Immunoprecipitable polypeptides specified by varicella-zoster virus

VIROLOGY

(1982)

1X3,86-95

lmmunoprecipitable

Polypeptides

Specified by Varicella-Zoster

CHARLES GROSE’ AND WILLIAM Departmats

Virus

E. FRIEDRICHS

of Pediatrics and Miorobiology, University of Texas Health Science Center, San Antonio, Texas 78284 Received August 27, 1981;accepted November 25, 1981

Polypeptides encoded by varicella-zoster virus (VZV) in infected cell cultures have been identified by radioimmune precipitation techniques. Detergent-solubilized extracts of VZV-infected cells were reacted with highly specific VZV antisera raised in strain-2 guinea pigs immunized with sonicates of syngeneic virus-infected cells. Fractionation of the immunoprecipitates in acrylamide slab gels demonstrated an average of 16 polypeptides, which ranged in molecular weight from 32,000to ~200,000. These included the three major immunogenic glycoproteins (gp 62, gp 98, and gp 118) and a prominent higher molecular weight nonglycosylated polypeptide at 155,000. One of the [‘%]methioninelabeled polypeptides comigrated with purified actin. Not all polypeptides were visible in any one particular Auorogram, but comparative analysis of polypeptide profiles derived from electrophoreses performed with different gel concentrations and different crosslinkers (methylene-bisacrylamide and N,N-diallyltartardiamide) clearly established a consistent and reproducible pattern of radioactive bands. A low background of radioactivity was nonspecifically precipitated by the antigen-antibody-protein A complexes; however, with the exception of a common band comigrating with actin, the electrophoretic profiles representing virus-specific and nonspecific immunoprecipitates were easily distinguished. INTRODUCTION

fected cell specific (ICS) glycopolypeptides correlate in molecular weight with VZVspecific immunoprecipitable glycoproteins (Grose et al., 1981a). To facilitate identification of the more numerous nonglycosylated gene products of viral origin, we have precipitated ICS polypeptides using highly specific VZV immune sera. These antisera raised in strain-2 guinea pigs (Grose, 1981) have proven to be extremely useful in the experiments described herein because they possessed little or no reactivity against cellular determinants or other human herpesviruses.

Varicella-zoster virus (VZV) is one of the most difficult human herpesviruses to characterize at a molecular level because it remains predominantly cell associated throughout the infectious cycle (Weller, 1953). Attempts at virus purification by conventional methods of density gradient sedimentation usually result in considerable aggregation with host cell elements as well as loss in structural integrity (Grose et al., 1979). Since VZV infection of cultured cells requires several cycles before cytopathic effect is complete, some host cell protein synthesis continues even when maximal infectivity is recoverable from the culture. Nevertheless, conditions have been defined for radiolabeling glycoproteins encoded by the VZV genome in cells exhibiting advanced cytopathology, when glycosylation of host cell proteins is extremely restricted (Grose, 1980). The in-

METHODS

Origin of cells and virus. The cell substrate for VZV propagation was the Mewo strain of human melanoma cells (HMC) (Grose et al., 1979). Monolayer cell cultures were grown in Eagle minimal essential medium supplemented with 2 mlM glutamine, 1% nonessential amino acids, pen-

1To whom reprint requests should be addressed. 0042-6822/82/050086-10$02.00/O C%wisbt 8 1082 by Academic Press, Inc. All riebta of mpmductIon in any form rewrved.

86

POLYPEPTIDES OF VARICELLA-ZOSTER TABLE 1 ANTIBODYRESPONSE OF STRAIN-2 GUINEA PIGS FOLLOWING VZV IMMUNIZATION FAMA” titer Weeks after booster immunization Guinea pig number i ii . ..

111

iv

1

3

6

1024 1024 512 512

512 512 256 512

512 512 128 256

’ Indirect fluorescence assay to detect antibody directed against VZV-induced membrane antigens (Grose, 1981). The titer is expressed as the reciprocal of the highest positive serum dilution.

icillin (100 U/ml), streptomycin (100 pg/ ml), and 8% fetal bovine serum (MEMFBS). The “VZV-32” strain of VZV was isolated from the vesicular fluid of a young boy with chickenpox (Grose et al., 1979) and serially passaged no more than 20 times by trypsin-dispersal of infected cells. Early-passage stocks of VZV-infected cells were cryopreserved in a freeze-dried state (Grose et al., 1981b). Virus

infection

and isotopic

labeling.

HMC monolayer cultures (25 and 75 cm’) were subcultivated at a 1:2 split ratio at 36”. Within 12 hr, subconfluent cultures were seeded with one-fourth equivalent monolayer of trypsin-dispersed VZV-infected cells and incubated at 32”. One day later the culture medium was replaced with either MEM-FBS containing 10 &i of tritiated fucose per ml or, alternatively, methionine-deficient medium supplemented with [%]methionine (10 &Ji/ ml). The cultures were incubated at 32” for an additional 24 hr, by which time cytopathic effect covered virtually the entire monolayer. VZV immune caviid sera. VZV-specific antisera were raised in four strain-2 guinea pigs immunized with VZV-32 strain passaged eight times in transformed caviid embryo cells, as previously described (Edmond et al., 1981; Grose, 1981). Antisera generated within this novel syngeneic sys-

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tem develop no reactivity against uninfected cellular determinants, nor do they cross-react with viral antigens present in cells infected with either herpes simplex virus I or human cytomegalovirus when tested by indirect immunofluorescence (titer <1:4). Three sets of sera were collected from the guinea pigs between 1 and 6 weeks after the booster inoculations. The titers of VZV-antibody as determined by indirect immunofluorescence are given in Table 1. Polyaqlamide gel electrophoresis and jlwrograph~. Slab gels were cast with ei-

ther 8 or 10% acrylamide and cross-linked with either 2.7% methylene-bisacrylamide (MBA) or an equal amount of NJdiallyltartardiamide (DATD). The radioactive samples were added to wells in a 4% acrylamide stacking gel and subjected to electrophoresis under constant voltage (100 V) or constant current (15 mA) in Trig-glycine buffer (pH 8.1) containing 0.1% sodium dodecyl sulfate as described (Maizel, 1971; Grose, 1980). Upon completion of electrophoresis, the slab gels were suffused with diphenyloxazole dissolved in dimethyl sulfoxide prior to drying (Bonner and Laskey, 1974). Exposure times of the dried gels to Kodak XRP-5 film at -70” ranged from 3 to 14 days. Immunoprecipitation. Virus-infected and uninfected HMC monolayers radiolabeled with [3H] fucose or [35S]methionine were washed and resuspended in modified Schwyzer’s buffer without detergents (Schwyzer, 1977). The suspensions were disrupted by sonication and centrifuged at low speed to remove debris (Grose et al., 1979). Solubilization was accomplished by the addition of detergents Nonidet P-40 (NP-40) and deoxycholate (DOC) to final concentrations of 1% (v/v) and 1% (w/v), respectively. After a 20-min incubation, insoluble material was removed by sedimentation at 85,000 g. The resulting clarified extracts from VZV-infected monolayers (75 cm2) contained -3.5 X lo5 cpm of [3H]fucose and -8.8 X lo5 cpm of [%Imethionine per 100 ~1, while uninfected monolayers (75 cm2) contained -6.0 X lo5 and -7.7 X lo5 cpm of [3H]fucose and [35S]methionine, respectively.

88

GROSE AND FRIEDRICHS

Direct immunoprecipitation was performed as previously described (Grose, 1980). For indirect immunoprecipitation, aliquots (100 ~1) of the solubilized antigens were incubated for 30 min at ambient temperature with 5- to 25-~1 samples of the appropriate antiserum, after which the antigen-antibody reaction mixture was diluted 1:3 with modified Schwyzer’s buffer containing 1% NP-40 and DOC (Schwyzer, 1977), adjusted to a final saline concentration of 200 mM, and incubated overnight at 4”. Preswollen protein A-sepharose CL 4B beads (110 ~1, Pharmacia Fine Chemicals, Inc., Piscataway, N. J.) were added to the reaction mixture, which was held for an additional 3 hr. The beads were washed three times with 2 ml of 0.1 M Tris-0.5 M LiCl (pH 8.8). Immune complexes were eluted at 100” into 110 ~1 of sample buffer (Grose et al., 1981a). The radioactive eluates were stored at -20”. Estimation of molecular weights. The molecular weights of the immunoprecipitable polypeptides were estimated by comparison of their migration in acrylamide relative to the distance travelled by standard proteins (Shapiro et al., 1967). The latter included myosin (200,000), phosphorylase b (93,000), bovine serum albumin (68,000), and ovalbumin (43,000) purchased from BioRad Laboratories, Richmond, Calif. In addition, actin from rabbit muscle (45,000) and cytochrome c from horse heart (12,000) were obtained from Sigma Chemical Company, St. Louis, MO. All the molecular weight standards were labeled with [‘4C]formaldehyde by reductive alkylation as described by Rice and Means (1971). Chemicals and isotopes. The reagents for polyacrylamide gel electrophoresis were purchased from Eastman Organic Chemicals, Rochester, N. Y. Dimethyl sulfoxide and DOC were obtained from Fisher Chemical (Fair Lawn, N. J.) and NP-40 was distributed by Particle Data Laboratories, Elmhurst, Ill., [asS]methionine (specific activity, 1350 Ci/mmol) was purchased from Amersham Corp., Arlington Heights, Ill., while [5,6-3H]fucose (specific activity, 56 Ci/mmol) and [‘“Clformaldehyde (specific activity, 10 mCi/

mmol) were supplied by New England Nuclear Corp., Boston, Mass. RESULTS

Spec$city

of the VZVImmune

Caviid Sera

Since VZV infection of cultured cells requires several cycles before CPE is complete, host-cell protein synthesis continues throughout the radiolabeling period. Presumably, therefore, the antigen preparations consist of varying ratios of radioactive cellular and virus-encoded polypeptides. In a series of cross-over immune precipitation experiments, preimmune caviid sera were mixed with [3Hlfucose-labeled VZV antigen, and postimmune sera were added to solubilized samples of uninfected cells grown in medium supplemented with tritiated sugar. In the former instance a mean of 0.6% of the radioactivity (M = 825 cpm) was precipitated, as compared with 15-19% (M = 64,454 cpm) for virus-specific antisera. Thus, neither the caviid serum (nor the Sepharose beads added to the reaction mixture) bound VZV antigens nonspecifically. In the latter case, an equally small number of counts (0.6%) was removed by high titer VZV antisera from the uninfected cell samples. These findings indicate that strain-2 guinea pig sera drawn after VZV immunization possessed little or no reactivity against glycosylated uninfected cell determinants. When similar immunoprecipitations were performed with [3SS]methionine-labeled antigen, the percentage recovery was considerably reduced. The initial four VZV immune caviid sera brought down 5.6 + 0.7% (M = 49,187 cpm) of the counts while the second and third sets precipitated 7.9 + 0.7% (M = 50,842 cpm) and 7.4 f 1.4% (M = 47,954 cpm), respectively. Thus, less than 10% of the ?S activity was accounted for in the precipitate, a finding similar to that reported with immunoprecipitation of closely cell-associated measles virus (Hayes et al., 1978). The effects of changes in the buffer system and pH also were analyzed to assure that conditions favorable to maximal recovery were being achieved. The most frequently used method (as described under Materials and

POLYPEPTIDES OF VARICELLA-ZOSTER

ABCDEFG

pl55 WI8 w98

w62

P45

FIG. 1. Immunoprecipitation profiles of preimmune and postimmune VZV-specific caviid antisera. Aliquots (5 pl) of preimmune and postimmune VZV antisera were incubated overnight with 100 ~1 of [g6S]methionine-labeled VZV antigen, after which protein A-coated sepharose beads were added to the reaction mixtures. Although the recovery of radioactivity was five- to eightfold higher with the postimmune sera (see text), similar numbers of cpm (-10,000) from the preimmune precipitates (lanes A-C) and postimmune precipitates (lanes E-G) were added to each well of the 8% acrylamide/MBA slab gel. Radioactive actin was added to lane D of the same gel as a marker protein. The major VZV-specific immunogenic glycoproteins are designated on the far right side of the figure; the [%]methioninelabeled bands comigrating with actin are indicated by their molecular weight (45,000).

Methods) was a modification of the procedure described by Schwyzer (1977); a change to a simpler phosphate buffer at a lower pH (6.4) did not appreciably affect the recovery of radioactivity (data not shown). In a series of immunoprecipitation analyses with different combinations of [?S]methionine-labeled viral antigen, preimmune caviid sera, and protein A-coated Sepharose beads, we always found a background level of nonspecific radioactivity slightly above 1% (1.3% + 0.1). To examine the differences in the polypeptide profiles of the specifically and nonspecifically precipitated samples, we subjected aliquots

VIRUS

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containing the same amount of radioactivity (10,000 cpm per well) to electrophoresis in the same slab gel (Fig. 1). The precipitation profiles of the preimmune and postimmune VZV antisera bore little resemblance to one another with a notable exception-the presence of prominent [35S]methionine-labeled polypeptides which comigrated with radioactive actin. Thus, actin appeared to be a component of the viral antigen preparation which was invariably present in both the specific and nonspecific reaction complex. Polypeptide Profiles The four initial postimmunization sera, which contained high titers of virus-specific antibody (rl:512), precipitated three major VZV glycoproteins (Fig. 2). These had previously been designated gp 62, gp 98, and gp 118 because of their apparent molecular weights as determined by relative migration in acrylamide (Grose, 1930). When the same four antisera were mixed with a detergent-solubilized VZVinfected cell extract of [%]methionine-labeled polypeptides, the immunoprecipitates contained at least 16 bands after fractionation in acrylamide (Fig. 2). The polypeptide profiles are presented in a diagrammatic format in the center of the fluorogram for ease of comparison, since some of the narrower bands did not reproduce well on the photographic plates. In an earlier study (Grose et al., 1981a), we discovered that the repertoire of virusspecific antibodies induced by VZV immunization in the guinea pig was, on occasion, incomplete in sera drawn 1 week postbooster. To assure maximal precipitation, we also used sera drawn 3 and 6 weeks after booster immunization when the humoral immune response always was found to be fully expressed. The electrophoretic profiles of the antigens precipitated by the later postbooster antisera (Fig. 3) did not differ from those pictured in Fig. 2, although it is apparent from examination of Fig. 3 that differences in avidity for particular polypeptides existed between the three sets of antisera. This increased binding was reflected in higher

GROSE AND FRIEDRICHS

90

X

ABC

Dy

E

F

G

H

-118 -98

-62

FIG. 2. Immunoprecipitable polypeptides of varicella-zoster virus. Detergent-solubilized extracts of VZV-infected cells were reacted with four different caviid VZV antisera obtained 1 week after booster immunizations (Table 1). Aliquots of the immunoprecipitates containing -8000 cpm % or -23,000 cpm 3H were subjected to electrophoresis at constant voltage in 8% acrylamide crosslinked with DATD. Polypeptides labeled with [35S]methionine are presented in lanes A to D, while the glycosylated polypeptides incorporating [3Hlfucose are pictured in lanes E to H. Lane x on the far left is the original [%]methionine-labeled antigen preparation; lane y in the middle is a schematic representation of the polypeptide profile pictured in lanes A to D. The major immunogenic glycoproteins are enumerated on the right side of the figure.

recoveries of radioactivity but not by the appearance of additional bands in the fluorograms. In Fig. 4, composite diagrammatic representations of three gels were placed side-by-side to facilitate recognition of common precipitable polypeptides. The molecular weights of the individual bands were assigned on the basis of their migration relative to that of the standard proteins and VZV glycoproteins. A very slowly migrating narrow band near the origin of the gel @200,000) was simply designated HMW polypeptide; this band was considerably heavier than cellular fibronectin (-230,000). Another prominent higher molecular weight polypeptide, which probably represents the major viral capsid protein commonly found in herpesvirus-infected cultures (Killington et al., 1977),

was assigned a value of 155,000. A 34,000 molecular weight polypeptide was identified in all three gels, while a slightly smaller polypeptide (32,000) was seen in two of three gels. Polypeptides <30,000 were not consistently identified under the conditions of these experiments. Inspection of this figure also illustrated that optimal fractionation of VZV immunoprecipitable polypeptides was not achieved in 10% acrylamide crosslinked with MBA, since the heavier polypeptides were poorly resolved in this gel system. Insoluble Proteins

The insoluble proteins which sedimented after high-speed centrifugation of the antigen extracts were also fractionated in acrylamide gels. As can be seen in

POLYPEPTIDES

I

OF VARICELLA-ZOSTER

JKLvMNOP

VIRUS

91

constant current rather than constant voltage may have accounted for some of these differences in resolution. VZV Glgcoproteins -118 Additiml -98 In an earlier publication we described an ICS glycopolypeptide designated gp 88 -62 which appeared to be labeled to a lesser extent by tritiated fucose than tritiated glucosamine (Grose, 1980). This glycoprotein was variably precipitated by several VZV antisera of human, rabbit, and guinea pig origin (Grose et al., 1981a). Following direct immunoprecipitation of [35S]methionine-labeled VZV extracts, we again observed the three major immunogenic glycoproteins and the capsid poly-

FIG. 3. Immunoprecipitable polypeptides of varicella-zoster virus. Extracts of VZV-infected cells grown in the presence of [86S]methionine were reacted with two sets of four different caviid VZV antisera obtained 3 and 6 weeks after booster immunizations (Table 1). The eight immunoprecipitates (-8000 cpm/well) were analyzed in an 8% acrylamide slab gel cross-linked with MBA. Lanes I to L contain the samples reacted with the “bweek” antisera and lanes M to P the “6-week” samples. Lane y is a schematic representation of the polypeptide profiles. The major immunogenic glycoproteins are enumerated on the right side of the figure.

Fig. 5 the major glycopolypeptides incorporating tritiated fucose were almost completely solubilized in the extraction buffer and thus absent from the gel, with one relative exception (lane A). The latter protein probably represents VZV-specific gp 62, since there are no cellular glycoproteins of similar molecular weight (Grose, 1980). Numerous [36S]methionine-labeled polypeptides also were evident in the postsedimentation pellet (lane B). Of particular interest was the relatively large amount of the 155,000 molecular weight protein which was not solubilized. In addition, the profile of the VZV immunoprecipitate in lane C was noteworthy because it distinguished so clearly three higher molecular weight VZV polypeptides (p 145, p 155, p 174), while some of the polypeptides <98,000 were less well visualized than in fluorograms previously presented in Figs. 2 and 3. Electrophoresis under

A

6

C

FIG. 4. Diagrammatic representations of VZVspecified polypeptide profiles. The schematic drawings from VZV immunoprecipitates fractionated in three different acrylamide gels: (lane A) 10% acrylamide/MBA, (lane B) 8% acrylamide/MBA, and (lane C) 8% acrylamide/DATD, are arranged sideby-side in order to aid in the identification of corresponding polypeptides. Molecular weights, as estimated by the method of Shapiro et al. (1967), are indicated on the right side of the figure. Not all polypeptides are present in each gel, e.g., one lower molecular band (-32,000) seen in lanes A and B was not visualized in lane C. A slowly migrating band below the origin (X200,000) was simply designated HMW (high molecular weight) polypeptide.

GROSE AND FRIEDRICHS

92

peptide (155,000); moreover, we also noted a polypeptide which comigrated with the 88,000 molecular weight glycoprotein (Fig. 6). This experiment reaffirmed the existence of this polypeptide and suggested that gp 88 may represent either an early precursor which incorporated little radioactive sugar during the labeling period or a glycopolypeptide with a relatively minor sugar moiety. Parenthetically, the fluorogram in Fig. 6 again illustrated the relative inefficiency of direct as compared with indirect immunoprecipitation except for the detection of major immunogenic

A

B

.p45

FIG. 5. Analysis of insoluble polypeptides. After addition of 1% NP-40 and DOC to VZV-infected cells, the samples were centrifuged at 85,000 g to remove insoluble proteins. Pellets recovered after detergent extraction of a monolayer incubated in the presence of tritiated fucose (lane A) and another from a culture labeled with P5S]methionine (lane B) were subjected to electrophoresis in an 8% acrylamide/MBA gel under constant current. For purpose of comparison, easily identifiable [35S]methionine-labeled polypeptides in a VZV immunoprecipitate (lane C) were designated in the right margin according to their molecular weights as enumerated in Fig. 4.

FIG. 6 Direct immunoprecipitation of [?S]methionine-labeled polypeptides. Detergent-solubilised extracts (0.1 ml) of uninfected and infected cells labeled with [SsS]methionine were directly precipitated, as described (Grose, 1980), with a 3-week postbooster VZV immune serum (0.1 ml) from guinea pig No. iii. A total of 17,680 cpm from the uninfected cell antigen immunoprecipitate was added to lane A and 19,800 cpm from the VZV-infected cell antigen immunoprecipitate was added to lane B. Electrophoresis was performed in an 8% acrylamide/MBA slab gel at constant current. The prominent radioactive hands in the virus sample, including an 88,006 MW polypeptide (lane B), were designated according to their previously established molecular weights (Figs. 2-4; Grose, 1980).

viral glycoproteins (cf. Spear, 19’76;Grose, 1980). DISCUSSION

The immunoprecipitation profiles of highly specific VW immune sera raised in inbred guinea pigs contained an average

POLYPEPTIDES

OF VARICELLA-ZOSTER

of 16 polypeptides. These ranged in molecular weight from 32,000 to &200,000 and included several prominent species, among which were three major immunogenic VZV glycoproteins, previously designated gp 62, gp 98, and gp 118 (Grose et al., 1981a); in addition, an ICS glycoprotein at MW 88,000 was identified in some of the immunoprecipitates, especially in the [?S]methionine-labeled viral samples. Since glycoproteins are highly soluble in a nonionic detergent such as NP-40, it seems likely that we have detected most VZV-specific glycoproteins by using varying radioimmune precipitation procedures. With the exception of one band at 45,000, additional glycoproteins were not seen in the electrophoretic analysis of the VZV-infected cells (Grose, 1980), so most viral glycoproteins also appeared to be good immunogens. It was much more difficult to assess the total number of virus-specified polypeptides, since the rate of synthesis of host polypeptides does not decline rapidly under the asynchronous conditions of in vitro VZV infection. Preliminary attempts to identify virus-encoded polypeptides by their increased rate of synthesis postinfection revealed only a few species which corresponded to those present in the immunoprecipitates (Grose, unpublished observation). Also, from review of experimental data with the prototype herpes simplex virus (Honess and Roizman, 1973; Powell and Courtney, 1975), we recognized that discrepancies between the numbers of ICS and immunoprecipitable [?S]methionine-labeled viral polypeptides may be a reflection of the relative insolubility of many nonglycosylated VZV-specified gene products. For the above reasons, we found it essential to perform numerous polyacrylamide gel analyses of the radioimmune precipitates under highly divergent electrophoretic conditions in order to define a consistent and reproducible fluorographic profile distinct from the host-cell background. The most easily identifiable nonglycosylated polypeptide corresponded in molecular weight to 155,000 and has been repeatedly observed in VZV immunoprecipitates (Grose, 1980; Grose et al., 1981a) and in a gradient-purified fraction

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93

of nucleocapsids, where it represented the major capsid protein (Zweerink and Neff, 1981). A slightly smaller polypeptide (-145,000) may correspond to an virus-induced DNA polymerase similar to that found in HSV-infected cells (Powell and Purifoy, 197’7),especially since no band of similar mobility is seen in a purified virion fraction (Grose, unpublished observation). A nonglycosylated protein comigrating with muscle actin was present to varying degree in most of the VZV immunoprecipitates; in this regard, VZV mimics other closely cell-associated virus systems (Wang et al., 1976). Investigators in three other laboratories have analyzed VZV polypeptides by immunoprecipitation techniques. Wolff (1978) distinguished 14 viral polypeptides in an immunoprecipitate of a partially purified VZV fraction. A 149,000 MW band (presumably the major capsid polypeptide) is easily visualized in his autoradiogram, but the glycoproteins are not readily distinguished, possibly because the antigen preparation was not extracted with detergents. Asano and Takahashi (1979) solubilized VZV-infected cultures in 1% Triton X-100 and 0.5% DOC. They found a total of 33 polypeptides, including 13 glycoproteins, which reacted with VZV immune sera raised in hyperimmunized guinea pigs and monkeys. Although their electrophoretic profiles (performed in 9% acrylamide with MBA crosslinkage) contained more bands than reported by others, several similarities are apparent, especially the presence of certain glycoproteins and the major capsid polypeptide. However, they probably included some cellular polypeptides in their enumeration. Zweerink and Neff (1981), using several human sera with different VZV antibody titers and an infected cell fraction solubilized with 1% Triton X-100 and 1% DOC, identified 14 virus-specified polypeptides, including one HMW band, after electrophoresis of the immunoprecipitates in 10% acrylamide (MBA-crosslinkage). These included three major glycoproteins and a prominent nonglycosylated band at MW 155,000. When all of the fluorograms are inspected side-by-side, a pattern emerges which is represented in tabular format in

GROSE AND FRIEDRICHS

94 TABLE 2

COMPARABLE PROMINENT VZV IMMUNOPRECIPITABLE POLYPEPTIDES IDENTIFIED IN THREE DIFFERENT LABORATORIES Asano and Takahashi (1979)

Zweerink and Neff (1981)

146’ 105* 96b 9ob 66-w

155 laob lx8

44 86

44-46 84.6

W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem 46, 83-88. EDMOND, B. J., GROSE, C., and BRUNELL, P. A. (1981). Varicella-zoster virus infection of diploid and chemically transformed quinea-pig embryo cells: Factors influencing virus replication. J. Gen. l&d 54,403-407. GROSE, C. (1989). The synthesis of glycoproteins in human melanoma cells infected with varicellazoster virus. Virology 101, 1-9. GROSE, C. (1981). Immunization of inbred guinea pigs with varicella-zoster virus grown in a syngeneic transformed embryo cell line. J. CZin.Microbial. 14, 229-231. GROSE, C., EDMOND, B. J., and FRIEDRICHS, W. E. (1981a). Immunogenic glycoproteins of laboratory and vaccine strains of varicella-zoster virus. Ir#eck Immun. 31,1044-1053. GROSE, C., FRIEDRICHS, W. E., and SMITH, K. 0. (1981b). Cryopreservation of varicella-zoster virions without loss of structural integrity or infectivity. Intervirology 15,&t-160. GROSE, C., PERROTTA, D. M., BRUNELL, P. A., and SMITH, G. C. (1979). Cell-free varicella-zoster virus in cultured human melanoma cells. J. Gen viral 43.1527. HAYES, E. C., WRIGHT, L. L., and ZWEERINK, H. J. (1978). Staphylococcal protein A-Sepharose columns and the characterization of measles virusspecific polypeptides in persistently infected cells. Anal Biochem 91,276-282. HONES& R. W., and ROIZMAN, B. (1973). Proteins specified by herpes simplex virus. XI. Identification and relative molar rates of synthesis of structural and non-structural herpesvirus polypeptides in infected cells. J. viral. 12, 1346-1365. BONNER,

Groae et al. (1981a) and

606

this paper

155 119’ 98” ssb 69 46” 84

’ Molecular weighta of the polypeptiden X l@. bGly.xxylati polypeptiden. NOTE: op 99 in the “Zweerink and Neff &mm is in the mrrect lo&ion, which mrresponds to the lesser of the two higher molecular weight prominent glycoproteins. c Cellular polypeptide, probably aetin.

Table 2. The characteristic features of the polypeptide profiles of VZV immunoprecipitates include major bands representing (i) the capsid protein, (ii) three immunogenic glycoproteins, (iii) actin, and (iv) a lower molecular weight polypeptide. With regard to the glycoproteins, some variability in their reported molecular weights may be attributable to synthesis and processing in different cell substrates (cf. Pereira et al., 1981). Nevertheless, recognition of this reproducible electrophoretie pattern will provide a means of examining for antigenic diversity between VZV strains of different origins and will facilitate further investigation of the biologic properties and immunochemistry of individual VZV specified polypeptides, including the characterization of antiviral monoclonal antibodies derived by hybridoma technology. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI 14604-04 from the National Institute of Allergy and Infectious Diseases and by awards from the Thrasher Research Fund (Salt Lake City, Utah), the Elsa U. Pardee Foundation (Midland, Mich.), and the Morrison Trust (San Antonio, Tex.). REFERENCES ASANO,Y., and TAKAHASHI, M. (1979). Studies on the polypeptides of varicella-zoster (V-Z) virus. 1. Detection of varicella-zoster virus polypeptides in infected cells. B&-en J. 22, 81-89.

KILLINGTON, R. A., YEO, L., HONESS, R. W., WATSON, D. H., DUNCAN, B. E., HALLIBURTON, I. W., and MUMFORD, J. (1977). Comparative analyses of the

proteins and antigens of five herpesviruses. J. Gen Viral 37, 297-310. MAIZEL, J. V. (1971). Polyacrylamide gel electrophoresis of viral proteins. In “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol. 5, Chap. 32, pp. 179-246. Academic Press, New York. PEREIRA, L., DONDERO, D., NORRILD, B., and ROIZMAN,B. (1981). Differential immunologic reactivity and processing of glycoproteins gA and gB of herpes simplex virus types 1 and 2 made in Vero and HEp-2 cells. Prrx. Nat. Awd sci. USA 78, 5202-5206. POWELL, K. L., and COURTNEY, R. J. (1975). Polypeptides synthesized in herpes simplex virus type 2 infected HEp-2 cells. virdogy 66, 217-228. POWELL, K. L., and PURIFOY, D. J. M. (1977). Nonstructural proteins of Herpes simplex virus. I. Pu-

POLYPEPTIDES

OF VARICELLA-ZOSTER

rification of the induced DNA polymerase. J. Viral 24,618-626. RICE, R. H., and MEANS, G. E. (1971). Radioactive labeling of proteins in vitro. J. Biol Chem 246,831832. SCHWYZER,M. (1977). Purification of SV, T-antigen by immunonaffinity chromatography on Staphylococcal protein A-sepharose. INSERM 69, 63-68. SHAPIRO,A. L., VINUELA, E., and MAIZEL, J. V. (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochew~ Biophys. Res. Commun 28,815-820. SPEAR,P. G. (1976). Membrane proteins specified by herpes simplex viruses. I. Identification of four glycoprotein precursors and their products in type l-infected cells. J. Viral. 17, 991-1008.

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WANG, E., WOLF, B. A., LAMB, R. A., CHOPPIN,P. W., and GOLDBERG,A. R. (1976). The presence of actin in enveloped viruses. In “Cell Motility, Book B” (R. Goldman, T. Pollard, and J. Rosenbaum, eds.), pp. 589-599. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. WELLER, T. H. (1953). Serial propagation in vitro of agents producing inclusion bodies derived from varicella and herpes zoster. Proc Sot. Exp. Biol Meat 33,340~346. WOLFF,M. H. (1978). The proteins of varicella-zoster virus. Med Microbid Immunol. 166,21-28. ZWEERINK,H. J., and NEFF, B. J. (1981). Immune response after exposure to varicella zoster virus: Characterization of virus-specific antibodies and their corresponding antigens. Iqfect. Immun 31, 436-444.