- Email: [email protected]
Primate cytomegalovirus assembly: Evidence that DNA packaging occurs subsequent to B capsid assembly
VIROLOGY
167,87-96
(1988)
Primate Cytomegalovirus Assembly: Evidence that DNA Packaging Subsequent to B Capsid Assembly JOANNA Virology
Laboratories,
Department
Y. LEE,’ ALICE IRMIERE,’
AND
WADE GIBSON3
of Pharmacology and Molecular Sciences, The Johns Hopkins 725 North Wolfe Street, Baltimore, Maryland 2 1205 Received
December
29, 1987; accepted
Occurs
University
School
of Medicine,
July 22, 1988
Results presented here show that when cytomegalovirus (strain Colburn)-infected cells are treated with the DNA synthesis inhibitor hydroxyurea or phosphonoformate, one type of intranuclear capsid accumulates. These particles appeared to contain symmetrically organized internal material, and had a protein composition and sedimentation rate characteristic of B capsids. Radiolabeling experiments provided evidence that a population of B capsids lacking DNA is present during the course of a normal infection. These capsids sedimented slightly slower than the peak of viral DNA in the same region of the gradient, and had a ratio of DNA/protein that was estimated to be sevenfold lower than that of the faster sedimenting C capsids. DNA in both the B and C capsid regions of such gradients was found to be relatively resistant to digestion with DNase. The possibility is considered that herpesvirus B capsids lacking DNA may be countero 1988Academic PWSS. IW. parts of unexpanded proheads in the bacteriophage assembly pathway.
proximately 20% of the capsid’s protein mass and is distinguished from the other capsid proteins in at least four ways: (i) it is phosphorylated, (ii) it has multiple forms and appears to undergo proteolytic cleavage, (iii) it exhibits unusual staining properties, and (iv) it is not present, as such, in the mature virion. This abundant protein, called VP22a (Gibson and Roizman, 1972, 1974) p40 (Heilman et al., 1979), NC-3 (Cohen et al., 1980) and ICP35e (Braun et al., 1984), is the HSV counterpart of the B capsid hallmark protein species, the assembly protein (lrmiere and Gibson, 1983, 1985). The gene encoding this HSV protein has been mapped genetically using inter-typic recombinants (Braun et a/., 1983) and a temperature-sensitive mutant, tsVP1201, whose genetic lesion maps to the same region has been described (Preston et al., 1983). At the restrictive temperature this mutant produces DNA and capsids, but is defective in assembly protein processing and DNA packaging. Cytomegalovirus (CMV)-infected cells also contain nuclear A and B capsids (Gibson, 1981; lrmiere and Gibson, 1985) and probable equivalents have been reported in equine herpesvirus types 1 (EHV-1) (Kemp et al., 1974; Perdue et al., 1975) and 3 (EHV-3) (Allen and Bryans, 1976) pseudorabies (PR) (Ben-Porat et a/., 1970; Ladin et al., 1980), and herpes zoster virus (HZV), (Friedrichs and Grosse, 1986) suggesting that these capsid forms are common among the herpesgroup viruses. The finding that the CMV assembly protein exhibits the same set of comparatively unusual characteristics (Gibson, 1981; lrmiere and Gibson, 1985) and that its gene is approximately colinear with
INTRODUCTION Assembly and maturation of the herpesgroup viruses begin in the nucleus of infected cells with the formation of capsids. These are typically about 100 nm in diameter with centers ranging in appearance from “empty” to “filled” with densely stained material. When DNA synthesis is inhibited, capsids with partially filled centers accumulate and then appear to give rise to completely filled particles after DNA synthesis resumes, suggesting that viral DNA is packaged into preformed capsids (Friedmann et al., 1975). This model is supported by results from pulse-chase radiolabeling experiments (O’Callaghan and Randall, 1976; Ladin et al., 1980) and studies using temperature-sensitive mutants (Ladin et a/., 1980; Preston et al., 1983). Experiments using herpes simplex virus (HSV) to study the sequence of events involved in herpesvirus assembly led to the recovery and characterization of two capsid forms, designated as A and B capsids, from infected cell nuclei. B capsids were distinguished by having a faster sedimentation rate, an internal core structure, a higher DNA content, and two proteins not found in A capsids. One of these proteins, VP21, is a minor constituent which also appears to be present in the mature virion (Gibson and Roizman, 1972, 1974). The second is an abundant species that represents ap’ Present address: The Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205. ’ Present address: Halvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, Boston, MA 021 15. ’ To whom requests for reprints should be addressed. 87
0042-6822/88
$3.00
Copyright 0 1998 by Academic Press, Inc. All rights of reproduction in any form resewed.
88
LEE,
IRMIERE,
that of HSV (Robson and Gibson, manuscript submitted for publication) underscores the similarities between HSV and CMV B capsids and suggests that they are functional equivalents. Additional insight into the involvement of B capsids in the assembly pathway, and the interaction of the assembly protein with these particles during nucleocapsid maturation, came from characterizing noninfectious enveloped particles (NIEPs) produced by human strains of CMV (Irmiere and Gibson, 1983). These particles are similar to virions in structure and protein composition, but are distinguished from them by the presence of the assembly protein and the absence of DNA. As discussed before (Irmiere and Gibson, 1983, 1985) these findings suggest that NlEPs represent enveloped B capsids, that the assembly protein becomes a capsid constituent before and independently of DNA packaging, and that elimination of the assembly protein is neither essential for nor a necessary consequence of acquiring the tegument and envelope layers. Since NlEPs do not contain DNA, the hypothesis that they represent enveloped B capsids predicted the existence of a population of CMV B capsids lacking DNA. Two approaches were taken to test this prediction. This first was to determine the relative efficiency of radiolabeling B capsids with DNA precursors, and the second was to determine whether B capsids are made in the presence of inhibitors of DNA synthesis. The results are consistent with the prediction that B capsids lacking DNA exist, and are discussed as they bear on the sequence of events surrounding capsid assembly and DNA packaging. MATERIALS
AND
METHODS
Cells and virus Human foreskin fibroblast cultures were prepared, maintained, and infected as described before (Gibson, 1981). Pertinent characteristics of the Colburn strain of cytomegalovirus have also been described (Gibson, 1981, 1983). Recovery
of virus particles
Details of the procedures used for fractionating infected cells and resolving virus particles by rate-velocity centrifugation have been presented before (Irmiere and Gibson, 1983) and are only summarized here. Cells from one or two 32-0~ bottles (approximately 5 x lo7 cells/bottle) were pelleted, resuspended in 0.5 ml of PB (40 mNI phosphate buffer, 150 mM NaCI, pH 7.4) containing 0.5% NP-40 and 1 mh/l phenylmethylsulfonyl fluoride (PMSF), and held on ice for 2 to 10 min, NP-40 nuclei were colleced by centrifugation and the
AND
GIBSON
supernatant fraction was saved as the NP-40 cytoplasm. Nuclei were resuspended in 0.5 ml of PB containing 1 mM PMSF and disrupted by sonication (D. Witt, personal communication) (5-10 set, setting 4, Branson Sonifier 185,0”), and the lysate was then clarified by centrifugation (1 OK g, 4”, 30 set). Clarified nuclear lysate or NP-40 cytoplasmic fractions were layered onto 15-5096 sucrose (w/w, in PB) gradients and subjected to centrifugation in a Beckman SW41 rotor (40,000 rpm, 4”, 20 min). Particles were recovered from the gradient by aspiration, or gradients were fractionated to record the optical absorbance (&,) pattern and measure radioactivity by scintillation spectrometry (50 ~1 sample, 4 ml Aquasol, New England Nuclear). Polyacrylamide
gel electrophoresis
Protein electrophoresis in sodium dodecyl sulfate (SDS)-containing polyacrylamide gels (SDS-PAGE) was done essentially as described by Laemmli (1970). Pierce SDS was used in the electrode buffer to resolve the Colburn CMV upper and lower matrix proteins (Weiner and Gibson, 1983). Gels were stained with Coomassie brilliant blue (Fairbanks et a/., 197 1) or ammoniacal silver (Wray et a/., 1981) and fluorography was done with diphenyloxazole (Bonner and Laskey, 1974) or calcium tungstate intensifying screens (Laskey and Mills, 1977). Densitometry was done using a transmission densitometer (E-C Apparatus Corp., St. Petersburg, FL). Electron
microscopy
Cells were fixed with 1% glutaraldehyde in cacodylate buffer, postfixed with 1% osmium tetroxide, stained enbloc with 1% aqueous uranyl acetate, dehydrated through a graded series of ethanol, embedded in Epon, sectioned using a Sorvall MT5000 ultramicrotome, stained with lead citrate and uranyl acetate, and examined and photographed using a Zeiss 1OA electron microscope. RESULTS Two series of experiments were done to determine whether CMV intranuclear B capsids lacking DNA could be identified. The first was to test the effect of DNA synthesis inhibitors on capsid production, with the expectation that only capsids which lack DNA would be made in their presence. The second approach was to grow infected cells in the presence of a radiolabeled DNA precursor, separate the intracellular capsid forms by centrifugation, and measure the radioactivity associated with each.
PRIMATE
Appearance of CMV particles produced presence of inhibitors of DNA synthesis
CMOMEGALOVIRUS
in the
This approach was prompted by the finding that inhibitors of DNA synthesis, such as phosphonoformate and hydroxyurea, at concentrations that reduced virion production by two to four orders of magnitude, had comparatively little effect on the production of NlEPs (i.e., enveloped B capsids) (Gibson and Irmiere, 1984). Furthermore, others have found that when DNA synthesis is inhibited, nuclear capsids with partially stained cores and of intermediate density continue to be produced (O’Callaghan et al., 1968; Friedmann et a/., 1975). These experiments were done to examine the possibility that capsids produced under such conditions correspond to B capsids lacking DNA. Hydroxyurea (HU, 5 mNI) was used in the first experiment. This drug is an inhibitor of ribonucleotide reductase, which catalyzes the rate-limiting step in the formation of deoxyribonucleoside triphosphates (reviewed by Cory, 1983) and results in an inhibition of DNA synthesis. Concentrations of HU ranging from 1 to 5 mM reduced the titer of Colburn CMV by lo- to 1O,OOO-fold (Anders et a/., 1986) and had differential effects on infected-cell protein synthesis (e.g., Fig. 3). At late times after infection in the presence of 5 mM HU, when extensive CPE was evident (e.g., 4-6 days), capsid proteins (e.g., MCP, 145-kDa major capsid protein; AP, 37-kDa assembly protein; mCP, 34-kDa minor capsid protein) were made in good yield but were incompletely transported to the nucleus, DB129 the Colburn early DNA-binding protein homolog of HSV ICP8 (Anders et a/., 1986, 1987; Kemble et al., 1987; Anders and Gibson, 1988) was overproduced, the tegument proteins (e.g., BPP, 1 19-kDa basic phosphoprotein; UM and LM, 69- and 66-kDa upper and lower matrix proteins, respectively) were grossly underrespected, and the synthesis of at least two host proteins (e.g., vimentin, actin) was unchanged (Fig. 3) (unpublished results). Similar effects on infected-cell proteins were produced by phosphonoformate (PFA, 200 pg/ml) (Gibson et al., 1984; Anders et a/., 1986) a selective inhibitor of the viral DNA polymerase (Huang, 1975). HU was added to the culture medium 36 hr after infection and 3 days later a drug-treated and nontreated culture were processed and examined by electron microscopy. Representative fields from the nuclear and cytoplasmic compartments of each culture demonstrate two major effects of the drug (Fig. 1). First, there was an absence of virus particles in the cytoplasm of drug-treated cells (Panel D). Enveloped capsids (e.g., virions), which are normally abundant in the cytoplasm of infected cells (Panel C), were found only rarely in cells treated with HU. Second, capsids present in the
89
ASSEMBLY
nucleus of drug-treated cells were comparatively uniform in appearance (Panel B). These particles characteristically showed a pattern of internally stained material that often appeared as a concentric six-beaded ring, approximately 55 nm outside diameter and 15 nm thick [seen better in Panel A, arrows, and, for HCMV, in Gibson et a/. (1984)]. Fewer, but otherwise indistinguishable nuclear particles were observed in infected cells treated with higher concentrations of hydroxyurea (10 mn/l) or with PFA (200 pg/ml) (data not shown). Nuclei of nontreated infected cells contained similar particles (see arrows, Panel A), but in contrast to drugtreated cells, also contained numerous capsids with densely stained internal material. Measurements of particles in the nucleus of nontreated cells (Panel A) indicated that the outside diameter of capsids with a beaded internal appearance (see arrows), like those in hydroxyurea-treated cells, is approximately 7% smaller than the others. Capsids position
in drug-treated of B capsids
cells sediment
close to
Infected cells were incubated in media containing HU (5 mM) or PFA (200 pg/ml), harvested, lysed, and subjected to rate-velocity sedimentation to identify intracellular virus particles. The infected cells were lysed in 0.1 M glycine (Hallauer and Kronauer, 1965) (pH 9.5, 37”, 75 min) to avoid the use of detergents and to obtain a whole cell lysate, rather than separate nuclear and cytoplasmic fractions. Optical absorbance profiles of the three gradients (Fig. 2) demonstrate that (i) drugtreated cultures showed significant absorbance only at the B capsid position; (ii) the HU-treated culture yielded approximately 40% more B capsids than nontreated cells; (iii) the PFA-treated culture gave the smallest yield of B capsids (i.e., about 50% that recovered from the nontreated culture); and (iv) C capsids were not recovered from either drug-treated culture. The broad zone of optical absorbance sedimenting ahead of C capsids was reduced in the drug-treated cultures. This region of the gradient normally contains substantial amounts of upper and lower matrix proteins (e.g., Fig. 3, top) but their synthesis is markedly reduced when viral DNA synthesis is inhibited (Fig. 3, bottom) (Gibson eta/., 1984; Anders eta/., 1986; additional unpublished results). When the pellet resulting from glycine extraction of HU-treated cells was further extracted with DOC, DNase, Brij 58, and urea (Gibson, 1981) additional B capsids (40-500~ amount extracted with glytine) and some A capsids were released (bottom panel, Fig. 2). Comparable results were obtained when these drugs, at the same respective concentrations, were added at the time of infection rather that 36 hr
LEE,
IRMIERE,
No Drug
FIG. 1. Thin sections of ceils infected with CMV in the presence or Drug) or presence (Hydroxyurea) of 5 mM hydroxyurea, as described in fields from the nuclear (Nucleus) and cytoplasmic (Cytoplasm) regions appearance that resemble those in (B). Arrow in (C) indicates virion containing vessicle. Bar in (B) indicates 100 nm.
after infection. However, under these conditions, unless the drug-treated cultures were allowed additional incubation time (e.g., l-2 days longer than nontreated cultures) the yield of B capsids was lower (data not shown). Capsids in H&treated protein composition
cells have B capsid-like
The protein composition of the capsids present in HU-treated cells was examined to determine their rela-
AND
GIBSON
H ydroxyurea
absence of hydroxyurea. Infected cells were grown in the absence (No the text, and were then processed for EM. Shown here are representative of each preparation. Arrows in (A) indicate capsids with a beaded internal envelope layer to distinguish it from the membrane of the larger virion-
tionship to nuclear B capsids. Infected cultures were grown either with 5 mM HU added at the time of infection or without drug, labeled with [35S]methionine (20 &i/ml) beginning 2 days after infection, harvested 5 days after infection, and treated with 0.5% NP-40 to prepare NP-40 nuclei. The nuclei were ruptured by sonication and the resulting clarified lysate was subjected to rate-velocity sedimentation in sucrose gradients. The gradients were fractionated and aliquots of each fraction were solubilized and analyzed by SDSPAGE. Fluorograms prepared from the resulting gels
PRIMATE
CYTOMEGALOVIRUS
91
ASSEMBLY
1983) from the +HU preparation. The upper panel also shows that the distribution in the gradient of several other proteins (e.g., BPP, 90K, and 78K) follows that of
Sedimentation
+
-MCP
-78~ rMPs
-D&i1 .45K .AP -mCP .28~
HU Pelleti DOC/DNase
-MCP -BPP
0
Sedimentation-
FIG. 2. Rate-velocity sedimentation of intracellular virus particles in drug-treated and nontreated cultures. CMV-infected cells maintained in the absence of added drug (No Drug), or in the presence of 5 mM hydroxyurea (+HU) or 200 &ml phosphoformic acid (+PFA), were glycine extracted and analyzed by rate-velocity sedimentation as described in the test. The glycine-extracted infected-cell pellet was further extracted with a combination of DOC and DNase and analyzed in parallel (HU Pellet + DOC/DNase). Shown here are absorbance (A280) patterns recorded from the resulting sucrose gradients. The positions of A, B, and C capsids are indicated.
-45K -AP
-28K
I
(Fig. 3) showed that the particles at the B capsid position in the “+HU” preparation had a characteristic B capsid protein composition; in particular they contained the assembly protein and its related 45-kDa form (45K) (Gibson, 1981). This figure also shows that the capsid proteins from the “no drug” preparation were more broadly distributed through the gradient, indicating greater heterogeneity in the structures present. A capsids (indicated in top panel) were detected only in the no drug preparation. The other notable difference between the two patterns is the absence of the DNAbinding protein DB51 (Gibson, 1983, 1984) and the matrix proteins (Weiner and Gibson, 1983; Gibson,
1
I
I,
I
5
II
I,
10
Fraction
I
I
*I,
I
15
ZII
20
Number
FIG. 3. Protein distribution in sucrose gradients following rate-velocity sedimentation of nuclear lysates from HU-treated and nontreated, infected cells. Nuclear lysates of HU-treated and nontreated, infected cultures were prepared and subjected to rate-velocity sedimentation as described in the text. Samples of the fractionated gradients were subjected to SDS-PAGE (10% gel). Shown here are fluorographic images of the resulting gels. Protein abbreviations in the right margin are explained elsewhere (Gibson, 1983) and are as follows: MCP (145-kDa major capsid protein), BPP (119-kDa basic phosphoprotein), 78K (78-kDa band), MPs (69 and 66-kDa upper and lower matrix proteins, respectively), 45K (45-kDa B capsid protein), AP (37-kDa assembly protein), mCP (34-kDa minor capsid protein, 28K (28.kDa capsid protein).
92
LEE,
IRMIERE.
A and B capsids, suggesting that they are specifically associated. An additional experiment was done to determine how this B capsid protein pattern was influenced by the time of HU addition. HU (5 mM) was added to pairs of cultures at the time of infection, or on Day 1 or Day 2 after infection. Two days after adding the drug (i.e., on Days 2, 3, and 4, respectively, after infection) one culture of each pair was labeled with [35S]methionine (20 &i/ml) and the other with 32Pi (200 &i/ml). A pair of cultures not treated with HU was labeled on Days 2 through 4 after infection and similarly processed. The infection progressed more rapidly in the cultures lacking HU and they were harvested and processed 4 days after infection. Cultures treated with HU on Days 0, 1, and 2 were harvested and processed on Days 4,5, and 6 after infection, respectively, times at which each showed strong cytopathic effect (e.g., cell rounding) but minimal cell detachment. Following rate-velocity sedimentation, the B capsid bands were recovered from the tubes by aspiration, concentrated by pelleting, and solubilized and subjected to SDS-PAGE. Fluorograms prepared from the resulting gel (Fig. 4) showed that the [35S]methionine-labeled preparations looked very similar. Preparations from HU-treated cultures, however, contained (i) decreased amounts of the basic phosphoprotein and matrix proteins, and (ii) increased amounts of the DNA-binding protein DB129 (see arrow) (Anders et a/., 1986) and a host cell protein indicated by a circle just above the 45K band (similarly indicated in Fig. 3). With the possible exception of DB129, these differences reflect gross changes in the amounts of these proteins throughout the gradient as seen with the matrix proteins and DB51 in Fig. 3. The 32P-labeled preparations showed that the level of assembly protein phosphorylation was not markedly influenced by adding HU to the cultures. This figure also demonstrates that the 38- and 39-kDa proteins (see circles just above assembly protein) (Irmiere and Gibson, 1985) and the 45K protein, all of which are related to the assembly protein (Gibson, 1981; unpublished immunological and peptide comparisons), are all phosphorylated. No attempt was made to quantify data from these preparations because of the differences in their radiolabeling interval. B capsids lacking DNA detected in presence absence of DNA synthesis inhibitors
or
These experiments were done in order to determine whether B capsids lacking DNA are produced during the course of a normal infection (i.e., no drug). Infected cells were radiolabeled with [3H]thymidine ([3H]TdR, 10 &i/ml), beginning 36 hr after infection, and intracellu-
AND
GIBSON
Day HU Added
BPF 78K- : MPs
AP mCP-
FIG. 4. Proteins in capsid preparations following separation by SDS-PAGE. CMV-infected cells were grown without drug (-) or in 5 mM HU added at the time of infection (0) or 1 or 2 days after infection [%]methionine (%-Met.) or ‘*/: was added as described in the text. Particles were recovered, concentrated, and subjected to SDSPAGE in a 10% gel. Shown here is a fluorographic image of the resulting gel. Protein abbreviations are as described in the legend to Fig. 3.
lar virus particles were analyzed 30 hr later. Cells were separated into nuclear and cytoplasmic fractions following treatment with NP-40, and the resulting nuclei were disrupted by sonication and clarified by centrifugation, all as described above and under Materials and Methods. The nuclear supernatant fraction was made 5 mhll in MgCI, and divided in half: DNase (10 pg/ml final concentration) was added to one half and both were incubated at 37” for 10 min and then subjected to rate-velocity centrifugation through sucrose gradients. Optical absorbance profiles were recorded as the gradients were collected, and portions of each resulting fraction were analyzed for their protein and DNA content, respectively, by SDS-PAGE and scintillation spectrometry.
PRIMATE
CYTOMEGALOVIRUS
93
ASSEMBLY
Sedimentation -
.07 .06
.02
.Ol I
.
10
. .
..,..
.
15
.
20
.
-
IO
15
20
Fraction Number FIG. 5. Effect of DNase treatment on distribution of nuclear capsids during rate-velocity sedimentation, A nuclear lysate of [3H]TdR-labeled infected cells was prepared; one half was treated with DNase and both were subjected to rate-velocity sedimentation as described in the text. Shown here are the absorbance patterns recorded as the gradients were collected. The positions of A, B. and C capsids are indicated.
The positions of A, B, and C capsids in each gradient were determined from the resulting absorbance (Fig. 5) and SDS-PAGE (Fig. 6) patterns. B capsids were most concentrated in fraction 14 of the nontreated preparation and fraction 13 of the nuclease-treated preparation, as shown by the relative abundance of the assembly protein (AP, Fig. 6). A capsids were located in fractions 11 and 12 (No DNase) or 11 (+DNase), and C capsids were present in fraction 20 (No DNase) or 19 (+DNase). The C capsid bands were coincident with peaks of radioactivity, indicating the presence of DNA (bottom panel, Fig. 6). The majority of the B capsid optical absorbance and protein staining, however, was in fractions on the trailing edge of the peak of [3H]TdR radioactivity, ratherthan coincident with it. Specifically, the peak of B capsid absorbance and protein staining was fraction 14 but the peak of radioactivity was in fraction 15 (No DNase). This separation is seen better in the +DNase preparation where the maximum of B capsid optical absorbance and protein staining was fracton 13 (a relative minimum in the radioactivity plot), while the radioactivity peak fraction was 14. Little, if any, DNA was present at the A capsid position. Interestingly, the basic phosphoprotein (BPP) was the only species noted whose abundance correlated well with the amount of DNA present in the capsid region (e.g., fractions 1O-22, +DNase) of such gradients. Calculations made from these data indicate that CMV B capsids contain about four times as much DNA as A capsids, and that C capsids contain approximately seven times as much DNA as B capsids (Table 1).
I ..*e-
-AP
-IncP
‘*t=
I
1
6
10
5
1
15
No DNase
1-u 1
5
10
15
20
25
30
' - '2b'
r
25'
'$0'
'
+ DNase
I I
1
5
10
15
20
25
30
Fraction Number FIG. 6. Protein and DNA distributions in gradients shown in Fig. 5. Portions of each fractionated gradient were analyzed by SDS-PAGE (10% gels) and scintillation spectrometry. The top two panels show the resulting silver stained polyacrylamide gels. Protein designations are as described in the legend to Fig. 3. Small circles in right margin indicate positions of upper and lower matrix proteins, respectively; smudginess in this region is due to a silver staining artifact band. Lower two panels show [3H]TdR distribution in these gradients. Arrow in right panel indicates that B capsids were more concentrated in fraction 13 than 14. Note that radioactivity is recorded on a logarithmic scale.
94
LEE,
IRMIERE, TABLE
AND
GIBSON
1
ESTIMATED RELATIVE DNA CONTENTOF
CMV
Capsid A Treatment No DNase + DNase Average Ratio Note. s cpm b AZsO c cpm d cpm
cpma
A 280b
650 387
2.6 1.2
CAPSID FORMS
form
B cPmh80
comC
250 322 286
22,200 25,552 B:A = 4.2
A 280 20.5 19.2
C cpmd
A 280
cpm/A28o
19,400 21,049 8,215 C:B = 6.8
2.5 2.4
7660 8770
wmlA280 1081 1334 1207
Calculations are based on measurements obtained from data in Figs. 5 and 6. rH]TdR in fractions 11 and 12 (No DNase) and fraction 11 (+DNase); background subtracted. was calculated as f (peak height X peak width at base); expressed in arbitrary units. [3H]TdR in fractions 13 and 14 (No DNase) and fractions 13 and 14 (+DNase); background subtracted. [3H]TdR in fraction 20 (No DNase) and fraction 19 (+DNase); background subtracted.
Under the conditions of the experiment described above, DNase treatment had no detected effect on the distribution of radioactivity in the gradients. At higher concentrations of DNase (e.g., 100 pg/ml), however, treated samples showed a greater percentage of the total radioactivity present at the top of the gradient. DNA in the B and C capsid peak areas remained comparatively resistent to the nuclease (i.e., 80-90% still present). Only viral DNA was present in the B and C capsid regions as determined by (i) its boyant density (1.71 g/cm3 relative to an internal marker of 1.703 g/ cm3); (ii) its DNA fragmentation pattern following cleavage with restriction endonuclease EcoRI; and (iii) the findings that neither particles recovered in the same way from cells labeled with [3H]TdR for 2 days prior to infection, and then infected and maintained in the absence of radiolabel, nor radiolabeled mock-infected cells showed peaks of radioactivity in these regions of the gradient. When similar experiments were done with HU-treated cultures (5 or 10 pg/ml, added beginning 24-48 hr after infection), essentially all of the DNA (i.e., [3H]TdR) was present at the top of the gradient; little if any sedimented coincident with the B capsid peak (data not shown). DISCUSSION This report shows that when cytomegalovirus (strain Colburn) RNA synthesis is inhibited with hydroxyurea or phosphonoformate (or aphidicolin or novobicin, data not shown), a comparatively homogeneous population of intranuclear capsids accumulate. These particles, recovered after lysing cells or nuclei by detergent treatment (Gibson, 1981) freezing and thawing (Irmiere and Gibson, 1985) sonication (Figs. 3-6) or glycine extraction (Fig. 2), were found to have the sedimentation
properties and distinguishing protein composition (i.e., presence of 37-kDa assembly protein) of B capsids. Since these particles are produced under conditions where viral DNA synthesis is inhibited, it is evident that the assembly protein can become a capsid component prior to DNA packaging, as concluded before (Irmiere and Gibson, 1983,1985). Sherman and Bachenheimer (1988) have drawn the same conclusion from studies using temperature-sensitive mutants of HSV-1. Evidence that B capsids lacking DNA are also produced during the course of a normal infection was provided by the observation that the peak of B capsid protein mass was not coincident with the peak of viral DNA in the same region of the gradient (Figs. 5 and 6). The sevenfold lower ratio of DNA to protein (i.e., cpm [3H]TdR/ODZ8J in the B capsid region of the gradient, compared to the C capsid region (Table l), is consistent with the presence of B capsids lacking DNA. It is unclear whether these empty B capsids are precursors of DNA-containing particles or represent abortive products of assembly. If they are precursors, it would be expected that they could be chased into DNA-containing particles after removing the block to DNA synthesis. Although we have been unable to demonstrate a precursor/product relationship by such experiments (unpublished results), this could be explained if the capsids were only transiently competent to package DNA, or if there is a tight coupling between viral DNA synthesis and encapsidation. The finding that HSV-1 DNA, produced by certain temperaturesensitive mutants grown at the restrictive temperature, could be chased into particles upon shift down to the permissive temperature, while preformed empty B capsids could not (Sherman and Bachenheimer, 1987, 1988) is consistent with our findings for CMV and is
PRIMATE
CYTOMEGALOVIRUS
more compatible with the possibility of a transient competence for DNA packaging than a requirement for concomitant DNA and capsid synthesis. The presence in nontreated, infected cells of capsids corresponding in appearance to those that accumulated in hydroxyurea-treated cells (Fig. 1) indicates that these particles are not simply artifacts of the drug treatment, and is consistent with the identification of empty B capsids in lysates of normally infected cultures (Fig. 6). Similar looking particles are commonly observed in the nucleus of herpesvirus-infected cells, and are consistently enriched for when viral DNA synthesis (Friedmann eta/., 1975; O’Callaghan, eta/., 1968) or packaging (Schaffer e? al., 1974; Ladin et a/., 1980, 1982; Preston et a/., 1983; Gibson et al., 1984) is inhibited. The absence of DNA in these CMV capsids establishes that their characteristic internal staining pattern, often appearing in cross-section as a beaded concentric ring, is not due to DNA. O’Callaghan and Randall (1976) have suggested that such internal staining of EHV intermediate density capsids is due to VPIII, the apparent EHV assembly protein counterpart. It is unresolved, however, whether the assembly protein is internal and itself constitutes the stained material or whether it is located elsewhere (e.g., surface) (Braun et al., 1984) and causes the staining pattern as an indirect effect (e.g., conformational alteration) of its interaction with other capsid constituents. Results presented here and elsewhere (Gibson and Roizman, 1972; O’Callaghan and Randall, 1976; Ladin, 1982; Preston et al., 1983; lrmiere and Gibson, 1983; Sherman and Bachenheimer, 1988) are compatible with the possibility that the assembly protein is the herpesvirus counterpart of the bacteriophage scaffolding protein. The scaffolding protein mediates the formation of an unexpanded prohead, and this structure is subsequently converted to an expanded prohead in conjunction with protein processing events, including proteolytic cleavage, that result in topological changes in the prohead’s structure (for review see Black and Showe, 1984). During this process the internally located scaffolding protein is either eliminated from the head and recycled (King and Casjens, 1974) or cleaved into small peptides (Laemmli, 1970). Resulting expanded proheads are competent to package DNA and become filled heads. Like the scaffolding protein, the herpesvirus assembly protein species (i) is an abundant constituent of capsids lacking a full complement of DNA (Figs. 3 and 6) (Perdue et a/., 1975; Sherman and Bachenheimer, 1988), (ii) is associated with capsids smaller in diameter than DNA-containing capsids (Fig. I), (iii) undergoes processing, probably including proteolytic cleavage (Gibson and Roizman, 1974; Ladin et a/., 1982; Preston et al., 1983; Braun et a/., 1984; Gibson
95
ASSEMBLY
et a/., submitted for publication: Robson and Gibson, submitted for publication), and (iv) is not present intact in the mature virion (Gibson and Roiz’man, 1972, 1974; Kemp et a/., 1974; Gibson, 1981; Ladin et al., 1982; lrmiere and Gibson, 1985).
ACKNOWLEDGMENTS We thank Donna Woods for excellent technical support with electron microscopy, and Glenn Sherman and Steve Bachenheimer for sharing their results with us prior to publication. This work was aided by Research Grants Al 13718, Al2271 1, and All 9373 from the National Institutes for Allergy and Infectious Diseases. J.Y.L. was a predoctoral fellow in the Pharmacology and Molecular Sciences Training Program and was supported by Public Health Service Training Grant GM07626. A.I. was a predoctoral fellow in the Biochemistry, Cellular, and Molecular Biology Training Program.
REFERENCES ALLEN, G. P., and BRYANS, J. T. (1976). Cell-free synthesis of equine herpesvirus type 3 nucleocapsid polypeptides. Virology 69, 751762. ANDERS, D. G., and GIBSON, W. (1988). Location, transcript analysis, and partial nucleotide sequence of the cytomegalovirus gene encoding an early DNA-binding protein with similarities to ICP8 of herpes simplex virus type 1. J. Viral. 62, 1364-l 372. ANDERS, D. G., IRMIERE, A., and GIBSON, W. (1986). Identification and characterization of a major early cytomegalovirus DNA-binding protein. J. Viral. 58, 253-262. ANDERS, D. G., KIDD, J. R., and GIBSON, W. (1987). immunological characterization of an early cytomegalovirus single-strand DNA binding protein with similarities to the HSV major DNA-binding protein. Virology 161, 579-588. BEN-P• RAT, T., SHIMONO, H., and KAPLAN, A. S. (1970). Synthesis of proteins in cells infected with herpes virus. IV. Analysis of the proteins in viral particles isolated from the cytoplasm and nucleus. Virology 41,256-264. BLACK, L. W., and SHOWE, M. K. (1984). Morphogenesis of the T4 head. In “Bacteriophage T4” (C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget, Eds.). pp. 219-245. ASM, Washington, DC. BLAIR, E. G., and HONESS, R. W. (1983). DNA-binding proteins specified by herpesvirus saimiri. 1. Gen. Viral. 64, 2697-2715. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyactylamide gels. Eur. J. Biochem. 46,83-88. BRAUN, D. K., PEREIRA, L., NORRILD. B., and ROIZMAN, B. (1983). Application of denatured, electrophoretically separated, and immobilized lysates of herpes simplex virus-infected cells for the detection of monoclonal antibodies for studies of the properties of viral proteins. J. Viral. 46, 103-l 12. BRAUN, D. K., ROIZMAN, B., and PEREIRA, L. (1984). Characterization of post-translational products of herpes simplexvirus gene 35 proteins binding to the surfaces of full capsids but not empty capsids. 1. Viral. 59, 142-l 53. COHEN, G. H., PONCE DE LEON, M., DIGGELMANN, H., LAWRENCE, W. C., VERNON. S. K., and EISENBERG, R. J. (1980). Structural analysis of the capsid polypeptides of herpes simplex virus types 1 and 2. J. Virol. 34, 521-53 1. CoRY, J. G. (1983). Role of ribonucleotide reductase in cell division, Pharmac. Ther. 21.265-276.
96
LEE,
IRMIERE,
FAIRBANKS, G., STECK, T. L., and WALLACH, D. F. (1971). ElectrophoretiC analysis of the major polypeptides of the human erythrocyte membrane. Biochemisfry 10,2606-2617. FRIEDMANN, A., COWARD, J. E., ROSENKRANZ, H. S.. and MORGAN, C. (1975). Electron microscopic studies on assembly of herpes simplex virus upon removal of hydroxyurea block. 1. Gen. Viral. 26, 171-181. FRIEDRICHS, W. E., and GROSE, C. (1986). Varicella-zoster p32/p36 complex is present in both the viral capsid and the nuclear matrix of the infected cell. J. Vifol. 57, 155-l 64. GIBSON, W. (1981). Structural and nonstructural proteins of strain Colburn cytomegalovirus. virology 111, 516-537. GIBSON, W. (1983). Protein counterparts of human and simian cytomegaloviruses. l/ifo/ogy 128, 39 l-406. GIBSON, W. (1984). Synthesis, structure and function of a cytomegalovirus major nonvirion nuclear protein. UCLA Symp. Mol. Cell. Biol. 21,423-440. GIBSON, W., and IRMIERE, A. (1984). Selection of particles and proteins for use as human cytomegalovirus subunit vaccines. In “Birth Defects: Original Article Series,” Vol. 20, pp. 305-324. A. R. Liss, New York. 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-l 052. GIBSON, W., and ROIZMAN, B. (1974). Proteins specified by herpes simples virus. X. Staining and radiolabeling properties of B capsid and virion proteins in polyactylamide gels. J. Viral. 13, 155-l 65. GIBSON, W., VAN BREEMEN, R., FIELDS, A., LAFEMINA, R., and IRMIERE, A. (1984). o,L-Difluoromethylornithine inhibits human cytomegalovirus replication. J. Viral. 50, 145-l 54. HALLAUER, C., and KRONAUER, G. (1965). Extraction of cell-associated virus without damage of the culture. Asch. Ges. Virusgorsch. 15, 433-440. HEILMAN, C. J., JR., ZWEIG, M., STEVENSON, J. R., and HAMPAR, B. (1979). Isolation of a nucleocapsid polypeptide of herpes simplex virus types 1 and 2 possessing immunologically type-specific and cross-reactive determinants. J. Viral. 29, 34-42. HUANG, E.-S. (1975). Human cytomegalovirus. IV. Specific inhibition of virus-induced DNA-polymerase activity and viral DNA replication by phosphonoacetic acid. J. Viral. 16, 1561-l 565. IRMIERE, A., and GIBSON, W. (1983). Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus. virology 131, 1 18-l 33. IRMIERE. A., and GIBSON, W. (1985). Isolation of human cytomegalovirus intranuclear capsids, characterization of their protein constituents, and demonstration that the B-capsid assembly protein is also abundant in noninfectious enveloped particles. J. Viral. 56, 277283. KEMBLE, G. W., MCCORMICK, A. L., PEREIRA, L., and MOCARSKI, E. S. (1987). A cytomegalovirus protein with properties of herpes simplex virus ICP8: Partial purification of the polypeptide and map position of the gene. J. Viral. 61, 3 143-3 15 1. KEMP, M. C., PERDUE, M. L., ROGERS, H. W., O’CALLAGHAN, D. J., and RANDALL, C. C. (1974). Structural polypeptides of the hamster strain of equine herpexvirus type 1: Products associated with purification. Virology 71,361-375. KING, J., BOTSTEIN, D., CASJENS, S., EARNSHAW, W., HARRISON, S., and LENK, E. (1976). Structure and assembly of the capsid of bacteriophage P22. Philos. Trans. R. Sot. London Ser. B 276,37-49. KING, J., and CASJENS, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature (London) 251, 112-l 19. LADIN, B. F., BLANKENSHIP. M. L.. and BEN-P• RAT, T. (1980). Replication of herpesvirus DNA. V. Maturation of concatameric DNA of
AND
GIBSON
pseudorabies virus to genome length is related to capsid formation. J. Viral. 33, 1151-l 164. LADIN, B. F., IHARA, S., HAMPL, H., and BEN-P• RAT, T. (1982). Pathway of assembly of herpesvirus capsids: An analysis using DNA+ temperature-sensitive mutants of pseudorabies virus. Virology 116, 544-561. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biothem. 56,335-341. LASKEY, R. A., and MILLS, A. D. (1977). Enhanced autoradiographic detection of 32P and ‘? using intensifying screens and hypersensitized film. FEBS Len 82,314-316. MURIALDO, H., and BECKER, A. (1978). Head morphogenesis of complex couble-stranded deoxyribonucleic acid and bacteriophages. Microbial. Rev. 47, 529-576. O’CALLAGHAN, D. J., HYDE, H. M., GENTRY, G. A., and RANDALL, C. C. (1968). Kinetics of viral deoxyribonucleic acid, protein, and infectious particle production and alterations in host macromolecular synthesis in equine abortion (herpes) virus-infected cells. 1. Viral. 36,104-114. O’CALLAGHAN, D. J., KEMP, M. C., and RANDALL, C. C. (1977). Properties of nucleocapsid species isolated from an in vivo herpesvirus infection. J. Gen. Viral. 37, 585-594. O’CALLAGHAN, D. J., and RANDALL, C. C. (1976). Molecular anatomy of herpesviruses: Recent studies. Pfog. Med. Viral. 22, 152-210. PERDUE, M. L., COHEN, J. C.. KEMP, M. C., RANDALL, C. C.. and O’CALLAGHAN, D. J. (1975). Characterization of three species of nucleocapsids of equine herpesvirus type-l (EHV-1). Virology 64, 187204. PERDUE, M., COHEN, J. C., RANDALL, C. C., and O’CALLAGHAN, D. J. (1976). Biochemical studies of the maturation of herpesvirus nucleocapsid species. Vifology74, 194-208. PRESTON, V. G., COATES, J. A. V., and RIXON, F. J. (1983). Identification and characterization of a herpes simplex virus gene product required for encapsidation of virus DNA. J. Viral. 456, 1056-l 064. RANDALL, R. E., HONESS, R. W., and O’HARE, P. (1983). Proteinsspecified by herpesvirus saimiri: identification and properties of virusspecific polypeptides in productively infected cells. 1. Gen. Viral. 64,19-35. SCHAFFER, P. A., BRUNSCHWIG, J. P., MCCOMBS, R. M., and BENYESHMELNICK, M. (1974). Electron microscopic studies of temperaturesensitive mutants of herpes simplexvirus type 1. Viro/ogy62,444457. SHERMAN, G., and BACHENHEIMER, S. L. (1988). Characterization of intranuclear capsids made by ts morphogenic mutants of HSV-1. Viro/ogy163,471-480. SMITH, J. D., and DE HARVEN, E. (1973). Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. I. Sequence of viral replication. J. Viral. 12,919-930. WAHREN, B., and OBERG, B. (1980). Reversible inhibition of cytomegalovirus replication by phosphonoformate. lntervirology 14, 7-l 5. WEINER, D., and GIBSON, W. (1983). Phosphotylation, maturational processing, and relatedness of strain Colburn matrix proteins. Virology 129, 155-169. WFLAY, W.. BOULIKAS, T., WRAY, V. P.. and HANCOCK, R. (1981). Silver staining proteins in polyacrylamide gels. Anal. Biochem. 118, 197203. ZWEIG, M.. HEILMAN, C. J. JR., RABIN, H., and HAMPAR, B. (1980). Shared antigenic determinants between two distinct classes of proteins in cells infected with herpes simplex virus. J. Viral. 35, 644-652.
167,87-96
(1988)
Primate Cytomegalovirus Assembly: Evidence that DNA Packaging Subsequent to B Capsid Assembly JOANNA Virology
Laboratories,
Department
Y. LEE,’ ALICE IRMIERE,’
AND
WADE GIBSON3
of Pharmacology and Molecular Sciences, The Johns Hopkins 725 North Wolfe Street, Baltimore, Maryland 2 1205 Received
December
29, 1987; accepted
Occurs
University
School
of Medicine,
July 22, 1988
Results presented here show that when cytomegalovirus (strain Colburn)-infected cells are treated with the DNA synthesis inhibitor hydroxyurea or phosphonoformate, one type of intranuclear capsid accumulates. These particles appeared to contain symmetrically organized internal material, and had a protein composition and sedimentation rate characteristic of B capsids. Radiolabeling experiments provided evidence that a population of B capsids lacking DNA is present during the course of a normal infection. These capsids sedimented slightly slower than the peak of viral DNA in the same region of the gradient, and had a ratio of DNA/protein that was estimated to be sevenfold lower than that of the faster sedimenting C capsids. DNA in both the B and C capsid regions of such gradients was found to be relatively resistant to digestion with DNase. The possibility is considered that herpesvirus B capsids lacking DNA may be countero 1988Academic PWSS. IW. parts of unexpanded proheads in the bacteriophage assembly pathway.
proximately 20% of the capsid’s protein mass and is distinguished from the other capsid proteins in at least four ways: (i) it is phosphorylated, (ii) it has multiple forms and appears to undergo proteolytic cleavage, (iii) it exhibits unusual staining properties, and (iv) it is not present, as such, in the mature virion. This abundant protein, called VP22a (Gibson and Roizman, 1972, 1974) p40 (Heilman et al., 1979), NC-3 (Cohen et al., 1980) and ICP35e (Braun et al., 1984), is the HSV counterpart of the B capsid hallmark protein species, the assembly protein (lrmiere and Gibson, 1983, 1985). The gene encoding this HSV protein has been mapped genetically using inter-typic recombinants (Braun et a/., 1983) and a temperature-sensitive mutant, tsVP1201, whose genetic lesion maps to the same region has been described (Preston et al., 1983). At the restrictive temperature this mutant produces DNA and capsids, but is defective in assembly protein processing and DNA packaging. Cytomegalovirus (CMV)-infected cells also contain nuclear A and B capsids (Gibson, 1981; lrmiere and Gibson, 1985) and probable equivalents have been reported in equine herpesvirus types 1 (EHV-1) (Kemp et al., 1974; Perdue et al., 1975) and 3 (EHV-3) (Allen and Bryans, 1976) pseudorabies (PR) (Ben-Porat et a/., 1970; Ladin et al., 1980), and herpes zoster virus (HZV), (Friedrichs and Grosse, 1986) suggesting that these capsid forms are common among the herpesgroup viruses. The finding that the CMV assembly protein exhibits the same set of comparatively unusual characteristics (Gibson, 1981; lrmiere and Gibson, 1985) and that its gene is approximately colinear with
INTRODUCTION Assembly and maturation of the herpesgroup viruses begin in the nucleus of infected cells with the formation of capsids. These are typically about 100 nm in diameter with centers ranging in appearance from “empty” to “filled” with densely stained material. When DNA synthesis is inhibited, capsids with partially filled centers accumulate and then appear to give rise to completely filled particles after DNA synthesis resumes, suggesting that viral DNA is packaged into preformed capsids (Friedmann et al., 1975). This model is supported by results from pulse-chase radiolabeling experiments (O’Callaghan and Randall, 1976; Ladin et al., 1980) and studies using temperature-sensitive mutants (Ladin et a/., 1980; Preston et al., 1983). Experiments using herpes simplex virus (HSV) to study the sequence of events involved in herpesvirus assembly led to the recovery and characterization of two capsid forms, designated as A and B capsids, from infected cell nuclei. B capsids were distinguished by having a faster sedimentation rate, an internal core structure, a higher DNA content, and two proteins not found in A capsids. One of these proteins, VP21, is a minor constituent which also appears to be present in the mature virion (Gibson and Roizman, 1972, 1974). The second is an abundant species that represents ap’ Present address: The Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205. ’ Present address: Halvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, Boston, MA 021 15. ’ To whom requests for reprints should be addressed. 87
0042-6822/88
$3.00
Copyright 0 1998 by Academic Press, Inc. All rights of reproduction in any form resewed.
88
LEE,
IRMIERE,
that of HSV (Robson and Gibson, manuscript submitted for publication) underscores the similarities between HSV and CMV B capsids and suggests that they are functional equivalents. Additional insight into the involvement of B capsids in the assembly pathway, and the interaction of the assembly protein with these particles during nucleocapsid maturation, came from characterizing noninfectious enveloped particles (NIEPs) produced by human strains of CMV (Irmiere and Gibson, 1983). These particles are similar to virions in structure and protein composition, but are distinguished from them by the presence of the assembly protein and the absence of DNA. As discussed before (Irmiere and Gibson, 1983, 1985) these findings suggest that NlEPs represent enveloped B capsids, that the assembly protein becomes a capsid constituent before and independently of DNA packaging, and that elimination of the assembly protein is neither essential for nor a necessary consequence of acquiring the tegument and envelope layers. Since NlEPs do not contain DNA, the hypothesis that they represent enveloped B capsids predicted the existence of a population of CMV B capsids lacking DNA. Two approaches were taken to test this prediction. This first was to determine the relative efficiency of radiolabeling B capsids with DNA precursors, and the second was to determine whether B capsids are made in the presence of inhibitors of DNA synthesis. The results are consistent with the prediction that B capsids lacking DNA exist, and are discussed as they bear on the sequence of events surrounding capsid assembly and DNA packaging. MATERIALS
AND
METHODS
Cells and virus Human foreskin fibroblast cultures were prepared, maintained, and infected as described before (Gibson, 1981). Pertinent characteristics of the Colburn strain of cytomegalovirus have also been described (Gibson, 1981, 1983). Recovery
of virus particles
Details of the procedures used for fractionating infected cells and resolving virus particles by rate-velocity centrifugation have been presented before (Irmiere and Gibson, 1983) and are only summarized here. Cells from one or two 32-0~ bottles (approximately 5 x lo7 cells/bottle) were pelleted, resuspended in 0.5 ml of PB (40 mNI phosphate buffer, 150 mM NaCI, pH 7.4) containing 0.5% NP-40 and 1 mh/l phenylmethylsulfonyl fluoride (PMSF), and held on ice for 2 to 10 min, NP-40 nuclei were colleced by centrifugation and the
AND
GIBSON
supernatant fraction was saved as the NP-40 cytoplasm. Nuclei were resuspended in 0.5 ml of PB containing 1 mM PMSF and disrupted by sonication (D. Witt, personal communication) (5-10 set, setting 4, Branson Sonifier 185,0”), and the lysate was then clarified by centrifugation (1 OK g, 4”, 30 set). Clarified nuclear lysate or NP-40 cytoplasmic fractions were layered onto 15-5096 sucrose (w/w, in PB) gradients and subjected to centrifugation in a Beckman SW41 rotor (40,000 rpm, 4”, 20 min). Particles were recovered from the gradient by aspiration, or gradients were fractionated to record the optical absorbance (&,) pattern and measure radioactivity by scintillation spectrometry (50 ~1 sample, 4 ml Aquasol, New England Nuclear). Polyacrylamide
gel electrophoresis
Protein electrophoresis in sodium dodecyl sulfate (SDS)-containing polyacrylamide gels (SDS-PAGE) was done essentially as described by Laemmli (1970). Pierce SDS was used in the electrode buffer to resolve the Colburn CMV upper and lower matrix proteins (Weiner and Gibson, 1983). Gels were stained with Coomassie brilliant blue (Fairbanks et a/., 197 1) or ammoniacal silver (Wray et a/., 1981) and fluorography was done with diphenyloxazole (Bonner and Laskey, 1974) or calcium tungstate intensifying screens (Laskey and Mills, 1977). Densitometry was done using a transmission densitometer (E-C Apparatus Corp., St. Petersburg, FL). Electron
microscopy
Cells were fixed with 1% glutaraldehyde in cacodylate buffer, postfixed with 1% osmium tetroxide, stained enbloc with 1% aqueous uranyl acetate, dehydrated through a graded series of ethanol, embedded in Epon, sectioned using a Sorvall MT5000 ultramicrotome, stained with lead citrate and uranyl acetate, and examined and photographed using a Zeiss 1OA electron microscope. RESULTS Two series of experiments were done to determine whether CMV intranuclear B capsids lacking DNA could be identified. The first was to test the effect of DNA synthesis inhibitors on capsid production, with the expectation that only capsids which lack DNA would be made in their presence. The second approach was to grow infected cells in the presence of a radiolabeled DNA precursor, separate the intracellular capsid forms by centrifugation, and measure the radioactivity associated with each.
PRIMATE
Appearance of CMV particles produced presence of inhibitors of DNA synthesis
CMOMEGALOVIRUS
in the
This approach was prompted by the finding that inhibitors of DNA synthesis, such as phosphonoformate and hydroxyurea, at concentrations that reduced virion production by two to four orders of magnitude, had comparatively little effect on the production of NlEPs (i.e., enveloped B capsids) (Gibson and Irmiere, 1984). Furthermore, others have found that when DNA synthesis is inhibited, nuclear capsids with partially stained cores and of intermediate density continue to be produced (O’Callaghan et al., 1968; Friedmann et a/., 1975). These experiments were done to examine the possibility that capsids produced under such conditions correspond to B capsids lacking DNA. Hydroxyurea (HU, 5 mNI) was used in the first experiment. This drug is an inhibitor of ribonucleotide reductase, which catalyzes the rate-limiting step in the formation of deoxyribonucleoside triphosphates (reviewed by Cory, 1983) and results in an inhibition of DNA synthesis. Concentrations of HU ranging from 1 to 5 mM reduced the titer of Colburn CMV by lo- to 1O,OOO-fold (Anders et a/., 1986) and had differential effects on infected-cell protein synthesis (e.g., Fig. 3). At late times after infection in the presence of 5 mM HU, when extensive CPE was evident (e.g., 4-6 days), capsid proteins (e.g., MCP, 145-kDa major capsid protein; AP, 37-kDa assembly protein; mCP, 34-kDa minor capsid protein) were made in good yield but were incompletely transported to the nucleus, DB129 the Colburn early DNA-binding protein homolog of HSV ICP8 (Anders et a/., 1986, 1987; Kemble et al., 1987; Anders and Gibson, 1988) was overproduced, the tegument proteins (e.g., BPP, 1 19-kDa basic phosphoprotein; UM and LM, 69- and 66-kDa upper and lower matrix proteins, respectively) were grossly underrespected, and the synthesis of at least two host proteins (e.g., vimentin, actin) was unchanged (Fig. 3) (unpublished results). Similar effects on infected-cell proteins were produced by phosphonoformate (PFA, 200 pg/ml) (Gibson et al., 1984; Anders et a/., 1986) a selective inhibitor of the viral DNA polymerase (Huang, 1975). HU was added to the culture medium 36 hr after infection and 3 days later a drug-treated and nontreated culture were processed and examined by electron microscopy. Representative fields from the nuclear and cytoplasmic compartments of each culture demonstrate two major effects of the drug (Fig. 1). First, there was an absence of virus particles in the cytoplasm of drug-treated cells (Panel D). Enveloped capsids (e.g., virions), which are normally abundant in the cytoplasm of infected cells (Panel C), were found only rarely in cells treated with HU. Second, capsids present in the
89
ASSEMBLY
nucleus of drug-treated cells were comparatively uniform in appearance (Panel B). These particles characteristically showed a pattern of internally stained material that often appeared as a concentric six-beaded ring, approximately 55 nm outside diameter and 15 nm thick [seen better in Panel A, arrows, and, for HCMV, in Gibson et a/. (1984)]. Fewer, but otherwise indistinguishable nuclear particles were observed in infected cells treated with higher concentrations of hydroxyurea (10 mn/l) or with PFA (200 pg/ml) (data not shown). Nuclei of nontreated infected cells contained similar particles (see arrows, Panel A), but in contrast to drugtreated cells, also contained numerous capsids with densely stained internal material. Measurements of particles in the nucleus of nontreated cells (Panel A) indicated that the outside diameter of capsids with a beaded internal appearance (see arrows), like those in hydroxyurea-treated cells, is approximately 7% smaller than the others. Capsids position
in drug-treated of B capsids
cells sediment
close to
Infected cells were incubated in media containing HU (5 mM) or PFA (200 pg/ml), harvested, lysed, and subjected to rate-velocity sedimentation to identify intracellular virus particles. The infected cells were lysed in 0.1 M glycine (Hallauer and Kronauer, 1965) (pH 9.5, 37”, 75 min) to avoid the use of detergents and to obtain a whole cell lysate, rather than separate nuclear and cytoplasmic fractions. Optical absorbance profiles of the three gradients (Fig. 2) demonstrate that (i) drugtreated cultures showed significant absorbance only at the B capsid position; (ii) the HU-treated culture yielded approximately 40% more B capsids than nontreated cells; (iii) the PFA-treated culture gave the smallest yield of B capsids (i.e., about 50% that recovered from the nontreated culture); and (iv) C capsids were not recovered from either drug-treated culture. The broad zone of optical absorbance sedimenting ahead of C capsids was reduced in the drug-treated cultures. This region of the gradient normally contains substantial amounts of upper and lower matrix proteins (e.g., Fig. 3, top) but their synthesis is markedly reduced when viral DNA synthesis is inhibited (Fig. 3, bottom) (Gibson eta/., 1984; Anders eta/., 1986; additional unpublished results). When the pellet resulting from glycine extraction of HU-treated cells was further extracted with DOC, DNase, Brij 58, and urea (Gibson, 1981) additional B capsids (40-500~ amount extracted with glytine) and some A capsids were released (bottom panel, Fig. 2). Comparable results were obtained when these drugs, at the same respective concentrations, were added at the time of infection rather that 36 hr
LEE,
IRMIERE,
No Drug
FIG. 1. Thin sections of ceils infected with CMV in the presence or Drug) or presence (Hydroxyurea) of 5 mM hydroxyurea, as described in fields from the nuclear (Nucleus) and cytoplasmic (Cytoplasm) regions appearance that resemble those in (B). Arrow in (C) indicates virion containing vessicle. Bar in (B) indicates 100 nm.
after infection. However, under these conditions, unless the drug-treated cultures were allowed additional incubation time (e.g., l-2 days longer than nontreated cultures) the yield of B capsids was lower (data not shown). Capsids in H&treated protein composition
cells have B capsid-like
The protein composition of the capsids present in HU-treated cells was examined to determine their rela-
AND
GIBSON
H ydroxyurea
absence of hydroxyurea. Infected cells were grown in the absence (No the text, and were then processed for EM. Shown here are representative of each preparation. Arrows in (A) indicate capsids with a beaded internal envelope layer to distinguish it from the membrane of the larger virion-
tionship to nuclear B capsids. Infected cultures were grown either with 5 mM HU added at the time of infection or without drug, labeled with [35S]methionine (20 &i/ml) beginning 2 days after infection, harvested 5 days after infection, and treated with 0.5% NP-40 to prepare NP-40 nuclei. The nuclei were ruptured by sonication and the resulting clarified lysate was subjected to rate-velocity sedimentation in sucrose gradients. The gradients were fractionated and aliquots of each fraction were solubilized and analyzed by SDSPAGE. Fluorograms prepared from the resulting gels
PRIMATE
CYTOMEGALOVIRUS
91
ASSEMBLY
1983) from the +HU preparation. The upper panel also shows that the distribution in the gradient of several other proteins (e.g., BPP, 90K, and 78K) follows that of
Sedimentation
+
-MCP
-78~ rMPs
-D&i1 .45K .AP -mCP .28~
HU Pelleti DOC/DNase
-MCP -BPP
0
Sedimentation-
FIG. 2. Rate-velocity sedimentation of intracellular virus particles in drug-treated and nontreated cultures. CMV-infected cells maintained in the absence of added drug (No Drug), or in the presence of 5 mM hydroxyurea (+HU) or 200 &ml phosphoformic acid (+PFA), were glycine extracted and analyzed by rate-velocity sedimentation as described in the test. The glycine-extracted infected-cell pellet was further extracted with a combination of DOC and DNase and analyzed in parallel (HU Pellet + DOC/DNase). Shown here are absorbance (A280) patterns recorded from the resulting sucrose gradients. The positions of A, B, and C capsids are indicated.
-45K -AP
-28K
I
(Fig. 3) showed that the particles at the B capsid position in the “+HU” preparation had a characteristic B capsid protein composition; in particular they contained the assembly protein and its related 45-kDa form (45K) (Gibson, 1981). This figure also shows that the capsid proteins from the “no drug” preparation were more broadly distributed through the gradient, indicating greater heterogeneity in the structures present. A capsids (indicated in top panel) were detected only in the no drug preparation. The other notable difference between the two patterns is the absence of the DNAbinding protein DB51 (Gibson, 1983, 1984) and the matrix proteins (Weiner and Gibson, 1983; Gibson,
1
I
I,
I
5
II
I,
10
Fraction
I
I
*I,
I
15
ZII
20
Number
FIG. 3. Protein distribution in sucrose gradients following rate-velocity sedimentation of nuclear lysates from HU-treated and nontreated, infected cells. Nuclear lysates of HU-treated and nontreated, infected cultures were prepared and subjected to rate-velocity sedimentation as described in the text. Samples of the fractionated gradients were subjected to SDS-PAGE (10% gel). Shown here are fluorographic images of the resulting gels. Protein abbreviations in the right margin are explained elsewhere (Gibson, 1983) and are as follows: MCP (145-kDa major capsid protein), BPP (119-kDa basic phosphoprotein), 78K (78-kDa band), MPs (69 and 66-kDa upper and lower matrix proteins, respectively), 45K (45-kDa B capsid protein), AP (37-kDa assembly protein), mCP (34-kDa minor capsid protein, 28K (28.kDa capsid protein).
92
LEE,
IRMIERE.
A and B capsids, suggesting that they are specifically associated. An additional experiment was done to determine how this B capsid protein pattern was influenced by the time of HU addition. HU (5 mM) was added to pairs of cultures at the time of infection, or on Day 1 or Day 2 after infection. Two days after adding the drug (i.e., on Days 2, 3, and 4, respectively, after infection) one culture of each pair was labeled with [35S]methionine (20 &i/ml) and the other with 32Pi (200 &i/ml). A pair of cultures not treated with HU was labeled on Days 2 through 4 after infection and similarly processed. The infection progressed more rapidly in the cultures lacking HU and they were harvested and processed 4 days after infection. Cultures treated with HU on Days 0, 1, and 2 were harvested and processed on Days 4,5, and 6 after infection, respectively, times at which each showed strong cytopathic effect (e.g., cell rounding) but minimal cell detachment. Following rate-velocity sedimentation, the B capsid bands were recovered from the tubes by aspiration, concentrated by pelleting, and solubilized and subjected to SDS-PAGE. Fluorograms prepared from the resulting gel (Fig. 4) showed that the [35S]methionine-labeled preparations looked very similar. Preparations from HU-treated cultures, however, contained (i) decreased amounts of the basic phosphoprotein and matrix proteins, and (ii) increased amounts of the DNA-binding protein DB129 (see arrow) (Anders et a/., 1986) and a host cell protein indicated by a circle just above the 45K band (similarly indicated in Fig. 3). With the possible exception of DB129, these differences reflect gross changes in the amounts of these proteins throughout the gradient as seen with the matrix proteins and DB51 in Fig. 3. The 32P-labeled preparations showed that the level of assembly protein phosphorylation was not markedly influenced by adding HU to the cultures. This figure also demonstrates that the 38- and 39-kDa proteins (see circles just above assembly protein) (Irmiere and Gibson, 1985) and the 45K protein, all of which are related to the assembly protein (Gibson, 1981; unpublished immunological and peptide comparisons), are all phosphorylated. No attempt was made to quantify data from these preparations because of the differences in their radiolabeling interval. B capsids lacking DNA detected in presence absence of DNA synthesis inhibitors
or
These experiments were done in order to determine whether B capsids lacking DNA are produced during the course of a normal infection (i.e., no drug). Infected cells were radiolabeled with [3H]thymidine ([3H]TdR, 10 &i/ml), beginning 36 hr after infection, and intracellu-
AND
GIBSON
Day HU Added
BPF 78K- : MPs
AP mCP-
FIG. 4. Proteins in capsid preparations following separation by SDS-PAGE. CMV-infected cells were grown without drug (-) or in 5 mM HU added at the time of infection (0) or 1 or 2 days after infection [%]methionine (%-Met.) or ‘*/: was added as described in the text. Particles were recovered, concentrated, and subjected to SDSPAGE in a 10% gel. Shown here is a fluorographic image of the resulting gel. Protein abbreviations are as described in the legend to Fig. 3.
lar virus particles were analyzed 30 hr later. Cells were separated into nuclear and cytoplasmic fractions following treatment with NP-40, and the resulting nuclei were disrupted by sonication and clarified by centrifugation, all as described above and under Materials and Methods. The nuclear supernatant fraction was made 5 mhll in MgCI, and divided in half: DNase (10 pg/ml final concentration) was added to one half and both were incubated at 37” for 10 min and then subjected to rate-velocity centrifugation through sucrose gradients. Optical absorbance profiles were recorded as the gradients were collected, and portions of each resulting fraction were analyzed for their protein and DNA content, respectively, by SDS-PAGE and scintillation spectrometry.
PRIMATE
CYTOMEGALOVIRUS
93
ASSEMBLY
Sedimentation -
.07 .06
.02
.Ol I
.
10
. .
..,..
.
15
.
20
.
-
IO
15
20
Fraction Number FIG. 5. Effect of DNase treatment on distribution of nuclear capsids during rate-velocity sedimentation, A nuclear lysate of [3H]TdR-labeled infected cells was prepared; one half was treated with DNase and both were subjected to rate-velocity sedimentation as described in the text. Shown here are the absorbance patterns recorded as the gradients were collected. The positions of A, B. and C capsids are indicated.
The positions of A, B, and C capsids in each gradient were determined from the resulting absorbance (Fig. 5) and SDS-PAGE (Fig. 6) patterns. B capsids were most concentrated in fraction 14 of the nontreated preparation and fraction 13 of the nuclease-treated preparation, as shown by the relative abundance of the assembly protein (AP, Fig. 6). A capsids were located in fractions 11 and 12 (No DNase) or 11 (+DNase), and C capsids were present in fraction 20 (No DNase) or 19 (+DNase). The C capsid bands were coincident with peaks of radioactivity, indicating the presence of DNA (bottom panel, Fig. 6). The majority of the B capsid optical absorbance and protein staining, however, was in fractions on the trailing edge of the peak of [3H]TdR radioactivity, ratherthan coincident with it. Specifically, the peak of B capsid absorbance and protein staining was fraction 14 but the peak of radioactivity was in fraction 15 (No DNase). This separation is seen better in the +DNase preparation where the maximum of B capsid optical absorbance and protein staining was fracton 13 (a relative minimum in the radioactivity plot), while the radioactivity peak fraction was 14. Little, if any, DNA was present at the A capsid position. Interestingly, the basic phosphoprotein (BPP) was the only species noted whose abundance correlated well with the amount of DNA present in the capsid region (e.g., fractions 1O-22, +DNase) of such gradients. Calculations made from these data indicate that CMV B capsids contain about four times as much DNA as A capsids, and that C capsids contain approximately seven times as much DNA as B capsids (Table 1).
I ..*e-
-AP
-IncP
‘*t=
I
1
6
10
5
1
15
No DNase
1-u 1
5
10
15
20
25
30
' - '2b'
r
25'
'$0'
'
+ DNase
I I
1
5
10
15
20
25
30
Fraction Number FIG. 6. Protein and DNA distributions in gradients shown in Fig. 5. Portions of each fractionated gradient were analyzed by SDS-PAGE (10% gels) and scintillation spectrometry. The top two panels show the resulting silver stained polyacrylamide gels. Protein designations are as described in the legend to Fig. 3. Small circles in right margin indicate positions of upper and lower matrix proteins, respectively; smudginess in this region is due to a silver staining artifact band. Lower two panels show [3H]TdR distribution in these gradients. Arrow in right panel indicates that B capsids were more concentrated in fraction 13 than 14. Note that radioactivity is recorded on a logarithmic scale.
94
LEE,
IRMIERE, TABLE
AND
GIBSON
1
ESTIMATED RELATIVE DNA CONTENTOF
CMV
Capsid A Treatment No DNase + DNase Average Ratio Note. s cpm b AZsO c cpm d cpm
cpma
A 280b
650 387
2.6 1.2
CAPSID FORMS
form
B cPmh80
comC
250 322 286
22,200 25,552 B:A = 4.2
A 280 20.5 19.2
C cpmd
A 280
cpm/A28o
19,400 21,049 8,215 C:B = 6.8
2.5 2.4
7660 8770
wmlA280 1081 1334 1207
Calculations are based on measurements obtained from data in Figs. 5 and 6. rH]TdR in fractions 11 and 12 (No DNase) and fraction 11 (+DNase); background subtracted. was calculated as f (peak height X peak width at base); expressed in arbitrary units. [3H]TdR in fractions 13 and 14 (No DNase) and fractions 13 and 14 (+DNase); background subtracted. [3H]TdR in fraction 20 (No DNase) and fraction 19 (+DNase); background subtracted.
Under the conditions of the experiment described above, DNase treatment had no detected effect on the distribution of radioactivity in the gradients. At higher concentrations of DNase (e.g., 100 pg/ml), however, treated samples showed a greater percentage of the total radioactivity present at the top of the gradient. DNA in the B and C capsid peak areas remained comparatively resistent to the nuclease (i.e., 80-90% still present). Only viral DNA was present in the B and C capsid regions as determined by (i) its boyant density (1.71 g/cm3 relative to an internal marker of 1.703 g/ cm3); (ii) its DNA fragmentation pattern following cleavage with restriction endonuclease EcoRI; and (iii) the findings that neither particles recovered in the same way from cells labeled with [3H]TdR for 2 days prior to infection, and then infected and maintained in the absence of radiolabel, nor radiolabeled mock-infected cells showed peaks of radioactivity in these regions of the gradient. When similar experiments were done with HU-treated cultures (5 or 10 pg/ml, added beginning 24-48 hr after infection), essentially all of the DNA (i.e., [3H]TdR) was present at the top of the gradient; little if any sedimented coincident with the B capsid peak (data not shown). DISCUSSION This report shows that when cytomegalovirus (strain Colburn) RNA synthesis is inhibited with hydroxyurea or phosphonoformate (or aphidicolin or novobicin, data not shown), a comparatively homogeneous population of intranuclear capsids accumulate. These particles, recovered after lysing cells or nuclei by detergent treatment (Gibson, 1981) freezing and thawing (Irmiere and Gibson, 1985) sonication (Figs. 3-6) or glycine extraction (Fig. 2), were found to have the sedimentation
properties and distinguishing protein composition (i.e., presence of 37-kDa assembly protein) of B capsids. Since these particles are produced under conditions where viral DNA synthesis is inhibited, it is evident that the assembly protein can become a capsid component prior to DNA packaging, as concluded before (Irmiere and Gibson, 1983,1985). Sherman and Bachenheimer (1988) have drawn the same conclusion from studies using temperature-sensitive mutants of HSV-1. Evidence that B capsids lacking DNA are also produced during the course of a normal infection was provided by the observation that the peak of B capsid protein mass was not coincident with the peak of viral DNA in the same region of the gradient (Figs. 5 and 6). The sevenfold lower ratio of DNA to protein (i.e., cpm [3H]TdR/ODZ8J in the B capsid region of the gradient, compared to the C capsid region (Table l), is consistent with the presence of B capsids lacking DNA. It is unclear whether these empty B capsids are precursors of DNA-containing particles or represent abortive products of assembly. If they are precursors, it would be expected that they could be chased into DNA-containing particles after removing the block to DNA synthesis. Although we have been unable to demonstrate a precursor/product relationship by such experiments (unpublished results), this could be explained if the capsids were only transiently competent to package DNA, or if there is a tight coupling between viral DNA synthesis and encapsidation. The finding that HSV-1 DNA, produced by certain temperaturesensitive mutants grown at the restrictive temperature, could be chased into particles upon shift down to the permissive temperature, while preformed empty B capsids could not (Sherman and Bachenheimer, 1987, 1988) is consistent with our findings for CMV and is
PRIMATE
CYTOMEGALOVIRUS
more compatible with the possibility of a transient competence for DNA packaging than a requirement for concomitant DNA and capsid synthesis. The presence in nontreated, infected cells of capsids corresponding in appearance to those that accumulated in hydroxyurea-treated cells (Fig. 1) indicates that these particles are not simply artifacts of the drug treatment, and is consistent with the identification of empty B capsids in lysates of normally infected cultures (Fig. 6). Similar looking particles are commonly observed in the nucleus of herpesvirus-infected cells, and are consistently enriched for when viral DNA synthesis (Friedmann eta/., 1975; O’Callaghan, eta/., 1968) or packaging (Schaffer e? al., 1974; Ladin et a/., 1980, 1982; Preston et a/., 1983; Gibson et al., 1984) is inhibited. The absence of DNA in these CMV capsids establishes that their characteristic internal staining pattern, often appearing in cross-section as a beaded concentric ring, is not due to DNA. O’Callaghan and Randall (1976) have suggested that such internal staining of EHV intermediate density capsids is due to VPIII, the apparent EHV assembly protein counterpart. It is unresolved, however, whether the assembly protein is internal and itself constitutes the stained material or whether it is located elsewhere (e.g., surface) (Braun et al., 1984) and causes the staining pattern as an indirect effect (e.g., conformational alteration) of its interaction with other capsid constituents. Results presented here and elsewhere (Gibson and Roizman, 1972; O’Callaghan and Randall, 1976; Ladin, 1982; Preston et al., 1983; lrmiere and Gibson, 1983; Sherman and Bachenheimer, 1988) are compatible with the possibility that the assembly protein is the herpesvirus counterpart of the bacteriophage scaffolding protein. The scaffolding protein mediates the formation of an unexpanded prohead, and this structure is subsequently converted to an expanded prohead in conjunction with protein processing events, including proteolytic cleavage, that result in topological changes in the prohead’s structure (for review see Black and Showe, 1984). During this process the internally located scaffolding protein is either eliminated from the head and recycled (King and Casjens, 1974) or cleaved into small peptides (Laemmli, 1970). Resulting expanded proheads are competent to package DNA and become filled heads. Like the scaffolding protein, the herpesvirus assembly protein species (i) is an abundant constituent of capsids lacking a full complement of DNA (Figs. 3 and 6) (Perdue et a/., 1975; Sherman and Bachenheimer, 1988), (ii) is associated with capsids smaller in diameter than DNA-containing capsids (Fig. I), (iii) undergoes processing, probably including proteolytic cleavage (Gibson and Roizman, 1974; Ladin et a/., 1982; Preston et al., 1983; Braun et a/., 1984; Gibson
95
ASSEMBLY
et a/., submitted for publication: Robson and Gibson, submitted for publication), and (iv) is not present intact in the mature virion (Gibson and Roiz’man, 1972, 1974; Kemp et a/., 1974; Gibson, 1981; Ladin et al., 1982; lrmiere and Gibson, 1985).
ACKNOWLEDGMENTS We thank Donna Woods for excellent technical support with electron microscopy, and Glenn Sherman and Steve Bachenheimer for sharing their results with us prior to publication. This work was aided by Research Grants Al 13718, Al2271 1, and All 9373 from the National Institutes for Allergy and Infectious Diseases. J.Y.L. was a predoctoral fellow in the Pharmacology and Molecular Sciences Training Program and was supported by Public Health Service Training Grant GM07626. A.I. was a predoctoral fellow in the Biochemistry, Cellular, and Molecular Biology Training Program.
REFERENCES ALLEN, G. P., and BRYANS, J. T. (1976). Cell-free synthesis of equine herpesvirus type 3 nucleocapsid polypeptides. Virology 69, 751762. ANDERS, D. G., and GIBSON, W. (1988). Location, transcript analysis, and partial nucleotide sequence of the cytomegalovirus gene encoding an early DNA-binding protein with similarities to ICP8 of herpes simplex virus type 1. J. Viral. 62, 1364-l 372. ANDERS, D. G., IRMIERE, A., and GIBSON, W. (1986). Identification and characterization of a major early cytomegalovirus DNA-binding protein. J. Viral. 58, 253-262. ANDERS, D. G., KIDD, J. R., and GIBSON, W. (1987). immunological characterization of an early cytomegalovirus single-strand DNA binding protein with similarities to the HSV major DNA-binding protein. Virology 161, 579-588. BEN-P• RAT, T., SHIMONO, H., and KAPLAN, A. S. (1970). Synthesis of proteins in cells infected with herpes virus. IV. Analysis of the proteins in viral particles isolated from the cytoplasm and nucleus. Virology 41,256-264. BLACK, L. W., and SHOWE, M. K. (1984). Morphogenesis of the T4 head. In “Bacteriophage T4” (C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget, Eds.). pp. 219-245. ASM, Washington, DC. BLAIR, E. G., and HONESS, R. W. (1983). DNA-binding proteins specified by herpesvirus saimiri. 1. Gen. Viral. 64, 2697-2715. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyactylamide gels. Eur. J. Biochem. 46,83-88. BRAUN, D. K., PEREIRA, L., NORRILD. B., and ROIZMAN, B. (1983). Application of denatured, electrophoretically separated, and immobilized lysates of herpes simplex virus-infected cells for the detection of monoclonal antibodies for studies of the properties of viral proteins. J. Viral. 46, 103-l 12. BRAUN, D. K., ROIZMAN, B., and PEREIRA, L. (1984). Characterization of post-translational products of herpes simplexvirus gene 35 proteins binding to the surfaces of full capsids but not empty capsids. 1. Viral. 59, 142-l 53. COHEN, G. H., PONCE DE LEON, M., DIGGELMANN, H., LAWRENCE, W. C., VERNON. S. K., and EISENBERG, R. J. (1980). Structural analysis of the capsid polypeptides of herpes simplex virus types 1 and 2. J. Virol. 34, 521-53 1. CoRY, J. G. (1983). Role of ribonucleotide reductase in cell division, Pharmac. Ther. 21.265-276.
96
LEE,
IRMIERE,
FAIRBANKS, G., STECK, T. L., and WALLACH, D. F. (1971). ElectrophoretiC analysis of the major polypeptides of the human erythrocyte membrane. Biochemisfry 10,2606-2617. FRIEDMANN, A., COWARD, J. E., ROSENKRANZ, H. S.. and MORGAN, C. (1975). Electron microscopic studies on assembly of herpes simplex virus upon removal of hydroxyurea block. 1. Gen. Viral. 26, 171-181. FRIEDRICHS, W. E., and GROSE, C. (1986). Varicella-zoster p32/p36 complex is present in both the viral capsid and the nuclear matrix of the infected cell. J. Vifol. 57, 155-l 64. GIBSON, W. (1981). Structural and nonstructural proteins of strain Colburn cytomegalovirus. virology 111, 516-537. GIBSON, W. (1983). Protein counterparts of human and simian cytomegaloviruses. l/ifo/ogy 128, 39 l-406. GIBSON, W. (1984). Synthesis, structure and function of a cytomegalovirus major nonvirion nuclear protein. UCLA Symp. Mol. Cell. Biol. 21,423-440. GIBSON, W., and IRMIERE, A. (1984). Selection of particles and proteins for use as human cytomegalovirus subunit vaccines. In “Birth Defects: Original Article Series,” Vol. 20, pp. 305-324. A. R. Liss, New York. 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-l 052. GIBSON, W., and ROIZMAN, B. (1974). Proteins specified by herpes simples virus. X. Staining and radiolabeling properties of B capsid and virion proteins in polyactylamide gels. J. Viral. 13, 155-l 65. GIBSON, W., VAN BREEMEN, R., FIELDS, A., LAFEMINA, R., and IRMIERE, A. (1984). o,L-Difluoromethylornithine inhibits human cytomegalovirus replication. J. Viral. 50, 145-l 54. HALLAUER, C., and KRONAUER, G. (1965). Extraction of cell-associated virus without damage of the culture. Asch. Ges. Virusgorsch. 15, 433-440. HEILMAN, C. J., JR., ZWEIG, M., STEVENSON, J. R., and HAMPAR, B. (1979). Isolation of a nucleocapsid polypeptide of herpes simplex virus types 1 and 2 possessing immunologically type-specific and cross-reactive determinants. J. Viral. 29, 34-42. HUANG, E.-S. (1975). Human cytomegalovirus. IV. Specific inhibition of virus-induced DNA-polymerase activity and viral DNA replication by phosphonoacetic acid. J. Viral. 16, 1561-l 565. IRMIERE, A., and GIBSON, W. (1983). Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus. virology 131, 1 18-l 33. IRMIERE. A., and GIBSON, W. (1985). Isolation of human cytomegalovirus intranuclear capsids, characterization of their protein constituents, and demonstration that the B-capsid assembly protein is also abundant in noninfectious enveloped particles. J. Viral. 56, 277283. KEMBLE, G. W., MCCORMICK, A. L., PEREIRA, L., and MOCARSKI, E. S. (1987). A cytomegalovirus protein with properties of herpes simplex virus ICP8: Partial purification of the polypeptide and map position of the gene. J. Viral. 61, 3 143-3 15 1. KEMP, M. C., PERDUE, M. L., ROGERS, H. W., O’CALLAGHAN, D. J., and RANDALL, C. C. (1974). Structural polypeptides of the hamster strain of equine herpexvirus type 1: Products associated with purification. Virology 71,361-375. KING, J., BOTSTEIN, D., CASJENS, S., EARNSHAW, W., HARRISON, S., and LENK, E. (1976). Structure and assembly of the capsid of bacteriophage P22. Philos. Trans. R. Sot. London Ser. B 276,37-49. KING, J., and CASJENS, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature (London) 251, 112-l 19. LADIN, B. F., BLANKENSHIP. M. L.. and BEN-P• RAT, T. (1980). Replication of herpesvirus DNA. V. Maturation of concatameric DNA of
AND
GIBSON
pseudorabies virus to genome length is related to capsid formation. J. Viral. 33, 1151-l 164. LADIN, B. F., IHARA, S., HAMPL, H., and BEN-P• RAT, T. (1982). Pathway of assembly of herpesvirus capsids: An analysis using DNA+ temperature-sensitive mutants of pseudorabies virus. Virology 116, 544-561. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biothem. 56,335-341. LASKEY, R. A., and MILLS, A. D. (1977). Enhanced autoradiographic detection of 32P and ‘? using intensifying screens and hypersensitized film. FEBS Len 82,314-316. MURIALDO, H., and BECKER, A. (1978). Head morphogenesis of complex couble-stranded deoxyribonucleic acid and bacteriophages. Microbial. Rev. 47, 529-576. O’CALLAGHAN, D. J., HYDE, H. M., GENTRY, G. A., and RANDALL, C. C. (1968). Kinetics of viral deoxyribonucleic acid, protein, and infectious particle production and alterations in host macromolecular synthesis in equine abortion (herpes) virus-infected cells. 1. Viral. 36,104-114. O’CALLAGHAN, D. J., KEMP, M. C., and RANDALL, C. C. (1977). Properties of nucleocapsid species isolated from an in vivo herpesvirus infection. J. Gen. Viral. 37, 585-594. O’CALLAGHAN, D. J., and RANDALL, C. C. (1976). Molecular anatomy of herpesviruses: Recent studies. Pfog. Med. Viral. 22, 152-210. PERDUE, M. L., COHEN, J. C.. KEMP, M. C., RANDALL, C. C.. and O’CALLAGHAN, D. J. (1975). Characterization of three species of nucleocapsids of equine herpesvirus type-l (EHV-1). Virology 64, 187204. PERDUE, M., COHEN, J. C., RANDALL, C. C., and O’CALLAGHAN, D. J. (1976). Biochemical studies of the maturation of herpesvirus nucleocapsid species. Vifology74, 194-208. PRESTON, V. G., COATES, J. A. V., and RIXON, F. J. (1983). Identification and characterization of a herpes simplex virus gene product required for encapsidation of virus DNA. J. Viral. 456, 1056-l 064. RANDALL, R. E., HONESS, R. W., and O’HARE, P. (1983). Proteinsspecified by herpesvirus saimiri: identification and properties of virusspecific polypeptides in productively infected cells. 1. Gen. Viral. 64,19-35. SCHAFFER, P. A., BRUNSCHWIG, J. P., MCCOMBS, R. M., and BENYESHMELNICK, M. (1974). Electron microscopic studies of temperaturesensitive mutants of herpes simplexvirus type 1. Viro/ogy62,444457. SHERMAN, G., and BACHENHEIMER, S. L. (1988). Characterization of intranuclear capsids made by ts morphogenic mutants of HSV-1. Viro/ogy163,471-480. SMITH, J. D., and DE HARVEN, E. (1973). Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. I. Sequence of viral replication. J. Viral. 12,919-930. WAHREN, B., and OBERG, B. (1980). Reversible inhibition of cytomegalovirus replication by phosphonoformate. lntervirology 14, 7-l 5. WEINER, D., and GIBSON, W. (1983). Phosphotylation, maturational processing, and relatedness of strain Colburn matrix proteins. Virology 129, 155-169. WFLAY, W.. BOULIKAS, T., WRAY, V. P.. and HANCOCK, R. (1981). Silver staining proteins in polyacrylamide gels. Anal. Biochem. 118, 197203. ZWEIG, M.. HEILMAN, C. J. JR., RABIN, H., and HAMPAR, B. (1980). Shared antigenic determinants between two distinct classes of proteins in cells infected with herpes simplex virus. J. Viral. 35, 644-652.