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Proteins of murine cytomegalovirus: Identification of structural and nonstructural antigens in infected cells
VIROLOGY
86,22-36
(1978)
Proteins
of Murine Cytomegalovirus: Nonstructural Antigens J. K. CHANTLER
Division
of Medical
Microbiology,
University Accepted
Identification in Infected
AND
and
J. B. HUDSON
of British Columbia, Canada November
of Structural Ceils
Vancouver,
British
Columbia,
V6T
1 WS,
28, 1977
The synthesis of viral proteins in tertiary mouse embryo cells infected with murine cytomegalovirus (MCMV) has been studied by polyacrylamide gel electrophoresis. Three immediate-early proteins were detected by 4 hr postinfection (p.i.), but between 4 hr and the onset of viral DNA synthesis at 8-10 hr, no further viral proteins could be distinguished. The major structural proteins appeared after viral DNA synthesis had commenced and continued to be made until 36 hr p.i. or later. Host translation occurred until 24 hr pi. but was inhibited between then and 30 hr p.i. at which time viral protein synthesis predominated. At this time 52 proteins could be labeled in infected cells and, of these, 30 could be precipitated with viral-specific antisera. The proteins precipitated included 22 with electrophoretic mobilities similar to structural viral proteins and a further 8 which were not present in purified MCMV. These, together with the three immediate-early proteins, were the only nonstructural proteins which were detected in the infected cell. INTRODUCTION
conditions for viral growth. Even then, it has proved difficult to identify a large number of viral proteins over the background of host synthesis without the use of antisera to precipitate viral antigens selectively. However, in this paper, the synthesis of 52 proteins late in the infection of mouse embryo cells with MCMV is reported. Of these, 29 are structural components of the virion, and a further 8 can be precipitated by antisera to infected cell antigens and are believed to be nonstructural proteins. The remainder, at present, are tentatively classified as host.
The genome of murine cytomegalovirus (MCMV) has the largest size documented for a member of the herpesvirus group at the present time (132 x lo6 daltons, Mosmann and Hudson, 1973). Despite this apparent genetic complexity, the virus appears to be dependent on certain functions in the host cell for replication as indicated by its restricted host range (Plummer, 1967) and its dependence on the cell cycle (Muller and Hudson, 1977a). In accordance with this idea, we report that host protein synthesis in MCMV-infected cells continues until late in the replicative cycle. This is in contrast to the situation described for herpes simplex or pig herpesviruses where host translation is rapidly inhibited in the first few hours postinfection (Roizman and Furlong, 1974; Ben-Porat and Kaplan, 1973). The continued synthesis of host proteins after MCMV infection has complicated the detection of virus-induced products. In addition, the dependence of the virus on host S-phase (Muller and Hudson, 1977a) has necessitated the use of partially synchronized cultures in order to obtain optimal
MATERIALS
METHODS
Cells. Mouse embryo cultures were prepared by trypsinization of 12- to 15-day embryos obtained from Swiss mice. They were propagated in Dulbecco’s modified Eagle’s medium (DMEM; Microcan) supplemented with 10% fetal calf serum and 20 pg/ml of gentamycin in a well-humidified atmosphere containing 5% COZ, at 37O. The cultures were used after two passages in vitro, i.e., at the tertiary stage (3ME). Virus. Stocks of murine cytomegalovirus (Smith strain, American Type Culture Col22
0042-6822/78/0861-0022$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
PROTEINS
OF
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lection) were prepared by infecting tertiary mouse embryo cells in roller bottles (120 X lo6 cells) at low multiplicity (CO.1 PFU/cell). The supernatant medium was harvested after 3 days and centrifuged at 3500 rpm in a Sorvall GSA rotor to remove cell debris, and then at 12,000 rpm for 2 hr to pellet the virus. This pellet was taken up in a small volume and used as high-titer viral stock (10’ + 10n PFU/ml). MCMV was assayed by plaque titration as described previously (Hudson et al., 197613). Infection and labeling of cells. Secondary cultures of ME cells which had been confluent for at least 2 days were subcultured into 60-mm petri dishes 16-20 hr prior to infection. The cells were infected with MCMV at 20-50 PFU/cell in 2 ml of DMEM + 2% fetal calf serum, in a centrifugal field of 800 g for 30 min, as described previously (Hudson et al., 197613). In the case of roller cultures, a standard infection (without centrifugation) was used, and the adsorption period was increased to 2 hr. After adsorption, the inoculum was removed and replaced with DMEM + 5% fetal calf serum and the cells were incubated at 37”. Cells were labeled in medium containing 20% of the normal concentration of methionine (DMEM l/5 Met; Microbiological Associates), and 5-20 PCi of [35S]methionine/ml. At harvest, the monolayers were rinsed with ice-cold phosphate-buffered saline (PBS) and then the cells were detached with a rubber policeman into a small volume of PBS, pelleted, and frozen at -20”. Purification of MCMV. Large quantities of radioactively labeled MCMV were prepared in roller cultures of tertiary mouse embryo cells infected at 1 PFU/cell. [35S]Methionine in DMEM l/5 Met (4 &X/ml) was added to the cultures at 24 hr postinfection and the supernatant medium was harvested at 3 days. Cell debris was removed and the virus was pelleted as described before for the preparation of viral stocks. The viral preparation was taken up in a small volume of PBS and sonicated briefly to disperse clumps (2 x 5 set at 40 W in a Biosonik II sonicator). Further purification was carried out as described by Kim et al. (1976). Briefly, the viral suspension was layered onto a 30-ml gradient of
CYTOMEGALOVIRUS
23
20-50s potassium tartrate in SSC (0.15 M NaC1; 0.015 M Na-citrate) and centrifuged at 25,000 rpm in a Beckman SW 27 rotor for 2.5 hr at 4”. The viral band was collected by fractionating the gradient from the bottom of the tube and, after dialysis against SSC, was rebanded on a second 20-50% potassium tartrate gradient centrifuged for 16 hr at 25,000 rpm. Fractions of 1 ml were collected and the absorbance at 660 mm was measured in a Beckman DB-G spectrophotometer. The distribution of radioactivity in the gradient was determined by counting lo+1 aliquots of each fraction in Aquasol (New England Nuclear) in a Searle Isocap/300 scintillation counter. Further aliquots were removed for examination under the electron microscope. The “peak” fractions were pooled, diluted lo-fold with distilled water, and centrifuged at 15,000 rpm in a Sorvall Type SS-34 rotor for 2 hr to pellet the purified virus. This pellet was stored at -20” before analysis on SDSpolyacrylamide gels. Preparation of antigens and antisera. A soluble extract enriched for viral proteins was prepared from infected cells, essentially as described by Purifoy and Powell (1976). Infected cells, harvested at 30 hr p.i. were taken up in PBS and disrupted by sonication for 2 x 20 set at 40 W. An equal volume of 3 M NaCl + 10 mA4 EDTA was added and the mixture was allowed to stand at 0” for 2 hr. DNA and insoluble material were then removed by centrifuging at 20,000 rpm in a Beckman Type 50 rotor for 1 hr at 4’. The supernatant fluid was dialyzed extensively against PBS at 4” and the slight precipitate which appeared was removed by centrifugation at 20,000 rpm as before. The resulting supernatant was used as soluble infected cell antigen (ICP/NaCl) for the preparation of antisera. Rabbits were injected intravenously with 0.5 ml of ICP/NaCl containing 2 mg/ml of protein. A further 0.5 ml emulsified with an equal volume of Freund’s adjuvant (Difco) was injected intramuscularly. Subsequent injections were done by the intravenous route only, at four weekly intervals. The animals were bled 10 days after the last inoculation, from an ear vein, and the whole procedure was repeated three times at intervals of 2 months.
24
CHANTLER
The antisera were adsorbed thoroughly against monolayer-s of mouse embryo tissue either air-dried or fixed in ethanol, in petri dishes. The IgG fraction was then precipitated by dropwise addition of an equal volume of saturated ammonium sulfate and was tested for specificity to viral components by agar gel immunodiffusion (Horwitz and Scharff, 1969) and immunofluorescence, before use in immune precipitation reactions. Immune precipitation. The conditions for maximal precipitation of radioactively labeled antigen were determined as described by Honess and Watson (1974). Subsequent experiments were carried out using a slight excess of antibody in the reaction mixture (5 ~1 of IgG; 175 vg of protein antigen in a total volume of 400 ~1). The IgG was reacted with a salt extract from infected or mock-infected cells, prepared in the same way as the ICP/NaCl antigen described above. The serum + antigen mixtures were incubated at 37” for 2 hr and were then centrifuged at 5000 rpm in a Sorval SS-34 rotor at 4’. The pellets were washed twice with 1 ml of ice-cold PBS and were then denatured and analyzed on SDS-polyacrylamide gels as described below. SDS-Polyacrylamide gel electrophoresis and autoradiography. The discontinuous buffer system of Laemmli (1970) as described by Spear and Roizman (1972) was employed. Prior to electrophoresis, proteins were denatured in 0.05 M Tris-HCl, pH 7,2% sodium dodecyl sulfate (SDS, BDH Biochemicals), 5% 2-mercaptoethanol (Sigma), 0.005% bromophenol blue, and 10% glycerol. In the case of wholecell pellets, the mixture was sonicated to decrease the viscosity caused by cellular DNA. All samples were heated at 100’ for 2 min and were then applied to a 10% polyacrylamide gel slab [0.375 M Tris-HCl, pH 8.8, 0.1% SDS, 0.03% (v/v) TEMED, 0.03% (w/v) ammonium persulfate, 10% acrylamide, 0.27% methylenebisacrylamide) of dimensions 14 cm x 12 cm x 1.5 mm. Electrophoresis was carried out for 5 hr in a Hoeffer slab gel apparatus at a constant current of 25 mA/gel slab. Following electrophoresis, the gels were stained in 0.2% Coomassie brilliant blue in a solution
AND
HUDSON
of 50% TCA. They were then destained in a solution of 5% methanol:7% acetic acid in distilled water (MAH). They were allowed to soak overnight in MAH containing 2% glycerol and were then dried onto filter paper (Whatman No. 1) or dialysis membrane using a Hoeffer slab gel drier. The dried gels were placed in contact with a sheet of Kodak RP/XR-1 X-ray film in a light-tight folder under pressure, for an exposure time of approximately 10 days. In some cases, the gels were soaked overnight in dimethylsulfoxide containing 2% diphenyloxazole (DMSO/PPO) as described by Bonner and Laskey (1974) and were then allowed to swell in 10% acetic acid:2% glycerol before being dried as before. In this case, autoradiography was for 24-50 hr at -70’. The films were developed in a Kodak RPX-O-Mat processor and were scanned with an adapted Helena quick-scan densitometer/integrator with a slit of 0.16 x 1.6 mm. Estimation of molecular weights of proteins. Molecular weights of viral proteins were determined by the procedure of Weber and Osborn (1969) using the following standard marker proteins of the indicated molecular weight: myosin, 212,000 (a gift from Dr. R. Dunn); /3-galactosidase, 130,006 (Worthington); phosphorylase b, 92,500 (Sigma); bovine serum albumin, 67,000 (Sigma); catalase, 60,000 (Sigma); aldolase, 40,000 (Pharmacia); ovalbumin, 44,500 (Pharmacia); chymotrypsinogen, 25,700 (Pharmacia); and cytochrome c, 13,500 (Pharmacia) . Electron microscopy. Samples of MCMV, before and after purification on potassium tartrate gradients, were dialyzed against PBS and then examined under a Philips EM-300 after staining with phosphotungstate at pH 6.4. Protein determinations. Protein concentrations were determined by the technique of Lowry et al. (1951). RESULTS
Optimal Conditions for Growth of MCMV MCMV is a cell cycle-dependent virus (Muller and Hudson, 1977a) and in order to synchronize the course of viral infection it is necessary to infect cells which are in the
PROTEINS
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same phase of the cell cycle. Suitable cultures of tertiary mouse embryo (3ME) cells growing in synchrony have routinely been obtained by subculturing a stationary monolayer at a fairly high density of 5 x lo6 cells/dish. These cells enter S-phase in a partially synchronous manner 16 hr later and continue to synthesize DNA for the next 8 hr. It has been reported elsewhere that infection of the synchronized cells immediately after subculture results in a prolonged latent phase with extracellular virus appearing at 20 hr p.i. or later (Muller and Hudson, 1977a). In contrast, infection of the cultures 16 hr after replating reduces the latent period to approximately 10 hr with >95% CPE apparent 2 hr earlier. In comparison, a randomly growing culture gives an asynchronous infection with only about 40% of the cells showing CPE at 8 hr. In this case, extracellular virus can be detected at 12 hr, but is present in lower amounts than in synchronized cells for up to 36 hr p.i., although final yields are comparable. Additional manipulation of the time of infection (e.g., at 12, 20, or 24 hr after replating) does not reduce the latent phase any further, and infection during the period 16-20 hr after subculture of a stationary monolayer has been found to give optimal conditions for viral growth. Purification
of Virus
Purification of MCMV was attempted on dextran, Ficoll, and potassium tartrate gradients, but in agreement with the findings of Kim et al. (1976) only the latter were found to give good yields of pure virus. The problem of host contamination was greatly reduced by using extracellular fluid as the starting material for viral purification and subjecting this to low-speed centrifugation to remove gross cell debris prior to gradient sedimentation. The presence of multicapsid virions in preparations of MCMV (Hudson et al., 1976a) has complicated the purification procedure and resulted in a broadening of the viral band in density gradients. This was particularly pronounced in dextran and Ficoll gradients but was also found with those of potassium tartrate. However attempts to subfractionate the band into single and multicapsids have so far not been successful and in most cases the whole band
25
CYTOMEGALOVIRUS
has been pooled prior to analysis by SDS-polyacrylamide gel electrophoresis. The degree of purification was monitored in two ways. (1) Negatively stained preparations of starting material and purified virus were examined under an electron microscope as shown in Fig. 1. A small amount of contamination with membrane fragments can be seen associated with the virions after two potassium tartrate gradients but it is clear that considerable purification has been achieved. The integrity of the virions was found to deteriorate during the second potassium tartrate gradient but this was unavoidable in order to reduce host contamination. (2) Mouse embryo cultures were labeled with [35S]methionine for 24 hr prior to infection, after which the monolayers were washed thoroughly and infected at 50 PFU/cell in the presence of twice the normal concentration of unlabeled methionine. Extracellular virus was harvested at 48 hr and was purified as before, each stage being monitored for loss of radioactive contaminating host material The result is shown in Table 1. Only a small amount of labeled material is found in the viral band of potassium tartrate gradients, and this may be due to turnover of a certain amount of host material and subsequent incorporation of the label into viral proteins. In agreement with this, polyacrylamide gel analysis of this viral band has indicated that the radioactivity is spread in trace amounts among a number of viral proteins and is not concentrated in select minor bands as would be expected if these represented contaminating host material. Protein
Composition
of MCMV
The purified preparations of MCMV were analyzed on 10% polyacrylamide gel slabs. After electrophoresis, the gels were dried and autoradiographed and the resulting pattern of viral polypeptides is shown in Fig. 2. In addition to the standard lo-day exposure of the autoradiogram, a very short exposure (3 days) and a long exposure (1 month) of sections of the gel are shown to aid identification of major or minor components. A total of 29 bands can be distinguished in this way, and these have been
26
CHANTLER
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HUDSON
FIG. 1. Electron micrographs of MCMV preparations stained with phosphotungstic acid before (A) and after (B) purification on 20-508 gradients of potassium tartrate. Both single and multicapsid virions can be seen in both samples, but most of the contaminating cellular material in A was removed during purification. x 65,090. TABLE 1 PURIFICATION OF MCMV FROM~UPERNATANT MEDIUMOFCULTURESPRELABELEDWITH [%‘~]METHIONINE Stage
of purification
Extracellular
Radioactivity ~---~~ (total (% cpm) cpm)
medium
Total PFU (log,0)
4.3 x 10’
100
11.4
5OOC g supernatant 5000 g pellet
4.0 x 106 3.9 x lo”
92 9
-
12,000 g supernatant 12,000 g pellet
3.6 x 10’ 2.4 x 10“
91 1
11.1
Band from gradient
5.2 x lo”
fast tartrate
Band from second trate gradient
tar-
9 x 10”
0.2
10.7
0.04
8.9
designated viral proteins (VP) l-29. As the viral preparations contain both single and multicapsid virions, the relative proportions of the viral proteins on acrylamide gels do not reflect their contribution to the structure of a single virion. This is
exemplified in the fact that there is always a great excess of VP 2-4, which are the major capsid proteins (unpublished observation) indicating that the multicapsids do not contain a full complement of envelope material. The electron micrographs of the multicapsids show that, together with the nucleocapsid structures, a considerable amount of amorphous material is present within the boundary membrane. This may include excess structural proteins which have not been assembled into nucleocapsids, further increasing the proportion of capsid components. In contrast to the situation reported for human cytomegalovirus, no dense bodies are seen in the supernatant fluid of cells infected with MCMV. Similar densely staining bodies are found in the nucleus and cytoplasm of infected cells, but these appear to be precursors to the multicapsid virions which are enveloped and released. The structural proteins of MCMV range in size from 261,000 to 22,500 daltons (Table 2) with two further components (a,b) of 285,000 and 270,000 daltons, respectively, which are frequently but not always de-
PROTEINS
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FK:. 2. Ten percent polyacrylamide-SDS gel of purified MCMV virions. The virus was prepared in roller cultures of 3ME cells and was labeled with ]‘“S]methionine (4 $X/ml) from 24 to 72 hr p.i. In addition to a lo-day exposure of the autoradiogram, a section of a 3-day and a l-month exposure are shown to aid identification of the major structural proteins and also minor components. Viral proteins have been numbered l-29.
tected. These may therefore represent material which has been insufficiently solubilized prior to electrophoresis. The number of major structural proteins identified is comparable with the report of Kim et al. (1976). However, differences have been found in the minor components and also in the molecular weights of the structural components estimated from their electrophoretic mobilities (Table 2). The results are also in line with published data on the
CYTOMEGALOVIRUS
27
composition of human cytomegalovirus in which 32 polypeptides of molecular weights ranging from 235,000 to 13,500 have been described (Gupta et al., 1977). The sum of the molecular weights of MCMV structural proteins is 2.15 X lo6 which is equivalent to 19,510 amino acids or 5.85 x lo4 nucleotides. Assuming the molecular weight of the MCMV genome to be 132 x lo6 (4.3 x lo5 nucleotides), 14% of the double-stranded DNA template is required to specify the structural proteins. A temporal analysis of proteins made in MCMV-infected 3ME cells was conducted to see whether a sequential pattern of synthesis of virus-coded products could be detected. In the cases of both herpes simplex and pseudorabies viruses, a complex system of regulation of viral translation has been described (Ben-Porat and Kaplan, 1973; Honess and Roizman, 1973, 1974). Viral proteins have been defined as immediateearly, early, or late (or (Y, p, or y) depending on their time of first appearance in the infected cell, or whether they are still expressed after treatment of the cells with certain metabolic inhibitors. Thus the immediate-early only are found after the reversal of a cycloheximide block imposed on the cells during the first few hours postinfection, while the distinction between early and late may be the onset of viral DNA synthesis (Swanstrom and Wagner, 1974). a. Early proteins. The kinetics of synthesis of MCMV proteins was studied in 3ME cells, infected with 20 PFU/cell and labeled for 4-hr periods up to 12 hr p.i. with [35S]methionine (20 @i/ml). The cell pellets obtained were solubilized and analyzed on 10% SDS-polyacrylamide gel slabs, and then autoradiography was performed on these. The results of one such experiment are shown in Fig. 3 and the first conclusion which can be drawn from the autoradiogram is that there is no rapid inhibition of host protein synthesis in the infected cell. This has made detection of small amounts of viral proteins extremely difficult but a number of observations can be made. During a 0- to 4-hr pulse, three virus-induced proteins can be distinguished (Fig. 3b, arrowheads). These are much more readily identified in infected cells which have been
TABLE CLASSIFICATION ICP
1 2 3 4 5 6 7 8 9 10 11 12 12a 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 34a 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 ” S, Structural;
Molecular
AND SIZE OF PROTEINS weight
Precipitated anti-(ICP/NaCl) I&
by
IN MCMV-INFECTED
3ME
Corresponding structural protein
VP 1
+/+++ +/++ ++ +
VP8 VP9 VP 10 VP 11
++ + +
VP 12 VP 13 VP 14
NS+S S S
+/+ +/++
VP 15 VP 16 VP 17
S S NS S
+/-
VP 18
S
VP3 VP4 VP5 VP6 VP 7
+ + + +/+/-
+ + + + ++++
VP VP VP VP
+ +/+ +/+ +/+ ++
19 20 21 22
S S S S NS
VP 23 VP 24
S S NS
NS
+ +
NS, nonstructural. 28
Immediateearly
S NS S NS S S S S S NS S S S S
VP2
CELLS
Classification” Late
285,000 270,000 261,000 230,000 172,000 154,ooO 138,000 123,000 109,000 94,000 91,500 86,500 83,000 76,000 74,000 72,500 70,500 69,OQO 67,500 66,500 66,000 6‘Goo 64,000 63,000 61,500 59,500 55,500 54,000 53,500 51,500 51,000 49,500 48,500 48,000 46,500 44,500 43,500 41,500 38,500 37,500 37,000 36,500 35,000 34,500 34,000 30,500 28,500 26,000 25,500 24,500 23,500 22,500
2
SYNTHESIZED
VP 25 VP 26
S S
VP 27
S
VP 28 VP 29
S S
NS
NS
NS
PROTEINS
FIG. 3. (a-d) infected; (b) O-4 infected (f) cells proteins can be molecular weight
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CYTOMEGALOVIRUS
29
A time course of synthesis of proteins in MCMV-infected cells between 0 and 12 hr. (a) Mockhr pi.; (c) 4-8 hr p.i.; (d) 8-12 hr p.i. In e and f the proteins synthesized in mock-infected (e) or after removal of a 3-hr cycloheximide block are shown (see text for details). Three early viral identified at O-4 hr (arrowheads). The continued synthesis of these plus an additional lower protein can be seen in f.
labeled after release from a cycloheximide block as shown in Fig. 3f. In this experiment, cells were treated with 50 pg/ml of cycloheximide from the time of viral ad-
sorption, until 3 hr p.i. The inhibitor was then removed by rinsing the culture five times in DMEM, and then the cells were labeled from 3 to 6 hr p.i. in DMEM l/5
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CHANTLER
Met containing 10 &i/ml of [“5S]methionine. A corresponding mock-infected culture shows only a low level of ongoing protein synthesis under these conditions (Fig. 3e). However, in the infected cells, the three early proteins are overproduced in a manner similar to that described for pseudorabies virus (Ben-Porat and Kaplan, 1973). They, and a fourth protein which has only been identified in the cycloheximidetreated cells (Fig. 3f) therefore appear to be immediate-early proteins (EP). The synthesis of the EP ceases shortly after 4 hr p.i. Even in the cycloheximidetreated cells their synthesis occurs for only approximately 3 hr after removal of the inhibitor. However in neither case is this early phase followed by a burst of viral protein synthesis as has been described for herpes simplex and pseudorabies viruses. A low level of synthesis of several viral proteins may occur but no viral bands can be detected with certainty on the autoradiogram. In view of the cell cycle dependency of MCMV, this apparently quiescent period may be due to a requirement for a host factor not present in the cells at this time. However, altering the time of infection relative to the host S-phase has not been found to be effective in reducing the latent period to less than 10 hr. In contrast, an infection in early G-l was shown to prolong the appearance of progeny virus to 20 hr or later (Muller and Hudson, 1977a). Further attempts to accelerate the course of infection by increasing the multiplicity to 50 PFU/cell were also ineffective, while any further increase in multiplicity was found to be toxic to the cells resulting in rapid appearance of CPE at 2-4 hr p.i. followed by cell death. b. Structural viral proteins. In Fig. 4 an autoradiogram representing a time course of infection up to 28 hr p.i. is shown. In this experiment, the first pulse was from 2 to 4 hr p.i. following a 2-hr adsorption period and only one EP could be detected. A closer examination of the first 6 hr p.i. has confirmed that two of the EP are maximally synthesized at O-2 hr p.i., while the third appears slightly later and continues to be made until 4-6 hr p.i. (unpublished result). The synthesis of viral structural proteins
AND HUDSON
can first be detected at 12 hr p.i. shortly after the onset of viral DNA replication. Only the major structural components (in particular, VP 2-58, and 26) can be distinguished up to 24 hr p.i., although thereafter host translation does decrease and by 30 hr p.i. viral protein synthesis predominates (Fig. 4). At this time 52 proteins (ICP) can be identified in infected cell extracts as shown in a densitometric scan of the autoradiogram in Fig. 5). Evidence for viral specificity of most of the ICP cannot be obtained by means of their electrophoretic mobility alone in view of the complex pattern of proteins labeled in mock-infected cells (Fig. 5). However, the following ICP are either absent prior to infection or are synthesized in much greater amounts in the infected cell: ICP 7-11, 13, 18-21, 28, 34, and 46. Estimates of the molecular weights of the ICP are given in Table 2. Identification of Virus-Induced Proteins by Immune Precipitation In order to identify further virus-induced proteins in MCMV-infected cells, use has been made of specific antisera to precipitate viral products selectively. The antisera for these experiments were prepared in rabbits against a high-speed supernatant of saltextracted infected cells (see Materials and Methods). The salt extraction was incorporated to solubilize additional viral antigens which would normally be lost during the centrifugation step. This finding was reported by Purifoy and Powell (1976) for preparation of extracts of HSV-2-infected cells for DNA-cellulose chromatography and agrees with results obtained from our laboratory. During fractionation of cells infected with MCMV or pig herpesvirus, the majority of viral proteins in the nucleus cosediment with host cell deoxynucleoprotein, even under low-speed centrifugation at 1500 g (Chantler and Stevely, 1973, and unpublished data). This association of viral macromolecules with host chromatin is not prevented by treatment of the nuclei with 0.35 M sodium chloride which is known to remove nonspecific host cell contaminants from chromatin (Johns and For-rester, 1969). While this may suggest a particular association between viral molecules and
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a
b
c
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d
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e
f
g
h
FK. 4. Autoradiogram showing the time course of synthesis of proteins in MCMV-infected cells between 0 and 24 hr pi. (a) Mock-infected, (b) O-4 hr p.i.; (c) 4-8 hr p.i.; (d) 8-12 hr pi.; (e) 12-16 hr p.i.; (f) 16-20 hr p.i.; (g) 20-24 hr p.i.; (h) 24-28 hr p.i. Host protein synthesis can be seen to continue until 24 hr pi. at least. In addition the presence of one immediate-early protein in b (arrowhead), and the appearance of the major structural protein VP 2 at 12 hr can be seen.
chromatin, it is also possible that cosedimentation of the two is merely due to the fact that the sites of viral assembly, the dense bodies, in the nucleus have a similar density to host cell nucleoprotein. Whether or not the association is specific, the end result is that only 20% of the viral proteins found after high salt extraction are present
in soluble antigen prepared by high-speed centrifugation alone. The antisera prepared against this enriched soluble infected cell antigen preparation (ICP/NaCl) were adsorbed thoroughly against 3ME cells prior to use. To reduce further nonspecific reactivity, the IgG fraction was selectively precipitated
CHANTLER
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MOCK
FIG. 5. Proteins from mock-infected (MOCK) or MCMV-infected @i/ml of [YS]methionine and separated on a 10% polyacrylamide autoradiogram obtained is shown. Infected-cell proteins are numbered
with ammonium sulfate. This antibody preparation was reacted against lysates of infected or mock-infected cells labeled 28-32 or O-4 hr p.i., respectively. These had undergone salt extraction and centrifugation in a manner identical to that used for the soluble antigen preparation described above. The immune precipitation reaction was carried out at 37’ for 2 hr as detailed under Materials and Methods and a typical result is shown in Table 3. A maximum of 41% of the input radioactivity could be re-
(INF) cells labeled 28-32 gel slab. A densitometric l-52.
hr p.i. with 4 scan of the
covered in the precipitates formed with infected cell extracts, but less than 5% with extracts from mock-infected cells (Table 3). These precipitates were denatured and analyzed on a 10% SDS-polyacrylamide gel slab, and in Fig. 6 a densitometric trace of the autoradiogram obtained is shown. Approximately 29 proteins can be identified in the precipitate formed with infected-cell extracts, while only one major peak can be seen in the control sample, and this comigrates with the largest peak (ICP 35) from
PROTEINS
OF
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RESULTS
OF IMMUNE
TABLE
..-
--_m_
Infected-cell antigen (ICP/NaCl) (PLP)
Mock-infected cell antigen (MockNaCl) (I%)
100 100 100 100
100 100 100 100
100 ’ Measured
a lo- or IOO-fold
dilution
OF REACTION
Total acid-insoluble cpm in ICP which were immune precipitated (a)
100 10 1” 0.1” 0.01”
100 using
3
PHECIPITATION
Anti-(ICP/NaCl) I& (d)
33
CYTOMEGALOVIRUS
l&l
26.5&l 35-+2
3 41+2
40.5+2
of the IgG preparation.
---Total acid-insoluble cpm in mock-infected cells which were immune precipitated (4) 3.5&l 2.55 1
3.0+ 1 4.1+1 3.92 1
5
INF
MOCK
‘7
FIG. 6. Densitometric antisera to their
scan of an autoradiogram comparing the proteins precipitated by virus-specific from infected cells (INF) and mock-infected cells (MOCK). The peaks for INF are numbered according equivalents in a scan of total infected-cell polypeptides (cf. Fig. 7).
infected cells. The control also contains a minor peak not present in infected-cell extracts and trace amounts of a number of other proteins. However precipitation by the virus-specific antiserum does seem to be extremely specific to infected-cell ex-
tracts and the major peaks in Fig. 6a can fairly certainly be identified as virus specific. In Fig. 7 a comparison is made of the proteins which are present in the immune precipitates from infected samples with the total spectrum of ICP and with structural
CHANTLER
FIG. 7. A comparison of the %-labeled an immune-precipitate formed by reacting MCMV. The samples were coelectrophoresed was scanned to give the profiles shown.
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HUDSON
proteins found in (A) infected-cell extracts labeled 28-32 hr pi.; (B) A with a slight excess of rabbit anti-MCMV IgG; and (C) purified on a 10% polyacrylamide gel slab and the resulting autoradiogram
viral polypeptides. Most of the major structural proteins are immune precipitated although not in the quantities expected from their concentration in the infected cell. In addition, the immune precipitates contain several other labeled species found in infected cells but not in MCMV preparations and these are defined as virus-induced nonstructural proteins. Details of the proteins precipitated including estimates of their molecular weights are given in Table 2.
DISCUSSION
The replicative cycle of MCMV in 3ME cells has been shown to be divided into three phases. During the first 4 hr p.i. there is synthesis of three immediate-early proteins of molecular weights 86,500, 74,000, and 69,000. The synthesis of these ceases shortly after 4 hr p.i. when the infection enters a quiescent period during which no viral protein synthesis is evident. A low level of synthesis of several viral enzymes
PROTEINS
OF
MURINE
may occur but cannot be detected over the background of continued host protein synthesis. The third phase commences after the onset of viral DNA replication, at approximately 10 hr p.i. with the appearance of a major structural protein, VP 2 (ICP 7). The apparent absence of early proteins is interesting in view of the known cell cycle dependency of the virus. In the case of herpes simplex virus, a large number of the early functions have been suggested to be enzymes involved in some way in viral DNA replication #chaffer, 1975). It is possible that these functions (or a portion of them) are lacking in MCMV and that continued host protein synthesis is necessary to the virus to enable its DNA replication to occur. Of interest in this respect is the finding that MCMV does not induce its own thymidine kinase postinfection (Muller and Hudson, 1977b) and that the virus is unable to replicate in G-l-arrested 3T3 cells (Muller and Hudson, manuscript submitted). This probable requirement for host enzymes does not explain why there is a lag of 8 hr or more before viral DNA replication commences when the cells are infected after they have entered S-phase, i.e., at a time when the host enzymes involved in DNA synthesis should already be present in the infected cell. It is possible that the virus also requires certain factors which it itself induces postinfection, but this is speculation at the present time. This dependence on the host cell suggests a defectiveness in the virus unexpected in view of the size of its genome (132 x 106, Mosmann and Hudson, 1973). Studies on viral DNA reassociation do not suggest the existence of tracts of repetitive sequences in the DNA but are indicative of a genetic complexity approximately equal to its size (Misra and Hudson, manuscript in preparation). However, analysis of the transcriptional patterns of the virus in the infected cell shows a maximum of 39% of the doublestranded DNA present as stable transcript late in infection (Misra et al., manuscript submitted). In addition, less than half of this (15-17s of the DNA) is found in the cytoplasm. As a high proportion of this information (over 90%) is required to code
35
CYTOMEGALOVIRUS
for the structural proteins of the virion, it appears that the absence of large numbers of nonstructural proteins is also reflected at the level of RNA synthesis. The function of the remaining 85% of the DNA, some of which is transcribed but not transported to the cytoplasm, is at present an enigma. ACKNOWLEDGMENTS The authors wish to thank Jessyca Suzuki and Theresa Sapp for excellent technical assistance. This project was supported in part by Grant MA 4762 from the Medical Research Council of Canada. REFERENCES
BEN-P• RAT, T., and KAPI.AN, A. S. (1973). Herpesviruses” (A. S. Kaplan, Academic Press, New York.
ed.), Chap.
In “The 6, p. 16.3.
BONNER, W. M., and LASKEY, R. A. (1974). detection method for tritium nucleic acids in polyacrylamide them. 46,83-88.
A film labelled proteins and gels. Eur. J. Bio-
CHANTLER, J. K., and STEVELY, W. S. (1973).
Virusinduced proteins in pseudorabies infected cells. 1. Acid-extractable proteins of the nucleus. J. Viral. 11,815-822. GIJPTA, P., ST. JEOR, S., and RAPP, F. (1977). Comparison of the polypeptides of several strains of human cytomegalovirus. J. Gen. Viral. 34,447-454. HONESS, R., and ROIZMAN, B. (1973). Proteins specified by herpes simplex virus. XI. Identification and relative molar rates of synthesis of structural and non-structural herpesvirus polypeptides in the infected cell. J. Virol. 12, 1347-1365. HONESS, R., and ROIZMAN, B. (1974). Regulation of herpesvirus macromolecular synthesis. 1. Cascade regulation of the synthesis of three groups of viral proteins. J. Virol. 14,8-19. HONESS, R., and WA’~SON, D. H. (1974). Herpes simplex virus-specific polypeptides studied by polyacrylamide gel electrophoresis of immune precipitates. J. Gen. Viral. 22, 171-183. HOHWITZ, N. S., and SCHARFF, M. D. (1969). In “Fundamental Techniques in Virology.” (K. Habel and N. P. Salzmann, eds.), Chap. 29, p. 297. Academic Press, New York. H~JDSON, J. B., MISIIA, V. M., and MOSMANN, T. R. (1976a). Properties of the multicapsid virions of murine cytomegalovirus. Virology 72,224-234. HUDSON, J. B., MISRA, V. M., and MOSMANN, T. R. (1976b). Cytomegalovirus infectivity: Analysis of the phenomenon of centrifugal enhancement of infectivity. Virology 72,235-243. JOHNS, E. W., and FORRESTER, S. (1969). Studies on nuclear proteins. The binding of extra acidic pro-
36
CHANTLER
teins to deoxyribonucleoprotein during the preparation of nuclear proteins. Eur. J. B&hem. 8, 547-551. KIM, K. S., SUPIENZA, V. I., CARP, R. I., and MOON, H. M. (1976). Analysis of the structural proteins of murine cytomegalovirus. J. Virol. 17,906-915. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-664. LOWRY, 0. H., ROSEBROLJGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MOSMANN, T. R., and HUDSON, J. B. (1973). Some properties of the genome of murine cytomegalovirus. Virology 54, 135-139. MULLER, M. T., and HUDSON, J. B. (1977a). Cell cycle dependency of murine cytomegalovirus in synchronized 3T3 cells. J. Viral. 22, 267-272. MULLER, M. T., and HUDSON, J. B. (1977b). Thymidine kinase activity in mouse 3T3 cells infected by murine cytomegalovirus. Virology 80,430-433. PLUMMEK, G. (1967). Comparative virology of the
AND
HUDSON
herpes group. Progr. Med. Virol. 9,302~340. PURIFOY, D. J. M., and POWELL, K. I. (1976). DNABinding proteins induced by herpes simplex type 2 in HE.p.2 cells. J. Viral. 19, 717-731. ROIZMAN, B., and FURLONG, D. (1974). In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, eds.), Vol. 3, Chap. 6, p. 229. Plenum Press, New York. SCHAFFER, P. A. (1975). Temperature-sensitive mutants of herpesviruses. Curr. Top. Microbial. Immunol. 70,52-K@. SPEAR, P. G., and ROIZMAN, B. (1972). Proteins specified by herpes simplex virus. V. Purification and structural proteins of the herpes virion. J. Viral. 9, 143-159. SWANSTROM, R. I., and WAGNER, E. K. (1974). Regulation of the synthesis of herpes simplex type 1 virus mRNA during productive infection. Virology 60,522-533. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determinations by dodecyl-sulphate polyacrylamide gel electrophoresis. J. Biol. Chem. 244,440~4412.
86,22-36
(1978)
Proteins
of Murine Cytomegalovirus: Nonstructural Antigens J. K. CHANTLER
Division
of Medical
Microbiology,
University Accepted
Identification in Infected
AND
and
J. B. HUDSON
of British Columbia, Canada November
of Structural Ceils
Vancouver,
British
Columbia,
V6T
1 WS,
28, 1977
The synthesis of viral proteins in tertiary mouse embryo cells infected with murine cytomegalovirus (MCMV) has been studied by polyacrylamide gel electrophoresis. Three immediate-early proteins were detected by 4 hr postinfection (p.i.), but between 4 hr and the onset of viral DNA synthesis at 8-10 hr, no further viral proteins could be distinguished. The major structural proteins appeared after viral DNA synthesis had commenced and continued to be made until 36 hr p.i. or later. Host translation occurred until 24 hr pi. but was inhibited between then and 30 hr p.i. at which time viral protein synthesis predominated. At this time 52 proteins could be labeled in infected cells and, of these, 30 could be precipitated with viral-specific antisera. The proteins precipitated included 22 with electrophoretic mobilities similar to structural viral proteins and a further 8 which were not present in purified MCMV. These, together with the three immediate-early proteins, were the only nonstructural proteins which were detected in the infected cell. INTRODUCTION
conditions for viral growth. Even then, it has proved difficult to identify a large number of viral proteins over the background of host synthesis without the use of antisera to precipitate viral antigens selectively. However, in this paper, the synthesis of 52 proteins late in the infection of mouse embryo cells with MCMV is reported. Of these, 29 are structural components of the virion, and a further 8 can be precipitated by antisera to infected cell antigens and are believed to be nonstructural proteins. The remainder, at present, are tentatively classified as host.
The genome of murine cytomegalovirus (MCMV) has the largest size documented for a member of the herpesvirus group at the present time (132 x lo6 daltons, Mosmann and Hudson, 1973). Despite this apparent genetic complexity, the virus appears to be dependent on certain functions in the host cell for replication as indicated by its restricted host range (Plummer, 1967) and its dependence on the cell cycle (Muller and Hudson, 1977a). In accordance with this idea, we report that host protein synthesis in MCMV-infected cells continues until late in the replicative cycle. This is in contrast to the situation described for herpes simplex or pig herpesviruses where host translation is rapidly inhibited in the first few hours postinfection (Roizman and Furlong, 1974; Ben-Porat and Kaplan, 1973). The continued synthesis of host proteins after MCMV infection has complicated the detection of virus-induced products. In addition, the dependence of the virus on host S-phase (Muller and Hudson, 1977a) has necessitated the use of partially synchronized cultures in order to obtain optimal
MATERIALS
METHODS
Cells. Mouse embryo cultures were prepared by trypsinization of 12- to 15-day embryos obtained from Swiss mice. They were propagated in Dulbecco’s modified Eagle’s medium (DMEM; Microcan) supplemented with 10% fetal calf serum and 20 pg/ml of gentamycin in a well-humidified atmosphere containing 5% COZ, at 37O. The cultures were used after two passages in vitro, i.e., at the tertiary stage (3ME). Virus. Stocks of murine cytomegalovirus (Smith strain, American Type Culture Col22
0042-6822/78/0861-0022$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
PROTEINS
OF
MURINE
lection) were prepared by infecting tertiary mouse embryo cells in roller bottles (120 X lo6 cells) at low multiplicity (CO.1 PFU/cell). The supernatant medium was harvested after 3 days and centrifuged at 3500 rpm in a Sorvall GSA rotor to remove cell debris, and then at 12,000 rpm for 2 hr to pellet the virus. This pellet was taken up in a small volume and used as high-titer viral stock (10’ + 10n PFU/ml). MCMV was assayed by plaque titration as described previously (Hudson et al., 197613). Infection and labeling of cells. Secondary cultures of ME cells which had been confluent for at least 2 days were subcultured into 60-mm petri dishes 16-20 hr prior to infection. The cells were infected with MCMV at 20-50 PFU/cell in 2 ml of DMEM + 2% fetal calf serum, in a centrifugal field of 800 g for 30 min, as described previously (Hudson et al., 197613). In the case of roller cultures, a standard infection (without centrifugation) was used, and the adsorption period was increased to 2 hr. After adsorption, the inoculum was removed and replaced with DMEM + 5% fetal calf serum and the cells were incubated at 37”. Cells were labeled in medium containing 20% of the normal concentration of methionine (DMEM l/5 Met; Microbiological Associates), and 5-20 PCi of [35S]methionine/ml. At harvest, the monolayers were rinsed with ice-cold phosphate-buffered saline (PBS) and then the cells were detached with a rubber policeman into a small volume of PBS, pelleted, and frozen at -20”. Purification of MCMV. Large quantities of radioactively labeled MCMV were prepared in roller cultures of tertiary mouse embryo cells infected at 1 PFU/cell. [35S]Methionine in DMEM l/5 Met (4 &X/ml) was added to the cultures at 24 hr postinfection and the supernatant medium was harvested at 3 days. Cell debris was removed and the virus was pelleted as described before for the preparation of viral stocks. The viral preparation was taken up in a small volume of PBS and sonicated briefly to disperse clumps (2 x 5 set at 40 W in a Biosonik II sonicator). Further purification was carried out as described by Kim et al. (1976). Briefly, the viral suspension was layered onto a 30-ml gradient of
CYTOMEGALOVIRUS
23
20-50s potassium tartrate in SSC (0.15 M NaC1; 0.015 M Na-citrate) and centrifuged at 25,000 rpm in a Beckman SW 27 rotor for 2.5 hr at 4”. The viral band was collected by fractionating the gradient from the bottom of the tube and, after dialysis against SSC, was rebanded on a second 20-50% potassium tartrate gradient centrifuged for 16 hr at 25,000 rpm. Fractions of 1 ml were collected and the absorbance at 660 mm was measured in a Beckman DB-G spectrophotometer. The distribution of radioactivity in the gradient was determined by counting lo+1 aliquots of each fraction in Aquasol (New England Nuclear) in a Searle Isocap/300 scintillation counter. Further aliquots were removed for examination under the electron microscope. The “peak” fractions were pooled, diluted lo-fold with distilled water, and centrifuged at 15,000 rpm in a Sorvall Type SS-34 rotor for 2 hr to pellet the purified virus. This pellet was stored at -20” before analysis on SDSpolyacrylamide gels. Preparation of antigens and antisera. A soluble extract enriched for viral proteins was prepared from infected cells, essentially as described by Purifoy and Powell (1976). Infected cells, harvested at 30 hr p.i. were taken up in PBS and disrupted by sonication for 2 x 20 set at 40 W. An equal volume of 3 M NaCl + 10 mA4 EDTA was added and the mixture was allowed to stand at 0” for 2 hr. DNA and insoluble material were then removed by centrifuging at 20,000 rpm in a Beckman Type 50 rotor for 1 hr at 4’. The supernatant fluid was dialyzed extensively against PBS at 4” and the slight precipitate which appeared was removed by centrifugation at 20,000 rpm as before. The resulting supernatant was used as soluble infected cell antigen (ICP/NaCl) for the preparation of antisera. Rabbits were injected intravenously with 0.5 ml of ICP/NaCl containing 2 mg/ml of protein. A further 0.5 ml emulsified with an equal volume of Freund’s adjuvant (Difco) was injected intramuscularly. Subsequent injections were done by the intravenous route only, at four weekly intervals. The animals were bled 10 days after the last inoculation, from an ear vein, and the whole procedure was repeated three times at intervals of 2 months.
24
CHANTLER
The antisera were adsorbed thoroughly against monolayer-s of mouse embryo tissue either air-dried or fixed in ethanol, in petri dishes. The IgG fraction was then precipitated by dropwise addition of an equal volume of saturated ammonium sulfate and was tested for specificity to viral components by agar gel immunodiffusion (Horwitz and Scharff, 1969) and immunofluorescence, before use in immune precipitation reactions. Immune precipitation. The conditions for maximal precipitation of radioactively labeled antigen were determined as described by Honess and Watson (1974). Subsequent experiments were carried out using a slight excess of antibody in the reaction mixture (5 ~1 of IgG; 175 vg of protein antigen in a total volume of 400 ~1). The IgG was reacted with a salt extract from infected or mock-infected cells, prepared in the same way as the ICP/NaCl antigen described above. The serum + antigen mixtures were incubated at 37” for 2 hr and were then centrifuged at 5000 rpm in a Sorval SS-34 rotor at 4’. The pellets were washed twice with 1 ml of ice-cold PBS and were then denatured and analyzed on SDS-polyacrylamide gels as described below. SDS-Polyacrylamide gel electrophoresis and autoradiography. The discontinuous buffer system of Laemmli (1970) as described by Spear and Roizman (1972) was employed. Prior to electrophoresis, proteins were denatured in 0.05 M Tris-HCl, pH 7,2% sodium dodecyl sulfate (SDS, BDH Biochemicals), 5% 2-mercaptoethanol (Sigma), 0.005% bromophenol blue, and 10% glycerol. In the case of wholecell pellets, the mixture was sonicated to decrease the viscosity caused by cellular DNA. All samples were heated at 100’ for 2 min and were then applied to a 10% polyacrylamide gel slab [0.375 M Tris-HCl, pH 8.8, 0.1% SDS, 0.03% (v/v) TEMED, 0.03% (w/v) ammonium persulfate, 10% acrylamide, 0.27% methylenebisacrylamide) of dimensions 14 cm x 12 cm x 1.5 mm. Electrophoresis was carried out for 5 hr in a Hoeffer slab gel apparatus at a constant current of 25 mA/gel slab. Following electrophoresis, the gels were stained in 0.2% Coomassie brilliant blue in a solution
AND
HUDSON
of 50% TCA. They were then destained in a solution of 5% methanol:7% acetic acid in distilled water (MAH). They were allowed to soak overnight in MAH containing 2% glycerol and were then dried onto filter paper (Whatman No. 1) or dialysis membrane using a Hoeffer slab gel drier. The dried gels were placed in contact with a sheet of Kodak RP/XR-1 X-ray film in a light-tight folder under pressure, for an exposure time of approximately 10 days. In some cases, the gels were soaked overnight in dimethylsulfoxide containing 2% diphenyloxazole (DMSO/PPO) as described by Bonner and Laskey (1974) and were then allowed to swell in 10% acetic acid:2% glycerol before being dried as before. In this case, autoradiography was for 24-50 hr at -70’. The films were developed in a Kodak RPX-O-Mat processor and were scanned with an adapted Helena quick-scan densitometer/integrator with a slit of 0.16 x 1.6 mm. Estimation of molecular weights of proteins. Molecular weights of viral proteins were determined by the procedure of Weber and Osborn (1969) using the following standard marker proteins of the indicated molecular weight: myosin, 212,000 (a gift from Dr. R. Dunn); /3-galactosidase, 130,006 (Worthington); phosphorylase b, 92,500 (Sigma); bovine serum albumin, 67,000 (Sigma); catalase, 60,000 (Sigma); aldolase, 40,000 (Pharmacia); ovalbumin, 44,500 (Pharmacia); chymotrypsinogen, 25,700 (Pharmacia); and cytochrome c, 13,500 (Pharmacia) . Electron microscopy. Samples of MCMV, before and after purification on potassium tartrate gradients, were dialyzed against PBS and then examined under a Philips EM-300 after staining with phosphotungstate at pH 6.4. Protein determinations. Protein concentrations were determined by the technique of Lowry et al. (1951). RESULTS
Optimal Conditions for Growth of MCMV MCMV is a cell cycle-dependent virus (Muller and Hudson, 1977a) and in order to synchronize the course of viral infection it is necessary to infect cells which are in the
PROTEINS
OF
MURINE
same phase of the cell cycle. Suitable cultures of tertiary mouse embryo (3ME) cells growing in synchrony have routinely been obtained by subculturing a stationary monolayer at a fairly high density of 5 x lo6 cells/dish. These cells enter S-phase in a partially synchronous manner 16 hr later and continue to synthesize DNA for the next 8 hr. It has been reported elsewhere that infection of the synchronized cells immediately after subculture results in a prolonged latent phase with extracellular virus appearing at 20 hr p.i. or later (Muller and Hudson, 1977a). In contrast, infection of the cultures 16 hr after replating reduces the latent period to approximately 10 hr with >95% CPE apparent 2 hr earlier. In comparison, a randomly growing culture gives an asynchronous infection with only about 40% of the cells showing CPE at 8 hr. In this case, extracellular virus can be detected at 12 hr, but is present in lower amounts than in synchronized cells for up to 36 hr p.i., although final yields are comparable. Additional manipulation of the time of infection (e.g., at 12, 20, or 24 hr after replating) does not reduce the latent phase any further, and infection during the period 16-20 hr after subculture of a stationary monolayer has been found to give optimal conditions for viral growth. Purification
of Virus
Purification of MCMV was attempted on dextran, Ficoll, and potassium tartrate gradients, but in agreement with the findings of Kim et al. (1976) only the latter were found to give good yields of pure virus. The problem of host contamination was greatly reduced by using extracellular fluid as the starting material for viral purification and subjecting this to low-speed centrifugation to remove gross cell debris prior to gradient sedimentation. The presence of multicapsid virions in preparations of MCMV (Hudson et al., 1976a) has complicated the purification procedure and resulted in a broadening of the viral band in density gradients. This was particularly pronounced in dextran and Ficoll gradients but was also found with those of potassium tartrate. However attempts to subfractionate the band into single and multicapsids have so far not been successful and in most cases the whole band
25
CYTOMEGALOVIRUS
has been pooled prior to analysis by SDS-polyacrylamide gel electrophoresis. The degree of purification was monitored in two ways. (1) Negatively stained preparations of starting material and purified virus were examined under an electron microscope as shown in Fig. 1. A small amount of contamination with membrane fragments can be seen associated with the virions after two potassium tartrate gradients but it is clear that considerable purification has been achieved. The integrity of the virions was found to deteriorate during the second potassium tartrate gradient but this was unavoidable in order to reduce host contamination. (2) Mouse embryo cultures were labeled with [35S]methionine for 24 hr prior to infection, after which the monolayers were washed thoroughly and infected at 50 PFU/cell in the presence of twice the normal concentration of unlabeled methionine. Extracellular virus was harvested at 48 hr and was purified as before, each stage being monitored for loss of radioactive contaminating host material The result is shown in Table 1. Only a small amount of labeled material is found in the viral band of potassium tartrate gradients, and this may be due to turnover of a certain amount of host material and subsequent incorporation of the label into viral proteins. In agreement with this, polyacrylamide gel analysis of this viral band has indicated that the radioactivity is spread in trace amounts among a number of viral proteins and is not concentrated in select minor bands as would be expected if these represented contaminating host material. Protein
Composition
of MCMV
The purified preparations of MCMV were analyzed on 10% polyacrylamide gel slabs. After electrophoresis, the gels were dried and autoradiographed and the resulting pattern of viral polypeptides is shown in Fig. 2. In addition to the standard lo-day exposure of the autoradiogram, a very short exposure (3 days) and a long exposure (1 month) of sections of the gel are shown to aid identification of major or minor components. A total of 29 bands can be distinguished in this way, and these have been
26
CHANTLER
AND
HUDSON
FIG. 1. Electron micrographs of MCMV preparations stained with phosphotungstic acid before (A) and after (B) purification on 20-508 gradients of potassium tartrate. Both single and multicapsid virions can be seen in both samples, but most of the contaminating cellular material in A was removed during purification. x 65,090. TABLE 1 PURIFICATION OF MCMV FROM~UPERNATANT MEDIUMOFCULTURESPRELABELEDWITH [%‘~]METHIONINE Stage
of purification
Extracellular
Radioactivity ~---~~ (total (% cpm) cpm)
medium
Total PFU (log,0)
4.3 x 10’
100
11.4
5OOC g supernatant 5000 g pellet
4.0 x 106 3.9 x lo”
92 9
-
12,000 g supernatant 12,000 g pellet
3.6 x 10’ 2.4 x 10“
91 1
11.1
Band from gradient
5.2 x lo”
fast tartrate
Band from second trate gradient
tar-
9 x 10”
0.2
10.7
0.04
8.9
designated viral proteins (VP) l-29. As the viral preparations contain both single and multicapsid virions, the relative proportions of the viral proteins on acrylamide gels do not reflect their contribution to the structure of a single virion. This is
exemplified in the fact that there is always a great excess of VP 2-4, which are the major capsid proteins (unpublished observation) indicating that the multicapsids do not contain a full complement of envelope material. The electron micrographs of the multicapsids show that, together with the nucleocapsid structures, a considerable amount of amorphous material is present within the boundary membrane. This may include excess structural proteins which have not been assembled into nucleocapsids, further increasing the proportion of capsid components. In contrast to the situation reported for human cytomegalovirus, no dense bodies are seen in the supernatant fluid of cells infected with MCMV. Similar densely staining bodies are found in the nucleus and cytoplasm of infected cells, but these appear to be precursors to the multicapsid virions which are enveloped and released. The structural proteins of MCMV range in size from 261,000 to 22,500 daltons (Table 2) with two further components (a,b) of 285,000 and 270,000 daltons, respectively, which are frequently but not always de-
PROTEINS
OF
MURINE
FK:. 2. Ten percent polyacrylamide-SDS gel of purified MCMV virions. The virus was prepared in roller cultures of 3ME cells and was labeled with ]‘“S]methionine (4 $X/ml) from 24 to 72 hr p.i. In addition to a lo-day exposure of the autoradiogram, a section of a 3-day and a l-month exposure are shown to aid identification of the major structural proteins and also minor components. Viral proteins have been numbered l-29.
tected. These may therefore represent material which has been insufficiently solubilized prior to electrophoresis. The number of major structural proteins identified is comparable with the report of Kim et al. (1976). However, differences have been found in the minor components and also in the molecular weights of the structural components estimated from their electrophoretic mobilities (Table 2). The results are also in line with published data on the
CYTOMEGALOVIRUS
27
composition of human cytomegalovirus in which 32 polypeptides of molecular weights ranging from 235,000 to 13,500 have been described (Gupta et al., 1977). The sum of the molecular weights of MCMV structural proteins is 2.15 X lo6 which is equivalent to 19,510 amino acids or 5.85 x lo4 nucleotides. Assuming the molecular weight of the MCMV genome to be 132 x lo6 (4.3 x lo5 nucleotides), 14% of the double-stranded DNA template is required to specify the structural proteins. A temporal analysis of proteins made in MCMV-infected 3ME cells was conducted to see whether a sequential pattern of synthesis of virus-coded products could be detected. In the cases of both herpes simplex and pseudorabies viruses, a complex system of regulation of viral translation has been described (Ben-Porat and Kaplan, 1973; Honess and Roizman, 1973, 1974). Viral proteins have been defined as immediateearly, early, or late (or (Y, p, or y) depending on their time of first appearance in the infected cell, or whether they are still expressed after treatment of the cells with certain metabolic inhibitors. Thus the immediate-early only are found after the reversal of a cycloheximide block imposed on the cells during the first few hours postinfection, while the distinction between early and late may be the onset of viral DNA synthesis (Swanstrom and Wagner, 1974). a. Early proteins. The kinetics of synthesis of MCMV proteins was studied in 3ME cells, infected with 20 PFU/cell and labeled for 4-hr periods up to 12 hr p.i. with [35S]methionine (20 @i/ml). The cell pellets obtained were solubilized and analyzed on 10% SDS-polyacrylamide gel slabs, and then autoradiography was performed on these. The results of one such experiment are shown in Fig. 3 and the first conclusion which can be drawn from the autoradiogram is that there is no rapid inhibition of host protein synthesis in the infected cell. This has made detection of small amounts of viral proteins extremely difficult but a number of observations can be made. During a 0- to 4-hr pulse, three virus-induced proteins can be distinguished (Fig. 3b, arrowheads). These are much more readily identified in infected cells which have been
TABLE CLASSIFICATION ICP
1 2 3 4 5 6 7 8 9 10 11 12 12a 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 34a 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 ” S, Structural;
Molecular
AND SIZE OF PROTEINS weight
Precipitated anti-(ICP/NaCl) I&
by
IN MCMV-INFECTED
3ME
Corresponding structural protein
VP 1
+/+++ +/++ ++ +
VP8 VP9 VP 10 VP 11
++ + +
VP 12 VP 13 VP 14
NS+S S S
+/+ +/++
VP 15 VP 16 VP 17
S S NS S
+/-
VP 18
S
VP3 VP4 VP5 VP6 VP 7
+ + + +/+/-
+ + + + ++++
VP VP VP VP
+ +/+ +/+ +/+ ++
19 20 21 22
S S S S NS
VP 23 VP 24
S S NS
NS
+ +
NS, nonstructural. 28
Immediateearly
S NS S NS S S S S S NS S S S S
VP2
CELLS
Classification” Late
285,000 270,000 261,000 230,000 172,000 154,ooO 138,000 123,000 109,000 94,000 91,500 86,500 83,000 76,000 74,000 72,500 70,500 69,OQO 67,500 66,500 66,000 6‘Goo 64,000 63,000 61,500 59,500 55,500 54,000 53,500 51,500 51,000 49,500 48,500 48,000 46,500 44,500 43,500 41,500 38,500 37,500 37,000 36,500 35,000 34,500 34,000 30,500 28,500 26,000 25,500 24,500 23,500 22,500
2
SYNTHESIZED
VP 25 VP 26
S S
VP 27
S
VP 28 VP 29
S S
NS
NS
NS
PROTEINS
FIG. 3. (a-d) infected; (b) O-4 infected (f) cells proteins can be molecular weight
OF
MURINE
CYTOMEGALOVIRUS
29
A time course of synthesis of proteins in MCMV-infected cells between 0 and 12 hr. (a) Mockhr pi.; (c) 4-8 hr p.i.; (d) 8-12 hr p.i. In e and f the proteins synthesized in mock-infected (e) or after removal of a 3-hr cycloheximide block are shown (see text for details). Three early viral identified at O-4 hr (arrowheads). The continued synthesis of these plus an additional lower protein can be seen in f.
labeled after release from a cycloheximide block as shown in Fig. 3f. In this experiment, cells were treated with 50 pg/ml of cycloheximide from the time of viral ad-
sorption, until 3 hr p.i. The inhibitor was then removed by rinsing the culture five times in DMEM, and then the cells were labeled from 3 to 6 hr p.i. in DMEM l/5
30
CHANTLER
Met containing 10 &i/ml of [“5S]methionine. A corresponding mock-infected culture shows only a low level of ongoing protein synthesis under these conditions (Fig. 3e). However, in the infected cells, the three early proteins are overproduced in a manner similar to that described for pseudorabies virus (Ben-Porat and Kaplan, 1973). They, and a fourth protein which has only been identified in the cycloheximidetreated cells (Fig. 3f) therefore appear to be immediate-early proteins (EP). The synthesis of the EP ceases shortly after 4 hr p.i. Even in the cycloheximidetreated cells their synthesis occurs for only approximately 3 hr after removal of the inhibitor. However in neither case is this early phase followed by a burst of viral protein synthesis as has been described for herpes simplex and pseudorabies viruses. A low level of synthesis of several viral proteins may occur but no viral bands can be detected with certainty on the autoradiogram. In view of the cell cycle dependency of MCMV, this apparently quiescent period may be due to a requirement for a host factor not present in the cells at this time. However, altering the time of infection relative to the host S-phase has not been found to be effective in reducing the latent period to less than 10 hr. In contrast, an infection in early G-l was shown to prolong the appearance of progeny virus to 20 hr or later (Muller and Hudson, 1977a). Further attempts to accelerate the course of infection by increasing the multiplicity to 50 PFU/cell were also ineffective, while any further increase in multiplicity was found to be toxic to the cells resulting in rapid appearance of CPE at 2-4 hr p.i. followed by cell death. b. Structural viral proteins. In Fig. 4 an autoradiogram representing a time course of infection up to 28 hr p.i. is shown. In this experiment, the first pulse was from 2 to 4 hr p.i. following a 2-hr adsorption period and only one EP could be detected. A closer examination of the first 6 hr p.i. has confirmed that two of the EP are maximally synthesized at O-2 hr p.i., while the third appears slightly later and continues to be made until 4-6 hr p.i. (unpublished result). The synthesis of viral structural proteins
AND HUDSON
can first be detected at 12 hr p.i. shortly after the onset of viral DNA replication. Only the major structural components (in particular, VP 2-58, and 26) can be distinguished up to 24 hr p.i., although thereafter host translation does decrease and by 30 hr p.i. viral protein synthesis predominates (Fig. 4). At this time 52 proteins (ICP) can be identified in infected cell extracts as shown in a densitometric scan of the autoradiogram in Fig. 5). Evidence for viral specificity of most of the ICP cannot be obtained by means of their electrophoretic mobility alone in view of the complex pattern of proteins labeled in mock-infected cells (Fig. 5). However, the following ICP are either absent prior to infection or are synthesized in much greater amounts in the infected cell: ICP 7-11, 13, 18-21, 28, 34, and 46. Estimates of the molecular weights of the ICP are given in Table 2. Identification of Virus-Induced Proteins by Immune Precipitation In order to identify further virus-induced proteins in MCMV-infected cells, use has been made of specific antisera to precipitate viral products selectively. The antisera for these experiments were prepared in rabbits against a high-speed supernatant of saltextracted infected cells (see Materials and Methods). The salt extraction was incorporated to solubilize additional viral antigens which would normally be lost during the centrifugation step. This finding was reported by Purifoy and Powell (1976) for preparation of extracts of HSV-2-infected cells for DNA-cellulose chromatography and agrees with results obtained from our laboratory. During fractionation of cells infected with MCMV or pig herpesvirus, the majority of viral proteins in the nucleus cosediment with host cell deoxynucleoprotein, even under low-speed centrifugation at 1500 g (Chantler and Stevely, 1973, and unpublished data). This association of viral macromolecules with host chromatin is not prevented by treatment of the nuclei with 0.35 M sodium chloride which is known to remove nonspecific host cell contaminants from chromatin (Johns and For-rester, 1969). While this may suggest a particular association between viral molecules and
PROTEINS
a
b
c
OF
MURINE
d
CYTOMEGALOVIRUS
e
f
g
h
FK. 4. Autoradiogram showing the time course of synthesis of proteins in MCMV-infected cells between 0 and 24 hr pi. (a) Mock-infected, (b) O-4 hr p.i.; (c) 4-8 hr p.i.; (d) 8-12 hr pi.; (e) 12-16 hr p.i.; (f) 16-20 hr p.i.; (g) 20-24 hr p.i.; (h) 24-28 hr p.i. Host protein synthesis can be seen to continue until 24 hr pi. at least. In addition the presence of one immediate-early protein in b (arrowhead), and the appearance of the major structural protein VP 2 at 12 hr can be seen.
chromatin, it is also possible that cosedimentation of the two is merely due to the fact that the sites of viral assembly, the dense bodies, in the nucleus have a similar density to host cell nucleoprotein. Whether or not the association is specific, the end result is that only 20% of the viral proteins found after high salt extraction are present
in soluble antigen prepared by high-speed centrifugation alone. The antisera prepared against this enriched soluble infected cell antigen preparation (ICP/NaCl) were adsorbed thoroughly against 3ME cells prior to use. To reduce further nonspecific reactivity, the IgG fraction was selectively precipitated
CHANTLER
AND
HUDSON
MOCK
FIG. 5. Proteins from mock-infected (MOCK) or MCMV-infected @i/ml of [YS]methionine and separated on a 10% polyacrylamide autoradiogram obtained is shown. Infected-cell proteins are numbered
with ammonium sulfate. This antibody preparation was reacted against lysates of infected or mock-infected cells labeled 28-32 or O-4 hr p.i., respectively. These had undergone salt extraction and centrifugation in a manner identical to that used for the soluble antigen preparation described above. The immune precipitation reaction was carried out at 37’ for 2 hr as detailed under Materials and Methods and a typical result is shown in Table 3. A maximum of 41% of the input radioactivity could be re-
(INF) cells labeled 28-32 gel slab. A densitometric l-52.
hr p.i. with 4 scan of the
covered in the precipitates formed with infected cell extracts, but less than 5% with extracts from mock-infected cells (Table 3). These precipitates were denatured and analyzed on a 10% SDS-polyacrylamide gel slab, and in Fig. 6 a densitometric trace of the autoradiogram obtained is shown. Approximately 29 proteins can be identified in the precipitate formed with infected-cell extracts, while only one major peak can be seen in the control sample, and this comigrates with the largest peak (ICP 35) from
PROTEINS
OF
MURINE
RESULTS
OF IMMUNE
TABLE
..-
--_m_
Infected-cell antigen (ICP/NaCl) (PLP)
Mock-infected cell antigen (MockNaCl) (I%)
100 100 100 100
100 100 100 100
100 ’ Measured
a lo- or IOO-fold
dilution
OF REACTION
Total acid-insoluble cpm in ICP which were immune precipitated (a)
100 10 1” 0.1” 0.01”
100 using
3
PHECIPITATION
Anti-(ICP/NaCl) I& (d)
33
CYTOMEGALOVIRUS
l&l
26.5&l 35-+2
3 41+2
40.5+2
of the IgG preparation.
---Total acid-insoluble cpm in mock-infected cells which were immune precipitated (4) 3.5&l 2.55 1
3.0+ 1 4.1+1 3.92 1
5
INF
MOCK
‘7
FIG. 6. Densitometric antisera to their
scan of an autoradiogram comparing the proteins precipitated by virus-specific from infected cells (INF) and mock-infected cells (MOCK). The peaks for INF are numbered according equivalents in a scan of total infected-cell polypeptides (cf. Fig. 7).
infected cells. The control also contains a minor peak not present in infected-cell extracts and trace amounts of a number of other proteins. However precipitation by the virus-specific antiserum does seem to be extremely specific to infected-cell ex-
tracts and the major peaks in Fig. 6a can fairly certainly be identified as virus specific. In Fig. 7 a comparison is made of the proteins which are present in the immune precipitates from infected samples with the total spectrum of ICP and with structural
CHANTLER
FIG. 7. A comparison of the %-labeled an immune-precipitate formed by reacting MCMV. The samples were coelectrophoresed was scanned to give the profiles shown.
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HUDSON
proteins found in (A) infected-cell extracts labeled 28-32 hr pi.; (B) A with a slight excess of rabbit anti-MCMV IgG; and (C) purified on a 10% polyacrylamide gel slab and the resulting autoradiogram
viral polypeptides. Most of the major structural proteins are immune precipitated although not in the quantities expected from their concentration in the infected cell. In addition, the immune precipitates contain several other labeled species found in infected cells but not in MCMV preparations and these are defined as virus-induced nonstructural proteins. Details of the proteins precipitated including estimates of their molecular weights are given in Table 2.
DISCUSSION
The replicative cycle of MCMV in 3ME cells has been shown to be divided into three phases. During the first 4 hr p.i. there is synthesis of three immediate-early proteins of molecular weights 86,500, 74,000, and 69,000. The synthesis of these ceases shortly after 4 hr p.i. when the infection enters a quiescent period during which no viral protein synthesis is evident. A low level of synthesis of several viral enzymes
PROTEINS
OF
MURINE
may occur but cannot be detected over the background of continued host protein synthesis. The third phase commences after the onset of viral DNA replication, at approximately 10 hr p.i. with the appearance of a major structural protein, VP 2 (ICP 7). The apparent absence of early proteins is interesting in view of the known cell cycle dependency of the virus. In the case of herpes simplex virus, a large number of the early functions have been suggested to be enzymes involved in some way in viral DNA replication #chaffer, 1975). It is possible that these functions (or a portion of them) are lacking in MCMV and that continued host protein synthesis is necessary to the virus to enable its DNA replication to occur. Of interest in this respect is the finding that MCMV does not induce its own thymidine kinase postinfection (Muller and Hudson, 1977b) and that the virus is unable to replicate in G-l-arrested 3T3 cells (Muller and Hudson, manuscript submitted). This probable requirement for host enzymes does not explain why there is a lag of 8 hr or more before viral DNA replication commences when the cells are infected after they have entered S-phase, i.e., at a time when the host enzymes involved in DNA synthesis should already be present in the infected cell. It is possible that the virus also requires certain factors which it itself induces postinfection, but this is speculation at the present time. This dependence on the host cell suggests a defectiveness in the virus unexpected in view of the size of its genome (132 x 106, Mosmann and Hudson, 1973). Studies on viral DNA reassociation do not suggest the existence of tracts of repetitive sequences in the DNA but are indicative of a genetic complexity approximately equal to its size (Misra and Hudson, manuscript in preparation). However, analysis of the transcriptional patterns of the virus in the infected cell shows a maximum of 39% of the doublestranded DNA present as stable transcript late in infection (Misra et al., manuscript submitted). In addition, less than half of this (15-17s of the DNA) is found in the cytoplasm. As a high proportion of this information (over 90%) is required to code
35
CYTOMEGALOVIRUS
for the structural proteins of the virion, it appears that the absence of large numbers of nonstructural proteins is also reflected at the level of RNA synthesis. The function of the remaining 85% of the DNA, some of which is transcribed but not transported to the cytoplasm, is at present an enigma. ACKNOWLEDGMENTS The authors wish to thank Jessyca Suzuki and Theresa Sapp for excellent technical assistance. This project was supported in part by Grant MA 4762 from the Medical Research Council of Canada. REFERENCES
BEN-P• RAT, T., and KAPI.AN, A. S. (1973). Herpesviruses” (A. S. Kaplan, Academic Press, New York.
ed.), Chap.
In “The 6, p. 16.3.
BONNER, W. M., and LASKEY, R. A. (1974). detection method for tritium nucleic acids in polyacrylamide them. 46,83-88.
A film labelled proteins and gels. Eur. J. Bio-
CHANTLER, J. K., and STEVELY, W. S. (1973).
Virusinduced proteins in pseudorabies infected cells. 1. Acid-extractable proteins of the nucleus. J. Viral. 11,815-822. GIJPTA, P., ST. JEOR, S., and RAPP, F. (1977). Comparison of the polypeptides of several strains of human cytomegalovirus. J. Gen. Viral. 34,447-454. HONESS, R., and ROIZMAN, B. (1973). Proteins specified by herpes simplex virus. XI. Identification and relative molar rates of synthesis of structural and non-structural herpesvirus polypeptides in the infected cell. J. Virol. 12, 1347-1365. HONESS, R., and ROIZMAN, B. (1974). Regulation of herpesvirus macromolecular synthesis. 1. Cascade regulation of the synthesis of three groups of viral proteins. J. Virol. 14,8-19. HONESS, R., and WA’~SON, D. H. (1974). Herpes simplex virus-specific polypeptides studied by polyacrylamide gel electrophoresis of immune precipitates. J. Gen. Viral. 22, 171-183. HOHWITZ, N. S., and SCHARFF, M. D. (1969). In “Fundamental Techniques in Virology.” (K. Habel and N. P. Salzmann, eds.), Chap. 29, p. 297. Academic Press, New York. H~JDSON, J. B., MISIIA, V. M., and MOSMANN, T. R. (1976a). Properties of the multicapsid virions of murine cytomegalovirus. Virology 72,224-234. HUDSON, J. B., MISRA, V. M., and MOSMANN, T. R. (1976b). Cytomegalovirus infectivity: Analysis of the phenomenon of centrifugal enhancement of infectivity. Virology 72,235-243. JOHNS, E. W., and FORRESTER, S. (1969). Studies on nuclear proteins. The binding of extra acidic pro-
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teins to deoxyribonucleoprotein during the preparation of nuclear proteins. Eur. J. B&hem. 8, 547-551. KIM, K. S., SUPIENZA, V. I., CARP, R. I., and MOON, H. M. (1976). Analysis of the structural proteins of murine cytomegalovirus. J. Virol. 17,906-915. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-664. LOWRY, 0. H., ROSEBROLJGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MOSMANN, T. R., and HUDSON, J. B. (1973). Some properties of the genome of murine cytomegalovirus. Virology 54, 135-139. MULLER, M. T., and HUDSON, J. B. (1977a). Cell cycle dependency of murine cytomegalovirus in synchronized 3T3 cells. J. Viral. 22, 267-272. MULLER, M. T., and HUDSON, J. B. (1977b). Thymidine kinase activity in mouse 3T3 cells infected by murine cytomegalovirus. Virology 80,430-433. PLUMMEK, G. (1967). Comparative virology of the
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herpes group. Progr. Med. Virol. 9,302~340. PURIFOY, D. J. M., and POWELL, K. I. (1976). DNABinding proteins induced by herpes simplex type 2 in HE.p.2 cells. J. Viral. 19, 717-731. ROIZMAN, B., and FURLONG, D. (1974). In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, eds.), Vol. 3, Chap. 6, p. 229. Plenum Press, New York. SCHAFFER, P. A. (1975). Temperature-sensitive mutants of herpesviruses. Curr. Top. Microbial. Immunol. 70,52-K@. SPEAR, P. G., and ROIZMAN, B. (1972). Proteins specified by herpes simplex virus. V. Purification and structural proteins of the herpes virion. J. Viral. 9, 143-159. SWANSTROM, R. I., and WAGNER, E. K. (1974). Regulation of the synthesis of herpes simplex type 1 virus mRNA during productive infection. Virology 60,522-533. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determinations by dodecyl-sulphate polyacrylamide gel electrophoresis. J. Biol. Chem. 244,440~4412.