic treatments and the sedimentation characteristics in neutral sucrose gradients of the released viral DNA were determined. Sedimentation crose gradients.

analysis

in neutral

su-

Neutral sucrose gradients (11.0 ml; l&30%, w/v, sucrose) containing 1 M NaCl; 0.01 M EDTA; 0.15% Sarkosyl NL-97; 0.05 M Tris-HCl buffer, pH 7.6, were overlaid with 0.8 ml of lysis solution (1 M NaCl, 0.01 M EDTA, 1% Sarkosyl NL-97, and 0.03 M PME in 0.05 M TrisHCl buffer, pH 7.6). An aliquot of cells (2-3 x 105)was applied and lysis was allowed to proceed as described above. The gradients were then centrifuged for 3 hr at 39,000 rpm and 20” (Spinco SW 41 rotor, L3-50 ultracentrifuge). The gradients were fractionated, 100 pg of carrier (BSA) was added to each fraction, and the samples were precipitated with 10% TCA (1 hr at 0”). The precipitates were collected on GF/ C filters, washed three times with 5% TCA, and dried. Samples were counted in a toluene-based scintillation mixture. Sedimentation coefficients were calculated according to Studier (1965) using adenovirus type 2 DNA (Burlingham and Doerfler, 1971) or other viral DNAs as sedimentation markers.

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DNA

tured calf thymus DNA. Chemicals and radioisotopes. Pronase was purchased from Calbiochem, La Jolla, Calif. A solution of 10 mg/ml was prepared in 10 mM Tris-HCl buffer, pH 7.6; 10 n&f EDTA and self-digested at 37” for 2 hr. Sarkosyl NL-97 was obtained from Geigy Chemical Co., Ardsley, N. Y. V4ClThymidine (51.3 mCi/mmol), [3H]thymidine (50.8 Ci/mmol), and [35S]methionine (283.8 Ci/ mmol) were purchased from the New England Nuclear Corp., Boston, Mass. RESULTS

Kinetics of DNA Cells

Synthesis

in Infected

The rate of incorporation of [3Hlthymidine into acid-precipitable radioactivity in intact virus-infected cells was several times greater (5- to 20-fold) than that measured in uninfected cells. In infected cells, viral DNA synthesis began at about 1 hr p.i., reached a peak between 2-3 hr, and then decreased by 4 hr p.i. (Esteban and Holowczak, 1977). Similar patterns of DNA synthesis have been reported previously in cells infected with vaccinia virus (Holowczak and Diamond, 1976; Joklik and Becker, 1964). At the multiplicities of infection employed in these studies, the inhibition of host DNA synthesis was almost complete by 3-5 hr postinfection (Joklik and Becker, 1964).

Fate of Parental Viral DNA and Replication of Vaccinia DNA by Analysis in Linear sucrose gradients Alkaline Sucrose Gradients (15-30%, w/v) containing 0.7 M NaCl, 0.3 Mouse L cells were infected with virions N NaOH, 0.01 M EDTA, and 0.15% Sarko-

Sedimentation crose gradients.

analysis

in alkaline

SU-

syl NL-97 were overlaid with 0.8 ml of lysis solution (0.7 M NaCl, 0.5N NaOH, 0.01 M EDTA, 1% Sarkosyl). After centrifugation, the gradients were fractionated and the fractions were processed under the conditions described above for neutral sucrose gradients. S-l nuclease assay. S-l nuclease was obtained from Sigma Chemical Co. One unit of enzyme degraded 10 pg of radioactively labeled, heat-denatured vaccinia or L-cell DNA to acid-soluble products in 10 min at 45”. The reaction mixtures contained enzyme (40 U/ml); labeled DNA in 0.03 M sodium acetate buffer, pH 4.5; 0.1 M NaCl; 1 m&f ZnSO,; and 10 pg/ml of heat-dena-

labeled in their DNA with [3H]thymidine. Under the conditions described here for virus infection, about 50% of the inoculum particles labeled with 13Hlthymidine became cell-associated. Cells infected with [3H]thymidine-labeled virions were then labeled with [14Clthymidine beginning at 1 hr postinfection. To check the effectiveness of viral infection, cells were plated on coverslips and stained with the bis-benzimide derivative Hoechst 33258 (American Hoechst Corp.) and the number of cells with factories were counted by fluorescent microscopy (Esteban, 1977). Over 90% of the infected cells contained virus factories by 3 hr after infection. Parental t3H-la-

312

ESTEBAN AND HOLOWCZAK

beled) and replicating (14C-labeled) viral DNA molecules were then analyzed from 2.5 to 7 hr p.i. in alkaline sucrose gradients after lysing 3 x lo5 cells on the surface of gradients, as indicated in Materials and Methods. At early times postinfection (Fig. 1B) newly synthesized DNA appeared as a heterogeneous population of “intermediate molecules” which sedimented more slowly than intact mature ssDNA (Type I, 70-728). As the infection cycle progressed, their size increased and finally cross-linked molecules could be detected 4-6 hr p.i. (Figs. lC, D, E, and F). No evidence for molecules sedimenting more rapidly than cross-linked viral DNA (Type III, 102-106 S) could be found at any time during viral DNA replication (Fig. l), indicating that polymerization was symmetrical and did not involve concatemeric forms (Gilbert and Dressler, 1968; Ihler and Thomas, 1970; Kelly and Thomas, 1969). Mouse L cells were infected with virions containing [3Hlthymidine-labeled genomes which contained cross-linked genomes (Fig. 1A). When the parental viral DNA molecules present in infected cells from 2.5 to 7 hr p.i. were analyzed by sedimentation in alkaline sucrose gradients, three different types of ssDNA molecules were detected (Fig. 1). Of the total cell-associated parental DNA, 20-25% appears to remain as Type III (102-106 S, two cross-links) molecules. Since replication of vaccinia DNA is semiconservative (Esteban and Holowczak, 1977) and would require the introduction of at least one nick in molecules bearing two cross-links, such molecules may not participate in the replication of viral DNA. Of the remaining parental viral DNA molecules, 4550% sedimented like Type II (92-94 S, one crosslink), and 20-23% like Type I (70-72 S, intact mature ssDNA) molecules. Such molecules could serve as templates for the semiconservative replication of vaccinia DNA. Fate and Replication of Vaccinia DNA as Determined by Sedimentation Analysis in Neutral Sucrose Gradients

Cells were infected with virions contain-

ing DNA labeled with 13Hlthymidine (as in Fig. 1) and 1 hr later [14Clthymidine was added to label newly synthesized viral DNA. At 1-hr intervals after infection 1 x lo6 cells were removed from the cultures and 2 x lo5 of these cells were lysed on top of the neutral sucrose gradients as described in Materials and Methods and Fig. 2. Analysis of the labeled parental viral DNA in neutral sucrose gradients showed that by 1 hr after infection over 90% of the cell-associated parental DNA was found as mature dsDNA (68-72 S, Fig. 2A). However, by 2 hr p.i., about 50% of the parental DNA sedimented as part of large aggregates or complexes (Fig. 2B; Dahl and Kates, 1970a, b; Joklik and Becker, 1964) with the bulk of host DNA (see legend of Fig. 2). By 3 hr p.i., under our conditions of analysis, the parental viral DNA molecules associated with the complexes could be released and sedimented predominantly as mature dsDNA (68-72 S; Fig. 20 A small fraction of the newly synthesized viral DNA (17%) sedimented more rapidly than unit-length mature viral DNA (68-72 S; Fig. 20. Molecules sedimenting from 80 to 120 S have been detected in the cytoplasm of vaccinia-infected HeLa cells after pulse-labeled replicating viral DNA molecules were analyzed by sedimentation into neutral sucrose gradients (Holowczak and Diamond, 19761, but the exact nature of these molecules has not been determined. Approximately 7-10% of total labeled viral DNA was found to be smaller in size than unitlength DNA. After 4 hr p.i., most of the parental DNA (90% by 5 hr p.i.; Fig. 2E) sedimented in a mature form (69-72 S), while only 5 10% remained pellet-associated. Analysis of the newly synthesized DNA under conditions of continuous labeling with [14Clthymidine from 1 hr p.i. showed that, at 2 hr p.i., 87% of the newly synthesized labeled DNA sedimented with the bulk of host DNA in the form of large complexes (Fig. 2B; and Table 3). A small fraction (11%) sedimented more slowly than unit-length mature dsDNA (Fig. 2B). As the labeling period was prolonged, from

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OF VACCINIA

DNA

313

LO

IO

20

IO

20

A

IO

‘;f;-..-,, 5

IO

I5

20

FRACTION

25

30

35

40 FkACilON

Fro. 1. Alkaline sucrose sedimentation analysis of parental and newly synthesized DNAs in infected L cells from 2.5 to 7 hr p.i. Conditions for infection of2 x 10’ cells with labeled virus (m.o.i. = 1000EB/cell; sp act, 4 X IO4cpm/pg DNA) were as described in Materials and Methods. Newly synthesized DNA was labeled with 2 &i/ml of [“Clthymidine beginning at 1 hr p.i. At the times indicated, 1.0 ml of cells was removed from the culture and washed in buffered saline, and 3 x lo5 cells were loaded onto a 15-30% alkaline sucrose gradient overlaid with an O.&ml layer of lysis solution. Lysis of cells on top of the gradients, centrifugation, and determination of radioactivity in each fraction were as described in Materials and Methods. 3H-labeled parental viral DNA (0) and “C-labeled newly synthesized DNA (0), analyzed at different times after infection: (A) Virion DNA (0.9 pg) from purified virus lysed on top of the gradient under conditions identical to those used for intact cells; (B) 2.5 hr p.i.; (C) 4 hr p.i.; (D) 5 hr p.i.; (E) 6 hr p.i.; and (F) 7 hr p.i. Total [Wthymidine incorporation was (counts per minute): (B) 4112; (C) 7929; (D) 8026; (E) 8845; and (F) 10,141. Approximately 1440 cpm of 3H-labeled parental DNA was cell-associated from 2.5 to 7 hr p.i. Less than 10% of the total radioactivity applied to alkaline sucrose gradients was recovered in the pellet fraction.

ESTEBAN

314

AND HOLOWCZAK

?

- I(

!O

n

-5

I(

-5

-10

-5

5

10

I5 20 FRACTION

25

30

35

40

FIG. 2. Neutral sucrose sedimentation analysis of parental and newly synthesized DNAs present in infected L cells from 1 to 6 hr p.i. Conditions for infection of 2 x 10’ cells with labeled virus (m.o.i. = 1000 EB./cell; sp act, 4 x 10’ cpm/pg DNA) were as described in Materials and Methods. Newly synthesized DNA was labeled with 3 &i/ml of YCIthymidine added at 1 hr pi. At the times indicated, 1.0 ml of cells was removed from the culture and washed in buffered saline, and 3 x 105cells were loaded onto a 15-301 neutral sucrose gradient overlaid with an O.&ml layer of lysis solution. Lysis of cells on top of the gradients, centrifugation, and determination of radioactivity in each fraction were as described in Materials and Methods. 3H-labeled parental viral DNA (0) and YJ-labeled newly synthesized DNA (C), analyzed at different times after infection: (A) 1 hr; (B) 2 hr, (C) 3 hr; (D) 4 hr; (E) 5 hr, and (F) 6 hr. Total [“Clthymidine incorporation was (counts per minute): (B) 6752; (C) 11,411; (D) 13,503; (E) 13,815; and (F) 13,866. Approximately 1400 cpm of SH-labeled parental DNA was cell-associated from 1 to 6 hr p.i. Of this amount 683 cpm (48% was recovered in the pellet fraction 2 hr p.i.

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r

A 312 s

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DNA

315

3 hr onwards, the amount of newly synthesized DNA that cosedimented with mature viral dsDNA (68-72 S) increased and represented 50-60% of the total labeled DNA recovered from the infected cells. (Figs. 2C, D, E, and F.). Molecules larger and smaller in size than mature dsDNA were detected at 3 hr p.i., but their relative amounts (10-X%) did not increase as the infection cycle continued (Fig. 2). Vaccinia dsDNA Replicates as Part of Large Complexes from Which “lntermediate Strands” and Mature Viral DNA Can Be Released

To determine the sequence of events leading to the association and release of replicating viral DNA from aggregates, infected and mock-infected cells were pulse-labeled at 2 hr p.i. for 10 min with [3Hlthymidine and chased with an excess of cold thymidine (5 n&f), and the labeled viral DNA present at 2-3 hr p.i. was analyzed by sedimentation in neutral sucrose gradients. Analysis of the data from the experiment shown in Fig. 3 (Table 1) showed that, after a lo-min pulse, 60% of the replicating viral DNA molecules in infected cells sedimented with the bulk of host DNA as part of large complexes and were recovered in the pellet. During a chase, viral DNA from these complexes could be dissociated under the conditions of analysis used, so that, after a 50-min chase, about 50% of the total labeled DNA was found sedimenting in neutral sucrose

FRACTION

3. Neutral sucrose sedimentation analysis of newly synthesized DNA in infected cells released from large aggregates. Infected and mock-infected cell cultures (1 x 10’ each) were prepared as described in Materials and Methods. At 2 hr p.i. cells were labeled with 10 &i/ml of [3H]thymidine and 10 min later were chased with 5 mM cold thymidine. At the indicated times, 1 x 1Og cells were removed from the cultures and washed in buffered saline, and 2 x 1oJ cells were loaded onto 15-30% neutral sucrose gradients overlaid with an 0.8ml layer of lysis solution. Lysis of the cells, centrifugaFIG.

tion, fractionation of gradients, and determination of the distribution of radioactivity of each fraction were as described in Materials and Methods. (A) Cells pulse labeled for 10 min; (B) cells pulse labeled for 10 min and chased up to 2.5 hr p.i.; (0 cells pulse labeled for 10 min and chased up to 3 hr p.i.; (0) infected cells; (0) mock-infected cells. Total incorporation was (counts per minute): A, 8153 (0) and.50,781 (0); B, 8050 (0) and 54,019 (0); C, 11,837 (0) and 62,747 (@). The fractions indicated in (B) were pooled, dialyzed extensively against 10 mM Tris-HCl buffer, pH 7.6; 1 mM EDTA, and treated with S-l nuclease as indicated in Materials and Methods. The height of each horizontal bar in (B) indicates the percentage of the 3H-labeled DNA from infected cells (0) that was found S-1 nucleaseresistant.

316

ESTEBAN

AND

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TABLE PERCENTAGE

OF TOTAL RADIOACTIVITY

Labeling

Sedimentation

period

Pulse (10 min) Pulse + 20-min chase Pulse + 50-min chase

1

RECOVERED IN NEWLY SYNTHESIZED IN NEUTRAL SUCROSE GRADIENTS”

in neutral

VIRAL DNA MOLECULES RESOLVED sucrose gradients

Pelleted

More rapid than intact viral DNA

As intact viral DNA

Slower than intact viral DNA

58.2 31.0 6.3

6.5 7.0 13.3

6.3 20.9 44.5

28.7 41.3 36.1

a The radioactivity was recovered in 3H-labeled DNA molecules from infected cells shown in Fig. 3. Larger than intact viral DNA, fractions 1 to 15; intact viral DNA, fractions 16 to 20; smaller than intact viral DNA, fractions 21 to 37. The pellet represents the radioactivity recovered in the bottom of the centrifuge tube.

gradients as mature viral dsDNA (68-72 S). The relative proportion of the newly synthesized DNA molecules found in different size classes is given in Table 1. The DNA molecules sedimenting more slowly than mature viral DNA were found to be “released” first from the aggregates, followed by unit-length mature viral DNA. This heterogeneous population of smallsize DNA molecules consists of precursors of the mature viral DNA since late in infection (-6 hr p.i.) they all became part of unit-length mature viral dsDNA (Figs. 1 and 2). To test whether ssDNA regions were present in the DNA molecules resolved after sedimentation in a neutral sucrose gradient, fractions from the gradient (shown in Fig. 3B) were pooled and treated with S-l nuclease. about 20% of the total labeled DNA throughout the gradient was S-l nuclease sensitive and therefore contained ssDNA regions. The more slowly sedimenting DNA species were more sensitive to the enzyme than more rapidly sedimenting viral DNA. Of interest was the fact that 92% of the molecules which sedimented slightly slower than unitlength mature DNA (fractions 21-25) were resistant to the enzyme and thus, by delinition, have a dsDNA conformation. Protein Synthesis and the Release of Viral DNA Molecules from Complexes

The amount of protein associated with the large aggregates was determined by labeling the cells with [35S]methionine, followed by sedimentation analysis in neutral sucrose gradients as described in Ma-

TABLE

2

OF TOTAL RADIOACTIVITY RECOVERED WITH LARGE AGGREGATES FROM [35SlM~~~~~~~~~LABELED CELIS AFFER ANALYSIS BY SEDIMENTATION IN NEUTRAL SUCROSE GRADIENTS” PERCENTAGE

Time (hr p.i.)

Total radioactivity (cpm)

Pelleted radioactivity (%)

310.02 298.08 299.06 579.08

0.21 0.18 0.17 0.21

1.5 2.0 2.5 Mock-infected

a Infected and mock-infected cells were prepared as described in Materials and Methods and in Fig. 1. Conditions for labeling the cells for 20 min with 10 &i/ml of [35S]methionine, lysis of top of 15-308 neutral sucrose gradients, centrifugation, and determination of radioactivity were as described in Materials and Methods. The pelleted fraction was the total radioactivity recovered at the bottom of the centrifuge tube. Mock-infected cells were labeled at 2.5 hr p.i.

terials and Methods. As shown in Table 2, less than 0.3% of the total [%Slmethioninelabeled proteins remained bound to the aggregates. Continuous protein synthesis was required for the release of the viral DNA molecules from the aggregates. As indicated in Table 3, when total protein synthesis was depressed (>98%) by addition of 100 pg/ml of cycloheximide to the culture 2 hr postinfection, 85-90% of total PHlthymidine-labeled DNA remained associated with the large aggregates as late as 4 hr p.i. Enzymatic

Digestion

of DNA Complexes

To investigate further the nature of the material which binds the replicating viral DNA molecules together in complexes, the

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3

PEBCENTAGE OF TOTAL RADIOACTIVITY RECOVERED WITH LARGE AGGREGATES FROM CYLOHEXIMIDETREATED CELLS APTER SEDIMENTATION ANALYSIS IN NEUTBAL SUCROSE GRADIENTS’ Experimental Time Total raTotal rasample (hr pi.) diT;tEy dioactivity C recovered in pellet fraction (% of total

cpm)

Mock-infected

2 4

Infected

2 2.5 3.0 4.0

13,432 23,914 84,135 140,016 168,168 204,458

95.5 90.2 90.1 (87) 90.2 88.3 (67) 85.8 (51)

’ Infected and mock-infected cells were prepared as described in Materials and Methods and in Fig. 3. At 1 hr p.i. cell cultures were labeled with 5 &i/ml of 13H1thymidine and, at 2 hr p.i., cycloheximide (100 pg/ml) was added. Procedures for lysing the cells (2.5 x lo5 cells) on top of 15-308 neutral sucrose gradients, centrifugation, and determination of radioactivity were as described in Materials and Methods. The pellet fraction refers to the total radioactivity recovered at the bottom of the centrifuge tube. The numbers in parentheses indicate the percentage of counts recovered in the pellet fraction from infected cells labeled in the same manner but with no cycloheximide added (Fig. 2).

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menting at 82-110 S with a peak at 104 S were released (Fig. 4D). Viral DNA molecules with similar sedimentation characteristics, labeled during short pulses, have been detected in the cytoplasm of infected HeLa cells (Holowczak and Diamond, 19761, and about 17% of replicating viral DNA molecules labeled continuously after infection (Fig. 2) were found to sediment in this region of neutral sucrose gradients. With higher levels of S-l nuclease (100 U/ ml) molecules sedimenting from about 20102 S (Fig. 4E) in neutral sucrose gradients, were released from complexes. Digestion with 1000 U/ml of S-l nuclease also released DNA molecules sedimenting at 20-102 S, and the release of viral DNA from complexes was complete (Table 4). These results and those presented in Fig. 1 indicated that replicating vaccinia DNA molecules are held together in complexes by alkali-labile material and that ssDNA species (or regions) may link or in some way facilitate the linking together of DNA molecules in the complexes. DISCUSSION

The fate of parental DNA and the nature of newly synthesized viral DNA molecules were studied in vaccinia-infected mouse L fibroblast cells. The conditions experiments described in Fig. 4 and Table employed appear to preserve the structure of all the intracellular forms of viral DNA. 4 were carried out. Viral DNA complexes were labeled by Not only was the opportunity for nuclease addition of [3Hlthymidine to infected cells activity minimized or abolished, but also for 1 hr after reversal of a HU block. In- all the viral DNA forms present in the fected cells containing such DNA com- infected cells were analyzed. Analysis of newly replicated vaccinia plexes were lysed and the lysates were treated with pancreatic RNase, Pronase, DNA by sedimentation in alkaline sucrose or S-l nuclease. As indicated in Table 4, gradients indicated that replication was digestion with pancreatic RNase did not discontinuous and that polymerization result in a significant release of DNA from was symmetrical and did not involve concomplexes (compare Figs. 4A and C). Pro- catemeric forms, in agreement with previnase digestion resulted in the release of ously published results (Esteban and Ho15-20% of the [3H]thymidine-labeled mole- lowczak, 1977; Holowczak and Diamond, cules associated with the complexes (Table 1976). We had previously demonstrated that 4). The majority of these “released” DNA parental DNA molecules, bearing two species, when analyzed by sedimentation into neutral sucrose gradients, sedimented cross-links (102-106 S) were nicked in the more rapidly than intact viral DNA (Table cytoplasm of infected cells, a step which 4, Fig. 4B). Analyses of lysates after diges- would be necessary for semiconservative tion with S-l nuclease (10 U/ml, 5 min, replication of vaccinia DNA molecules 25”) showed that DNA molecules sedi- (E&ban Holowczak, 1977). In the studies

ESTEBAN

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HOLOWCZAK

5 t

0

5

IO

15

20

25

FRACTION

30

!

35 (TOP)

0

5

IO

I5

20

25

30

35 (TOP)

FRACTION

FIG. 4. Sedimentation analysis in neutral sucrose gradients of vaccinia DNA released from DNA complexes by enzymatic digestion. Mouse L fibroblasts (1 x 10’ cells) were infected at a multiplicity of 1000 EB/cell with vaccinia virus and at the end of the adsorption period diluted into medium containing 5 m&f UH. Four hours later, the HU block was reversed as described in Materials and Methods and the infected cells were resuspended in fresh medium (1 x lo6 cells/ml) and labeled for 1 hr with PHlthymidine (12 &i/ ml). Aliquots of the infected culture containing 2 x lo5 cells were removed and treated as described in Materials and Methods and in the footnotes to Table 4. After the appropriate treatment, aliquots of the cell lysates containing about 2.0 x lo5 cell equivalents were layered onto 15-30% neutral sucrose gradients which had been overlaid with 0.7 ml of lysis solution as described in Fig. 2. The gradients were then incubated for 16 hr at 4” in the dark, centrifuged as described in Fig. 2, and fractionated, and the distribution of acid-insoluble radioactivity was determined. (A) Distribution of viral DNA molecules in infected cells labeled for 1 hr after HU reversal. The majority of the DNA molecules are associated with complexes which pellet under these conditions of analysis (Table 4). A similar distribution of radioactivity was found in samples manipulated as described in Table 4 in preparation for treatment with enzymes, except that no exogeneous enzymes were added. (Bl Sample digested with Pronase (1 mg/mll (Table 4, footnote d) before analysis. (Cl Sample digested with 100 pg/ml of pancreatic ribonuclease (Table 4, footnote c) before analysis. (D) Sample digested with 10 U/ml (5 min, 25”) of S-l nuclease (Table 4, footnote e) before analysis. (El Sample digested with 100 U/ml (5 min, 25”) of S-1 nuclease (Table 4, footnote e.) The amount of viral DNA which pelleted and therefore was complex-associated after enzymatic digestion is indicated in Table 4. A DNA (-33 S) labeled with [‘*C]thymidine was included as a sedimentation marker in all samples analyzed. In parallel gradients, PHlthymidine-labeled vaccinia DNA (68-72 S), SV40 DNA, form I (-21 59, and PClthymidine-labeled DNA (-33 S) were analyzed. The sedimentation values indicated are relative to these markers.

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DNA

4

EFFECT OF ENZYMATIC DIGESTION ON VACCINIA VIRUS DNA COMPLEXES Experimental

samples0

Total radioactivity analyzed

(cpm)

Infected Infected Infected Infected Infected Infected

(not treated)* +RNase (100 ~g/ml)’ +Pronase (1 mg/mDd +S-1 nuclease (10 U/mDe +S-1 nuclease (lo* U/mDe +S-1 nuclease (lo3 U/ml)”

161,350 138,576 203,928 145,230 143,466 140,038

Distribution of radioactivity after analysis in neutral sucrose gradients’ (% of total recovered) In pellet (DNA cornplexes)

Sedimenting more rapidly than intact viral DNA (>68-72 S)

Sedimenting as intact viral DNA (6872 S)

Sedimenting more slowly than intact viral DNA (~68-72 S)

91.8 89.5 76.6 78.5 35.9 8.4

1.1 1.7 10.8 13.7 14.4 26.0

2.4 3.5 6.1 3.2 26.7 38.1

4.8 5.2 6.5 4.6 22.9 34.6

a Mouse L fibroblasts (1 x 10’ cells) infected with vaccinia virus were diluted at the end of the adsorption period into medium containing 5 mM HU. At 4 hr p.i. the cells were collected by centrifugation and washed to reverse the HU block as described in Materials and Methods. The cells were resuspended in fresh medium without inhibitor (1 x lo8 cells/ml) and labeled with [SH]thymidine (12 &i/ml, 1 hr, 37”). Aliquots of the cultures containing 2 x lo5 cells were harvested, washed with buffered saline, and prepared for analysis. Control, mock-infected cells similarly treated incorporated less than 5000 cpm when labeled in this manner after HU reversal. b Five aliquots of infected cells containing 2 x 10” cells were processed. One aliquot was lysed directly on the surface of a neutral sucrose gradient and analyzed as described in Materials and Methods and in Fig. 2. The remaining aliquots were treated as described below for samples which were digested with the various enzymes except that no enzyme was added. These served as controls to test the effect of adding various ions or detergents, incubation at >4O, or shaking on the sedimentation properties of the viral DNA complexes. In general, these manipulations in the absence of enzymatic digestion did not significantly alter the distribution of radioactivity as shown in Fig. 4A and summarized in Table 4. c Pancreatic RNase (100 fig/ml) was added to 2 x IO5 infected cells labeled as described above, and the mixture was layered onto a preformed 15-308 neutral sucrose gradient which had been overlaid with 0.7 ml of lysis solution (see Materials and Methods). The gradients were incubated at room temperature for 60 min and then at 4” for 16 hr in the dark before centrifugation. d Samples containing 2 x lo5 infected cells labeled as described above were lysed in 1.0 ml of lysis solution, Pronase (1 mg/ml) was added, and the mixture was incubated for 1 hr at 37” before being overlaid on 15-30% neutral sucrose gradients for incubation at 4” for 16 hr followed by centrifugation. e Infected cells were resuspended in 20 mM Tris-HCl buffer, 0.3 M NaCl, warmed to 25”; SDS (0.2%) added and the samples were shaken at room temperature for 10 min. To the lysate, 0.1 vol of 10X buffer (0.3 M sodium acetate, pH 4.5; 10 mM ZnSO,) was added and the mixture was gently rolled for 2 min. S-l nuclease (10, 100, or 1000 U/ml) was added and incubation with shaking was continued for 5 min. The samples were then rapidly mixed with lysis solution and layered onto 15-301 neutral sucrose gradients. ’ All samples, after being treated as described above, were incubated at 4” in the dark on the surface of 1530% neutral sucrose gradients which had been overlaid with 0.7 ml of lysis buffer as described in Materials and Methods. Gradients were then centrifuged and fractionated, and the distribution of radioactivity was determined (Fig. 2).

described here we have extended those initial observations and have followed the fate of cell-associated cross-linked parental DNA molecules throughout the infection cycle. From the results presented here, we have concluded: (a) Cell-associated parental DNA molecules are not degraded to acid-soluble products; even as late as 5-7 hr p.i. more than 90% of the labeled, cell-associated parental genomes

can be recovered. (b) Parental DNA molecules which sedimented at 102-106 or 9092 S in alkaline sucrose gradients appropriate for molecules bearing two crosslinks or a single cross-link (Geshelin and Berns, 1974; Holowczak, 1976) are nicked; molecules sedimenting at 70-72 S were detected in the cytoplasm of infected cells but further “degradation” of the parental molecules was not observed. (c) Analysis in

320

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alkaline sucrose gradients showed that parental and replicating viral DNA molecules are held together in complexes by alkali-labile material (compare Figs. 1 and 2).

One can speculate that one of the nicked forms of parental viral DNA detected in the cytoplasm of infected cells serves as the template for the semiconservative replication of vaccinia DNA (Esteban and Holowczak, 1977). Molecules sedimenting at 90-92 S (Type II, one nick) appear to accumulate with time after infection and may function as the template. However the matter is complex; nicking and repair of such nicks could readily occur during DNA replication so that the exact nature of the parental DNA molecules which function as templates during vaccinia DNA replication remains to be determined. Analysis by sedimentation in neutral sucrose gradients indicated that both parental DNA and newly synthesized viral DNA in the early stages of replication are present in the cell as part of large complexes which pelleted in neutral sucrose gradients, under the conditions of analysis described here. From 87 to 100% of the newly synthesized viral DNA was associated with these complexes at 2 hr p.i. (Fig. 1B). Approximately 50% of the cell-associated parental genomes were detected in such complexes at 2 hr p.i., but not at 1 hr p.i. (Figs. 2A and B). Thus, the initial event afier uncoating of the genome appears to be the association of viral DNA into large aggregates where replication begins. It may be that viral proteins synthesized early after infection, recovered in virosomes from vaccinia-infected cells (Dahl and Kates, 1970a; Polisky and Kates, 1975) play a role in forming these complexes. However, after the analysis described here, less than 0.3% of the total [35Slmethionine-labeled proteins remained associated with the aggregates (Table 2). This suggested that proteins did not play a major role in the structural integrity of these complexes or, alternatively, that proteins with a low methionine content were involved. Enzymatic digestion of cell lysates containing- replicating DNA in the form of _

HOLOWCZAK

complexes (Fig. 4, Table 4) showed that S1 nuclease, but not pancreatic RNase or Pronase, caused the complete release of DNA molecules from complexes. Polisky and Kates (1972) had demonstrated that Pronase digestion would cause the release of DNA molecules from complexes present in the cytoplasm of HeLa cells 3 hr p.i. We have been able to demonstrate that in L cells infected with vaccinia virus, DNA begins to be released after complexes at 2 hr p.i., in the absence of exogenous enzymes. It may be that at 3 hr p.i., as genomes whose replication is completed begin to associate with protein in preparation for viral assembly, the integrity of the complexes can be altered by Pronase digestion. Our studies were conducted with complexes formed 1 hr after HU reversal, at a time when no virion assembly occurs and Pronase digestion did not result in significant release of viral DNA molecules from such complexes (Table 4). The results of the experiments reported here indicated that ssDNA species could bind replicating DNA molecules together or, alternatively, serve as the site at which other kinds of molecules may bind to hold the complexes together. The results presented in Fig. 1 showed that the molecules involved in such binding were alkali labile. Since RNase digestion did not release DNA molecules from the complexes, one can conclude that proteins with affinity for ssDNA and relatively resistant to Pronase digestion or ssDNA regions per se are involved in the binding. The proteins found associated with complexes were poorly labeled with [“Slmethionine (Table 2). Further characterization of the “binding material” is required before a final conclusion concerning its nature can be reached. A “brush-like” model has recently been proposed for the intracellular T4 chromosome in which the DNA is held together at roughly genome-length intervals by some non-DNA core material which was not removed by detergents or protease treatments (Curtis and Alberts, 1976);the gene32 product (TCDNA unwinding protein) appears to play a role in forming these structures (Curtis and Alberts, 1976). Work is now in progress to determine if a

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virion-associated protein or a protein synthesized early in the infection cycle can be detected in vaccinia-infected cells, i.e., a protein which could bind to ssDNA regions in the vaccinia genome (Geshelin and Berns, 1974) resulting in complex formation. ACKNOWLEDGMENTS

The expert technical assistance of Ms. Domenica Bucolo is gratefully acknowledged. This investigation was supported by Public Health Service Research Grant CA-11027-08 from the National Cancer Institute. J.A.H. is a recipient of Public Health Service Research Career Development Award CA-70458 from the National Cancer Institute. We thank Dr. R. W. Schlesinger for critical reading of the manuscript. REFERENCES BECKER, Y., and JOKLIK, W. K. (1964). Messenger RNA in cells infected with vaccinia virus. Proc. Nat. Acad. Sci. USA 51, 577-585. BOLDEN,A., ANC~ER, J., and WEISSBACH,A. (1975). Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid. J. Viral. 16, 1584-1592. BIJRLINGHAM, B. T., and DOERFLER, W. (1971). Three size-classes of intracellular adenovirus deoxyribonucleic acid. J. Viral. 7, 707-719. CAIRNS, J. (1960). The initiation of vaccinia infection. Virology 11, 603-623. CURTIS, M., and ALBERTS, B. (1976). Studies on the structure of intracellular bacteriophage T4 DNA. J. Mol. Biol. 102, 793-816. DAHL, R., and KATES, J. R. (1970a). Intracellular structures containing vaccinia DNA: Isolation and characterization. Virology 42, 453-462. DAHL, R., and KATES, J. R. (1970b). Synthesis of vaccinia virus “early” and “late” messenger RNA in vitro with nucleoprotein structures isolated from infected cells. Virology 42, 463-472. DALES, S., and SIMINOVITCH,L. (1961). The development of vaccinia virus in Earle’s L strain cells as examined by electron microscopy. J. Biophys. Biohem. Cytol. 10, 475-502. ESTEBAN,M. (1977). Rifampicin and Vaccinia DNA. J. Viral. 21, 796-801. ESTEBAN, M., and HOUIWCZAK, J. A. (1977). Replication of vaccinia DNA in Mouse L cells. I. In uiuo DNA synthesis. Virology 78, 57-75. ESTEBAN, M., and METZ, D. H. (1973). Early virus protein synthesis in vaccinia virus infected cells. J. Gen. Viral. 19, 201-216. FIL, W., HOL~WCZAK, J. A., FLARES, L., and THOMAS, V. (1974). Biochemical and electron mi-

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croscopic observations of vaccinia virus morphogenesis in HeLa cells after hydroxyurea reversal. Virology 61, 376-396. GAFF~RD,L. G., and RANDALL, C. (1976). Virus specific RNA and DNA in nuclei of cells infected with fowlpox virus Virology 69, l-. GESHELIN, P., and BERNS, K. I. (1974). Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA. J. Mol. Biol. 88, 785-796. GILBERT, W., and DRE~SLER, D. (1968). DNA replication: The rolling circle model. Cold Spring Harbor Symp. Quant. Biol. 23, 473-484. HARFORD, C. G., HAMLIN, A., and RIEDERS, E. (1966). Electron microscopic autoradiography of DNA synthesis in cells infected with vaccinia virus. Exp. Cell. Res. 42, 50. HOL~WCZAK, J. A. (1976). Poxvirus DNA. I. Studies on the structure of the vaccinia genome. Virology 72, 121-133. HOLOWCZAK, J. A., and DIAMOND, L. (1976). Poxvirus DNA. II. Replication of vaccinia virus DNA in the cytoplasm of HeLa cells. Virology 72, 134-146. HOL~WCZAK, J. A., and JOKLIK, W. K. (1967). Studies on the proteins of vaccinia virus. I. Structural proteins of the virion and core. Virology 33, 717725. IHLER, G. M., and THOMAS, C. A. (1970). Equal incorporation of both parental bacteriophage T7 deoxyribonucleic acid strands into intracellular concatemeric DNA. J. Viral. 6, 877-879. JOKLIK, W. K., and BECKER, Y. (1964). The replication and coating of vaccinia DNA. J. Mol. Biol. 10, 452-474. JUNGWIRTH, C., and LAUNER, J. (1968). Effect of poxvirus infection on host cell deoxyribonucleic acid synthesis. J. Viral. 2, 401-408. KATO, S., TAKAHASHI, M., KAMEYAMA, S., and KAMAHORA, J. (1959). A study on the morphological and cytoimmunological relationship between the inclusions of variola, cowpox, rabbitpox, vaccinia (variole origin) and vaccinia IHD and a consideration of the term “Guarnieri body.“Biken J. 2,353363. KATO, S., OGAWA, M., and MYAMOTO, M. (1964). Nucleocytoplasmic interaction in poxvirus infected ceils. I. Relationship between inclusion formation and DNA metabolism of the cells. Biken J. 7, 45-56. KELLY, T. J., JR., and THOMAS, C. A. (1969). An intermediate in the replication of bacteriophage T7 DNA molecules. J. Mol. Biol. 44, 459-475. LA COLLA, P., and WEISSBACH, A. (1975). Vaccinia virus infection of HeLa cells. I. Synthesis of vaccinia DNA in host cell nuclei. J. Viral. 15, 305-315. POGO,B. G. T., and DALES, S. (1971). Biogenesis of vaccinia: Separation of early stages from maturation by means of hydroxyurea. Virology 43, 144151.

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Poco, B. G. T., and DALES, S. (1974a). Biogenesis of poxvirus: Inactivation of host DNA polymerase by a component of the invading inoculum particle. Proc. Nat. Acad. Sci. USA 70, 1726-1729. POGCJ, B. G. T., and DALES, S. (1974b). Biogenesis of poxvirus: Further evidence for inhibition of host and virus DNA synthesis by a component of the invading inoculum particle. Virology 58, 377-386. Pooo, B. G. T., KATZ, J. R., and DALES, S. (1975). Biogenesis of poxvirus: Synthesis and phosphorylation of a basic protein associated with the DNA. Virology 64, 531-543. POLISKY, B., and KATES, J. R. (1972). Vaccinia virus

intracellular

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complex: Biochemical

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characteristics of associated protein. Virology 49, 68-79. POLISKY, B., and KATES, J. R. (1975). Viral specific polypeptides associated with newly replicated vaccinia DNA. Virology 66, 128-139. POLISKY, B., and KATES, J. R. (1976). Interaction of vaccinia DNA-binding proteins with DNA in uitro Virology 69, 143-147. SAROV, I., and JOKLIK, W. K. (1973). Isolation and characterization of intermediates in vaccinia virus morphogenesis. Virology 52, 223-233. STUDIER, F. W. (1965). Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11, 373-390. WALEN, K. H. (1971). Nuclear involvement in poxvirus infection. Proc. Nat. Acad. Sci USA 68, 165168.