Structural and nonstructural proteins of strain Colburn cytomegalovi

VIROLOGY

111,

Structural

516-537 (1981)

and Nonstructural

Proteins WADE

Department

of Pharmacology School

of Strain Colburn

GIBSON’

alzd Experimental Therapeutics, The Johns of Medicine, Baltimore, Maryland 21205 Accepted

Cytomegalovil

December

Hopkins

University,

8, 1980

Studies to investigate the involvement of cytomegalovirus (CMV, strain Colburn) proteins in the infection process have led to the following observations. First, four types of virus particles can be recovered from infected cells. These particles are distinguished on the basis of their intracellular compartmentalization, sedimentation properties in rate-velocity sucrose gradients, protein composition, and infectivity. By analogy with their apparent counterparts in herpes simplex virus (HSV)-infected cells, these particles have been designated as A-, B-, C-capsids and virions. A-capsids, composed of three protein species (i.e., 145K, 34K, and 28K daltons), have the simplest structure; vu-ions, containing at least 20 protein species ranging in molecular weight from approximately 200K to 20K, are structurally the most complex. Among the protein constituents of these virus particles, the 145K was found to be the major structural element of the capsid; a 45K and a 37K B-capsidspecific protein were shown to be closely related and, like their HSV counterparts, absent from the mature virion; and a 66K protein is suggested to serve as a matrix, interfacing the nucleocapsid with the outer envelope. Second, evidence based on peptide analyses, pulsechase radiolabeling experiments, and comparisons of intracellular viral proteins with those present in virus particles, indicates that several strain Colburn proteins undergo posttranslational modifications. And third, these studies have established that, in addition to the proteins identified as structural elements of virus particles, strain Colburn-infected cells contain at least five nonstructural proteins. Most of the structural as well as these nonstructural proteins partitioned strongly with the NP-40 nuclear fraction, but were found to enter that fraction at markedly different rates. INTRODUCTION

Cytomegalovirus (CMV) is structurally typical of the herpes group viruses. It is composed of a linear, double-stranded DNA molecule, approximately 150 x lo6 in molecular weight (Kilpatrick and Huang, 19’77; DeMarchi et al., 1978; Geelen et al., 1978), that is replicated and packaged into an icosahedral capsid within the nucleus of the infected cell, and surrounded by an envelope most probably acquired as the nucleocapsid “buds” through the nuclear membrane into the cytoplasm (Morgan et al., 1959; Kanich and Craighead, 1972; Iwasaki et al., 1973; Smith and deHarven, 1973). In contrast to herpes simplex virus (HSV) and other strongly cytocidal herpesviruses, CMV has a longer growth cycle (Rapp and Benyesh-Melnick, 1963), is gen1 To whom reprint requests should be addressed. 0042-6822/81/080516-22$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

erally considered to be more host cell type specific, and its infectivity appears to be more strongly cell associated. Although these last characteristics have slowed molecular studies of human isolates of CMV (HCMV), it is well documented that morphological and ultrastructural changes take place in the cell as a consequence of infection. Among the most dramatic of these is the appearance of large cyand intranuclear inclusions, toplasmic which recent studies suggest may be areas of virus assembly (Iwasaki et al., 1973; Smith and deHarven, 1973; Fumiko et al., 1979). With the development of better culturing techniques a number of laboratories have been successful in recovering and characterizing the mature virion (Sarov and Abady, 1975; Kim et al., 1976; Fiala et al., 1976; Stinski, 1976; Gupta et al., 1977), and have begun to identify and characterize 516

STRUCTURAL

AND

NONSTRUCTURAL

HCMV proteins in infected cells (Stinski, 1977; Gupta and Rapp, 1978). As yet, however, there is little information available concerning the functional involvement of these various intracellular inclusions, particles, and proteins during the infection process. The studies described here were intended to approach this question by developing a better understanding of the intrinsic physical and biochemical characteristics of specific CMV proteins, and investigating their intracellular associations. For this purpose, experiments were done to determine (i) when during the replication cycle specific viral proteins appear; (ii) how these proteins are compartmentalized within the infected cell; and (iii) which of them are structural components of virions and intracellular capsids. A recently isolated strain of CMV (strain Colburn) has been used in these studies since it grows comparatively fast and to high titer in human fibroblast cells. Strain Colburn was isolated from a brain biopsy of a child with clinical encephalopathy (Charamella et al., 1973); exhibits strong DNA sequence homology with simian CMV (Kilpatrick et al., 1976; Huang et al., 1978); and is antigenically cross-reactive with both simian and human CMVs (Huang et al., 1978; J. L. Waner, personal communication; Weiner and Gibson, manuscript in preparation). (A preliminary report of this work was presented at the Herpesvirus Workshop held in Cambridge, England, August 2025, 1978.) MATERIALS

AND

METHODS

Human foreskin fibroblast (HFF) cell cultures were prepared from foreskins by mascerating the tissue using scissors and allowing fibroblasts to grow out in Dulbecco’s modified Eagle’s medium (DMEM; No. 430-2100, Gibco, Grand Island, N. Y.) containing 10% fetal calf serum (Rehatuin F. S., Reheis Chemical Co., Phoenix, Ariz.) and 1% penicillinstreptomycin (Gibco No. 6005140). Cells were maintained for approximately 12 divisions in the presence of penicillin and streptomycin, and subsequently propagated Cells and virus.

PROTEINS

OF

CYTOMEGALOVIRUS

517

without antibiotics. Primary cell cultures were grown in 6-cm plastic petri dishes (Corning No. 25010, Corning Glass Works, Corning, N. Y.) containing 5 ml of medium and maintained in a 5% CO, atmosphere at 37”. Subsequent passages of these cells were grown in 32-0~ glass bottles (Brockway Glass Co., Brockway, Pa.) containing 40 ml of medium and flushed with a gas mixture of 5% CO$95% air. The cytomegalovirus strain used in these studies was obtained from Dr. Milan Fiala at the Harbor General Hospital in Los Angeles, California. The identity of this virus as strain Colburn, originally isolated and described by Charamella et al. (1973), was confirmed by DNA fragment analysis using several DNA restriction enzymes (G. Hayward, personal communication). Virus stocks were initially derived by infecting cells with a dilute suspension of virions recovered from sucrose gradients, as described below, and overlaying the infected cells with media containing 0.3% agarose. Material from a single plaque beneath the agarose overlay was then used to infect just subconfluent cell cultures. The infection was allowed to progress until strong cytopathic effect (c.p.e.> was evident. The cultures were then shaken vigorously and the resulting suspension was frozen in loto 20-ml aliquots at - 70”. Virus infectivity was measured by plaque assay (Dulbecco and Vogt, 1953) or by c.p.e. endpoint titration, both using HFF cells. Infection of cells and radiolabeling. Cell cultures were infected by incubating them with an appropriate dilution of the virus stock to give a multiplicity of infection of approximately 20. After 60 min of intermittent rocking, the inoculum was removed, replaced with maintenance medium, and the cultures returned to incubation at 37”. Biosynthetic radiolabeling was done by replacing the maintenance medium with medium containing: l/lOth the normal levels of those amino acids included in the labeling mixture, 2% fetal calf serum, and a synthetic mixture of 12 14C-amino acids (NEC 445, New England Nuclear Corp., Boston, Mass.). The amount of radioactivity and the labeling interval varied with the experiment and are detailed in the text.

WADE

‘IG. 1. CMV particles separated in rate-velocity su3e gradients. Infected cells and supernatant mem were processed and analyzed by rate-velocity imentation in sucrose gradients, all as described ler Materials and Methods. Shown here is a comite photograph of the resulting gradients that were ially overlayed with: extracellular (A), cytoplasmic and nuclear (C) preparations. Incandescent illulation from the top of the tube revealed single light;tering bands in the extracellular and cytoplasmic parations, and two bands in the nuclear preparaL As explained in the text, these respective bands s designated as “V” (virions), “C” (C-capsids), “A” “B” (A- and B-capsids).

lecovery of virus particles from infected

!s. The procedures used for recovering IV particles from virus-infected cells re essentially the same as described ewhere (Gibson and Roizman, 1972). utinely, cells were scraped from the cule vessel into the medium when extene cytopathic effect was observed (3 to 4 7s after infection). The dislodged cells re then collected by low-speed centrifu;ion (1500 g, 4”, 10 min). ntracellular virus particles were recov!d by subjecting the resulting infected 1 pellet to the following sequence of ‘atments. First, cytoplasmic membranes re dispersed by incubating the pelleted 1s at 0” for 10 min in 0.04 M phosphate ffer, pH 7.4, containing 0.15 M NaCl and ;% (v/v) Nonidet P-40 (NP-40 PB; 0.5 ‘32-0~ bottle). Following separation by v-speed centrifugation, the supernatant

(:IBSON

cytoplasmic fraction was held on ice. The nuclear pellet fraction was resuspended (200 pll32-oz bottle) in the above buffer lacking NP-40 (PB) and warmed to 25”. Deoxycholate (DOC, lo%, w/w, in H,O) was added dropwise to the nuclear suspension until the solution became viscous (generally 2 to 3 drops or 20 to 50 ~1/32-oz bottle). The viscosity was reduced by adding DNAase I (5 mg/ml H,O) to a linal concentration of 50 to 100 pug/ml. One-tenth volume of urea (5 M in H,O) was then added, followed by an amount of Brij 58 (10% w/w, in H,O) equal to the amount of DOC used above. The lysate was then cooled to 4” and clarified of large particulate material by low-speed centrifugation. The resulting cytoplasmic and nuclear preparations were subjected to a final lowspeed centrifugation and layered above 15 to 50% (w/w> continuous gradients of sucrose in PB. Preparative gradients were formed in 38-ml cellulose nitrate tubes, overlayed with 0.3 to 1.0 ml of lysate, and spun for 15 min at 20,000 rpm and 4” in a Sorvall TV850 vertical rotor or 60 min at 20,000 rpm in a Sorvall AH 627 swinging bucket rotor. Analytical gradients were formed in 17-ml cellulose nitrate tubes, overlayed with 100 to 300 ~1 of lysate, and spun for 60 min at 20,000 rpm and 4” in a Sorvall AH 627 rotor accelerated at 45 psi using the slow start mode of a Sorvall OTD 50 ultracentrifuge. Particle bands were visualized by light scattering and collected by aspiration from the side of the tube using a 23-gauge hypodermic needle. The material was then either diluted 2: 1 (PB: sample) for rebanding, or diluted 5 : 1 (PB : sample), overlayed with PB, and spun for 2 hr at 25,000 rpm and 4” in an AH 627 or Spinco SW 27.1 rotor. Particles concentrated by “pelleting” in this manner were immediately solubilized by heating at 100” for 2 min in a solution containing 2% sodium dodecyl sulfate (SDS), 10% 2-mercaptoethanol, 10% glycerol, and 50 mM Tris, pH 7.0, and stored at - 70” until needed. Extracellular virus particles were recovered from the supernatant medium. After infected cells had been removed by lowspeed centrifugation, the medium was further clarified by high-speed centrifugation

STRUCTURAL

AND

NONSTRUCTURAL

PROTEINS

OF CYTOMEGALOVIRUS

519

c

FIG. 2. Electron micrographs of CMV particles recovered from sucrose gradients. A-, B-, and Ccapsids and virions, respectively, were adsorbed to grids from aliquots of the aspirated gradient bands shown in Fig. 1. Shown here are electron micrographs of A-capsids (A), B-capsids (B), C-capsids (C), and virions (D), negatively stained with 2% sodium phosphotungstate, pH 7.0, containing 0.01% bovine serum albumin. For purposes of reference, A-capsids were estimated to have a diameter of 113 nm. Note apparent internal structures in A-capsids and irregular “coating” on C-capsids. An internal structure, similar to those seen in A-capsids, is evident in the stain-penetrated top member of the virion triplet shown in Panel D.

(10,000 r-pm, 4”, 10 min) in a Sorvall SS-34 rotor. Except where indicated, particles were collected from the supernatant by centrifugation for 60 min at 20,000 rpm and 4” in either a Sorvall AH 627 or Spinco SW 27 rotor. After decanting the supernatant, the resulting pellet of virus particles was resuspended (200 @pellet from one 32-0~

bottle) in either PB or maintenance medium, Dounce homogenized x 4 strokes with a tight-fitting (A) piston, and cleared of large particulate material by low-speed centrifugation. The preparation was then layered above 15 to 50% sucrose gradients and subjected to centrifugation and band collection as described above.

520

WADE

GIBSON

Particle

r

1

B

A

C

Vir.

-205 -163

J- 48,47,46 -42 -40 -36

27

a

b

c

d

FIG. 3. Protein constitutuents of CMV particles. Radiolabeled A-, B-, C-capsids, and virions were solubilized and subjected to electrophoresis in a 14% polyacrylamide slab gel. Shown here is a photograph of a fluorogram prepared from the resulting gel; channels contain the proteins of Bcapsids (a), A-capsids (b), C-capsids (c), and virions (d). An appropriate amount of each sample

STRUCTURAL

Polyacrylamide

AND

gel

NONSTRUCTURAL

electrophoresis.

Polyacrylamide gel electrophoresis was done essentially as described by Laemmli (1970). Details of the system used in this study are as follow: (i) resolving gels were 14% acrylamide, crosslinked with diallyltartardiamide (DATD) in a ratio of 1.09 : 28 (w/w, DATD : acrylamide; Anker, 1970), (ii) stacking gels were 5% acrylamide, crosslinked with DATD in a ratio of 1: 5.7 (w/w, DATD:acrylamide), as suggested by Baumann and Chrambach (1976), (iii) resolving gels were 14 cm long x 14 cm wide x 1.0 mm thick, and (iv) electrophoresis was carried out at a regulated current of 30 mA/gel at room temperature. Following electrophoresis the gels were stained (Fairbanks et al., 1971) and processed for fluorography (Bonner and Lasky, 1974; Lasky and Mills, 1975). Densitometric scans of the resulting fluorograms were made using an EC 910 transmission densitometer (E-C Apparatus Corporation, St. Petersburg, Fla. > equipped with a 540-nm interference filter. Peptide comparisons. Two-dimensional tryptic peptide comparisons were done as previously described (Gibson, 1974). Reagents. Detergents used were from the following sources: sodium dodecyl sulfate (SDS), Bio-Rad Laboratories, Richmond, California; Nonidet P-40 (NP-40) and BRIJ 58, BDH Chemicals, Ltd., Poole, England; deoxycholate (DOC), SchwarzMann, Orangeburg, New York. Coomassie brilliant blue R-250 stain (CBB) was from Pierce Chemical Company, Rockford, Illinois, and RNAase-free deoxyribonuclease I (DNAase) was from Worthington Biochemical Corporation, Freehold, New Jersey. Most solutions were passed through 0.45-pm filters (Millipore Corp., Bedford, Mass.) prior to use. RESULTS

Recovery

of Particles from Infected

Cells

Human foreskin fibroblast (HFF) cell cultures infected with cytomegalovirus

PROTEINS

OF

CYTOMEGALOVIRUS

521

(CMV) were processed, as described under Materials and Methods, to recover intracellular particles from the nucleus and cytoplasm, and extracellular particles from the maintenance medium. As shown in Fig. 1, rate-velocity sedimentation of these preparations through sucrose gradients resolved two predominant light-scattering bands in the nuclear fraction, one in the cytoplasmic fraction, and one in the extracellular fraction. On the basis of (i) their relative sedimentation properties, (ii) the fraction of the infected cells from which they were recovered, and (iii) their morphology and molecular composition (discussed below), particles in these bands have been designated provisionally as A-, B-, C-capsids and virions following the nomenclature for their apparent herpes simplex virus (HSV) counterparts (Gibson and Roizman, 1972). A-, B-, and C-capsids sedimented as tight, well-separated bands, while extracellular virions sedimented as a somewhat broader band. Inspection of material from each band by use of an electron microscope (Fig. 2) showed that: A-capsids have an estimated diameter of 113 nm, and appeared only sparingly penetrated by stain (Panel A). B-capsids were estimated to be 117 nm in diameter, and excluded stain (Panel B). Ccapsids, estimated to be 139 nm in diameter, appeared freely permeable to stain (Panel C). And virions, having a uniform diameter estimated to be 159 nm, largely excluded stain and had the general appearance of enveloped herpes virus particles (Panel D). With the exception of virions, the estimated size of these particles correlated well with their relative sedimentation position in sucrose gradients. Since estimates of both size and structural complexity (see below) indicate that virions represent the largest of these particles, their sedimentation position suggests that they are at, or approaching, their equilibrium density in these gradients. Apparent internal structures (Panel A) were noted fre-

was applied to give approximately the same intensity 145K band (major capsid protein). Numbers in the left- and right-hand margins indicate the molecular weights (X10m3) of capsid and extracapsid proteins, respectively. Densitometric scans prepared from this gel are shown in Fig. 4 to better demonstrate the underexposed and overexposed bands.

522

WADE

GIBSON

FIG. 4. Densitometric scans prepared from the fluorogram shown in Fig. 3. The ordinate in each panel represents increasing (bottom to top) optical absorbance and the abscissa reflects increasing (left to right) distance from the top of the gel. Shown here is a photograph of a collage prepared from Xerox copies of the actual scans of A-capsid (A), C-capsid (B), B-capsid (C), and virion (D) proteins. The numbers indicate the proteins’ molecular weight (X 10m3).Proteins numbered in the upper panels are not renumbered in the lower panels.

quently in A- and B-capsid preparations. Similar structures seen in negatively stained HSV capsids were shown, by examination of thin-sectioned preparations, to be physically distinguishable substructures of the capsid (Gibson and Roizman, 1972). Of these four particle types, only virions were infectious. Protein

Constituents

of CMV Particles

Disruption of these CMV particles followed by electrophoretic separation of their protein constituents in polyacrylamide gels, as described under Materials and Methods, established that each has a characteristic protein composition (Fig. 3). A-capsids (Channel b) are composed of

three predominant protein species having estimated molecular weights of 145K, 34K, and 28K, respectively. B-capsids (Channel a) contain all three A-capsid proteins and two additional species-a major component of approximately 37K daltons, and a minor component of approximately 45K daltons. Small amounts of a protein migrating at the position of the 66K C-capsid band (Channel d), and two or three other proteins migrating just slower than the 37K band (see trailing small shoulder at base of 37K peak, Figs. 4C, 9A and C), are routinely detected in B-capsid preparations. The 37K proximal bands are always present in about the same relative amounts; however, variability from preparation to

STRUCTURAL

A

AND

NONSTRUCTURAL

B

PROTEINS

523

OF CYTOMEGALOVIRUS

shared by the apparent counterpart protein of HSV B-capsids (i.e., 39K protein, Gibson and Roizman, 1974). This shared property of the 45K and 37K CMV proteins suggested that they may be related. To examine this possibility, the two proteins were radiolabeled in vitro with lz51 and processed for tryptic peptide comparison. Two-dimensional separations of the resultFluores.

CBB

45

-37 -34 -28

37 FIG. 5. A- and B- capsid proteins stained with Coomassie brilliant blue stain. A- and B- capsids were prepared as described in the text, solubilized, and subjected to electrophoresis in a 14% polyacrylamide gel. Following electrophoresis the gel was stained, destained, and dried onto Whatman 3MM filter paper as usual. Shown here is a photograph prepared from the dried down gel, indicating the estimated molecular weight (X 1O-3) of each capsid protein. The 145K bands contain approximately 5 kg of protein.

preparation in the amount of 66K protein present suggests that it is not an integral capsid constituent. A gel showing the electrophoretically separated A- and B-capsid proteins stained with Coomassie brilliant blue (CBB; Fig. 5), demonstrates that these preparations do not contain significant amounts of additional nonradiolabeled proteins. Intense illumination of such a stained gel revealed that the 45K and 37K B-capsid proteins exhibit a pink fluorescence (Fig. 6)-a characteristic property

34

a

b

FIG. 6. B-capsid 45K and 37K proteins fluoresce. Bcapsid proteins were separated in a 14% polyacrylamide gel and stained with Coomassie brilliant blue stain. When illuminated at an oblique incidence angle with incandescent light, and above a dark background, the 45K and 3’7K bands emitted a pink fluorescence. Neither the 34K band, shown here for comparison, nor the other capsid proteins exhibited this property. Shown here are the same CBB-stained 45K, 37K, and 34K bands illuminated from above using incandescent light against a dark background (A) and from below using transmitted fluorescent light (B). The 37K band contained approximately 5 fig of protein.

524

WADE

GIBSON

37k

45k

FIG. 7. Peptide comparison of 45K and 3’7K B-capsid proteins. B-capsid proteins were radiolabeled in vitro using intact B-capsids and chloramine-T-catalyzed iodination, and separated in a 14% polyacrylamide gel. The 45K and 37K bands were excised from the resulting gel and processed for peptide analysis. Shown here are two-dimensional separations of tryptic hydrolysates prepared from the two proteins. The origin appears in the extreme lower left-hand corner of each panel; electrophoresis was from left to right, followed by chromatography from bottom to top.

ing preparations yielded similar peptide distributions (Fig. 7), indicating that the 45K and 3’7K proteins must share at least some sequences in common. Cytoplasmic C-capsids contain all three A-capsid proteins (i.e., 145K, 34K, and 28K) but lack both B-capsid-specific species (i.e., 37K and 45K). C-capsids also contain at least 12 protein species absent from intranuclear capsids. Among these, the 205K protein stands out as the largest of the CMV structural proteins, and the 66K doublet as the predominant C-capsid band. The protein composition of extracellular virions differs from that of intracellular capsids in at least two ways. First, virions appear to be missing the 28K protein found in A-, B-, and C-capsids. And second, virions contain at least 12 additional protein species not detected, as such, in intracellular capsids (i.e., 163K, 97K, ‘78K, 65K, 59K, 46K, 40K, 39K, 36K, 35K, 27K, and 23K). These results, and estimates of the relative molar amounts of each protein based on the densi-

tometric scans shown in Fig. 4, are summarized in Table 1. Stability

of Virus Particles

A major concern in evaluating the role of these virus particles in the life cycle of CMV was the possibility that they are generated as artifacts of the isolation procedure used to obtain them. Two experiments were done to examine this possibility. The frrst of these was intended to determine whether A-, B-, C-capsids, and virions, isolated as described under Materials and Methods, are stable to subsequent detergent -enzyme treatment. This was done as follows. All four particle types were recovered from two 32-0~ cultures of CMV-infected cells, combined with 2 vol of 0.15 M NaCl, 0.04 M phosphate buffer, pH 7.4 (PB), and subjected to the sequential addition of DOC, DNAase, urea, and Brij 58 to final concentrations of 0.5%, 50 pg/ml, 0.5 M, and 0.5%. respectively. The

STRUCTURAL

AND

NONSTRUCTURAL

PROTEINS TABLE

RELATIVE

AMOUNTS

OF SPECIFIC

A-Capsid

205 163 145 127 119 97 90 79 78 66 65 59 48 47 46 45 42 40 39 37 36 35 34 28 27 23

Mass”

IN FOUR

6.69

46

Mass”

0.06

1.24

0.67 0.20

20 7

0.68 0.21

OF CMV

PARTICLES

C-Capsid

Abundanceb

6.69

SPECIES

type

B-Capsid

Abundance*

525

CYTOMEGALOVIRUS

1

PROTEINS

Particle

Protein size (MW x 1O-3)

OF

46

Mass”

Virion

Abundanceb

0.26

1

6.69 0.58 0.58

46 5 5

0.90 0.56

10 7

6.65

100

Mass”

I

0.51 3.09 6.69

2 19 46

12.82c

104*

0.57 0.55

6 6

1.94 11.49 0.85 1.86

25 174 13 32

I

1

Abundance*

0.2T

5*

0.15 0.07 0.04

4 2 1

0.22 1.05 0.66

6 30 19

2.51

93

34

20 8

0.67 0.15

20 5

(L Relative values calculated from measurements made from the densitometric scans shown in Fig. 4 and normalized to the 145K, major capsid protein. b Calculated by: dividing the relative mass (see footnote a) by the molecular weight x lo-‘. c Indicated bands not independently measurable; value shown represents combined peak area. * Calculated by dividing combined mass value (see footnote c) by the average MW of the combined bands.

“treated” and “nontreated” preparations were then layered above 20 to 50% linear gradients of sucrose (w/w in PB) and subjected to centrifugation as described. Visual inspection of the resulting lightscattering bands showed that: (i) such treatment of A-, B-, and C-capsids did not noticably change their respective sedimentation characteristics. (ii) No additional bands were generated as a consequence of treating the intracellular capsid forms. And, (iii) the virion band was eliminated following this treatment, or by exposure to

the comparatively milder detergent, NP-40 (Fig. 8). A second experiment was next done to examine the possibility that the A-, B-, and recovered from detergentC-capsids treated infected cells may represent stripped forms of more complex particles. The approach used was to compare the protein compositions of intracellular particles recovered in the absence of detergents with those of particles recovered in the Alternative presence of detergents. methods of cell disruption were investi-

526

WADE GIBSON

FIG. 8. CMV vu-ions are disrupted by NP-40. CMV virions were prepared as described under Materials and Methods and diluted 2: 1 with PB (PB: sample). Half of the preparation was held at 0”; the other half was made 0.5% in NP-40 and held at 0” for 10 min. Both samples were then layered above 20 to 50% sucrose gradients prepared and spun as described under Materials and Methods. The resulting gradients were collected from the top using a Model 185 density gradient fractionator and monitored at 280 nm using a Model UA-5 absorbance monitor, both from ISCO, Lincoln, NB. Shown here is a photograph of a collage prepared from Xerox copies of the actual gradient recordings of the NP-40treated preparation (A) and the nontreated preparation (B). Sedimentation was from left (top) to right (bottom). The strong absorbance at the top of the gradient containing the detergent-treated preparation is due to the presence of NP-40 which absorbs at this wavelength.

gated for this purpose, and the following procedures were adopted. Intracellular virions and C-capsids were obtained by swelling the pelleted, infected cells in hypotonic buffer (0.04 M phosphate buffer, 1 mM 2-mercaptoethanol, pH 7.4) for 10 min at 0“; rupturing the swollen cells by Dounce homogenization; clearing the lysate of large particulate material by low-speed centrifugation; and subjecting the resulting supernatant to separation in 15 to 50% sucrose gradients as usual. Intranuclear B-capsids were obtained from the Dounce pellet fraction of infected cells, described above, by resuspending it in PB; lysing it by three cycles of freezing and thawing, or three 5-set sonic pulses using a Branson “Cell Disruptor 185” at a setting of 4; and subjecting the clarified (low-speed cen-

trifugation) lysate to separation in 15 to 50% sucrose gradients, as described above. Results of this second experiment can be summarized as follows. (i) C-capsids were released only poorly by Dounce homogenization of infected cells. They could be recovered readily, however, from the Dounce pellet fraction following its treatment with NP-40. (ii) Similarly, A-capsids were not recovered in good yield by physical lysis of infected cell nuclei. They were recovered readily from the resulting nuclear pellet fraction, however, following its subsequent treatment with DOC, urea, DNAase, and Brij 58. (iii) Like extracellular virions, intracellular CMV virions were disrupted by NP-40 into material that remained at the top of sucrose gradients. (iv) B-capsids recovered using detergents contained all of

STRUCTURAL

AND NONSTRUCTURAL

PROTEINS

527

OF CYTOMEGALOVIRUS

B C4aDsids

37 -I*-22 1..-34

JL205 D

I45

-I2I -s 3-L LAA:.

J!

-65 -24 -28

FIG. 9. Comparison of B- and C-capsids recovered in the presence or absence of detergents. Radiolabeled B- and C-capsids were recovered as described in the text; solubilized and subjected to electrophoresis in a 14% polyacrylamide slab gel. A fluorogram of the resulting gel was used to prepare these densitometric scans showing the protein consitiuents of B-capsids recovered in the absence (A) or or presence (C) of detergents, and C-capsids recovered in the absence (B) or presence (D) of detergents. This figure was prepared as described in the legend to Fig. 4.

the proteins present in those recovered in the absence of detergents (Fig. 9, compare Panels A and C). However, densitometric quantitation revealed that detergenttreated B-capsids contained only 40 to 50% as much of the 45K and 3’7K protein species as nontreated B-capsids. And, (v) two differences were noted between the protein compositions of detergent-treated and nontreated C-capsids. First, nontreated C-capsids were found to contain eight-fold more of a protein having an estimated molecular weight of 48K (seen in Panel B between the 66K and 34K peaks), and approximately 20% more of the 205K, 127K, and 90K bands. And second, the amount of 119K,

79K and 66K protein present in detergentrecovered C-capsids was 50, 25, and 40% more, respectively, than in the Douncereleased C-capsids. This unexpected observation may reflect a detergent-sensitive membrane interaction of the C-capsid through these proteins. Identijication Cells

of Viral

Proteins

in Infected

Two experiments were done to investigate protein metabolism in CMV-infected cells. In the fist, one culture each of CMVinfected and mock-infected cells was grown in the presence of 14C-amino acids (2

528

WADE

@Z/ml) from 48 to 72 hr postinfection (p.i.). The cells were then separated into nuclear and cytoplasmic fractions using NP-40, solubilized, and then examined by polyacrylamide gel electrophoresis. Figure 10 shows a photograph of a fluorogram prepared from the resulting gel, and contains the following information. First, with the possible exception of the 119K structural protein, all A-, B-, and C-capsid proteins were present in the nuclear fraction of CMV-infected cells (compare Channels c, d, and e with b). Second, by this time after infection only a few radiolabeled host cell proteins were detected in the nuclear fraction of infected cells (compare Channels a and b). Third, the nuclear fraction contains at least five proteins (i.e., 94K, 51K, 48K, 42K, and 32K) that are not present, as such, in CMV particles (compare Channels c to g, with b). Fourth, there are at least five proteins present in mature enveloped virus particles (i.e., 163K, 119K, 78K, 65K, and 59K, Channel f) that were either absent or present in greatly reduced amounts in the nuclear (Channel b) and cytoplasmic (Channel h) fraction of infected cells. And finally, the 27K virion protein (darkest low-molecularweight band in Channel f) was found in greater relative amount in the cytoplasmic fraction than in the nuclear fraction (compare Channels h and b). Some of these points are better demonstrated by the densitometric scans of gel Channels a, b, h, and i that are shown in Fig. 11, Panels C, A, B, and D, respectively. The second experiment to examine protein metabolism in CMV-infected cells was intended to establish when CMV proteins can first be detected, whether some proteins appear earlier than others, and whether post-translational processing of CMV proteins occurs. The experiment was done as follows. Sets of infected and mockinfected cells were labeled using 14C-amino acids (2 pCi/ml) for 20-min intervals (“pulse”) beginning 18 hr p.i. After radiolabeling, one culture each of infected and mock-infected cells was harvested; separated into nuclear and cytoplasmic fractions using NP-40; solubilized and stored at - 70” for subsequent analysis. A second set of infected and mock-infected cultures, ra-

GIBSON

diolabeled during the same 20-min interval, was incubated in the absence of radiolabel (“chase”) for an additional 2-hr period at 37”, and then similarly processed for analysis by polyacrylamide gel electrophoresis. Additional samples were prepared in the same way at 40 and 63 hr after infection; the 63-hr time point included samples “chased” for 2, 8, and 21 hr. Results of this experiment are shown in Fig. 12, and can be summarized as follows. (i) Synthesis of the 94K and 51K nonstructural proteins was detected by 18 hr p.i.-prior to that of other viral proteins. The relative amount of 94K was maximal at the earliest time point and decreased at later times; it was distributed approximately equally between the cytoplasmic and nuclear fractions. Synthesis of the 51K nuclear protein, although detectable by 18 hr p.i., was not maximal until the 40-hr interval, and then decreased by nearly 50% during the 63-hr labeling period. Evidence that these proteins represent the strain Colburn immedate-early protein (94K) and “quasi-late,” DNA-binding nuclear protein (51K) will be presented elsewhere (Jeang and Gibson, 1980; Gibson, Murphy, and Roby, 1981). (ii) A group of infected cell proteins, including most notably the 145K, 79K, and 37K structural proteins, were detected in the nuclear fraction only after a “chase” interval. Appearance of the 129K, 66K doublet, and 51K proteins in that fraction was rapid, by comparison. Measurements made from densitometric scans of the fluorogram shown in Fig. 12, indicate that at the end of the 63-hr “pulse” time point, essentially all of the newly synthesized 145K protein was in the cytoplasmic fraction; the 129K was distributed about equally between the cytoplasmic and nuclear fractions; and greater than 90% of the 66K doublet and 51K protein was in the nuclear fraction. The 79K and 37K bands were not seen in either the cytoplasm or the nucleus following the 20-min “pulse,” suggesting that they may be derived, during the “chase” interval, from other proteins. In this connection, we are investigating the possibility that a 76K and a 40K protein found predominantly in the “pulse” sample, may be precursors of the 79K and 37K proteins,

STRUCTURAL

AND NONSTRUCTURAL

Nut.

‘u

7.d

t

c

PROTEINS

Particle

OF CYTOMEGALOVIRUS

4

529

cyto.

B A C Vir. D.B.‘I

d

e

f

g

h

i

FIG. 10. Identification of CMV-specific structural and nonstructural proteins in infected cells. Cytoplasmic and nuclear fractions of radiolabeled CMV-infected and mock-infected cells were prepared as described in the text and subjected to electrophoresis in a 14% polyacrylamide slab gel, along with a sample of each species of CMV particle so far identified. Shown here is a photograph of a fluorogram prepared from the resulting gel; the sample order is: nuclear fraction of mock-infected (a) and CMV-infected (b) cells, B-capsids (c), A-capsids (d), C-capsids (e), virions (f), “dense bodies” (g), and the cytoplaamic fraction of CMV-infected (h) and mock-infected (i) cells. Channels “c” to “f’ are from the same fluorogram as shown in Fig. 3, channels “a” to “d,” respectively. Infected-cell-specific proteins without counterparts among the five CMV particles are indicated in the leff-hand margin; CMV structural proteins so far not detected in infected cells are indicated in the right-hand margin. The numbers shown indicate the estimated molecular weight (x 10m3) of the corresponding protein.

respectively. Following the 2-hr “chase” period, 80% of the 145K protein was still in the NP-40 cytoplasm, while essentially all of the 66K, 51K, and 37K was in the nuclear fraction. Not until the 8-hr chase period had a substantial amount (85%) of

the pulse-labeled 145K protein moved into the nuclear fraction. (iii) At least three bands decreased in intensity during the “chase” intervals. The largest of these (i.e., 129K), and a band (76K) seen just below the position of the 79K protein, were de-

530

WADE GIBSON

tected in both the cytoplasmic and nuclear “pulse” fractions; the smallest of these proteins (i.e., approximately 20K) was present only in the cytoplasmic fractions. And (iv), the 66K doublet bands became progressively broader and more diffuse during the “chase” periods. This effect was especially dramatic for the upper band which doubled in thickness (i.e., leading to trailing edge) by the end of the 21-hr “chase” period. DISCUSSION

Herpesviruses are the only viruses so far identified that mature by budding through

the nuclear membrane. This feature of their life cycle affords a simple and efficient means of distinguishing between immature forms (e.g., intranuclear capsids) and mature forms (e.g., cytoplasmic and extracellular virions) of these viruses. In addition, since the viral DNA is replicated, transcribed, and packaged in the nucleus, attendant regulatory functions are also predicted to be located in that fraction, while virus products influencing protein synthesis and membrane structure would be expected to be located in the cytoplasm. Conthat enable sequently, techniques separation of infected cells into nuclear and

B Cytoplasm

FIG. 11. Desitometrie scans prepared from the fluorogram shown in Fig. 10. The ordinate in each panel represents increasing (bottom to top) optical absorbance and the abscissa reflects increasing (left to right) distance from the top of the gel. Shown here is a photograph of a collage prepared from Xerox scans of the nuclear fractions of infected (A) and mock-infected (C) cells, and the cytoplaamic fractions of infected (B) and mock-infected (D) cells. These traces correspond, respectively, with channels h, a, h, and i shown in Fig. 10. The numbers indicate the proteins’ molecular weight (x 107.

STRUCTURAL

AND NONSTRUCTURAL N.

PROTEINS

Cvto.Fractions Cyto. Fractic

I

‘pCpCP~cc”PCPePcct ‘P c P c P c c c”P

c

Nut. Fractions

I I

hf.

Mock hf.

P ----c P c mm--

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hf.

Mock hf.

‘P c P c P c c c’ ‘P c ‘PCPCPccc”PcPCPCCC’

c t

531

OF CYTOMEGALOVIRUS

P c P c c c’ - -- .?. -

i.a

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i

II - - ” 1” L _. %I

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1 b0 1 c3 1 1 2

2

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-

Ir

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I 1

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2 8 21

2

1

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FIG. 12. Protein metabolism in CMV-infected cells. CMV-infected and mock-infected cells were radiolabeled; separated into cytoplasmic and nuclear fractions; and solubilized for gel electrophoresis as described in the text and under Materials and Methods. Channels containing samples from the 26min “pulse” radiolabeling intervals (i.e., 18, 40, and 63 hr p.i.) are indicated at the top of this figure by the letter “I”‘; channels containing samples from the “chase” periods (i.e., 2, 8, or 21 hr in maintenance medium) are indicated by the letter “C.” The time after infection of the three 26min radiolabeling periods is indicated immediately beneath the fluorogram (“Hr. P.i. Pulsed:“), and the length of the corresponding “chase” periods (“Hrs. Chased:“), is indicated on the bottom line of this figure. The numbers in the right-hand margin indicate the proteins’ molecular weight (x 10-9; also shown for reference is the position of actin. The three lines in the space separating the cytoplasmic from the nuclear fractions indicate the respective positions of the 145K, 66K, and 20K protein bands.

cytoplasmic fractions should aid in identifying both intermediates in the herpesvirus assembly pathway, as well as nonstructural proteins that could be candidates for specific enzymatic functions required by the virus. Results of the studies reported here demonstrate the utility of such an approach applied to cytomegalovirus (CMV, strain Colburn)-infected cells, and provide information concerning: (i) the protein composi-

tion of CMV virions and intracellular capsid forms; (ii) the identification of several “nonstructural” proteins in CMV-infected cells; and (iii) the intracellular distribution and metabolism of CMV proteins in human foreskin fibroblast cells. Structural

Proteins

of CMV

Four types of virus particles have been recovered from CMV-infected human fore-

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skin fibroblast cells. Two of these, designated as A- and B-capsids, are confined to the nuclear fraction; a third, designated as Ccapsids, is present in the cytoplasmic fraction; and the fourth, designated as virions, can be recovered either from cells by Dounce homogenization or from the extracellular medium. In addition to their characteristic intracellular distribution, these particles were distinguished on the basis of their sedimentation behavior in rate-velocity sucrose gradients (Fig. l), their appearance in the electron microsccjpe following negative staining (Fig. 2), and their respective protein compositions (Figs. 3 and 4). Evidence obtained through a series of experiments to investigate the stability of these virus particles indicates that none is a simple degradation product of another. Only virions were found to be infectious. Results of these experiments have provided evidence for the architectural involvement of specific CMV proteins in the virus structure and revealed close parallels at the molecular level in the assembly pathways of CMV and HSV. It was demonstrated that the simplest CMV capsid (i.e., A-capsid) is comprised of the 145K, 34K, and 28K proteins, and that each of the intracellular capsid forms contain these same protein species in a molar ratio of approximately 7 : 3 : 1, respectively. The 145K protein, constituting about 90% of the A-capsid protein mass, is the major structural element of the capsid. The 28K protein, although the smallest and least abundant of the capsid proteins, appears to play an important role in maintaining the structural integrity of the particle since the virion nucleocapsid, which does not contain an intact 28K protein, is disrupted by relatively mild detergents that were without detected effect of A-, B-, and C-capsids (Fig. 8). Aside from small differences in the sizes of their respective protein constituents and the presence in HSV capsids of one additional protein (i.e., 53K), CMV and HSV A-capsids are similar in structure and composition. A 37K protein that distinguishes B-capsids from A-capsids (Figs. 3-Q is the CMV counterpart of the 39K HSV B-capsid

GIBSON

protein that has been suggested to be involved in DNA packaging and/or nucleocapsid envelopment (i.e., VP22a, Gibson and Roizman, 1972, 1974). Like its HSV counterpart (Zweig et al., 1979), this CMV protein is closely related to a higher molecular weight protein (i.e., 45K) that is also contained in B-capsids, but in much lower molar amounts (Fig. 7, Table 1). Although the topological location of these proteins has not been established, results of surface radiolabeling experiments using intact particles and bead-bound lactoperoxidase show that the 45K and 37K proteins are preferentially iodinated, suggesting that they are exposed at the surface of the capsid (unpublished results). The observation that approximately 50% of the 45K and 37K protein bound to B-capsids was released during their recovery in the presence of detergents and DNAase (Fig. 9) is also compatable with a surface location, and suggests that these proteins may be bound to the capsid through cooperative (removed by detergents/DNAase) as well as direct (stable to detergents/DNAase) interactions. Parenthetically, the presence of a protein coating with exposed hydrophobic groups on the nucleocapsid surface (e.g., 37K), is mechanistically attractive since it could be expected to facilitate interaction of the nucleocapsid with more hydrophobic membrane elements, thereby promoting nucleocapsid envelopment. The fluorescence exhibited by the CBB-stained 37K protein is still an unexplained phenomenon, but provides a convenient means of identifying it, and may be a general property of this class of herpesvirus proteins (Fig. 6; Gibson and Roizman, 1974). Notable among the C-capsid.proteins absent from intranuclear A- and B-capsids (i.e., 205K, 127K, 119K, 90K, 79K, 66K), are the 205K and 66K bands. The 205K protein is the largest Colburn structural protein and the apparent analog of the 275K HSV C-capsid and virion protein (Spear and Roizman, 1971; Gibson and Roizman, 1972, 1973). The 66K protein constitutes approximately 70% of the non-A-capsid protein mass; is also a predominant constituent of virions; and is frequently detected

STRUCTURAL

AND

NONSTRUCTURAL

in small amounts in B-capsid preparations (Fig. 3). Further, as shown in Figs. 10 (Channel b) and 11 (Panel A), an electrophoretically indistinguishable protein is present in large amounts in the NP-40 nuclear fraction of infected cells. Recent studies (Weiner and Gibson, manuscript is preparation) show that this protein is phosphorylated and shares a number of other properties in common with the cellular nuclear matrix or lamina proteins (Berezney and Coffey, 1974; Keller and Riley, 1976; Gerace et al., 1978). Its apparent topological location, between the nucleocapsid and the putative outer envelope proteins discussed below, is consistent with that of other virus matrix proteins (reviewed by Lenard and Compans, 1974), and suggestive that the 66K protein may function to interface the viral nucleocapsid and envelope constituents. Virions contain at least seven proteins (i.e., 163K, 97K, 78K, 65K, 59K, 35K, 27K) that were not found in A-, B-, or C-capsids and, therefore, are considered likely to be constituents of the outer envelope structure (Fig. 3, Channel d). The 119-127K doublet band is also thought to be an envelope component since it is present in lo-fold higher amounts in virions than in C-capsids (Fig. 4, Table l), and it is selectively radiolabeled following bead-bound lactoperoxidase-catalyzed iodination of the intact particle (unpublished observations). The presence in CMV virions of several proteins that are more abundant in the particle than within the infected cell (Fig. 10) suggests that these proteins may be derived from others by slow, postassembly modifications. The protein compostition of purified CMV virions has been the subject of several recent studies (Sarov and Abady, 1975; Kim et al., 1976; Fiala et al., 1976; Stinski, 1976; Gupta et al., 1977). Although the virus strain, host cell type, and method of purification used in most of these studies differed, and the proteins of strain Colburn are somewhat smaller than their AD169, Davis and Towne counterparts (Gibson, manuscript in preparation), the results are in general agreement. In particular, all show approximately 20 protein species

PROTEINS

OF CYTOMEGALOVIRUS

533

ranging in molecular weight from about 20K to 2OOK, and demonstrate a predominant 60- to 7OK-dalton band. Also in agreement with results of earlier studies (Sarov and Abady, 1975; Fiala et al., 1976; Kim et al., 1976), the predominant protein constituent of CMV “dense bodies” was found to comigrate with the 60-70K band (Fig. 10, Channel g). Concerning the involvement of these CMV particles in the assembly pathway, the following observation can be made. First, intracellular capsid forms similar to the CMV particles described here are commonly observed, using an electron microscope, in cells infected by herpesgroup viruses (for reviews see Heine and Dalton, 1972; Watson, 1973; Roizman and Furlong, 1975; O’Callaghan and Randall, 1976). In addition, capsids having similar physical and compositional characteristics have been recovered from cells infected by other herpesgroup viruses, including pseudorabies (Ben-Porat et al., 1970; Ladin et al., 1980), HSV (Gibson and Roizman, 1972, 1974; Powell and Watson, 1975; Zweig et al., 1979; Cohen et aZ., 1980), EHV-1 (Perdue et al., 1975; Kemp et al., 1974), EHV-3 (Allen and Bryans, 1976), and HZV and EBV (our unpublished observations). Although the specific involvement of these intracellular particles in the herpesvirus assembly pathway is not yet established, a recent study (Ladin et al., 1980) using temperature-sensitive mutants of pseudorabies virus provides strong evidence that empty capsids (A-capsid counterpart?) are percursoral forms of DNA-containing particles (B- or C-capsid and virion counterparts?). Second, absence of the 37K B-capsid protein from virions indicates that if B-capsids are precursors to virions, then this protein must be modified or eliminated from the particle -presumably during nucleocapsid envelopment. In this regard, CMV virions like those of HSV, contain a diffuse protein band that migrates slightly faster than the major B-capsid-specific protein (i.e., 35K in CMV, Figs. 3 and 4; in HSV, protein 22, Fig. 4, Gibson and Roizman, 1972; protein 12, Fig. 5, Powell and Watson, 1975). Based on a number of shared biochemical

534

WADE

characteristics, it has been proposed that this diffuse protein represents a modified from the 39K B-capsid protein in HSV (Gibson and Roizman, 1972, 1974). And finally, it has been suggested that cytoplasmic HSV C-capsids represent stripped forms of intracellular vu-ions. This suggestion is based on the observation that nonionic detergents strip the envelope from HSV virions (Olshevsky and Becker, 1970; Spear and Roizman, 1971; Gibson and Roizman, 1972) producing a particle that appears to be the same as that recovered from the cytoplasmic fraction of detergentdisrupted cells. Based on the following lines of evidence presented in this report, however, it seems unlikely that CMV capsids are analogous detergent-stripped remains of virions: (i) C-capsids can be recovered from infected cells without using detergents (Fig. 9B); (ii) C-capsids contain at least one protein (i.e., 28K) absent from vu-ions (Figs. 3 and 4); and, (iii) detergent treatment of CMV virions results in their structural disintegration (Fig. 8). The possibility that these capsids represent a distinct class of immature particles is consistent with the observation of “coated” nucleocapsids in the perinuclear region of the cytoplasm (Smith and DeHarven, 1973). In addition, based on the fact that the 66K protein constitutes nearly 70% of the non-A-capsid protein mass of these particles, it can be speculated that this C-capsid coating may be composed largely of the 66K protein. Intracellular

Metabolism

of CMV Proteins

Most of the structural proteins described above are readily identified in lysates prepared from infected cells (Fig. 10). The 163K, 119K, 78K, 65K, and 59K virion proteins are exceptions, and were difficult to detect in such lysates. It is suggested that these proteins undergo slow postassembly modifications that account for this discrepancy. At the other extreme, the 45K protein is present in much greater amount within the cell than in virus particles, compatable with the possibility that it represents a nonstructural precursor of the related (Fig. 7) 37K B-capsid protein. In

GIBSON

addition to these structural proteins, cells infected with strain Colburn CMV were found to contain at least five protein species (i.e., 94K, 51K, 48K, 42K, 32K) that were not constituents of any of the virus particles examined. Of these, the 94K protein has been established as a Colburn immediate-early protein (Jeang and Gibson, 1980), and the 51K protein seems an attractive candidate to be involved in the novel nucleosome-like structures that Kierszenbaum and Huang (1979) have described, since: (i) it is present in the nuclear fraction in appropriately large amounts, (ii) it has a comparatively strong net basic charge, and (iii) it is a DNA-binding protein (Gibson, Murphy, and Roby, 1981). A number of further observations relating to the metabolism of CMV proteins emerged from the pulse-chase experiment summarized in Fig. 12. First, it was found that in addition to the 94K immediate-early protein, synthesis of the 51K protein could be demonstrated earlier than that of most other viral proteins. Although the 51K protein is not an “early function, since its synthesis requires preceding viral protein and DNA synthesis (Jeang and Gibson, 1980; Gibson, Murphy, and Roby, 1981), it does appear to be among the first of the “late” proteins made (e.g., “quasi-late,” O’Farrell and Gold, 1973; “beta’‘-class, Honess and Roizman, 1974). Secondly, results of this experiment showed that viral proteins accumulating in the NP-40 nuclear fraction of infected cells enter that fraction at markedly different rates. One group, including most notably the 119K, 66K, and 51K proteins, partitioned strongly with the nuclear fraction immediately following a 20-min radiolabeling interval. A second group of proteins appeared in the nuclear fraction only following a lag period. Most notable of these are the capsid proteins (e.g., 145K and 37K). Although these differences have not been further investigated, it may be relevant that all three of the rapidly transported proteins (i.e., 119K, 66K, and 51K) are phosphorylated (Weiner and Gibson, manuscript in preparation). And third, this study has provided evidence of both rapid and slow post-transla-

STRUCTURAL

AND NONSTRUCTURAL

tional modifications of CMV proteins. Such modifications were best exemplified by the 66K and 20K proteins (Fig. 12). The 66K doublet, most dramatically the upper band, became progressively more diffuse during an extended chase period (i.e., 21 hr). Since other studies have established that this protein is phosphorylated at multiple sites (Weiner and Gibson, manuscript in preparation), it is suggested that the increased band width reflects an increased molecular heterogeneity resulting from phosphorylation. Glycosylation, a common modification of cell membrane and virus envelope proteins including several CMV proteins (Kim, et al., 19’76; Stinski, 19’77), could also account for this heterogeneity. However, since the 66K protein appears to be an intranuclear molecule, and since glycosylation is not a common modification of nuclear proteins, this possibility seems less attractive. In contrast to the slow, apparently intranuclear modification of the 66K protein, the 20K protein is processed rapidly and in the cytoplasm. Disappearance of this band, in the absence of a corresponding intensity increase in another band, suggests that it is proteolytically degraded. Finally, it should be emphasized that strain Colburn is not typical of the human CMV isolates. Although recovered from human cell cultures infected with brain biopsy material from a child with clinical encephalopathy (Charamella et al., 1973), strain Colburn shows stronger immunological cross-reactivity and DNA restriction fragment similarities to simian strains of CMV (Huang et al., 1978). Our recent comparisons of both structural and nonstructural proteins of simian and human CMV isolates likewise have shown that, while clearly distinct from rhesus and vervet CMVs, strain Colburn is more like the simian isolates than the human (Weiner and Gibson; Gibson, manuscripts submitted and in preparation, respectively). ACKNOWLEDGMENTS I thank Dr. Micheline McCarthy, of the Department of Neurology at Johns Hopkins, for generous assistance in looking at virus specimens using the electron microscope, and for preparing the electronmicro graphs shown in Fig. 2. I also thank Barbara Hit-

PROTEINS

OF CYTOMEGALOVIRUS

535

chens, Lynette Davies, and Michael Murphy for excellent technical assistance at different times during the course of these studies, and Barbara Ireland for help in typing the manuscript. In addition, I would lie to acknowledge a series of preliminary collaborative experiments with Dr. Milan Fiala (Seattle, Wash.) that stimulated my curiosity about cytomegalovirus, and thank Dr. Walter E&hart (Salk Institute, La Jolla, Calif.) for permitting this work to begin in his laborabrY. These studies were initially aided by Institutional Research Grants from the U. S. Public Health Service (RR-5378) and the American Cancer Society (lNllP), and Grant VC-242 from the American Cancer Society; and are now supported by Public Health Service grant AI13718 from the National Institute for Allergy and Infectious Diseases, and Research Grant 1-613 from the National Foundation for Birth Defects. REFERENCES ALLEN, G. P., and BRYANS, J. R. (1976). Cell-free synthesis of equine herpesvirus type 3 nucleocapsid polypeptides. Virology 69, 751-762. ANKER, H. S. (1970). A solubilizable acrylamide gel for electrophoresis. Fed. Eur. Biochem. Sot. L&t. 7, 293. BAUMANN, G., and CHRAMBACH, A., (1976). A highly cross-linked transparent polyacrylamide gel with improved mechanical stability for use in isoelectric focusing and isotachophoresis. Anal. Biochm. 70, 32-38. BEN-P• RAT, R., SHIMONO, H., and KAPLAN, A. S. (1970). Synthesis of proteins in cells infected with herpesvirus. IV. Analysis of the proteins in viral particles isolated from the cytoplasm and the nucleus. Virology 41,256-264. BEREZNEY, R., and COFFEY, D. S. (1977). Nuclear matrix. Isolation and characterization of a framework structure from rat liver nuclei. J. Cell Biol. 73,616~637. BONNER, W., and LASKEY, R. A. (1974). A fdm detection method for tritium labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88. CHARAMELLA, L. J., REYNOLDS, R. B., C&EN, L. T., and ALFORD, C. A. (1973). Biologic characterization of an unusual human cytomegalovirus (CMV) isolated from brain. In “Abstracts of the Annual Meeting of the American Society for Microbiology,” p. 256. American Society for Microbiology, Washington, D. C. COHEN, G. H., PONCE DELEON, M., DIGGEUUANN, H., LAWRENCE, W. C., VERNON, S. K., and Em SENBERG, R. J. (1980). Structural analysis of the capsid polypeptides of herpes simplex virus types 1 and 2. J. Viral. 34, 521-531. DE MARCHI, J. M., BLANKENSHIP, M. L., BROWN, G.

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D. and KAPLAN, A. 8. (1978). Size and complexity of hyman cytomegalovirus DNA. Virology 89, 643646. DULBECCO, R., and VOGT, M. (1953). Some problems of animal virology as studied by the plaque technique. Cold Spring Harbor Symp. Quant. Biol. 18, 273-279.

FAIRBANKS, G., STECK, T. L., and WALLACH, D. F. H. (1971). Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry

10, 2606-2617.

FIALA, M., HONESS, R. W., HEINER, D. C., HEINE, J. W., MURNAME, J., WALLACE, R., and GUZE, L. B. (1976). Cytomegalovirus proteins. I. Polypeptides of virions and dense bodies. J. Viral. 19,243254. GEELEN, J. L. M. C., WALIG, C., WERTHEIN, P., and VAN DER NOORDAA, J. (1978). Human cytomegalovirus DNA. I. Molecular weight and infectivity. J. Virol. 26, 813-816. GERACE, L., BLUM, A., and BLOBEL, G. (1978). Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. J. Cell

Biol.

79, 546-566.

GIBSON, W. (1974). Polyoma virus proteins: A description of the structural proteins of the virion based on polyacrylamide gel electrophoresis and peptide analysis. Virology 62, 319-336. GIBSON, W., and ROIZMAN, B. (1972). Proteins specified by herpes simplex virus. VIII. Characterization and composition of multiple capsid forms of subtypes 1 and 2. J. Viral. 10, 1044-1052. GIBSON, W., and ROIZMAN, B. (1973). The structural and metabolic involvement of polyamines with herpes simplex virus. In “Polyamines and Cancer.” Raven Press, New York. GIBSON, W., and ROIZMAN, B. (1974). Proteins specified by herpes simplex virus. X. Staining and radiolabeling properties of B-capsid and virion proteins in polyacrylamide gels. J. Virot. 13, 155-165. GIBSON, W., MURPHY, T. L., and ROBY, C. (1981). Cytomegalovirus-infected cells contain a DNA-binding protein. Virology 111,251-261. GUPTA, P., ST. JEOR, S., and RAPP, F. (1977). Comparison of the polypeptides of several strains of human cytomegalovirus. J. Ga. Virob S&447454. GUPTA, P., and RAPP, F. (1978). Cyclic synthesis of human cytomegalovirus-induced proteins in infected cells. Virology 84, 199-202. HEINE, U. I., and DALTON, A. J. (1974). Ultrastructural analysis of herpes-type viruses, In “Molecular Studies in Viral Neoplasia,” pp. 63-96. Williams and Wilkins, Baltimore, Md. HONESS, R. W., and ROIZMAN, B. (1974). Regulation of herpesvirus macromolecular synthesis. I. Caacade regulation of the synthesis of three groups of viral proteins. J. Viral. 14, 8-19.

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HUANG, E.-S., KILPATRICK, B., LAKEMAN, A., and ALFORD, C. A. (1978). Genetic analysis of a cytomegalovirus-like agent isolated from human brain. J. Viral. 26, 718-723. IWASAKI, Y., FURUKAWA, T., PLOTKIN, S., and KoPROWSKI, H. (1973). Ultrastructural study on the sequence of human cytomegalovirus infection of human diploid cells. Arch. Gesamte Virusforsch. 40, 311-324. JEANG, K.-T., and GIBSON, W. (1980). A cycloheximide-enhanced protein in cytomegalovirus-infected cells. Virology 107, 362-374. KANICH, R. E., and CRAIGHEAD, J. E. (1972). Human cytomegalovirus infection of cultured fibroblasts. I. Cytopathologic effects induced by an adapted and wild strain. Lab. Invest. 27, 263-272. KEMP, M. C., PERDUE, M. L., ROGERS, H. W., O’CALLAGHAN, D. J., and RANDALL, C. C. (1974). Structural polypeptides of hamster strain of equine herpes virus type I: Produces associated with purification. Virology 61, 361-375. KIERSZENBAUM, A. L., and HUANG, E.-S. (1978). Chromatin pattern consisting of repeating bipartite structures in WI-38 cells infected with human cytomegalovirus. J. Virol. 28, 661-664. KILPATRICK, B. A., HUANG, E.-S., and PAGANO, J. S. (1976). Analysis of cytomegalovirus genomes with restriction endonucleases Hin D,,, and Eco R,. J. Viral.

18, 1095-1105.

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