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Biogenesis of vaccinia: Relationship of the envelope to virus assembly
VIROLOGY
75, 242-255
Biogenesis
(1976)
of Vaccinia:
WILLIAM
Relationship Assembly STERN
AND
With the Assistance Department
of Cytobiology,
of the Envelope
SAMUEL of Tellervo
The Public Health Research Institute New York, New York 10016
to Virus
DALES Huima of the City of New York, Inc.,
Accepted June 30,1976 The surface tubule protein p58 was found to be a useful marker to follow vaccinia membrane biogenesis as it is related to virus assembly. Synthesis of p58 does not occur in the absence of DNA replication and is maximal between 8-12 hr postinfection. During normal vaccinia development, ~58 as well as other proteins located at or near the surface is among the last components to be integrated into developing particles. Although envelopes can be assembled around immature particles in the absence of DNA and ~58 synthesis, combined biochemical and electron microscopic autoradiography studies reveal that such envelopes are either not utilized at all or only inefficiently in the formation of mature virions, once DNA replication has been restored. These observations imply that vaccinia assembly is a tightly coupled process in which internal components are integrated before the completion of the envelope. INTRODUCTION
integration of identifiable components into progeny. The isolation and identification of ST as described in the accompanying article (Stern and Dales, 1976) have provided us with a convenient marker of the vaccinia envelope. In the current study we have used ST to correlate the synthesis and assembly of the vaccinia envelope with virus development.
Biochemical and morphological studies have shown that poxvirus development proceeds through a series of intermediate stages (Morgan et al., 1954; Dales and Siminovitch, 1961; Dales and Mosbach, 1968; Sarov and Joklik, 1973). Virus maturation begins with the formation of the lipoprotein membrane or envelope, which can be formed even in the absence of DNA synthesis (Rosenkranz et al., 1966; Pogo and Dales, 1971). Once membrane assembly appears to be complete, a number of structural changes occurs at the surface and within the particle resulting in the formation of mature virus which possess the characteristic nucleoid, lateral bodies, and surface tubules (ST) (Dales and Mosbath, 1968). For any study of virus assembly a useful approach is to follow the synthesis and
MATERIALS
Radioactive
labeling
of virus
and
cells.
Monolayer cultures were infected with lo20 plaque-forming units (PFU)/cell at 4” 242
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
With the exception of methods specific for this article, all procedures utilized were identical to those mentioned in the accompanying article (Stern and Dales, 1976). Chemicals and isotopes. L-[4,5-“Hlleutine (60 Ci/mmol), [2-3Hlglycerol (6.6 Ci/ mmol), [33P]P04”-, and L-[35S]methionine (100-300 Ci/mmol) were purchased from New England Nuclear Corporation. Hydroxyurea (HU) was purchased from Sigma Chemical Company.
1 Present address: Department of Bacteriology and Immunology, University of Western Ontario, London, Ontario, N6A 5Cl Canada. Author to whom reprint requests should be addressed. Copyright AI1 rights
AND
VACCINIA
ENVELOPE
for 1 hr (Pogo and Dales, 1971). Virus proteins were labeled by incubating infected HeLa cells (4 x lo6 tells/60-mm petri dish) for 30 min in 1 ml of leucine-free medium (LFM) or methionine-free (MFM) Eagle’s medium and subsequently for 1 hr in 0.4 ml of LFM with C3Hlleucine or MFM with [35Slmethionine. The pulse was terminated by washing and incubating the cells with complete Eagle’s medium supplemented with 2% fetal calf serum (FCS). In some experiments the cells were harvested 24 hr after infection, in others at various times during the chase period. Prior to the isolation of virus, aliquots equivalent to 2 x lo7 infected cells or 200 pg of pure virus were added to each sample. In addition to unlabeled carrier virus, equal aliquots from a suspension of 4 x lo6 infected cells exposed continuously for 16 hr to [14Clleucine or 13Hlleucine (25 PCi in 4 ml of LFM supplemented with 2% FCS) were added to the labeled cells. The radioactively labeled virus was purified as described in the accompanying paper (Stern and Dales, 1976). Virus lipids were labeled with [“HIglycerol and/or 3P and extracted as previously described (Stern and Dales, 1974). Isotopically labeled extracts of cell cytoplasm were prepared by incubating infected monolayer cultures in 35mm petri dishes for 30 min in MFM and subsequently for the periods indicated (usually 60 min) in 0.2 ml of MFM containing 5 PCi of [35Slmethionine. The pulse labeling was terminated by removing the radioactive medium and adding warmed MEM for the time indicated. The cells were harvested by squirting them off the petri dish, concentrated by centrifugation, and resuspended in 0.1 ml of distilled water. Aliquots of 5 ~1 were applied to filter paper disks (Whatman 3MM), washed sequentially with 10 and 5% trichloroacetic acid (TCA), and counted in toluene scintillation fluid. The remainder of the sample was stored at -20” for analysis. Electrophoresis of cytoplasmic extracts and virus proteins. Cells and virus were prepared for polyacrylamide gel electrophoresis (PAGE) by mixing one volume of the suspension of material with an equal volume of dissociating buffer, and the
243
IN ASSEMBLY
samples were analyzed on 11% gels containing sodium dodecyl sulfate (SDS) (Stern and Dales, 1976). Autoradiograms of the gels were performed on a JoyceLoebl MKIIIC Microdensitometer. Virus and cell proteins were identified by their molecular weight (Stern and Dales, 1976). RESULTS
Kinetics
of ST Formation
During the transition from the immature to the mature form of vaccinia, the spicules on the external surface of immature particles disappear and are replaced by a coat of ST (Dales and Mosbach, 1968). To ascertain whether spicules and surface tubules are different polymeric forms of the same protein, the time course of ST synthesis was determined. In one type of experiment, HeLa cells were infected in the presence or absence of HU and labeled for 1 hr with [3S]methionine at 5, 8, and 11 hr postinfection. Immediately after being labeled the cells were harvested and analyzed by a combination of PAGE and autoradiography. In the presence of HU, DNA synthesis is inhibited (Young and Hodas, 19641, and virus development is arrested at the immature stage (Rosenkranz et al., 1966; Pogo and Dales, 1971). Since under these conditions only early proteins are labeled (Holowczak and Joklik, 1967b), it is possible to determine whether the formation of ST requires DNA replication. An autoradiogram of a gel which contained the extracts of cells labeled in the presence and absence of HU is shown in Fig. 1. Although the structural proteins of vaccinia appear to be richer in leucine than methionine (Fig. 11, [35S]methionine was used instead of [14C]leucine in most experiments since the specific activity of the former was much higher, and one could obtain satisfactory autoradiographic data in a shorter time. Preliminary experiments indicated that the polypeptide composition of infected cells which were labeled with [35S]methionine was the same as those labeled with [l”Clleucine. The gel in Fig. 1 showed that host protein synthesis in our cell-virus system was not completely arrested until the formation of late proteins had occurred; however, it was possible to identify the early proteins. At 5
24 4
STERN
A
B
AND
DALES
CDEFGHI
FIG. 1. Autoradiogram of a slab gel of virus and infected HeLa cells labeled with [Wmethionine. One x 10” tells/35-mm petri dish, infected in the presence or absence of HU with 10 PFUicell, were labeled with 10 ,xCi of [%]methionine for 1 hr at 5, 8, and 11 hr postinfection and then harvested for SDS-PAGE. The numbers at the top of the gel refer to the time postinfection when the label was administered in the presence (+) or absence (-1 of HU. (A) Uninfected cells; (B-G) infected cells; (H) [Wlmethionine-labeled virus; (I) [‘4Clleucine-labeled virus.
VACCINIA
ENVELOPE
hr after infection the same early virus polypeptides were labeled in the presence and absence of HU. Virus structural polypeptides that were synthesized at this time included ~145, 140, and 33. These proteins presumably correspond to VSP-1 and VSP6 of Holowczak and Joklik (1967a) which are produced in the presence of cytosine arabinoside, another inhibitor of DNA synthesis (Holowczak and Joklik, 1967b). P145 and 140 occur in the virus core, whereas p33 is presumably located at the surface because it is soluble in NP40. Another surface protein, ~58, was absent from the extracts of HU-treated cells, but it appeared in the extracts of untreated cells at 11 hr after infection. The absence of ST in HU-treated cells was also indicated by the fact that ~58 antiserum (Stern and Dales, 1976) did not react in Ouchterlony plates with cells incubated in the presence of HU, but did react with cells which were not treated with the drug. To determine the kinetics of ~58 synthesis, pulse-labeled with cells were [3H]leucine at various times postinfection, harvested at the end of the infectious cycle, and mixed with a standard amount of infected cells continuously labeled with [14Clleucine. Virus was purified from the mixture and analyzed by PAGE. Individual stained bands were cut out, eluted, and their radioactive content determined. Initial experiments indicated that the yield of mature virus labeled with [3H]leucine was not influenced by the time during the growth cycle when the radioactive label was administered. Using 14Clabeled virus as a baseline, variations in the ratio of 3H/14C in mature virus labeled at various times during the infectious cycle paralleled changes in the specific activity of virus t3H cpm/pg of protein). Table 1 summarizes changes in the ratio 3H/14C of several major virion proteins including ~58 during the virus growth cycle. The data are expressed as a percentage of the maximum 3H/14C for each polypeptide in order to readily compare changes in the ratio of 3H/14C. The results presented in Table 1 showed that the rate of incorporation of 3H into ~58 was similar to that of ~62, 60, 33, 24, and 18 in that the maximum incorporation of 3H occurred between 8 and 12 hr
245
IN ASSEMBLY TABLE
1
TIME COURSE OF [3H]L~~~~~~ IINCORPORATION INTO VIRAL PROTEINS DURING INFECTION” Virus PolyylP
Percentage maximum 3H/14C Time of labeling (hr) 3-4
p145 140 62 60 58 40 33 24 18
5-6
8-9
11-12
l&15
17-18
100
51
43
34
7
6
11 9 14 25 12 15 12
49 53 40 100 25 62 58
100 100 97 75 90 100 100
79 87 100 61 100 84 91
50 54 52 48 69 29 66
24 28 35 31 51 28 32
n Infected cells (4 x lo6 tells/60-mm plate) were labeled for 1 hr at the indicated times with 16 &i/ ml and harvested 24 hr postinfection. The cells were mixed with infected cells labeled with [‘%]leucine as described in Materials and Methods and virus was purified from the mixture. After analyzing the samples of virus by PAGE, the gel was stained with Coomassie blue; the stained bands were cut out, eluted in NCS, and counted.
after infection. By contrast, the early proteins ~145 and 140 were produced maximally at 3 to 4 hr postinfection and thereafter the rate of their synthesis declined. In order to rule out the possibility that spicules coating the external surface of immature particles are a high molecular weight precursor of ST, infected cells were labeled for 1 hr with 13%lmethionine and were either harvested immediately after pulsing or placed in MEM for 4 hr before harvesting. Cytoplasmic extracts of the pulse and chase samples were analyzed by PAGE and autoradiography. Densitometric tracings of the autoradiograms (Fig. 2) revealed that the peaks corresponding to four major virus polypeptides ~62, 60, 24, and 18 were absent at the end of the pulse but appeared in the chase sample. On the other hand, there was no change in the absorbance of the peak corresponding to ~58. These results suggest that ~62, 60,24, and 18 were formed by post-translational cleavage whereas ~58 was not. The data from the above experiments, when taken together, suggest that the spicules of immature particles are not the same as the surface tubules of mature virus.
246
STERN
AND
DALES
4
3
CHASE
-
3-
clrtqm
109876
5 MOLECULAR
2
I.5 Dye ’ fmnt
WEIGHT x Iti
FIG. 2. Pulse-chase of infected HeLa cells labeled with [%lmethionine. One x 10” cells/35mm petri dish were labeled for 1 hr at 15 hr postinfection with 10 &i of [““Slmethionine in 0.2 ml of MFM. At the end of labeling period the radioactive medium was removed; the sample designated as “pulse” was immediately harvested and the sample designated as “chase” was incubated for 4 more hr in MEM. The samples were analyzed by SDS-PAGE and the resulting autoradiograms were scanned by a densitometer. Long arrow indicates position of ~58; short arrows indicate polypeptides that appeared during the chase.
Integration of p58 into Vaccinia Virus Assembly
during
cation. The data summarized in Table 2 showed that during the 17-hr chase the 3H/ 14C in whole virus increased 11-fold, with Since p58 is a late protein and apparthe greatest change in this ratio occurring ently different from the spicules of immaduring the first 4 hr of the chase. Table 2 ture particles, we set out to determine also shows that immediately after pulsing, when ~58 was integrated into the virus. 3H/‘4C of the outer proteins of vaccinia was Initial experiments were conducted to 2.9 times greater than that of the cores and study the kinetics of integration of outer that during the chase the difference beproteins (i.e., those solubilized by NP40tween the two ratios declined. Furthermercaptoethanol) and core proteins. Inmore, at the end of the pulse 3H counts fected cells were pulse labeled with associated with the outer proteins ac13Hlleucine at 7-8 hr after infection, counted for 40% the total radioactivity in placed in chase medium, and harvested at the virus, but at the end of 17 hr the provarious times thereafter. The cells were portion of 3H counts in the outer proteins mixed with a standard quantity of 14C- was only 19%. This result implies that labeled infected cells prior to virus purifisoon after their synthesis the outer pro-
VACCINIA
ENVELOPE
teins of vaccinia were incorporated into virus at a higher rate than the core proteins. Alternatively, the nascent outer proteins were added to virus factories which already contained the DNA and the core proteins. A parallel experiment to determine when ~58 was integrated into vaccinia was performed using [35Slmethionine as a precursor. Labeled virus was subjected to PAGE and autoradiography, and data were obtained by densitometric analysis of the autoradiogram. The tracings in Fig. 3 showed that the amount of 35Slabel in all polypeptides increased with time of chase. At the end of the pulse the peak height of ~58 relative to other proteins was much higher than at later times during the chase. The actual amount of label in the individual virus proteins was determined by cutting out the bands and counting them in a liquid spectrometer. Only bands for which corresponding ones were evident in the 0 time sample were considered for analysis. The data in Table 3 show the distribution of YS label among vaccinia proteins as a function of the period of the chase. The percentage of 35Slabel in ~58, TABLE
2
INCORPORATION OF 13HlL~uc1~~
INTO NASCENT
VIRUS= Time
Whole 8 8.5 9 10 12 25
Percentage
3H/‘4C
postinfection (hr)
solubilizedd
O&X” proteins
Cores proteins
3H
Y!
virus
0.32 0.41 0.80 1.60 2.34 3.52
0.46 0.60 1.02 1.77 2.38 3.01
0.16 0.25 0.58 1.28 2.00 2.95
47 40 32 31 27 19
23 23 21 24 23 19
u Between 7 and 8 hr postinfection, 4 x lo6 cells per sample were labeled with 5.5 @X/ml of [3H11eucine. At the indicated times the samples were mixed with infected cells labeled with [14C]leucine, as described in Materials and Methods, and virus was purified from the mixture. b Proteins which were solubilized by NP40-mercaptoethanol. r, Proteins which were insoluble in NP40-mercaptoethanol. d The percentage refers to those counts that were solubilized by NP40 and mercaptoethanol.
247
IN ASSEMBLY
33, and 29, which are outer proteins, decreased with time. Another outer protein, ~14, also decreased with time but not as much. The glycoprotein (Holowczak, 1970; Garon and Moss, 1971), ~40, which is near the virus surface, was virtually unchanged. The amount of label in the core polypeptides p62,60, and 18 increased with time, while p24 remained unchanged. In the case of two other core polypeptides, p92 and 65, the relative amount of label declined with time. These data suggest that in line with the results in Table 2, ~58 and at least some other outer proteins are initially incorporated more rapidly into virus than the major core proteins. Although the decline in core polypeptide ~50 cannot be explained, the decrease in core polypeptides p92 and 65 could be accounted for if, in fact, they are the precursors of p62 and 60 and their decline reflects post-translational cleavage. This suggestion is based on the apparent similarity between the molecular weights of p92 and 65 and to those of the precursor proteins in cellular extracts (Fig. 2). Vaccinia Assembly Removal
following
HU
The data in the previous section suggested that envelopes are added onto virus at a late stage during assembly. However, electron microscopic studies indicate that envelopes are the first virus structure to appear (Dales and Mosbach, 19681, and that internal structures become evident after the completion of the envelope (Dales and Mosbach, 1968). In the presence of HU immature particles with complete envelopes and no internal structures are formed (Rosen-kranz et al., 1966; Pogo and Dales, 1971). After the removal of HU a nucleoid and lateral bodies appear inside completed immature particles (Pogo and Dales, 1971). Since most of the virus proteins are late functions (Holowczak and Joklik, 1967b), it might be presumed that the internal constituents of the mature virus are inserted into completed immature particles. To determine whether vaccinia assembly is a tightly coupled process or involves the insertion of late proteins through the membrane of immature parti-
248
STERN
7 own
10967
6
5
4
AND
DALES
3
MOLECULAR
WEIGHT x Id’
FIG. 3. Radioautogram of virus isolated from labeled cells at various times after administration of the pulse. Four x lo6 cells per sample were labeled between 8 and 8.5 hr postinfection with 125 &i of [3”]methionine. At the times after pulse-labeling as indicated in the upper left-hand corner, the samples were mixed with infected cells labeled with PHlleucine, as described in Materials and Methods, and virus was purified from the mixture. Equal amounts of protein in each sample were electrophoresed, subjected to autoradiography, and scanned by a densitometer. Arrow indicates position of ~58.
cles, the relationship of HU-induced immature particles and mature virus was studied. HeLa cells were infected and maintained in the presence of HU for 16 hr at which time they were labeled for 1 hr with [“YSlmethionine. Afterwards, the cells were exposed to HU for up to 6 hr. Each sample was then incubated for 20 hr in MEM without HU. The cells were harvested and mixed with vaccinia labeled with [3Hlleucine and virus containing both labels was purified. The data in Table 4 showed that the ratio of 35S/3H was highest in virus obtained from cells which were maintained in medium from which HU was removed immediately after pulse la-
beling. The ratio of 35S/3H decreased in proportion to the time the cells were maintained in the presence of HU after being labeled with [35Slmethionine. The cells that were kept in HU for 6 hr following 35Slabeling produced virus whose 35S/3H ratio was half the value of virus originating from cells maintained in medium from which HU was removed immediately after labeling. The decline in 35S/3H did not reflect decreased virus production since removal of HU at various times after pulselabeling did not alter the final yields of mature virus harvested 20 hr after removal of HU. When HU was removed at 18 hr postinfection the virus yield 20 hr later
VACCINIA TABLE
ENVELOPE
3
DISTRIBUTION OF [Y~IMETHIONINE IN VIRAL PROTEINS AT VARIOUS TIMES AFTER PULSELABELING*
Virus polypeptide P92 65 62 60 58 50 40 33 31 29 24 18 14
T
Percentage
7.2’ 6.0 4.8 9.6 7.2 4.6 7.3 9.2 6.5 9.9 7.0 8.2 12.1
0.5
1
2
4
6.4 6.2 8.6 15.7 4.4 3.1 5.6 6.2 5.4 6.6 6.6 9.1 15.7
4.7 4.9 15.0 18.1 3.5 2.6 4.7 5.2 3.3 4.2 7.0 12.4 14.2
1.6 2.4 18.0 26.9 1.9 2.0 5.8 4.0 2.6 3.1 6.3 12.2 13.0
1.2 2.7 19.0 32.6 1.5 0.9 5.3 3.0 2.0 2.4 6.1 11.7 11.5
1.4 2.4 .9.8 10.3 1.7 0.6 5.5 2.2 1.5 2.3 7.5 !5.7 8.8
n See Fig. 3. * Time of chase in hours. (’ Percentage total counts per minute.
was 1.6 x 10y PFU or 100 pg of protein/lo7 cells, when removed at 20 hr the yield was 1.0 x lo9 PFU or 120 ,ug of protein/lo7 cells, and when removed at 22 hr the yield was 1.2 x 10g PFU or 100 pg/lOT cells. Furthermore, this decline was not the result of increased protein turnover due to the presence of HU, since the specific activity of total protein of cells “chased” in the presence and absence of HU was the same. A more direct approach involved a combination of electron microscopy (EM) and autoradiography. L cells were infected in the presence of HU and labeled at 18 hr after infection for 1 hr with 13Hlleucine in the presence of the drug. One sample was subsequently maintained in the presence of HU for 3 hr and then for another 5 hr in its absence, another sample was chased in the absence of HU for 8 hr. The results based on counts of silver grains and virusrelated structures observed in thin sections showed that cells which were chased in the presence of HU for 3 hr and then in its absence did not have any grains associated with mature particles (Table 5 and Fig. 5). On the other hand, cells which were chased in the absence of HU contained labeled mature particles (Table 5 and Fig. 6). Sample 2 had fewer mature
249
IN ASSEMBLY
particles than sample 3 of Table 5, probably due to the fact that the cells in sample 2 were incubated in the absence of HU for 5 hr, whereas those in sample 3 were incubated in the absence of HU for 8 hr, allowing more time for mature progeny to be formed. In both cases the number of grains per cell and number of grains per immature particle were about the same. An alternative approach for studying the participation of immature forms of vaccinia in assembly and maturation involved an experiment using EM-autoradiography in which the process of morphogenesis was interrupted by the drug rifampicin. In the presence of rifampicin most or all the major virion polypeptides and DNA are synthesized (Moss et al., 1971) but membrane assembly and morphogenesis are arrested (Grimley et al., 1970; Nagayama et al., 1970). Data from the present study, summarized in Table 6, showed that when cells were labeled in the presence of rifampicin grains became associated with aberrant structures of the type expected to be formed in the presence of the drug (Table 6 and Fig. 7). However, when the cells were chased in the absence of rifampicin the aberrant factories disappeared (Fig. 8) and the grains were associated with immature and mature particles and virus factories of normal appearance (Table 6) (Moss et al., 1971). These results indicate that under conditions in which TABLE
4
INCORPORATION OF [Y~IMETHIONINE INTO VIRUS FROM CELLS LABELED IN THE PRESENCE OF HLJ”
Chase time in the presence of HU (hr)
““S,:‘H
0 2 4 6
2.94 2.07 1.83 1.34
o Cells (1 x 10” cells per sample) were infected and maintained in HU for 16 hr. The cells were pulse-labeled with 20 PCi of [:‘“Slmethionine for 1 hr at 16 hr postinfection in the presence of HU and subsequently maintained for the indicated times in the presence of HU plus MEM. HU was removed at the indicated times and the infection for each sample was allowed to proceed for another 20 hr. The ceils were mixed with 140 pg of pure vaccinia which contained 50,000 cpm of [?H]leucine.
FIG. 4. EM autoradiography corresponding to sample 1 of Table 5. Note the prominent factory with immature particles and absence of mature virus. Silver grains are scattered throughout the cytoplasm as well as over the factory. M, mitochondria. x 17,600. FIG. 5. As above, corresponding to sample 2 in Table 5. Immature and mature progeny are present but silver grains occur only over immature virions (arrows). N, nucleus; M, mitochondria. x 17,600. 250
VACCINIA
ENVELOPE
viral envelope formation occurs prior to the insertion of viral DNA and late proteins, maturation of particles assembled before DNA synthesis cannot occur even if late proteins are subsequently synthesized. In contrast to HU, reversal of rifampicin is effective in allowing maturation, since rifampicin creates a block of the enveloping process and not the synthesis of at least the majority of vaccinia polypeptides. Kinetics
of Lipid
duced into virus, it was of interest to determine whether the lipids of the envelope were integrated with similar kinetics. In order to perform experiments such as those described in Table 2, it was necessary to determine whether one could distinguish between phospholipid exchange, known to occur during vaccinia morphogenesis (Stern and Dales, 19741, and the net transfer of lipid into virus undergoing assembly. To discriminate between preexisting and nascent lipids in the virus, infected cells prelabeled with 33P were pulselabeled for 1 hr with [3Hlglycerol at 5 hr after infection. At various times during the chase the cells were harvested, mixed with unlabeled infected cells, and virus was purified from the mixture. Since the carrier virus was in excess, the specific activities of the virus lipid provided a measure of the amount of nascent virus formed. The data in Table 7 showed that the specific activities of 33P and 3H lipids increased with the time of chase; however, the ratio of 3H/33P of viral lipids was relatively constant during the chase. Since the cellular lipids were labeled with 33P before infection and with 3H after infection, the absence of change in the ratio of the two labels suggests that nascent lipids and preexisting lipids were randomized. Therefore, one cannot discriminate between phospholipid exchange and the net transfer of lipid to virus undergoing assembly. From the above it is evident that one cannot measure the kinetics of lipid integration into the envelope in a manner analogous to that of proteins.
Integration
Since the envelope proteins appear to be among the last components to be introTABLE INCORPORATION AND MATURE
Sample Gyeisi
5
OF [“HILEUCINE INTO IMMATURE VIRUS IN THE PRESENCE AND ABSENCE OF HU”
Grains/;~~scell
pro- Particles/100 cell profiles
Facto- Immaries ture 1 2 3
12 14 14
25 101 166
12 47 59
Mature
Immature
Mature
0 0 36
159 754 997
1 180 677
a L cells (4 x lo6 tells/60-mm petri dish) were infected with 15 PFUicell of vaccinia in the presence of 5 mM HU. At 18 hr postinfection, samples 1, 2, and 3 were labeled for 1 hr with 20 FCi of [SH]leucine in 0.4 ml of LFM + 5 mM HU. At the end of the pulse, samples 1 and 2 were washed and maintained in MEM with HU, whereas sample 3 was washed and maintained in MEM without HU. After being incubated in MEM + HU for another 3 hr, HU was removed from sample 2. All samples were harvested and prepared for electron microscopy at 27 hr after infection. TABLE INCORPORATION
OF [SH]L~~~~~~
6
INTO NORMAL AND ABERRANT VIRUS ABSENCE OF RIFAMPICI~~
Sample
Grains/l00
251
IN ASSEMBLY
STRUCTURES
cell profiles
IN TRE PRESENCE
AND
Particles/l00
cell profiles
Immature
Mature
685 1288 610 1210
9 492 12 354
GZi?si Particles
Factories Normal 1 2 3 4
14 13 16 21
0 188 4 258
Rifampicin 343 10 520 ‘76
Immature 20 46 30 114
Mature 0 40 2 78
n L cells (4 x 10fi tells/60-mm petri dish) were infected with 10 PFU/cell of vaccinia in the presence of 100 pg/ml of rifampicin. At 4 to 5 hr (samples 1 and 2) and 8 to 9 hr (samples 3 and 4) after infection, the cells were labeled with 20 WCi of [3H]leucine in 1 ml of LFM + rifampicin. At the end of the pulse, samples 1 and 3 were maintained in MEM + rifampicin, while the drug was removed from samples 2 and 4. At 12 hr after infection, all of the samples were harvested.
252
STERN
FIG. 6. As above, corresponding grains
occur over both immature
AND
DALES
to sample 3 in Table 5. Immature and mature progeny are absent. Silver and mature virions (arrows). x 15,000.
FIG. 7. EM autoradiography corresponding to sample 3 in Table 6. Note the prominent labeling of the aberrant factories and associated immature particles (arrows). x 12,500. FIG. 8. As above, corresponding to sample 4 in Table 6. Note that following removal of rifampicin, numerous immature and mature progeny of normal appearance are labeled (arrows). x 12,500. 253
254
STERN
AND
DALES TABLE
DISCUSSION
Morphologic studies have shown that the envelope is the earliest identifiable component of vaccinia to be formed during normal virus development (Dales and Mosbach, 1968) and can be assembled to enclose immature virus in the absence of DNA synthesis (Rosenkranz et al., 1966; Pogo and Dales, 1971). The transition from immature to mature virus involves a differentiation within the completed envelopes of immature particles (Dales and Mosbach, 1968). Based on these and other observations Pogo and Dales (1971) proposed two models of virus assembly. In the first model, the membranes of immature particles are completed prior to the insertion of late internal proteins through the sealed envelope. In the second, the envelope constituents assemble within factories around quanta of DNA and internal proteins. According to the latter model the envelope would not be completely sealed until all the necessary polypeptides are in place. One may envisage that additional internal proteins such as the precursors of p62 (Katz and Moss, 1970), ~60, ~24, and p18 accumulate within the developing envelope to be processed as the interior is reorganized into the core and lateral bodies of mature virus. The results from the present study favor the second model since it appears that immature particles, whose envelopes are completed in the presence of HU, are unlikely to progress to maturity when DNA and late protein synthesis are resumed. The absence of DNA in immature particles formed in the presence of HU probably does not account for this result because when DNA and the majority of the late polypeptides are made, as in the presence of rifampicin, removal of the drug at the time when further protein synthesis is blocked by streptovitacin A results in the assembly of DNA containing immature forms of virus which fail to progress into mature forms (Nagayama et al., 1970). In addition to changes that occur within the envelope, the virus surface also undergoes a transformation in which the spicules that cover immature particles disappear and ST appear. The spicules, however, are neither precursors nor a different
INCORPORATION
Time postinfection (hr) 6 6.5 7 8 9
I
OF [3HlG~~~~~~~ NASCENT VIRU@
Lipid cpm/mg protein 3H
33P
3138 2931 3677 5898 9479
4062 4552 4194 9458 14246
INTO LIPIDS
OF
3H/33P
0.77 0.64 0.86 0.62 0.66
a Uninfected cells (4 x lo6 cells per sample) were labeled for 15 hr before infection with 8 &i of 33P. At 5 hr after infection, the cells were incubated for 1 hr with 17 &i of [3H]glycerol. At the indicated times a sample was removed and mixed with 2 x 10’ unlabeled infected cells. The lipids of pure virus were extracted as previously described (Stern and Dales, 1975).
form of the surface tubules, since ~58 is not synthesized in the absence of DNA replication and is not produced via the cleavage of high molecular weight precursor. Furthermore, immunological studies have shown that ferritin-conjugated antibody which binds to the surface of virus fails to react with the spicule-coated surface of immature particles (Morgan et al., 1962). The dissimilarity between spicules and ST can also be seen from studies on conditional lethal mutants of vaccinia which show that the spicules are lost from the surface of immature virus when internal differentiation occurs and are replaced by the surface tubules (Stern et al., 1975). Data from the present study and those of Sarov and Joklik (1973) indicate that ~58 like other proteins found at or near the surface are among the last to be integrated into maturing virus. However, Sarov and Joklik hypothesized that vaccinia virus assembly progresses via intermediate particles. By contrast, data from our past and present work together with studies on conditional lethal mutants (Stern et al., 1975) indicate that vaccinia morphogenesis occurs in a tightly coupled fashion in which the internal constituents are assembled as a complex of DNA and polypeptides at about the time the envelope condenses around this complex. ACKNOWLEDGMENTS This investigation was supported in part by U.S. Public Health Service Grant No. AI 07477. We
VACCINIA thank
Vladimir
Milovanovich
for competent
ENVELOPE assist-
REFERENCES DALES, S., and MOSBACH, E. H. (1968). Vaccinia as a model for membrane biogenesis. Virology 35,564583. 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, 475-503. GARON, C. F., and Moss, B. (1971). Glycoprotein synthesis in cells infected with vaccinia virus. II. A glycoprotein component of the virion. Virology 46, 233-246. GRIMLEY, P. M., ROSENBLUM, E. N., MIMS, S. J., and Moss, B. (1970). Interruption by rifampin of an early stage in vaccinia virus morphogenesis: Accumulation of membranes which are precursors of virus envelopes. J. Viral. 6, 519-533. HOLOWCZAK, J. A. (1970). Glycopeptides of vaccinia virus. I. Preliminary characterization and hexosamine content. Virology 42, 87-99. HOLOWCZAK, J. A., and JOKLIK, W. K. (1967a). Studies of the structural proteins of vaccinia virus. I. Structural proteins of virions and cores. Virology 33, 717-725. HOLOWCZAK, J. A., and JOKLIK, W. K. (196713). Studies of the structural proteins of vaccinia virus. II. Kinetics of the synthesis of individual groups of structural proteins. Virology 33, 726-739. KATZ, E., and Moss, B. (1970). Vaccinia virus structural polypeptide derived from a high molecular weight precursor: Formation and integration into virus particles. J. Viral. 6, 717-726. MORGAN, C, RIFKIND, R. A., and ROSE, H. M. (1962). The use of ferritin-conjugated antibodies in electron microscopic studies of influenza and vaccinia viruses. Cold Spring Harbor Symp. Quant. Biol. 27, 57-65.
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255
MORGAN, C., ELLISON, S. A., ROSE, H. M., and MOORE, D. H. (1954). Structure and development of viruses observed in the electron microscope. II. Vaccinia and fowlpox viruses. J. Exp. Med. 100, 301-310. Moss, B., ROSENBLUM, E. N., and GRIMLEY, P. M. (1971). Assembly of vaccinia virus particles from polypeptides made in the presence of rifampicin. Virology 45, 123-134. NAGAYAMA, A., POGO, B. G.-T., and DALES, S. (1970). Biogenesis of vaccinia: Separation of early stages from maturation by means of rifampicin. Virology 40, 1039-1051. POGO, B. G.-T., and DALES, S. (1971). Biogenesis of vaccinia: Separation of early stages from maturation by means of hydroxyurea. Virology 43, 144151. ROSENKRANZ, H. S., ROSE, H. M., MORGAN, C., and Hsu, K. C. (1966). Effect of hydroxyurea on virus development. II. Vaccinia virus. Virology 28, 510519. SAROV, I., and JOKLIK, W. K. (1973). Isolation and characterization of intermediates in vaccinia virus morphogenesis. Virology 52, 223-233. STERN, W., and DALES, S. (1974). Biogenesis of vaccinia: Concerning the origin of the envelope phospholipids. Virology 62, 293-306, STERN, W., WEINTRAUB, S. B., and DALES, S. (1975). Relationship between vaccinia membrane formation and virus maturation: Analysis by means of temperature-sensitive mutants. J. Cell Biol. 67, 837a. STERN, W., and DALES, S. (1976). Biogenesis of vaccinia: Isolation and characterization of a surface component that elicits neutralizing antibody suppressing infectivity and cell-cell fusion. Virology 75, 232-241. YOUNG, C. W., and HODAS, S. (1964). Hydroxyurea: Inhibitory effect on DNA metabolism. Science 146, 1172-1174.
75, 242-255
Biogenesis
(1976)
of Vaccinia:
WILLIAM
Relationship Assembly STERN
AND
With the Assistance Department
of Cytobiology,
of the Envelope
SAMUEL of Tellervo
The Public Health Research Institute New York, New York 10016
to Virus
DALES Huima of the City of New York, Inc.,
Accepted June 30,1976 The surface tubule protein p58 was found to be a useful marker to follow vaccinia membrane biogenesis as it is related to virus assembly. Synthesis of p58 does not occur in the absence of DNA replication and is maximal between 8-12 hr postinfection. During normal vaccinia development, ~58 as well as other proteins located at or near the surface is among the last components to be integrated into developing particles. Although envelopes can be assembled around immature particles in the absence of DNA and ~58 synthesis, combined biochemical and electron microscopic autoradiography studies reveal that such envelopes are either not utilized at all or only inefficiently in the formation of mature virions, once DNA replication has been restored. These observations imply that vaccinia assembly is a tightly coupled process in which internal components are integrated before the completion of the envelope. INTRODUCTION
integration of identifiable components into progeny. The isolation and identification of ST as described in the accompanying article (Stern and Dales, 1976) have provided us with a convenient marker of the vaccinia envelope. In the current study we have used ST to correlate the synthesis and assembly of the vaccinia envelope with virus development.
Biochemical and morphological studies have shown that poxvirus development proceeds through a series of intermediate stages (Morgan et al., 1954; Dales and Siminovitch, 1961; Dales and Mosbach, 1968; Sarov and Joklik, 1973). Virus maturation begins with the formation of the lipoprotein membrane or envelope, which can be formed even in the absence of DNA synthesis (Rosenkranz et al., 1966; Pogo and Dales, 1971). Once membrane assembly appears to be complete, a number of structural changes occurs at the surface and within the particle resulting in the formation of mature virus which possess the characteristic nucleoid, lateral bodies, and surface tubules (ST) (Dales and Mosbath, 1968). For any study of virus assembly a useful approach is to follow the synthesis and
MATERIALS
Radioactive
labeling
of virus
and
cells.
Monolayer cultures were infected with lo20 plaque-forming units (PFU)/cell at 4” 242
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
With the exception of methods specific for this article, all procedures utilized were identical to those mentioned in the accompanying article (Stern and Dales, 1976). Chemicals and isotopes. L-[4,5-“Hlleutine (60 Ci/mmol), [2-3Hlglycerol (6.6 Ci/ mmol), [33P]P04”-, and L-[35S]methionine (100-300 Ci/mmol) were purchased from New England Nuclear Corporation. Hydroxyurea (HU) was purchased from Sigma Chemical Company.
1 Present address: Department of Bacteriology and Immunology, University of Western Ontario, London, Ontario, N6A 5Cl Canada. Author to whom reprint requests should be addressed. Copyright AI1 rights
AND
VACCINIA
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for 1 hr (Pogo and Dales, 1971). Virus proteins were labeled by incubating infected HeLa cells (4 x lo6 tells/60-mm petri dish) for 30 min in 1 ml of leucine-free medium (LFM) or methionine-free (MFM) Eagle’s medium and subsequently for 1 hr in 0.4 ml of LFM with C3Hlleucine or MFM with [35Slmethionine. The pulse was terminated by washing and incubating the cells with complete Eagle’s medium supplemented with 2% fetal calf serum (FCS). In some experiments the cells were harvested 24 hr after infection, in others at various times during the chase period. Prior to the isolation of virus, aliquots equivalent to 2 x lo7 infected cells or 200 pg of pure virus were added to each sample. In addition to unlabeled carrier virus, equal aliquots from a suspension of 4 x lo6 infected cells exposed continuously for 16 hr to [14Clleucine or 13Hlleucine (25 PCi in 4 ml of LFM supplemented with 2% FCS) were added to the labeled cells. The radioactively labeled virus was purified as described in the accompanying paper (Stern and Dales, 1976). Virus lipids were labeled with [“HIglycerol and/or 3P and extracted as previously described (Stern and Dales, 1974). Isotopically labeled extracts of cell cytoplasm were prepared by incubating infected monolayer cultures in 35mm petri dishes for 30 min in MFM and subsequently for the periods indicated (usually 60 min) in 0.2 ml of MFM containing 5 PCi of [35Slmethionine. The pulse labeling was terminated by removing the radioactive medium and adding warmed MEM for the time indicated. The cells were harvested by squirting them off the petri dish, concentrated by centrifugation, and resuspended in 0.1 ml of distilled water. Aliquots of 5 ~1 were applied to filter paper disks (Whatman 3MM), washed sequentially with 10 and 5% trichloroacetic acid (TCA), and counted in toluene scintillation fluid. The remainder of the sample was stored at -20” for analysis. Electrophoresis of cytoplasmic extracts and virus proteins. Cells and virus were prepared for polyacrylamide gel electrophoresis (PAGE) by mixing one volume of the suspension of material with an equal volume of dissociating buffer, and the
243
IN ASSEMBLY
samples were analyzed on 11% gels containing sodium dodecyl sulfate (SDS) (Stern and Dales, 1976). Autoradiograms of the gels were performed on a JoyceLoebl MKIIIC Microdensitometer. Virus and cell proteins were identified by their molecular weight (Stern and Dales, 1976). RESULTS
Kinetics
of ST Formation
During the transition from the immature to the mature form of vaccinia, the spicules on the external surface of immature particles disappear and are replaced by a coat of ST (Dales and Mosbach, 1968). To ascertain whether spicules and surface tubules are different polymeric forms of the same protein, the time course of ST synthesis was determined. In one type of experiment, HeLa cells were infected in the presence or absence of HU and labeled for 1 hr with [3S]methionine at 5, 8, and 11 hr postinfection. Immediately after being labeled the cells were harvested and analyzed by a combination of PAGE and autoradiography. In the presence of HU, DNA synthesis is inhibited (Young and Hodas, 19641, and virus development is arrested at the immature stage (Rosenkranz et al., 1966; Pogo and Dales, 1971). Since under these conditions only early proteins are labeled (Holowczak and Joklik, 1967b), it is possible to determine whether the formation of ST requires DNA replication. An autoradiogram of a gel which contained the extracts of cells labeled in the presence and absence of HU is shown in Fig. 1. Although the structural proteins of vaccinia appear to be richer in leucine than methionine (Fig. 11, [35S]methionine was used instead of [14C]leucine in most experiments since the specific activity of the former was much higher, and one could obtain satisfactory autoradiographic data in a shorter time. Preliminary experiments indicated that the polypeptide composition of infected cells which were labeled with [35S]methionine was the same as those labeled with [l”Clleucine. The gel in Fig. 1 showed that host protein synthesis in our cell-virus system was not completely arrested until the formation of late proteins had occurred; however, it was possible to identify the early proteins. At 5
24 4
STERN
A
B
AND
DALES
CDEFGHI
FIG. 1. Autoradiogram of a slab gel of virus and infected HeLa cells labeled with [Wmethionine. One x 10” tells/35-mm petri dish, infected in the presence or absence of HU with 10 PFUicell, were labeled with 10 ,xCi of [%]methionine for 1 hr at 5, 8, and 11 hr postinfection and then harvested for SDS-PAGE. The numbers at the top of the gel refer to the time postinfection when the label was administered in the presence (+) or absence (-1 of HU. (A) Uninfected cells; (B-G) infected cells; (H) [Wlmethionine-labeled virus; (I) [‘4Clleucine-labeled virus.
VACCINIA
ENVELOPE
hr after infection the same early virus polypeptides were labeled in the presence and absence of HU. Virus structural polypeptides that were synthesized at this time included ~145, 140, and 33. These proteins presumably correspond to VSP-1 and VSP6 of Holowczak and Joklik (1967a) which are produced in the presence of cytosine arabinoside, another inhibitor of DNA synthesis (Holowczak and Joklik, 1967b). P145 and 140 occur in the virus core, whereas p33 is presumably located at the surface because it is soluble in NP40. Another surface protein, ~58, was absent from the extracts of HU-treated cells, but it appeared in the extracts of untreated cells at 11 hr after infection. The absence of ST in HU-treated cells was also indicated by the fact that ~58 antiserum (Stern and Dales, 1976) did not react in Ouchterlony plates with cells incubated in the presence of HU, but did react with cells which were not treated with the drug. To determine the kinetics of ~58 synthesis, pulse-labeled with cells were [3H]leucine at various times postinfection, harvested at the end of the infectious cycle, and mixed with a standard amount of infected cells continuously labeled with [14Clleucine. Virus was purified from the mixture and analyzed by PAGE. Individual stained bands were cut out, eluted, and their radioactive content determined. Initial experiments indicated that the yield of mature virus labeled with [3H]leucine was not influenced by the time during the growth cycle when the radioactive label was administered. Using 14Clabeled virus as a baseline, variations in the ratio of 3H/14C in mature virus labeled at various times during the infectious cycle paralleled changes in the specific activity of virus t3H cpm/pg of protein). Table 1 summarizes changes in the ratio 3H/14C of several major virion proteins including ~58 during the virus growth cycle. The data are expressed as a percentage of the maximum 3H/14C for each polypeptide in order to readily compare changes in the ratio of 3H/14C. The results presented in Table 1 showed that the rate of incorporation of 3H into ~58 was similar to that of ~62, 60, 33, 24, and 18 in that the maximum incorporation of 3H occurred between 8 and 12 hr
245
IN ASSEMBLY TABLE
1
TIME COURSE OF [3H]L~~~~~~ IINCORPORATION INTO VIRAL PROTEINS DURING INFECTION” Virus PolyylP
Percentage maximum 3H/14C Time of labeling (hr) 3-4
p145 140 62 60 58 40 33 24 18
5-6
8-9
11-12
l&15
17-18
100
51
43
34
7
6
11 9 14 25 12 15 12
49 53 40 100 25 62 58
100 100 97 75 90 100 100
79 87 100 61 100 84 91
50 54 52 48 69 29 66
24 28 35 31 51 28 32
n Infected cells (4 x lo6 tells/60-mm plate) were labeled for 1 hr at the indicated times with 16 &i/ ml and harvested 24 hr postinfection. The cells were mixed with infected cells labeled with [‘%]leucine as described in Materials and Methods and virus was purified from the mixture. After analyzing the samples of virus by PAGE, the gel was stained with Coomassie blue; the stained bands were cut out, eluted in NCS, and counted.
after infection. By contrast, the early proteins ~145 and 140 were produced maximally at 3 to 4 hr postinfection and thereafter the rate of their synthesis declined. In order to rule out the possibility that spicules coating the external surface of immature particles are a high molecular weight precursor of ST, infected cells were labeled for 1 hr with 13%lmethionine and were either harvested immediately after pulsing or placed in MEM for 4 hr before harvesting. Cytoplasmic extracts of the pulse and chase samples were analyzed by PAGE and autoradiography. Densitometric tracings of the autoradiograms (Fig. 2) revealed that the peaks corresponding to four major virus polypeptides ~62, 60, 24, and 18 were absent at the end of the pulse but appeared in the chase sample. On the other hand, there was no change in the absorbance of the peak corresponding to ~58. These results suggest that ~62, 60,24, and 18 were formed by post-translational cleavage whereas ~58 was not. The data from the above experiments, when taken together, suggest that the spicules of immature particles are not the same as the surface tubules of mature virus.
246
STERN
AND
DALES
4
3
CHASE
-
3-
clrtqm
109876
5 MOLECULAR
2
I.5 Dye ’ fmnt
WEIGHT x Iti
FIG. 2. Pulse-chase of infected HeLa cells labeled with [%lmethionine. One x 10” cells/35mm petri dish were labeled for 1 hr at 15 hr postinfection with 10 &i of [““Slmethionine in 0.2 ml of MFM. At the end of labeling period the radioactive medium was removed; the sample designated as “pulse” was immediately harvested and the sample designated as “chase” was incubated for 4 more hr in MEM. The samples were analyzed by SDS-PAGE and the resulting autoradiograms were scanned by a densitometer. Long arrow indicates position of ~58; short arrows indicate polypeptides that appeared during the chase.
Integration of p58 into Vaccinia Virus Assembly
during
cation. The data summarized in Table 2 showed that during the 17-hr chase the 3H/ 14C in whole virus increased 11-fold, with Since p58 is a late protein and apparthe greatest change in this ratio occurring ently different from the spicules of immaduring the first 4 hr of the chase. Table 2 ture particles, we set out to determine also shows that immediately after pulsing, when ~58 was integrated into the virus. 3H/‘4C of the outer proteins of vaccinia was Initial experiments were conducted to 2.9 times greater than that of the cores and study the kinetics of integration of outer that during the chase the difference beproteins (i.e., those solubilized by NP40tween the two ratios declined. Furthermercaptoethanol) and core proteins. Inmore, at the end of the pulse 3H counts fected cells were pulse labeled with associated with the outer proteins ac13Hlleucine at 7-8 hr after infection, counted for 40% the total radioactivity in placed in chase medium, and harvested at the virus, but at the end of 17 hr the provarious times thereafter. The cells were portion of 3H counts in the outer proteins mixed with a standard quantity of 14C- was only 19%. This result implies that labeled infected cells prior to virus purifisoon after their synthesis the outer pro-
VACCINIA
ENVELOPE
teins of vaccinia were incorporated into virus at a higher rate than the core proteins. Alternatively, the nascent outer proteins were added to virus factories which already contained the DNA and the core proteins. A parallel experiment to determine when ~58 was integrated into vaccinia was performed using [35Slmethionine as a precursor. Labeled virus was subjected to PAGE and autoradiography, and data were obtained by densitometric analysis of the autoradiogram. The tracings in Fig. 3 showed that the amount of 35Slabel in all polypeptides increased with time of chase. At the end of the pulse the peak height of ~58 relative to other proteins was much higher than at later times during the chase. The actual amount of label in the individual virus proteins was determined by cutting out the bands and counting them in a liquid spectrometer. Only bands for which corresponding ones were evident in the 0 time sample were considered for analysis. The data in Table 3 show the distribution of YS label among vaccinia proteins as a function of the period of the chase. The percentage of 35Slabel in ~58, TABLE
2
INCORPORATION OF 13HlL~uc1~~
INTO NASCENT
VIRUS= Time
Whole 8 8.5 9 10 12 25
Percentage
3H/‘4C
postinfection (hr)
solubilizedd
O&X” proteins
Cores proteins
3H
Y!
virus
0.32 0.41 0.80 1.60 2.34 3.52
0.46 0.60 1.02 1.77 2.38 3.01
0.16 0.25 0.58 1.28 2.00 2.95
47 40 32 31 27 19
23 23 21 24 23 19
u Between 7 and 8 hr postinfection, 4 x lo6 cells per sample were labeled with 5.5 @X/ml of [3H11eucine. At the indicated times the samples were mixed with infected cells labeled with [14C]leucine, as described in Materials and Methods, and virus was purified from the mixture. b Proteins which were solubilized by NP40-mercaptoethanol. r, Proteins which were insoluble in NP40-mercaptoethanol. d The percentage refers to those counts that were solubilized by NP40 and mercaptoethanol.
247
IN ASSEMBLY
33, and 29, which are outer proteins, decreased with time. Another outer protein, ~14, also decreased with time but not as much. The glycoprotein (Holowczak, 1970; Garon and Moss, 1971), ~40, which is near the virus surface, was virtually unchanged. The amount of label in the core polypeptides p62,60, and 18 increased with time, while p24 remained unchanged. In the case of two other core polypeptides, p92 and 65, the relative amount of label declined with time. These data suggest that in line with the results in Table 2, ~58 and at least some other outer proteins are initially incorporated more rapidly into virus than the major core proteins. Although the decline in core polypeptide ~50 cannot be explained, the decrease in core polypeptides p92 and 65 could be accounted for if, in fact, they are the precursors of p62 and 60 and their decline reflects post-translational cleavage. This suggestion is based on the apparent similarity between the molecular weights of p92 and 65 and to those of the precursor proteins in cellular extracts (Fig. 2). Vaccinia Assembly Removal
following
HU
The data in the previous section suggested that envelopes are added onto virus at a late stage during assembly. However, electron microscopic studies indicate that envelopes are the first virus structure to appear (Dales and Mosbach, 19681, and that internal structures become evident after the completion of the envelope (Dales and Mosbach, 1968). In the presence of HU immature particles with complete envelopes and no internal structures are formed (Rosen-kranz et al., 1966; Pogo and Dales, 1971). After the removal of HU a nucleoid and lateral bodies appear inside completed immature particles (Pogo and Dales, 1971). Since most of the virus proteins are late functions (Holowczak and Joklik, 1967b), it might be presumed that the internal constituents of the mature virus are inserted into completed immature particles. To determine whether vaccinia assembly is a tightly coupled process or involves the insertion of late proteins through the membrane of immature parti-
248
STERN
7 own
10967
6
5
4
AND
DALES
3
MOLECULAR
WEIGHT x Id’
FIG. 3. Radioautogram of virus isolated from labeled cells at various times after administration of the pulse. Four x lo6 cells per sample were labeled between 8 and 8.5 hr postinfection with 125 &i of [3”]methionine. At the times after pulse-labeling as indicated in the upper left-hand corner, the samples were mixed with infected cells labeled with PHlleucine, as described in Materials and Methods, and virus was purified from the mixture. Equal amounts of protein in each sample were electrophoresed, subjected to autoradiography, and scanned by a densitometer. Arrow indicates position of ~58.
cles, the relationship of HU-induced immature particles and mature virus was studied. HeLa cells were infected and maintained in the presence of HU for 16 hr at which time they were labeled for 1 hr with [“YSlmethionine. Afterwards, the cells were exposed to HU for up to 6 hr. Each sample was then incubated for 20 hr in MEM without HU. The cells were harvested and mixed with vaccinia labeled with [3Hlleucine and virus containing both labels was purified. The data in Table 4 showed that the ratio of 35S/3H was highest in virus obtained from cells which were maintained in medium from which HU was removed immediately after pulse la-
beling. The ratio of 35S/3H decreased in proportion to the time the cells were maintained in the presence of HU after being labeled with [35Slmethionine. The cells that were kept in HU for 6 hr following 35Slabeling produced virus whose 35S/3H ratio was half the value of virus originating from cells maintained in medium from which HU was removed immediately after labeling. The decline in 35S/3H did not reflect decreased virus production since removal of HU at various times after pulselabeling did not alter the final yields of mature virus harvested 20 hr after removal of HU. When HU was removed at 18 hr postinfection the virus yield 20 hr later
VACCINIA TABLE
ENVELOPE
3
DISTRIBUTION OF [Y~IMETHIONINE IN VIRAL PROTEINS AT VARIOUS TIMES AFTER PULSELABELING*
Virus polypeptide P92 65 62 60 58 50 40 33 31 29 24 18 14
T
Percentage
7.2’ 6.0 4.8 9.6 7.2 4.6 7.3 9.2 6.5 9.9 7.0 8.2 12.1
0.5
1
2
4
6.4 6.2 8.6 15.7 4.4 3.1 5.6 6.2 5.4 6.6 6.6 9.1 15.7
4.7 4.9 15.0 18.1 3.5 2.6 4.7 5.2 3.3 4.2 7.0 12.4 14.2
1.6 2.4 18.0 26.9 1.9 2.0 5.8 4.0 2.6 3.1 6.3 12.2 13.0
1.2 2.7 19.0 32.6 1.5 0.9 5.3 3.0 2.0 2.4 6.1 11.7 11.5
1.4 2.4 .9.8 10.3 1.7 0.6 5.5 2.2 1.5 2.3 7.5 !5.7 8.8
n See Fig. 3. * Time of chase in hours. (’ Percentage total counts per minute.
was 1.6 x 10y PFU or 100 pg of protein/lo7 cells, when removed at 20 hr the yield was 1.0 x lo9 PFU or 120 ,ug of protein/lo7 cells, and when removed at 22 hr the yield was 1.2 x 10g PFU or 100 pg/lOT cells. Furthermore, this decline was not the result of increased protein turnover due to the presence of HU, since the specific activity of total protein of cells “chased” in the presence and absence of HU was the same. A more direct approach involved a combination of electron microscopy (EM) and autoradiography. L cells were infected in the presence of HU and labeled at 18 hr after infection for 1 hr with 13Hlleucine in the presence of the drug. One sample was subsequently maintained in the presence of HU for 3 hr and then for another 5 hr in its absence, another sample was chased in the absence of HU for 8 hr. The results based on counts of silver grains and virusrelated structures observed in thin sections showed that cells which were chased in the presence of HU for 3 hr and then in its absence did not have any grains associated with mature particles (Table 5 and Fig. 5). On the other hand, cells which were chased in the absence of HU contained labeled mature particles (Table 5 and Fig. 6). Sample 2 had fewer mature
249
IN ASSEMBLY
particles than sample 3 of Table 5, probably due to the fact that the cells in sample 2 were incubated in the absence of HU for 5 hr, whereas those in sample 3 were incubated in the absence of HU for 8 hr, allowing more time for mature progeny to be formed. In both cases the number of grains per cell and number of grains per immature particle were about the same. An alternative approach for studying the participation of immature forms of vaccinia in assembly and maturation involved an experiment using EM-autoradiography in which the process of morphogenesis was interrupted by the drug rifampicin. In the presence of rifampicin most or all the major virion polypeptides and DNA are synthesized (Moss et al., 1971) but membrane assembly and morphogenesis are arrested (Grimley et al., 1970; Nagayama et al., 1970). Data from the present study, summarized in Table 6, showed that when cells were labeled in the presence of rifampicin grains became associated with aberrant structures of the type expected to be formed in the presence of the drug (Table 6 and Fig. 7). However, when the cells were chased in the absence of rifampicin the aberrant factories disappeared (Fig. 8) and the grains were associated with immature and mature particles and virus factories of normal appearance (Table 6) (Moss et al., 1971). These results indicate that under conditions in which TABLE
4
INCORPORATION OF [Y~IMETHIONINE INTO VIRUS FROM CELLS LABELED IN THE PRESENCE OF HLJ”
Chase time in the presence of HU (hr)
““S,:‘H
0 2 4 6
2.94 2.07 1.83 1.34
o Cells (1 x 10” cells per sample) were infected and maintained in HU for 16 hr. The cells were pulse-labeled with 20 PCi of [:‘“Slmethionine for 1 hr at 16 hr postinfection in the presence of HU and subsequently maintained for the indicated times in the presence of HU plus MEM. HU was removed at the indicated times and the infection for each sample was allowed to proceed for another 20 hr. The ceils were mixed with 140 pg of pure vaccinia which contained 50,000 cpm of [?H]leucine.
FIG. 4. EM autoradiography corresponding to sample 1 of Table 5. Note the prominent factory with immature particles and absence of mature virus. Silver grains are scattered throughout the cytoplasm as well as over the factory. M, mitochondria. x 17,600. FIG. 5. As above, corresponding to sample 2 in Table 5. Immature and mature progeny are present but silver grains occur only over immature virions (arrows). N, nucleus; M, mitochondria. x 17,600. 250
VACCINIA
ENVELOPE
viral envelope formation occurs prior to the insertion of viral DNA and late proteins, maturation of particles assembled before DNA synthesis cannot occur even if late proteins are subsequently synthesized. In contrast to HU, reversal of rifampicin is effective in allowing maturation, since rifampicin creates a block of the enveloping process and not the synthesis of at least the majority of vaccinia polypeptides. Kinetics
of Lipid
duced into virus, it was of interest to determine whether the lipids of the envelope were integrated with similar kinetics. In order to perform experiments such as those described in Table 2, it was necessary to determine whether one could distinguish between phospholipid exchange, known to occur during vaccinia morphogenesis (Stern and Dales, 19741, and the net transfer of lipid into virus undergoing assembly. To discriminate between preexisting and nascent lipids in the virus, infected cells prelabeled with 33P were pulselabeled for 1 hr with [3Hlglycerol at 5 hr after infection. At various times during the chase the cells were harvested, mixed with unlabeled infected cells, and virus was purified from the mixture. Since the carrier virus was in excess, the specific activities of the virus lipid provided a measure of the amount of nascent virus formed. The data in Table 7 showed that the specific activities of 33P and 3H lipids increased with the time of chase; however, the ratio of 3H/33P of viral lipids was relatively constant during the chase. Since the cellular lipids were labeled with 33P before infection and with 3H after infection, the absence of change in the ratio of the two labels suggests that nascent lipids and preexisting lipids were randomized. Therefore, one cannot discriminate between phospholipid exchange and the net transfer of lipid to virus undergoing assembly. From the above it is evident that one cannot measure the kinetics of lipid integration into the envelope in a manner analogous to that of proteins.
Integration
Since the envelope proteins appear to be among the last components to be introTABLE INCORPORATION AND MATURE
Sample Gyeisi
5
OF [“HILEUCINE INTO IMMATURE VIRUS IN THE PRESENCE AND ABSENCE OF HU”
Grains/;~~scell
pro- Particles/100 cell profiles
Facto- Immaries ture 1 2 3
12 14 14
25 101 166
12 47 59
Mature
Immature
Mature
0 0 36
159 754 997
1 180 677
a L cells (4 x lo6 tells/60-mm petri dish) were infected with 15 PFUicell of vaccinia in the presence of 5 mM HU. At 18 hr postinfection, samples 1, 2, and 3 were labeled for 1 hr with 20 FCi of [SH]leucine in 0.4 ml of LFM + 5 mM HU. At the end of the pulse, samples 1 and 2 were washed and maintained in MEM with HU, whereas sample 3 was washed and maintained in MEM without HU. After being incubated in MEM + HU for another 3 hr, HU was removed from sample 2. All samples were harvested and prepared for electron microscopy at 27 hr after infection. TABLE INCORPORATION
OF [SH]L~~~~~~
6
INTO NORMAL AND ABERRANT VIRUS ABSENCE OF RIFAMPICI~~
Sample
Grains/l00
251
IN ASSEMBLY
STRUCTURES
cell profiles
IN TRE PRESENCE
AND
Particles/l00
cell profiles
Immature
Mature
685 1288 610 1210
9 492 12 354
GZi?si Particles
Factories Normal 1 2 3 4
14 13 16 21
0 188 4 258
Rifampicin 343 10 520 ‘76
Immature 20 46 30 114
Mature 0 40 2 78
n L cells (4 x 10fi tells/60-mm petri dish) were infected with 10 PFU/cell of vaccinia in the presence of 100 pg/ml of rifampicin. At 4 to 5 hr (samples 1 and 2) and 8 to 9 hr (samples 3 and 4) after infection, the cells were labeled with 20 WCi of [3H]leucine in 1 ml of LFM + rifampicin. At the end of the pulse, samples 1 and 3 were maintained in MEM + rifampicin, while the drug was removed from samples 2 and 4. At 12 hr after infection, all of the samples were harvested.
252
STERN
FIG. 6. As above, corresponding grains
occur over both immature
AND
DALES
to sample 3 in Table 5. Immature and mature progeny are absent. Silver and mature virions (arrows). x 15,000.
FIG. 7. EM autoradiography corresponding to sample 3 in Table 6. Note the prominent labeling of the aberrant factories and associated immature particles (arrows). x 12,500. FIG. 8. As above, corresponding to sample 4 in Table 6. Note that following removal of rifampicin, numerous immature and mature progeny of normal appearance are labeled (arrows). x 12,500. 253
254
STERN
AND
DALES TABLE
DISCUSSION
Morphologic studies have shown that the envelope is the earliest identifiable component of vaccinia to be formed during normal virus development (Dales and Mosbach, 1968) and can be assembled to enclose immature virus in the absence of DNA synthesis (Rosenkranz et al., 1966; Pogo and Dales, 1971). The transition from immature to mature virus involves a differentiation within the completed envelopes of immature particles (Dales and Mosbach, 1968). Based on these and other observations Pogo and Dales (1971) proposed two models of virus assembly. In the first model, the membranes of immature particles are completed prior to the insertion of late internal proteins through the sealed envelope. In the second, the envelope constituents assemble within factories around quanta of DNA and internal proteins. According to the latter model the envelope would not be completely sealed until all the necessary polypeptides are in place. One may envisage that additional internal proteins such as the precursors of p62 (Katz and Moss, 1970), ~60, ~24, and p18 accumulate within the developing envelope to be processed as the interior is reorganized into the core and lateral bodies of mature virus. The results from the present study favor the second model since it appears that immature particles, whose envelopes are completed in the presence of HU, are unlikely to progress to maturity when DNA and late protein synthesis are resumed. The absence of DNA in immature particles formed in the presence of HU probably does not account for this result because when DNA and the majority of the late polypeptides are made, as in the presence of rifampicin, removal of the drug at the time when further protein synthesis is blocked by streptovitacin A results in the assembly of DNA containing immature forms of virus which fail to progress into mature forms (Nagayama et al., 1970). In addition to changes that occur within the envelope, the virus surface also undergoes a transformation in which the spicules that cover immature particles disappear and ST appear. The spicules, however, are neither precursors nor a different
INCORPORATION
Time postinfection (hr) 6 6.5 7 8 9
I
OF [3HlG~~~~~~~ NASCENT VIRU@
Lipid cpm/mg protein 3H
33P
3138 2931 3677 5898 9479
4062 4552 4194 9458 14246
INTO LIPIDS
OF
3H/33P
0.77 0.64 0.86 0.62 0.66
a Uninfected cells (4 x lo6 cells per sample) were labeled for 15 hr before infection with 8 &i of 33P. At 5 hr after infection, the cells were incubated for 1 hr with 17 &i of [3H]glycerol. At the indicated times a sample was removed and mixed with 2 x 10’ unlabeled infected cells. The lipids of pure virus were extracted as previously described (Stern and Dales, 1975).
form of the surface tubules, since ~58 is not synthesized in the absence of DNA replication and is not produced via the cleavage of high molecular weight precursor. Furthermore, immunological studies have shown that ferritin-conjugated antibody which binds to the surface of virus fails to react with the spicule-coated surface of immature particles (Morgan et al., 1962). The dissimilarity between spicules and ST can also be seen from studies on conditional lethal mutants of vaccinia which show that the spicules are lost from the surface of immature virus when internal differentiation occurs and are replaced by the surface tubules (Stern et al., 1975). Data from the present study and those of Sarov and Joklik (1973) indicate that ~58 like other proteins found at or near the surface are among the last to be integrated into maturing virus. However, Sarov and Joklik hypothesized that vaccinia virus assembly progresses via intermediate particles. By contrast, data from our past and present work together with studies on conditional lethal mutants (Stern et al., 1975) indicate that vaccinia morphogenesis occurs in a tightly coupled fashion in which the internal constituents are assembled as a complex of DNA and polypeptides at about the time the envelope condenses around this complex. ACKNOWLEDGMENTS This investigation was supported in part by U.S. Public Health Service Grant No. AI 07477. We
VACCINIA thank
Vladimir
Milovanovich
for competent
ENVELOPE assist-
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