Identification and preliminary mapping with monoclonal antibodies of a herpes simplex virus 2 glycoprotein lacking a known type 1 counterpart

VIROLOGY

133, 242-247 (1984)

Identification and Preliminary Mapping with Monoclonal Antibodies of a Herpes Simplex Virus 2 Glycoprotein Lacking a Known Type 1 Counterpart BERNARD ROIZMAN,*!~ BODIL NORRILD,~ CYNTHIA CHAN,$

AND

LENOREPEREIRA~

*The Marjorie B. Kc&r Viral Oncology Laboratories, The University of Chicago, 910 East 58th Street, Chicago, Illinois 60637;TDepartment of Medical Microbiology, The University of Copenhgen, Cope-nhq~en,Denmark; and *Viral and Rickettsial Disease Laboratory, California Department of Public Health, Berkeley, California Received October 17, 1983;accepted December 9, 1983 The properties of herpes simplex virus 2 (HSV-2)-specific proteins reactive with monoclonal antibody H966 derived from mice immunized with HSV-2 strain G are reported. The reactive proteins contained in infected cell lysates subjected to electrophoresis in denaturing gels and transferred to nitrocellulose sheets form a relatively sharp band characteristic of M, 124,000 proteins and a diffuse, more slowly migrating band. Antigens reactive with H966 were detected on the surface of viable, unfixed cells. The electrophoretic mobility of the H966-reactive proteins made in the presence of tunicamycin was more rapid than that of the proteins made in the absence of the drug. Direct evidence that the HSV-2-specific antigen was a glycoprotein emerged from purification of [“clglucosamine-labeled proteins with similar electrophoretic mobilities by immunoabsorption to H966 bound to Sepharose beads. Analyses of the reactivity of HSV-1 X HSV-2 recombinants indicated the gene specifying the glycoprotein maps in the S component of the DNA. The glycoprotein detected by H966 has no known counterpart in HSV-1 and corresponds to the glycoprotein previously designated as gC of HSV-2 and reported to map to the right of gC specified by HSV-1. Inasmuch as an HSV-2 gene colinear with HSV1 gC has been reported to specify a glycoprotein currently designated as gC of HSV-2 by Para et al. [J. Viral. 45,1223-1227 (1%33)], the glycoprotein identified by H966 should be designated as gG.

In a report comparing the structural proteins of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), Cassai et aC (1) noted that some of the HSV-2 glycoproteins differed from HSV-1 glycoproteins, designated previously according to decreased electrophoretic mobility as VP8, Vr7, and VP8.5, VP18-19 (2,3). Subsequently, Spear (4) renamed the HSV-1 glycoproteins VPS, VP7, VP8.5, and VP18-19 as gC, gB, gA, and gD, and presented evidence suggesting that (i) both gA and gD of HSV-1 might share immunologic determinants with HSV-2 glycoproteins having similar but not identical electrophoretic mobilities; (ii) that an HSV-2 glycoprotein of intermediate size might share antigenic determinants with either one of the high-mo1 To whom reprint

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lecular-weight B or C components of HSV1; and (iii) that HSV-1 and HSV-2 might each specify one antigenically unique glycoprotein. Still later, Ruyechan et cd (5) extended the letter designations to HSV2 glycoproteins with corresponding electrophoretic mobilities and mapped the location of the genes specifying the HSV-1 and HSV-2 glycoproteins on the basis of analyses of HSV-1 X HSV-2 recombinants. Those studies showed that, within the limits of detection of the HSV-1 X HSV-2 crossover sites, the HSV-1 and HSV-2 genes specifying the glycoproteins &;A, gB, and gD were colinear whereas those specifying gC were not. The HSV-1 gC gene was mapped within map positions 0.58-0.70 whereas the HSV-2 gC was mapped to the right, between 0.70 and the junction of the L-S components in the prototype arrangement of the DNA. Recent studies employ242

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FIG. 1. The reactivity of monoelonal antibody H966 with electrophoretically separated, transferred polypeptides from HSV infected cells. Lanes l-5: Reactivity of H966 with electrophoretically separated, transferred polypeptides from HEp-2 cells infected with HSV-l(F), HSV-2(G), and HSV-1 X HSV-2 recombinants. HEp-2 cells grown in 25-cm2 flasks were infected as indicated below at multiplicities of 5 pfu/ cell. After 1 hr of adsorption the inoculum was aspirated and replaced with a maintenance medium consisting of mixture 199 supplemented with 1% calf serum. At 18 hr postinfection, the cells were harvested, washed with phosphate-buffered saline, solubilized in disruption buffer (IO), subjected to electrophoresis in 9.25% polyacrylamide gels, and transferred to nitrocellulose sheets as described by Braun et al. (II). The transferred polypeptides were reacted first for 15 hr at room temperature with monoclonal antibody H966 contained in ascites fluid diluted l:lOO, and then with horseradish peroxidase coupled rabbit anti-mouse serum diluted 1:800 (Miles, Elkhart, Ind.). Lane 1, cells infected with HSV-l(F); lane 2, cells infected with HSV-2(G); lane 3, cells infected with HSV-2(G) and maintained in the presence of tunicamycin (3 pg/ ml of medium; Sigma, St. Louis, MO.) from the end of the adsorption interval until the cells were harvested at 18 hr postinfection; lane 4, cells infected with HSV-l(mP) X HSV-2(G) recombinant R50BG6 (12); lane 5, cells infected with HSV-l(mP) X HSV2(G) recombinant R50BG13 (12); lanes 6-9, the same as above except that Vero cells were used. At 16 hr postinfection the medium was replaced with a maintenance medium in which the methionine normally present in the medium was replaced with 20 pg of [%S]methionine (1266 Ci/miW, New England Nu-

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ing monoclonal antibodies detected a smaller HSV-2 glycoprotein initially designated as gF (6, 7). Mapping studies have shown that the gene specifying gF maps in a position colinear with gC of HSV-1 and shares with that glycoprotein antigenie determinant sites (8,9). The question, therefore, arises as to the origin and properties of the slowly migrating glycoprotein designated as gC of HSV-2 and mapped by Ruyechan et al. (5) to the right of gC of HSV-1. In this paper we report on the properties of a HSV-2 glycoprotein reactive with a monoclonal antibody (H966) derived from Balb/c mice immunized with lysates of HSV-Z(G)-infected cells. The monoclonal antibody-producing cells were selected on the basis of the specificity of the antibody for HSV-2-infected cells in immunofluorescence tests. The relevant results of our studies are as follows: (i) The reactivity of H966 was HSV-2 specific. H966 reacted with HSV-2-specific polypeptides but not with HSV-l-specific polypeptides when exposed to electrophoretically separated proteins of HSV-l(F)and HSV-B(G)-infected cells electrically transferred to nitrocellulose sheets (Fig. 1, lanes 1 and 2). Thus H966 reacted with polypeptides with an apparent molecular weight >128,000 forming a diffuse band, with a polypeptide with an apparent molecular weight corresponding to 128,000 forming a sharp band, and with a more rapidly migrating antigen with an apparent molecular weight of approximately 75,000 present in lysates of mock- (not shown) and HSV-l-infected cells. It is likely that the M, 75,000 protein corresponds to a host protein which shares an antigenic determinant site with the virusspecific proteins. It should be noted that preliminary data indicate that the proteins clear, Boston, Mass.). After 2 hr of incubation in labeling medium, the cells were harvested and processed as described above. Lanes 6 and ‘7, immune reactivity of H966 with electrophoretically separated, transferred polypeptides from HSV-Z(G)- and HSV-l(F)infected Vero cells, respectively. Lanes 8 and 9, autoradiographic images of lanes 6 and 7.

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13 FIG. 2. Maps of were constructed the S component DNA sequences,

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HSV-2 X HSV-2 recombinants R50BG6, R50BG8, and R50BG13. The recombinants and mapped as described by Tognon et al. (12). Crossover sites were detected in as shown. The upper and lower of the double lines represent HSV-1 and HSV-2 respectively. The heavy line represents the DNA sequences in the recombinants.

purified by immunoabsorption to the H966 react with human sera from patients infected with HSV-2 but not with sera from patients infected with HSV-1 (L. Pereira, M. Colmann, and A. Nahmias, studies in progress). (ii) Studies on HSV-1 X HSV-2 recombinants indicate that the gene specifying the protein reactive with H966 maps in the S component of the HSV-2 genome. This is illustrated by the reactivity of the monoclonal antibody with the recombinants R50BG13, R50BG6 (Fig. l), and R50BG8 (not shown). In these recombinant viruses (1.2), the HSV-2 DNA sequences are contained entirely within the S component (Fig. 2). (iii) The H966 antibodies bind to the surface of unfixed, HSV-B(G)-infected cells in both immunofluorescence (Fig. 3) and biotin-avidin-amplified reactions with anti-mouse immunoglobulin coupled to horseradish peroxidase. In the latter studies (not shown), HEp-2 monolayer cell cultures were infected with approximately 100 PFU of either HSV-l(F), HSV-B(G), R50BG6, R50BG8, or R50BG13. After 3 days of incubation the infected cultures were reacted with the H966 antibody and visualized by biotin-avidin-amplified reaction with anti-mouse immunoglobulin coupled to horseradish peroxidase. This reaction has been used to visualize viral glycoproteins accumulating on the surface of infected cells. H966 reacted with HSV2(G)-, R50BG6-, R50BG8-, and R50BG13-

infected cells but not with cells infected with HSV-l(F). (iv) In the presence of tunicamycin the HSV-2 proteins reactive with H966 exhibited a lower apparent molecular weight. In these experiments, HSV-Z(G)-infected cells were treated with tunicamycin (3 pg/ml) immediately after exposure to the virus. At 18 hr postinfection, the cells were harvested and lysed. The proteins contained in the lysates were electrophoretically separated in denaturing polyacrylamide gels, electrically transferred to nitrocellulose sheets, and reacted with H966. As shown in Fig. 1, the accumulation of rapidly migrating forms was decreased and new reactive bands corresponding to proteins of lower molecular weights became apparent. These results suggest that tunicamycin inhibits the processing of the HSV-2 proteins reactive with H966. (v) Direct evidence that the HSV-2 proteins reactive with H966 are glycosylated emerged from the purification of [14Clglucosamine-labeled infected cell proteins by immunoabsorption to H966 bound to Sepharose beads (Fig. 4). (vi) We have previously reported that HSV glycoproteins are cleaved by a host cell protease present in Vero cells (16, I?‘). As described in Fig. 1, analyses of proteins extracted from HSV-Z(G)-infected Vero and HEp-2 cells show that the HSV-2 glycoprotein identified by H966 is also subject to cleavage by the Vero cell protease (Fig. 1, panel C).

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FIG. 3. Photomicrographs of HSV-l(F)and HSV-Z(G)-infected cells stained by immunofluorescenee with monoelonal antibodies H966, HCl, and HDl. Type-specific monoclonal antibody HCl reacts with glycoprotein gC of HSV-1; HDl reacts with gD specified by both HSV-1 and HSV-2 (13). Procedures for immunofluorescenee were published elsewhere (14).

These studies indicate

the following:

(i) H966 reacts specifically with an HSVZ(G)-specific glycoprotein which appears to correspond in electrophoretic mobility to a glycoprotein migrating more slowly than gC of HSV-1, and was identified as gC of HSV-2 by Ruyechan et al. (5). (ii) Current studies indicate that this glycoprotein maps in the S component of HSV-2 DNA, and therefore it maps farther

to the right than previously reported by Ruyechan et al. (5). Reexamination of the data of Ruyechan et ah (3) indicates that most of the recombinants permitted the mapping of this glycoprotein to the right of 0.70 and only a few established the right border of the presumed location of the gene. The results of that study indicated that the accumulation of this glycoprotein in cells infected with HSV-1 X HSV-2 recombinants did not appear to be required

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FIG. 4. Autoradiograms of electrophoretically separated glycoproteins from HSV-l(F)and HSV-2(G)infected cell extracts reactive with H966 and HCl monoclonal antibodies. HEp-2 cell monolayer cultures were labeled with [i4Clglucosamine from 6 to 18 hr postinfection with 10 pfu of HSV-l(F) or HSV-2(G) per cell. The left-most lane shows HSV-2 polypeptides from HSV-e-infected cells labeled with [“S]methionine from 6 to 18 hr postinfection. Lane 2 from left shows the HSV-2 polypeptides labeled with [‘“Clglucosamine. The glycoprotein reactive with H966 and designated as gG is displayed in the third lane; it was purified by affinity chromatography from lysates of HSV-2-infected cells labeled with [14Clglucosamine using H966 monoclonal antibody bound to Sepharose beads as described elsewhere (15). The rightmost lane shows HSV-1 gC and its precursor (pgC) immune precipitated from lysates of HSV-linfected cells labeled with glucosamine. Electrophoresis was done in 9.25% polyacrylamide gels in the presence of sodium dodecyl sulfate (10).

for infectivity. Consequently, additional undetected crossover sites in the S component within the domain of this gene could have precluded its expression; this may explain the failure to place correctly the right border of the gene. Current studies place the gene in the S component of HSV DNA. Marsden et a.!. (18) reported that HSV2 specified a glycoprotein with an apparent molecular weight of 92,000,migrating more rapidly than the HSV-2 glycoprotein described in this and previous studies. However, electrophoretic mobilities of glycoproteins in denaturing gels vary considerably from one gel system to another, and the apparent molecular weights are not reliable markers of identity when different systems are compared. For example, the relative position of the bands of the high-molecular-weightHSV-lglycoprotein in polyacrylamide gels crosslinked with N,N,N,N’-diallyltartardiamide is reversed from that seen in gels crosslinked with bisacrylamide (19). However, mapping studies reported by H. Marsden at the International Herpesvirus Workshop in Oxford suggest that the 1M, 92,000 glycoprotein described by Marsden et al. (18) corresponds to the one described in this study. Because the next available letter designation (gF) has been withdrawn, the designation of this glycoprotein should be gG. It is noteworthy that an HSV-1 glycoprotein corresponding to gG of HSV-2 has not been reported to date. If none is found, it would support the hypothesis that this glycoprotein does not appear to be essential for viral replication, inasmuch as HSV-1 X HSV-2 recombinants which do not appear to specify this glycoprotein appear to be viable (5). The function of this protein is not clear. We may speculate that, inasmuch as in the human population HSV2 infection usually follows HSV-1 infection, and inasmuch as many of the glycoproteins share antigenic determinant sites, a function of the glycoproteins, such as gG, exposing in its native conformation typespecific determinant sites, might be to shield HSV-2 from the host immune response directed to HSV-1.

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ACKNOWLEDGMENTS These

studies

were

aided

by grants

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stitute Merieux, France, to the University of Chicago, the University of Copenhagen, and to the California Public Health Foundation. REFERENCES

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