Intracellular structures containing vaccinia DNA: Isolation and characterization

VIROLOGY

42,453-462

Intracellular

(1970)

Structures

Containing

Vaccinia

DNA:

Isolation

and

Characterization R. DAHL Department

of

Chemistry,

University Accepted

J. R. KATES

AND

of Colorado, June

Boulder,

Colorado

80302

24, 1970

A sucrose density gradient technique is described which is effective in the partial purification and analysis of vaccinia DNA complexes isolated from infected cells. The structures containing viral DNA are separated primarily on the basis of their densities. The viral DNA complexes undergo dramatic alterations in density as a function of time during the infection cycle. The low density of several components suggests that some viral DNA components are associated with cellular membranes.

Intracellular vaccinia DNA was studied in some detail (Becker and Joklik, 1964a) at Intracellular viral DNA appears to be various times after infection by fractionaassociated with proteins which function in tion of the cytoplasmic fraction on the basis the transcription and regulation of viral genes (Snyder and Geiduschek, 1968; Hay- of size by sucrose density gradient centrifuward and Green, 1969). Recent studies have gation. In the latter study it was observed that newly replicated viral DNA occurred substantiated the value of experimentation with native viral DNA-protein complexes in large aggregates which were heterogeneous isolated from infected cells (Snyder and in size and which were readily disrupted to, Geiduschek, 1968; Hayward and Green, apparently, smaller aggregates by removal of 1969). magnesium from the cell breakage buffer. In this and the adjoining communication It was proposed that a protein might have we present the results of initial studies on been responsible for maintenance of the agthe isolation and characterization of vacgregated condition of the viral DNA and cinia DNA-protein complexes from infected that magnesium, in some manner, might HeLa cells. Since these complexes are the have served to preserve this aggregate. probable sites of gene transcription and Below we describe a methodology for the duplication, it follows that in vitro studies of their structure and function may elucidate characterization and partial purification of vaccinia DNA-containing complexes. These their role in the above processes. Little information is available concerning isolated structures retain the ability to the state of vaccinia DNA in the infected synthesize viral messenger RNA in vitro. cell complex. Autoradiographic studies Since viral DNA complexes are quite (Cairns, 1960) indicated that viral DNA poorly defined biochemically it seems apreplication occurs in a few discrete cyto- properiate to refer to such structures by the plasmic foci. Electron microscopic studies general term “virosomes” (viral body), in confirmed Cairns’s conclusions by demonstrating large cytoplasmic inclusions con- analogy with chromosomes,in order to avoid taining viral DNA and possessinga complex more restricted nomenclature (e.g., viral configuration (Dales, 1963). Assembly of DNA, DNA “factories,” etc.), which may, viral progeny was observed at the periphery in fact, create a misleading impression conof the viral DNA inclusions. cering their composition and function. INTRODUCTION

453

454

DAHL MATERIALS

AND

AND

KATES

METHODS

Virus and cells. The WR strain of vaccinia virus and HeLa S-3 cells, grown in suspension culture, were used in the present studies. Cell growth and virological procedures were as previously described (Becker and Joklik, 1964b). Preparation of cytoplasmic extracts. Cells were harvested by centrifugation and washed once in 0.15 M NaCl containing 0.05 M Tris-HCI, pH 8.0, and EDTA, 0.005 M. The cells were then resuspended in 0.01 M TrisHCI pH S.0 containing 0.005 M EDTA and 0.01 31 KC1 at a concentration of 3 X lo7 per milliliter and kept at 0°C for 5 min. Cell breakage was carried out in a Dounce homogenizer which was calibrated with respect to number of strokes to give greater t(han 90 % cell disruption. The cell lysate was then centrifuged at SO0 g for 2 min to remove the nuclei. The cytoplasmic fraction was carefully removed, taking care not to disturb the nuclear pellet. Sucrose density gradient centrifugation. Biological grade RNase-free sucrose was purchased from Schwarz (Orangeburg, New York). Sucrose density gradients were made up in Tris-HCl buffer, 0.01 M, pH 8.0, containing 0.05 M KC1 and 0.005 M EDTA (final concentration in sucrose solution). Gradients ranging from 38 to 50% sucrose w/v over a 5 ml of SO% sucrose cushion were used for analysis of the cytoplasmic fraction. Approximately 2 ml of the cytoplasmic fraction was layered on top of the gradients and they were centrifuged at 27,000 rpm at 5” in the SW 27 rotor of the Spinco ultracentrifuge for at least 2 hours and in some cases for 12 hours. For additional purification of viral DNA complexes 50-80% w/v sucrose density gradients were used and the conditions were the same as described above except that the Spinco SW41 rotor was used at 35,000 rpm for at least 12 hours. Fractions were collected from the bottom of the tubes and the radioactivity in DNA was estimated after 5% trichloroacetic acid precipitation and collection on Whatman GF/C fiberglass filters. Alteration of the ionic composition of the breakage medium or in the sucrose density gradient buffer has marked effects on the

fractionation of the observed viral DNA components. Determination of DNase sensitivity of viral DNA complexes. Gradient fractions were made 0.01 M with respect to MgCl, and were incubated at 37” with 50 pg pancreatic DNase per milliliter for 30 min. The sample was then precipitated with 5% trichloroacetic acid at 0” and filtered. The amount of DNA remaining acid insoluble was determined by liquid scintillation spectrometry. Preparation of radioactive virus and cores. Virus labeled with thymidine-H3 was grown with 1 mCi of thymidine-H3 (20 Ci/mMole) per liter of medium containing 4 X IO* cells. When labeling was carried out with thymidine-U4, 50 &i/liter of infected cells was used. Virus was prepared 24 hour post-infection by previously described procedures (Joklik, 1962). Vaccinia cores were prepared from purified virus preparations using the 2-mercaptoethanol and nonionic detergent (Nonidet P-40 or Triton-X-100) as previously described (Kates and Beeson, 1970). RESULTS

Cytoplasmic Nuclease Acitivity Since our aim was to isolate vaccinia DNA complexes in a state closely resembling their native, in vivo condition, one of our prime concerns was to avoid exposure of the complexes to nuclease digestion during the isolation procedure. Numerous deoxyribonuclease activities have been characterized in vaccinia-infected HeLa cells (&Auslan, 1965; Eron and ?rIcAuslan, 1966). It may be seen in Fig. 1 that radioactively labeled viral DNA is degraded extensively when the cytoplasmic fraction from infected HeLa cells is incubated in buffer containing 2.5 mM MgCl,. Although this degradation is reduced at 2’ it is not completely suppressed and extensive degradation of the viral DNA occurs during the course of normal manipulations. Cytoplasmic extracts prepared in 5 mM EDTA and lacking Mgz+ result in no detectable degradation of viral DNA to acid-soluble components. Thus EDTA was used routinely during the isolation and initial purification of the vaccinia DNA complexes. After the first sucrose gradient

INTRACELLULAR

STRUCTURES

x

h 0

3

i

OU 0

MINUTES FIG. 1. Kndogenous nuclease degradation of vaccinia DNA in HeLa cytoplasmic extracts. Viral DNA was labeled in infected cells from 1.5 to 3 hours post-infection. Cytoplasmic extracts were prepared at 3 hours post-infection and incubated at 37”. The amount of viral DNA not degraded to acid-soluble fragments was determined at each time point by 5y0 TCA precipitation, collection of the precipitate on Whatman GF/A filters, and counting of the filters by liquid scintillation spectrometry: O---O, cytoplasm prepared in 0.01 M Tris-HCl pH 8.0, 0.0025 MgCl, 0.01 M NaCl; O--O, cytoplasm prepared in 0.01 M Tris-HCl pH 8.0, 0.005 M EDTA, 0.01 M KCl; H, Viral DNA complexes purified on sucrose gradient (see Fig. 5) then incubated in 0.01 M Tris-HCl pH 8.0 containing 0.005 M MgCl, and 0.01 M KCl.

fractionation of the “virosomes,” which separated them from the cytoplasmic soluble protein, little if any nuclease activity was observed when they were incubated in the presence of 5 mM nJg+. Xeclimentation of Vaccinia Virosomes Previous sedimentation studies of newly replicated vaccinia DNA present in cytoplasmic extracts of HeLa cells indicated that the DNA occurred in structures which were heterogeneous in size and possessed high sedimentation coefficient (Becker and Joklik, 1964a). The latter investigators observed that the large vaccinia DNA aggregates underwent reversible disaggregation into smaller fragments if Mg2+ was removed from the cytoplasmic extract. To test

CONTAINING

VACCINIA

455

DNA

whether or not Mg2+ was critical for maintenance of the aggregates, we infected HeLa cells and labeled the viral DNA for 60 min with thymidine-H3 between 1.5 and 2.5 hours post-infection. The cytoplasmic fraction was then prepared in a buffer containing no hIg2+ and 5 mM EDTA. Centrifugation of a portion of the cytoplasmic extract on a 2540% w/v sucrose gradient for 20 min at 20,000 rpm revealed that all the viral DNA had sedimented to the 80% sucrosecushion at the bottom of the tube (Fig. 2). This indicated a very high sedimentation rate for the viral DNA complexes even in the absence of Mg2+. The latter observation is not inconsistent with the results of Becker and Joklik. We suggest, however, that perhaps some other parameter, in addition to Mg2+ concentration, is critical for maintenance of aggregation since even in the absence of n4g+ large aggregates exist. Sonication of

0

5

10

15

FRACTION 2. Sedimentation of vaccinia-DNA plexes. Vaccinia-infected HeLa cells were labeled with thymidine-3H and cytoplasmic extracts were prepared at 3 hours post-infection in 0.01 M TrisHCl, pH 8.0, 0.01 M KCl, 0.005 M EDTA. Samples of these extracts were centrifuged for 10 min at 10” through a 2540y0 w/v sucrose gradient over an 80% sucrose cushion in 0.01 M Tris-HCl, pH 8.0, 0.05 M KCl, 0.005 M EDTA, at 20,000 rpm on the Spinco SW 27 rotor for 20 min. Fractions were collected from the bottom of the tube, and radioactivity in DNA was determined after 5y0 trichloroacetic acid precipitation; a--@, regular cytoplasmic fraction; A--A, cytoplasm son icated on MSE sonicator, 8 p amplitide for 15 set, prior to centrifugation. FIG.

456

DAHL

AND

KATES

the cytoplasmic fraction prior to sedimentation analysis on sucrose gradients resulted in a nearly complete disruption of the large aggregates (Fig. 2). Density Equilibrium Analysis of Vaccinia ‘LVirosomes” in Sucrose Gradienfs at Di$erent Stages of the Viral Growth Cycle In view of the heterogeneous size distribution of vaccinia DNA-complexes (Becker and Joklik, 1964a) we have utilized a density centrifugation method which exploits the high sedimentation, moderate density, and great stability in sucrose of the “virosomes” to separate them from other cytoplasmic components and to analyze changes in their structure during the viral growth cycle. This technique (see Materials and Methods) has enabled us to resolve several different structures which contain viral DNA and which possess characteristic densities in sucrose. Initially we addressed ourselves to the fate of radioactively labeled parental input DNA. It was known from previous studies (Dales, 1963, 1965; Joklik, 1964) that a moderate percentage on the input viral DNA is uncoated shortly after the introduction of the internal component, the core, into the cytoplasm of the infected cell. We wished therefore to determine the position of cores and uncoated parental viral DNA complexes in our gradient system. Cytoplasmic fractions prepared at various times after infection with thymidine-3H-labeled virus were analyzed on 38-50% w/v sucrose density gradients over an 80 % sucrose cushion after centrifugation at 27,000 rpm for 12 hours in the Spinco SW 27 rotor at 5”. Since the band positions of input DNA did not change as a function of time after infection, only the 2hour pattern is illustrated in Fig. 3. Two major components of density 1.157 and 1.175 are observed in the upper part of the gradient. The minor component floating on 80% sucrose is sometimes absent and probably represents an artifact of the preparative procedure resulting from disruption of the upper components (see Fig. 3b). Only the heavier (1.175 g/ml) component shows some sensitivity to DNase, and this sensi-

tivity varies from 30 to 50% of the DNA being rendered acid soluble at 2 hours postinfection (Fig. 3a). If cells are infected in the presence of 15 pg/ml of Streptovitacin, an inhibitor of protein synthesis and viral uncoating (Dales, 1965), both components remain entirely resistant to DNase digestion (Fig. 3b). Treatment of the cytoplasmic fraction with 0.05 % (final cont.) Triton- X 100 results in extensive conversion of the two light bands to heavier particles that float on the 80% sucrose cushion. Vaccinia cores purified from vaccinia virus particles also float on the SO% sucrose (Fig. 3b). It is likely, therefore, on the basis of previous studies (Cairns, 1960; Joklik, 1964; Dales, 1965) that the light input bands contain viral cores which may be associated with a cellular membrane, and that following disruption of the membrane by Triton X-100 the cores sediment to the 80% sucrose. The denser component (1.175 g/ml) may be a mixed peak containing uncoated DNA and cores, both associated with a membrane. Analysis of cytoplasmic extracts from cells which were labeled during the period of viral DNA replication (progeny DNA labeled) resulted in a different pattern of density banding. In these experiments infected cells were labeled continuously from 1 hour post-infection. The cytoplasmic extract prepared at 2 hours showed DNA distributed in two peaks (Fig. 4a). The light component banded near the position of the denser parental DNA peak while a denser component floated on the 80% sucrose cushion. In order to determine if the light, replicating DNA complex was isodense with the denser of the two parental components, the cytoplasm of cells infected with thymidine-H3 labeled virus and having thymidine14C labeled replicating DNA, was analyzed. Figure 4b shows that the replicated, progeny DNA possesses a slightly greater density than the input component in question (1.180 us 1.175). The replicated DNA complex isolated at 3 hours post-infection occurs almost entirely on the 80% sucrose cushion (Fig. 5) and very little of a lighter component is evident. The recovery of nearly all the newly replicated DNA in the heavy component at about

INTRACELLULAR

STRUCTURES

(1) 5

I

0

5

10

15

FRACTION

;c

P 2 x

0

5

10

15

FRACTION FIG. 3. Sucrose density gradient density equilibrium banding of vaccinia parental DNA components. (A) HeLa cells were infected with 300 elementary particles of thymidine-3H-labeled vaccinia virus per cell (lo4 cpm per lOlo vaccinia particles). The cytoplasmic fraction was prepared at 2 hours post-infection (see “Materials and Methods”) and layered over a 30 ml 38-50~0 w/v sucrose gradient over a 5 ml 80% w/v sucrose cushion (the sucrose solution contained 0.005 M EDTA, 0.05 M KCl, and 0.05 M Tris-HCl buffer, pH 8.0). The gradients were centrifuged on the Spinco SW 27 rotor at 27,000 rpm for 12 hours at 5”. Fractions of approximately 2 ml were collected from the bottom of the tube and assayed for acidinsoluble radioactivity after precipitation with 57, trichloroacetic acid. O-0, parental DNA; A----A, parental DNA after treatment of each

CONTAINING

VACCINIA

DNA

457

3 hours post-infection occurs almost entirely on the 80% sucrose cushion (Fig. 5) and very little of a lighter component is evident. The recovery of nearly all the newly replicated DNA in the heavy component at 3 hours post-infection has been consistently observed. This indicates that the lighter component containing progeny DNA is converted to the heavier component sometime between 2 and 3 hours post-infection. Progeny DNA is more than 90 % sensitive to DNase digestion between 2 and 3 hours post-infection (see Figs. 4a and 5). The distribution of viral DNA at 5 hours post-infection is considerably more complex (Fig. 6). In order to resolve all the components in this mixture, a combination of sedimentation and density equilibrium centrifugation is required. If the cytoplasmic extract is centrifuged for 2 hours, one observes about one-half of the amount of heavy component present at 3 hours, and in addition two components at the top of the gradient. Both of the upper components are about 50% resistant to DNase digestion. If the same sample is centrifuged for 12 hours the fraction of the uppermost component which is DNase sensitive observed in the 2 hours centrifugation is recovered on the 80% sucrose cushion. This indicates that it originally occurred on top of the gradient due to its small size rather than its true density. These results indicate that between 3 and 5 hours post-infection [after DNA replication has terminated (Becker and Joklik, 1964a; Kates and McAuslan, 1967)] the dense DNA component is converted to lighter particles fraction with 50 pm/ml pancreatic DNase at 37” for 30 min in presence of 0.01 M MgClz (B) HeLa cells were infected with thymidine3H-labeled vaccinia virus in the presence of 15 pg/ml Streptovitacin A. Extracts prepared at 2 hours were subjected to sucrose gradient analysis, as in (A), above: O-0, parental DNA; O--O, parental DNA after DNaee treatment of each gradient fraction; A----A, cytoplasmic fraction was made 0.1% v/v with respect to TritonX-100 prior to centrifugation; H, vaccinia virus cores labeled with thymidine-14C and run on a sucrose density gradient. This figure contains the combined results of two parallel gradients.

4.58

DAHL

AND

KATE

and also to smaller particles. These new components probably represent stages of viral maturation which have been observed in previous studies (Becker and Joklik,

0

5

10

15

20

FRACTION

0

5

10

15

20

FRACTION

10

FIG. 5. Sucrose density gradient analysis of a-hours post-infection vaccinia DNA complexes. Infected cells were labeled with thymidine-i4C from 1 to 3 hours post-infection. At 3 hours the cytoplasmic fraction was prepared and analyzed as described in legend to Fig. 3. o-0, 1% viral DNA; A.. . . .A, i4C viral DNA after treatment. of each gradient fraction with DNase.

6

0

5

10

15

20

FRACTION 4. Sucrose density gradient centrifugation of vaccinia-DNA complexes. (A) Cells were infected and progeny viral DNA was labeled with thymidine-14C until 2 hours post-infection. The cytoplasmic fraction was analyzed on 38-50y0 sucrose gradients as described in legend of Fig. 3. O-0, radioactive DNA; A-A, radioactive DNA after DNase treatment; O-O, cytoplasm from uninfected cells. (B) Cells were infected with thymidine-%Hlabeled vaeeinia virus and the replicating viral DNA was labeled with thymidine-W. The cytoplasmic fraction was prepared at 2.25 hours postinfection and analyzed by sucrose gradient centrifugation as in Fig. 3A. O-0, W replicating DNA; A---& 3H parental input DNA. FIG.

0

5

10

15

20

fItACTION FIG. 6. Sucrose density gradient analysis of vaccinia DNA complexes at 5 hours post-infection. Infected cells were labeled with thymidine-‘*C throughout the course of viral DNA synthesis. Cytoplasmic extracts were prepared at 5 hours post-infection and analyzed by sucrose gradient centrifugation. O-0, centrifugation was for 2 hours; A--A, centrifugation for 2 hours and each fraction was treated with DNase; w - 4, centrifugation for 12 hours.

INTRACELLULAR TABLE INHIBITION

OF DNA

INTO

MATURING

PARTICLES”

Amount of DNA in dense component (cpm) 3 Hr

None Streptovitacin IBT

1

PACKAGING

VIRUS

Treatment

STIWCTURES

8350 8430 8170

r~,~nr&

6 Hr 1204 7057 G103

at 6 hr 14.4 83.7 74.8

0 Infected cells were labeled from the beginning of infection with thymidineJ4C (2.5 pCi/ liter; sp. act. 25 mCi/mmole). After 3 hours one culture received 15 pg/ml Streptovitacin A, a second culture was treated with 50 & isatin@-thiosemicarbazone (IBT), and a third culture was not treated with drugs. Samples of the cytoplasmic fraction (prepared immediately after addit,ion of the drugs at 3 hours, and also at 6 hours post-infection) from each culture were analyzed on 38-50% w/v sucrose gradients (see legend of Fig 3). The amount of radioactivity in the dense viral DNA component is shown at 3 hours and 6 hours post-infection.

1964a;

Iiates et al., 1968). Since viral is inhibited by streptovitacin, an inhibitor of protein synthesis Dales, 1965) and by isatin-beta-thiosemicarbazone, an inhibitor of vaccinia late functions (Woodson and Joklik, 1965), the latter drugs were tested for their ability to inhibit the conversion of the heavy viral DNA complex to the upper gradients components. Table 1 indicates that both of the drugs were effective at inhibiting this conversion. Thus it is likely that the conversion of the heavy replicated DNA component to upper components is a process which requires the synthesis of late viral proteins, probably viral capsid proteins.

CONTAINING

VACCINIA

I>NA

459

1.75 hour post-infection. They were then labeled with thymidine-3H for 1 min and 2 min. The cytoplasmic extracts were then analyzed on the sucrose gradient. Figure 7 illustrates that most of the 14C DNA occurs in the upper component, indicating that most of the DNA replicating between 1 and 1.75 hours is present in this lighter fraction, The 3H-DNA, on the other hand, occurs primarily in the dense fraction. These results demonstrate that newly replicated DNA occurs in both peaks. The 3H/14C ratio is 25 times greater in the denser component. Since most of the progeny DiSA is found in the dense component at 3 hours post-infection, it is likely that some of the DNA present in the lighter component is later converted to the heavier component. Further Puri$cotion Complex

of the Dense Viral DNA

The 3-hour dense vaccinia DNA complex accounted for more than 90% of the total viral intracellular DNA. It is therefore likely that this structure is the site of transcription of late viral genes, and evidence for

maturation

On the Site of Viral DNA

Replication

Two distinct fractions of newly replicated viral DNA were observed on analysis of 2 hours cytoplasmic extracts. Between 2 and 3 hours post-infection the lighter of the two components appeared to be converted into the denser one. It was therefore of interest to determine whether DNA replication was associated with only the upper component or with both of the structures. Infected cells were labeled with thymidine-14C from 1 to

0

s

10

15

FRACTION FIG. 7. Sucrose density gradient centrifugatiog of cytoplasmic components containing replicatinn vaccinia DNA. jnfected cells were labeled with thymidine-YJ fram 1 to 1.75 hours post-infection. The cells were t,hen labeled for 1 min and 2 min with thymidine-tH. Cytoplasmic extracts were subjected to sucrose density gradient centrifugation as described in Fig. 3: O--O, X-DNA long-term label; O---O, I-min thymidine-rH pulse; A.--A, 2cmin thymidine-3H pulse.

460

DAHL

AND

KATES

this conclusion is presented in the adjoining paper (Dahl and Hates, 1970). It was therefore of great interest to purify and characterize this structure further. The dense component collected from the 80% sucrose cushion of the first gradient was centrifuged through a 50-80% w/v sucrose gradient. Figure 8 shows that most of the DNA banded as a single peak of density 1.280. The material isolated from this band contained a protein:DNA ratio of approximately 3: 1 using the Lowry protein assay (Lowry et al., 1951) and the Burton DNA assay

(Burton, 1955) standardized against bovine serum albumin and calf thymus DNA, respectively. About 2% of the original total cytoplasmic protein was recovered in this fraction. In order to estimate the degree of association of HeLa cell proteins with the virosomes, cells were labeled for 20 hours prior to infection with leucine-3H in Eagle’s F-14 medium containing reduced leucine concentration (0.52 mg/liter). Just before infection the cells were washed and resuspended into new regular F-14 medium. The X-hour virosomes were purified through the 50-80% sucrose gradient and then analyzed for content of radioactively labeled host cell proteins. Table 2 indicates that the final component obtained in the 50-80% sucrose gradient contains 1.3% of the total labeled cytoplasmic protein. This indicates that a nearly 57-fold purification relative to HeLa cell protein has been achieved. Other procedures, including nonionic detergent treatment of the virosomes, are currently being explored and preliminary results indicate that most of the associated host protein can be removed without loss of the ability to synthesize RNA. DISCUSSION

FRACTION FIG. 8. Density equilibrium banding of vaccinia DNA-complex from 3 hours post-infection. The vaccinia DNA component from the 80% sucrose cushion of the 38-50y0 sucrose gradient (see Fig. 5) was layered on a 5GSOa/, sucrose gradient and centrifuged for 24 hours at 35,000 rpm at. 5°C on the Spinco SW 41 rotor. Each fraction was assayed for radioactive DNA after precipitation with 57Z0 trichloroacetic acid.

The results presented above indicate that vaccinia DNA in cytoplasmic extracts prepared by gentle lysis of infected cells occurs in several distinct species with characteristic densities in sucrose gradients. Furthermore, the types of components observed on gradients change as a function of time during the viral growth cycle. Although detailed electron microscope

TABLE PURIFICATION 14C Viral Purification

step

(1 Recovery of viral DNA 3 hours vaccinia “virosomes” in crude cytoplasmic extract.

5.1 4.5 3.8

x x x

DNA

2 ‘TIROSOMES”~ 3H HeLa

Percent

Cpm Crude cytoplasm 3&50$& gradient 50~307~ gradient

OF VACCINIA

104 104 104

100 89 74

Qm 2.2 2.3 2.8

x 106 X lo5 x 104

cell protein Percent 100 10.5 1.3

and HeLa cell protein as a function of the purification is given (for details see text). Percentages are based

-

Purification

0 9 57

steps applied to on amount present

INTRACELLULAR

STRUCTUR.ES TABLE

I)ESCRIPTIVE

SUMMARY

OF

VARIOUS

CONTAINING

461

DNA

3

COMPONENTS

‘~~~;’

VACCINIA

CONTAINING

Density

Parental DNA

DNase Sensitivity

1.157

+

None

Not sensitive

1.175

+

None

30-5Oa/, sensitive

1.180 1.285

Trace

+ +

~100yo sensitive ~100yo sensitive

Heterogeneous upper components 4 hours + post-infection

Trace

+

Variable

S’ACCINIA

DNA

Probable structure

__.~

studies of vaccinia replication have been carried out (Dales, 1963), it is not possible at the present time to correlate our observations with definite structures observed in infected cells. Furthermore, doubts arise concerning the effects of cell lysis on viral structures existing in the cytoplasm. With these reservations in mind, it might still be useful to speculate about the possible origin of the components which were resolved on the sucrose gradients (see Table 3). The 3hour post-infection DNA component appears to correspond to the “DNA-factories” described by Cairns (1960), studied by Becker and Joklik (1964a), and the cytoplasmic DNA inclusions observed by Dales (1963) after DNA synthesis was completed. The loss of this component during viral maturation is consistent with the above interpretation. Furthermore, the high sedimentation observed for this component agrees with the observed size of viral DNA aggregates in electron micrographs of infected cells. The dual peaks observed with labeled input viral DNA are somewhat puzzling. Even when viral uncoating is blocked by drugs and all input virus should accumulate as cores in the cytoplasm, two peaks are observed. Only the denser peak ultimately contains uncoated parental DNA. It is possible that intracytoplasmic viral cores become membrane associated with two different types of membranes or structures, and that only one of these associations(the dense component, 1.175 g/ml) is productive in the uncoating process. It might also explain the

Viral cores bound t,o a membrane Viral cores and uncoated DNA membrane-bound Viral cytoplasmic DNA inclusions (Cairns, 1960; Dales, 1963) Various states of maturation of virions

numerous failures to obtain viral DNA uncoating in vitro, where membrane associations might not occur (unpublished results). The shift in density observed between parental DNA and the dense, replicated DNA component may be quite significant from a functional point of view. It is known that in tivo the “early” parental DNA aggregate synthesized only a restricted classof messenger RNAs, while the “late” viral DNA complex synthesize both “early” and “late” classesof viral RNA (Oda and Joklik, 1967). The structural difference may therefore be directly related to the observed functional difference. We have shown that the dense complexes isolated at 3 hours postinfection synthesize both “early” and “late” classesof viral RNA in vitro. ACKNOWLEDGMENT This research was supported by P.H.S. Grant 1 ROl AI08413-02. We are grateful to Mr. J. Beeson for his valuable technical assistance. REFERENCES Y., and JOKLIK, W. K. (1964a). The replication and coating of vaccinia DNA. J. Mol. Biol. 10, 452-474. BECKER, Y., and JOKLIK, W. K. (196413). Messenger RNA in cells infected with vaccinia virus. Proc. Nat. Acad. Aci. U. S. 51, 577-585. BURTON, K. (1955). A study of the conditions and mechanisms of the diphenylamine reaction for the calorimetric estimation of DNA. Biochemistry 62, 315-322. CAIRNS, H. J. F. (1960). The initiation of vaccinia infection. Virology 11, 6033623. BECKER,

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AND

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DOLES, 6. (1963). The uptake and development of vaccinia virus in strain L cells followed with labelled viral DNA. J. Cell Biol. 18, 51-72. DALES, S. (1965). Effects of Streptovitacin A on the initial events in the replication of vaccinia and reovirus. Proc. Nat. Acad. Sci. U. S. 54, 462-468. ERON, L. J., and MCAUSL~N, B. It. (1966). The nature of poxvirus-induced deoxyribonucleases. Biochem. Biophys. Res. Commun. 22, 518-523. H.IYIV~RD, W. S., and GREEN, M. H. (1969). Effect of lambda repressor on the binding of RNA polymerase to DNA. Proc. Nut. Acad. Sci. U. S. 64, 962-969. JOKLIK, W. K. (1962). The preparation and characteristics of highly purified radioactively labelled poxvirus. Biochim. Biophys. Acta 61, 290-301. JOICLIK, W. K. (1964). The intracellular uncoating of poxvirus DNA. J. iMoZ. Biol. 8, 263-288. K.YTES, J. R., and MCAUSL~N, B. R. (1967). Relationship between protein synthesis and viral DNA synthesis. J. Viral. 1, 110-114. KATICS, J. R., and BEESON, J. (1970). Ribonucleic

acid synthesis in vaccinia virus. J. Mol. BioZ. in press. KATES, J., DAHL, R., and MIELKE, M. (1968). Synthesis and intracellular localization of vaccinia virus DNA-dependent RNA polymerase. J. Viral. 2, 894-900. LOWRY, 0. H., ROSEBROUGH, N. J., FUR, A. L., and RANDALL, R. J. (1951). Protein measurement with Folin phenol reagent. J. BioZ. Chem. 193, 265-275. MCAUSL‘ZN, B. R. (1965). Deoxyribonuclease activity of normal and poxvirus-infected HeLa cells. Biochem. Biophys. Res. Commun. 19, 1520. ODA, K., and JOKLIK, W. K. (1967). Hybridization and sedimentation studies on “early” and “late” vaccinia messenger RNA. J. Mol. BioZ. 27, 395-419. SNYDER, L., and GEIDUSCHEK, E. P. (1968). In vitro synthesis of Td late messenger RNA. Proc. Nat. Acad. Sci. U. S. 59, 459-466. WOODSON, B., and JOKLIK, W. K. (1965). The inhibition of vaccinia virus multiplication by isatin-fl-thio-semicarbozone. Proc. Nut. Acad. Sci. U. S. 54, 462-468.