Vol. 66, No. 5

JOURNAL OF VIROLOGY, May 1992, p. 3048-3055

0022-538X/92/053048-08$02.00/0 Copyright © 1992, American Society for Microbiology

Identification and Characterization of Pseudorabies Virus Glycoprotein H BARBARA G. KLUPP,' NICO VISSER,2 AND THOMAS C.

METTENLEITER`*

Federal Research Centre for Virus Diseases of Animals, P.O. Box 1149, D-7400 Tubingen, Germany,' and Intervet International B. V., 5830AA Boxmeer, The Netherlands2 Received 20 November 1991/Accepted 31 January 1992

On the basis of DNA sequence analysis, it has recently been shown that the pseudorabies virus (PrV) genome encodes a protein homologous to glycoprotein H (gH) of other herpesviruses (B. Klupp and T. C. Mettenleiter, Virology 182:732-741, 1991). To obtain antibodies specific for gH(PrV), rabbits were immunized with synthetic peptides representing two potential epitopes on gH(PrV) as predicted by computer analysis. The antipeptide sera recognized the gH precursor polypeptide pgH translated in vitro from an in vitro-transcribed mRNA. Western blot (immunoblot) analyses of purified pseudorabies virions using these antisera revealed specific reactivity with a protein with an apparent molecular mass of 95 kDa. Specificity of the reaction could be demonstrated by competition experiments with respective peptides. Analysis of PrV deletion mutants defective in genes encoding known glycoproteins proved that gH(PrV) constitutes a novel PrV glycoprotein not previously found. Treatment of purified virion preparations with endoglycosidase H reduced the apparent molecular mass of gH(PrV) to 90 kDa, indicating the presence of N-linked high-mannose (or hybrid) carbohydrates in mature virions. Removal of all N-linked carbohydrates by N-glycosidase F resulted in a product of 76 kDa. In summary, our results demonstrate the existence of gH in PrV as a structural component of the virion.

Pseudorabies virus (PrV) belongs to the subfamily Alphaherpesvirinae of neurotropic herpesviruses and is the causative agent of Aujeszky's disease in pigs (1). Seven glycoprotein genes have been localized and sequenced in the PrV genome so far. All known PrV glycoproteins exhibit homologies to proteins of other herpesviruses. Glycoproteins gI, gIll, gp63, and gX are dispensable for virus replication in cell culture, as are their homologs in herpes simplex virus (HSV), gE, gC, gI, and gG (reviewed in reference 31). In contrast, gll and gp5O, homologs to HSV gB and gD, respectively, constitute essential proteins (40). However, no protein product has been described for the gene that encodes a PrV protein homologous to the gH of other herpesviruses (26). In HSV, gH represents one of the glycoproteins that have been identified and characterized in more detail only recently (4, 17, 30). Genes that encode glycoproteins homologous to gH(HSV) have been found in alpha-, beta-, and gammaherpesviruses (7, 16, 17, 20, 23, 25, 26, 34, 37, 39), and they constitute the second most highly conserved group of herpesviral glycoproteins, surpassed only by the gB homologs (13). Antibodies against gH(HSV) have been shown to possess complement-independent neutralizing (13, 15) and in vivo protecting activities (12). Complementindependent neutralizing antibodies specific for gH-homologous glycoproteins of Epstein-Barr virus (gp85; 35), varicella-zoster virus (gpIII; 36), and human cytomegalovirus (gp86; 7) have also been isolated. This already points to an important function that gH glycoproteins perform in the life cycles of the respective viruses. Analysis of a temperaturesensitive HSV mutant deficient in the expression of functional gH proved that gH(HSV) constitutes an essential glycoprotein (8). Further studies implicated gH(HSV) and gH(EBV) in viral entry and direct cell-cell spread of the virus, both processes constituting membrane fusion events *

(13, 19, 35). These results indicate common functions of the gH proteins and establish gH as an important immunogen and an essential viral protein. In PrV, we recently described the sequence of a gene that encodes a potential protein product of approximately 72 kDa with significant homology to the gH proteins of other herpesviruses (26). In vitro translation of either hybrid-selected or in vitro-transcribed RNA led to the demonstration of a 71-kDa polypeptide representing the nonglycosylated primary translation product pgH (26). However, since antibodies that specifically recognize gH(PrV) were not available, the existence of mature gH in either PrV-infected cells or pseudorabies virions could not be demonstrated. We describe here the preparation and use of rabbit antisera directed against peptides representing two predicted epitopes on gH(PrV). These sera detected gH(PrV) in purified virion preparations, establishing the existence of gH(PrV) as a novel structural virion component. Enzymatic analyses showed the presence of N-linked carbohydrates on gH(PrV). MATERIALS AND METHODS Cells and viruses. PrV strain Ka was used throughout this study as a prototypic wild-type strain (24). Ka-derived deletion mutants lacking essential glycoprotein gIl (41) or gpSO (40) and quadruple-deletion mutant 234, lacking all known nonessential PrV glycoproteins (32), have already been described. Wild-type PrV and mutant 234 were propagated on pig kidney (PSEK) cells. For growth of the gll and gp5O deletion mutants, complementing cell lines MT3, which expresses gll, and MT50-3, which expresses gp5O, were used (40, 41). To obtain stocks of virions lacking gll or gp5O, noncomplementing cells were infected with phenotypically complemented deletion mutants at high (gIl- PrV) or low (gp50- PrV) multiplicity (40). Computer-based protein sequence analysis. Sequence analysis was performed by using the software package of the

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University of Wisconsin Genetics Computer Group, VAX/ VMS version 5.3-1 (9). Protein secondary structure was predicted by the method of Chou and Fasman (6), and the antigenic index was calculated as described by Jameson and Wolf (22). Preparation of peptide antisera. Peptide 1 (pep 1), ARGAP QGGPPSPQGGPAPTA, representing amino acids 28 to 47 of the gH open reading frame, and peptide 2 (pep 2), SFVFTRRRPDCGPAYTLG, corresponding to amino acids 510 to 527, were synthesized by Cambridge Research Biochemicals, Northwich, United Kingdom. For immunization, the peptides were used as such or coupled to keyhole limpet hemocyanin (KLH; Calbiochem, San Diego, Calif.) by using the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate (Pharmacia, Uppsala, Sweden) essentially as recommended by the supplier. Free peptides at a final concentration of 1 mg/ml were emulsified in incomplete Freund's oil adjuvant. KLH-coupled peptides were also mixed with incomplete Freund's adjuvant at a final concentration of 100 ,ug of peptide per ml. For immunization, three specific-pathogen-free rabbits each were inoculated intramuscularly with 1 ml of peptide emulsion per dose. Animals were injected four times at 4-week intervals and bled 2 weeks after each immunization. The sera described in Results were obtained after the third and/or fourth immunization. In vitro translation, immunoprecipitation, and immunofluorescence. In vitro-transcribed gH-mRNA was translated in vitro in the presence of [35S]methionine (26) with or without inclusion of canine pancreatic microsomal membranes (Amersham-Buchler, Braunschweig, Germany). Immunoprecipitation of in vitro translation products was performed as previously described (33). For immunofluorescence, Vero cells in microtiter plates were infected with PrV, fixed 1 day postinfection in 96% ethanol at -70'C for 1 h, sequentially incubated with rabbit antipeptide or preimmune serum and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G, and analyzed. Virus purification. Cells were infected at a multiplicity of infection of 1. After a complete cytopathic effect had been induced, the supernatant was harvested and clarified by low-speed centrifugation. Virions were purified by centrifugation on a discontinuous gradient of 9 ml each of 50, 40, and 30% sucrose in 10 mM Tris-HCl (pH 7.4)-0.2 mM EDTA in a Beckman SW28 rotor for 2 h at 18,000 rpm. Virions accumulated at the interphase between the 40 and 50% sucrose layers. They were collected by aspiration, diluted 10-fold in phosphate-buffered saline (PBS), and pelleted in the same rotor at 20,000 rpm for 1 h. Pelleted virions were resuspended in PBS and stored at -70°C. Electron microscopic examination revealed the presence of a highly purified virion preparation (data not shown). Protein concentration was determined by the method of Lowry et al. (29). Western blotting. Proteins of purified virions (50 ,ug of protein per lane) were electrophoretically separated in sodium dodecyl sulfate (SDS)-8% polyacrylamide gels (28) under reducing or nonreducing conditions. Proteins were then electrotransferred onto nitrocellulose membranes for 5 h at 1 A in 25 mM Tris base-192 mM glycine-0.01% SDS (45). Filters were incubated for 2 h in 3% bovine serum albumin (BSA) dissolved in PBS, and reacted overnight with rabbit serum at a dilution of 1:50 or ascitic fluid of monoclonal antibodies (MAb) against PrV glycoproteins at a dilution of 1:100. MAb against PrV glycoproteins gI (MAb 3/6) and gll (MAb 5/14) were kindly provided by H.-J. Rziha. Monospecific anti-gp5O serum was collected after immunization of

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rabbits with a vaccinia virus-gp5O recombinant (25a). For peptide competition experiments, antisera were mixed with 50 ,g of peptide prior to addition to the filters. After overnight incubation, filters were washed in 1% BSA-0.1% Triton X-100 in PBS for 30 min, then for 5 min in 1 M NaCl in PBS, and finally for 30 min in 1% BSA-0.1% Triton X-100 in PBS. Filters were then reacted with alkaline phosphataseconjugated goat anti-rabbit antibodies (Dianova, Hamburg, Germany) for 2 h. After two additional washes, bound antibodies were visualized after incubation with 3,3'-diaminobenzidine and H202. Enzyme digestions. To remove high-mannose or hybrid forms of N-linked carbohydrates, purified virions were incubated in 1% SDS for 3 min at 95°C and then with 10 mU of endo-3-N-acetylglucosaminidase H (endo H) in 50 mM sodium citrate-0.1% SDS for 18 h. For complete removal of all N-linked carbohydrates, purified virion preparations were incubated for 3 min at 95°C in 1% SDS and digested for 18 h in 50 mM potassium phosphate buffer (pH 7.4)-0.6% 3 - [(3 - cholamidopropyl) - dimethyl- ammonio] - 1 - propanesulfonate (CHAPS)-0.1% SDS-25 mM EDTA with 2 U of N-glycosidase F (Boehringer, Mannheim, Germany) prior to gel electrophoresis and Western blotting. RESULTS Production of anti-gH peptide sera. Monospecific antisera that recognize the protein to be analyzed are a major prerequisite for detailed studies on glycoprotein structure and function. After determination of the complete sequence of the gH gene of PrV strain Ka, computer analysis of the deduced protein product allowed the prediction of several putative epitopes (Fig. 1). One site close to the amino terminus of the predicted polypeptide (Fig. 1, pepl) and one site in a more carboxy-terminal location (Fig. 1, pep2) were chosen for analysis. Peptide 1, encompassing 20 amino acids corresponding to positions 28 to 47 of the predicted gH primary translation product, and peptide4,2, of 18 amino acids, corresponding to residues 510 to 527, were synthesized and used for immunization of three rabbits, either uncoupled or conjugated to KLH. Sera collected after the third and fourth boosts were analyzed in detail. In the following experiments, only sera against KLH-coupled pepl (1193 and 1194) or pep2 (1198, 1199, and 1200) showed a positive reaction, whereas serum 1156, also prepared by immunization with KLH-coupled pepl, did not specifically react in any of the tests. In indirect immunofluorescence analyses, sera 1193 and 1194, as well as serum 1200, showed PrV-specific staining (data not shown). None of the sera obtained after immunization of rabbits with free peptide recognized a PrV-specific protein. Anti-gH peptide sera recognize pgH. Until now, the only known gH(PrV) gene product was the pgH detected after in vitro translation of either hybrid-selected or in vitro-transcribed RNA (26). We therefore tested whether antipeptide sera were able to recognize pgH after in vitro translation by radioimmunoprecipitation tests. Only anti-pepl sera 1193 (Fig. 2, lane 2) and 1194 (Fig. 2, lane 4) clearly recognized in vitro-translated pgH (Fig. 2, lane 1), compared with precipitation with the respective preimmune sera (Fig. 2, lanes 3 and 5). However, comparison of lanes 2 and 4 with the total translation assay before immunoprecipitation in lane 1 shows that the reaction was weak. Sera 1198, 1199, and 1200, directed against KLH-coupled pep2, exhibited even weaker reactivity with pgH (data not shown), whereas the other sera proved negative. When total RNA isolated from

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NH2

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FIG. 1. Predictions of secondary structure and antigenicity of gH(PrV). Secondary structure and antigenicity were predicted from the deduced amino acid sequence of the gH open reading frame (26) by the methods of Chou and Fasman (6) and Jameson and Wolf (22), respectively. Octagons indicate local antigenic indices higher than 1.2. Pepl and Pep2 denote regions contained in the corresponding oligopeptides used for immunization of rabbits.

infected cells was used for in vitro translation, no polypeptide could be demonstrated after immunoprecipitation with any rabbit serum. In summary, antibodies against pepl and, to a lesser extent, those against pep2 were able to specifically recognize nonglycosylated pgH after in vitro translation. Anti-gH peptide sera detect mature gH in Western blots. For further characterization of the antisera, Western blot analyses were performed. Purified Pr virions were lysed, and proteins were separated in SDS-8% polyacrylamide gels, transferred to nitrocellulose, and probed with rabbit sera at a 1:50 dilution. Positive results were obtained with sera 1193 and 1194, directed against pepl, and 1198 and 1199, directed against pep2 (Fig. 3). As is evident by comparison between the antiserum (lanes 2) and the respective preimmune serum (lanes 1), a 95-kDa protein was specifically recognized by sera 1193 (Fig. 3A), 1194 (Fig. 3B), 1198 (Fig. 3C), and 1199 (Fig. 3D). Generally, sera against pepl showed little background staining (Fig. 3A and B), whereas with both anti-pep2 sera, significant nonspecific staining was detected (Fig. 3C and D). These results show that antisera against pepl and pep2 were able to specifically recognize a protein of 95 kDa in purified pseudorabies virion preparations after Western blotting, proving the existence of PrV gH as a structural virion component. Western blot analyses of PrV-infected cells did not show specific reaction with any serum (data not

shown). Peptide competition eliminates gH recognition. To test the

specificity of the antisera, peptide competition experiments were performed. Nitrocellulose filters onto which proteins from purified pseudorabies virions had been transferred were incubated with either serum 1193, directed against pepl (Fig. 4A), or 1198, directed against pep2 (Fig. 4B). For competition, 50 ,ug of either pepl (lanes 2) or pep2 (lanes 3) was included in the reaction. As a positive control, in lanes 1 only the respective antiserum, with no added peptide, was used. As is evident in Fig. 4A, lane 2, free pepl efficiently interfered with recognition of gH by serum 1193 whereas pep2 did not reduce the gH-specific signal (Fig. 4A, lane 3). On the other hand, detection of gH by serum 1198 was inhibited by pep2 (Fig. 4B, lane 3) but not pepl (Fig. 4B, lane 2), demonstrating the specificity of the observed reaction. gH constitutes a novel PrV glycoprotein. The existence of six PrV glycoproteins, gI, gll, gIII, gpSO, gp63, and gX, has previously been demonstrated. To test whether the polypeptide recognized by our antipeptide sera actually constitutes a novel PrV glycoprotein not related to any of the known glycoproteins, mutant PrV deficient in the synthesis of either essential glycoprotein gll (Fig. 5, lanes 2) or gpSO (Fig. 5, lanes 3) or unable to express all four known nonessential glycoproteins, gI, glll, gp63, and gX (mutant 234 [32]; Fig. 5, lanes 4), was analyzed in comparison with wild-type pseudorabies virions (Fig. 5, lanes 1). Purified virion preparations were electrotransferred to nitrocellulose after polyacrylamide gel electrophoresis under reducing (Fig. SA, C, and D) or nonreducing (Fig. SB) conditions. In Fig. 5B, nonreducing

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FIG. 2. Precipitation of pgH by anti-gH peptide sera. In vitrotranscribed gH-mRNA (26) was translated in a rabbit reticulocyte lysate, and products were precipitated with anti-pepl serum 1193 (lane 2) or 1194 (lane 4). In lane 1, total translation products are shown. Lanes 3 and 5 show precipitation with respective preimmune sera 1193 and 1194. Positions of molecular mass markers are indicated on the left in kilodaltons (K).

conditions were employed to detect the disulfide-linked glycoprotein complex gll more clearly. Filters were probed with MAb 5/14, directed against gll (Fig. SB); a monospecific anti-gpSO rabbit serum (Fig. SC); MAb 3/6, directed against gI (Fig. SD); or anti-gH serum 1193 (Fig. 5A). As expected, gII was lacking only in the gIl- virion preparation (Fig. SB, lane 2) whereas gpSO was not detectable in the gpSO- PrV preparation (Fig. SC, lane 3). Glycoprotein gI was absent from the mutant lacking all four nonessential glycoproteins (Fig. SD, lane 4). All virus mutants, however, showed the presence of the gH-specific 95-kDa protein band (Fig. SA). This demonstrates that gH(PrV), as recognized by the antipeptide sera, actually constitutes a novel PrV glycoprotein not related to any of the known PrV glycoproteins. Analysis of carbohydrates in gH(PrV). Recently, we showed that in vitro processing after in vitro translation resulted in a size increase of pgH from 71 to 85 kDa, suggesting the addition of carbohydrate moieties (26). Sequence analysis showed the presence of seven consensus sequences for N-linked glycosylation (Asn-X-Thr/Ser), of which three have been proposed to be likely glycosylation sites because of the absence of proline and aspartic acid (21, 26, 27). To test whether the mobility shift after in vitro processing was due to the addition of N-linked carbohydrates, in vitro-translated pgH (Fig. 6, lane 2) was processed in vitro (Fig. 6, lane 3) and then incubated with (Fig. 6, lane 4) or without (Fig. 6, lane 5) endo H in citrate buffer containing 0.1% SDS. In Fig. 6, lane 1, products of in vitro translation without exogenous RNA are shown. After endo H digestion, an electrophoretic mobility similar to that found before processing was observed (Fig. 6, lane 4). This shows

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FIG. 3. Western blot analysis with anti-gH peptide sera. Purified pseudorabies virions were lysed and proteins were separated in SDS-8% polyacrylamide gels and electrotransferred to nitrocellulose membranes. Filters were probed with anti-pepl sera 1193 (A, lane 2) and 1194 (B, lane 2) or anti-pep2 sera 1198 (C, lane 2) and 1199 (C, lane 2). In lanes 1, reactions with the respective preimmune sera are shown. The arrows point to the 95-kDa gH specifically recognized by the antipeptide sera. Positions of molecular mass markers are indicated on the left in kilodaltons (K).

that in vitro processing of pgH results in the addition of approximately 14 kDa of endo H-sensitive N-linked carbohydrate moieties. To analyze glycosylation of gH in pseudorabies virions, purified virion preparations were either treated with endo H (Fig. 7, lanes 2) or N-glycosidase F (Fig. 7, lanes 3) or left untreated (Fig. 7, lanes 1). The presence of gH(PrV) was detected after Western blotting by anti-pepl serum 1193 (Fig. 7A). As a control, gI was visualized after incubation with MAb 3/6 (Fig. 7B). Digestion with endo H resulted in increased mobility of gH(PrV) (Fig. 7A, lane 2) compared with untreated virion proteins (Fig. 7A, lane 1). The size reduction was calculated to amount to approximately 5 kDa. Complete removal of N-linked carbohydrates by digestion with N-glycosidase F resulted in a gH(PrV) size decrease to approximately 76 kDa (Fig. 7A, lane 3). Glycoprotein gI did not show different migration after digestion by endo H (Fig. 7B, lane 2), indicating the absence of endo H-sensitive N-linked carbohydrates. Digestion by N-glycosidase F, however, resulted in a size decrease from 130 to 90 kDa (Fig. 7B, lane 3), demonstrating the presence of approximately 40 kDa of N-linked carbohydrates in gI. These results show that (i) gH(PrV), as detected by our antisera in purified virion preparations, does indeed constitute a glycosylated protein; (ii) approximately S kDa of endo H-sensitive carbohydrate moieties is present on gH incorporated into pseudorabies virions; and (iii) all N-linked carbohydrates contribute approximately 19 kDa to mature gH(PrV). DISCUSSION Many proteins that are translocated into the endoplasmic reticulum are altered by addition of carbohydrate residues

3052

KLUPP ET AL. 1

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attached through either N or 0 linkages. Enveloped viruses generally use this cellular pathway to synthesize membrane glycoproteins that mature in the endoplasmic reticulum and the Golgi compartments (2, 5, 44). PrV, as a member of the herpesvirus family of enveloped viruses containing linear double-stranded DNA genomes, encodes at least seven glycoproteins. Four of them, gI, glll, gp63, and gX, have been shown to be dispensable for viral replication in vitro (32), whereas gIl and gpSO have recently been demonstrated as essential for viral infectivity (40). Here we report the detection and characterization of gH of PrV, whose existence had been predicted after sequence determination of a PrV gene whose deduced protein product exhibited significant amino acid homologies to the gH proteins of other herpesviruses (16, 26). Computer-based prediction of antigenic sites led to the selection of two potential epitopes that were included in two synthetic peptides containing 20 and 18 amino acids, respectively. Somewhat surprisingly, antisera against both predicted epitopes recognized gH(PrV) in preparations of purified pseudorabies virions, emphasizing the value of computer-based epitope prediction. Only peptides that had been coupled to KLH prior to immunization of rabbits produced sera that showed specific reactivity. Whether the contribution of KLH has been solely that of an immunogenic carrier or whether coupling also influenced conformation of the peptides is not known. Of the KLHcoupled peptides, those corresponding to amino-terminal epitope 1 (pepl) exhibited the strongest reactivity in all of the tests. However, the failure of serum 1156, also directed against KLH-coupled pepl, to specifically detect an antigen clearly shows variation in individual immune responses to identical immunogens. Sera directed against the more carboxy-terminal epitope 2 (pep2) generally showed a weaker reaction (and higher background staining). Whether this is

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FIG. 5. gH(PrV) constitutes a novel PrV glycoprotein. Proteins from purified virions of wild-type PrV (lanes 1) or mutants lacking essential glycoproteins gll (lanes 2) or gp5O (lanes 3) or all nonessential glycoproteins, gI, gp63, gIll, and gX (lanes 4), were separated in SDS-8% polyacrylamide gels and transferred to nitrocellulose. Filters were reacted with anti-pepl serum 1193 (A), anti-gIl MAb 5/14 (B), a gpSO-specific polyclonal antiserum (C), or anti-gI MAb 3/6 (D). For panel B, the gel was run under nonreducing conditions to demonstrate more clearly the 155-kDa disulfide-linked glycoprotein complex gll. For panels A, C, and D, gels were run under reducing conditions. The arrow marks the position of gH, the open triangle indicates gll, the closed triangle points to gp5O, and the asterisk marks the position of gI. Positions of molecular mass markers are indicated on the left in kilodaltons (K).

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). In contrast, gI did not show different mobility after endo H digestion (--_), whereas N-glycosidase F treatment resulted in a size reduction to approximately 90 kDa (z1).

the size of mature gH(PrV), 95 kDa, is in the range of that found for other gH polypeptides, e.g., 110-kDa gH(HSV) (4) and 85-kDa gH(EBV) (38). Analysis of N-linked carbohydrate moieties of gH(PrV) showed that nonglycosylated pgH is processed in vitro by addition of approximately 14 kDa of endo H-sensitive N-linked glycans. Mature gH(PrV), as found in virions, is surprisingly still sensitive to endo H digestion. This indicates that N-linked carbohydrates on gH(PrV) were not completely modified into complex structures in the Golgi apparatus. Virion glycoprotein gI, in contrast, does not contain endo H-sensitive carbohydrates. Interestingly, the gH proteins of HSV (4), varicella-zoster virus (36), and Epstein-Barr virus (10) have also been shown to contain endo H-sensitive N-glycans. Complete removal of N-linked carbohydrates by N-glycosidase F resulted in the emergence of a gH(PrV) of approximately 76 kDa. This size is very close to that reported for the nonglycosylated gH precursor protein pgH (71 kDa). Whether any 0-linked carbohydrates are present on gH(PrV) is currently under investigation. It is, however, clear that elimination of N-linked glycans did not abolish recognition of gH(PrV) by the antipeptide sera showing the continued presence of the epitope even after (partial) deglycosylation. In this report, the first demonstration of mature gH(PrV) is presented. Herpesviral gH polypeptides have been shown to execute essential functions during the viral replicative cycle (8, 11, 19) in membrane fusion events during infection and viral entry and direct viral spread by cell-cell fusion (13, 19, 35). It therefore appears reasonable to speculate that gH(PrV) also constitutes an essential virion component that is necessary for penetration of the virus into target cells or fusion of PrV-infected cells with adjacent noninfected cells. Functional complementation of a gII-negative PrV mutant by glycoprotein gI of bovine herpesvirus 1, both homologous to gB(HSV), has recently been shown (41). Regarding the

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KLUPP ET AL.

significant amino acid conservation between members of the gH family, it is tempting to analyze whether similar complementation could also be achieved among the gH proteins. Work is in progress to answer some of these questions. ACKNOWLEDGMENTS

This study was supported by grant Me 854/2-2 from the Deutsche Forschungsgemeinschaft and a grant from Intervet International B.V. We thank Axel Karger for technical advice and Gunther Keil for

helpful suggestions. REFERENCES 1. Ben-Porat, T., and A. S. Kaplan. 1985. Molecular biology of pseudorabies virus, p. 105-173. In B. Roizman (ed.), The herpesviruses, vol. III. Plenum Publishing Corp., New York. 2. Berger, E. C., E. Buddecke, J. Kamerling, A. Kobata, J. Paulson, and J. Vliegenthart. 1982. Structure, biosynthesis and functions of glycoprotein glycans. Experientia 38:1129-1162. 3. Blacklaws, B., S. Krishna, A. Minson, and A. Nash. 1990. Immunogenicity of herpes simplex virus type 1 glycoproteins expressed in vaccinia virus recombinants. Virology 177:727736. 4. Buckmaster, E. A., U. Gompels, and A. Minson. 1984. Characterization and physical mapping of an HSV-1 glycoprotein of approximately 115 x 103 molecular weight. Virology 139:408413. 5. Campadelli-Fiume, G., and F. Serafini-Cessi. 1985. Processing of the oligosaccharide chains of herpes simplex virus type 1 glycoproteins, p. 357-382. In B. Roizman (ed.), The herpesviruses, vol. III. Plenum Publishing Corp., New York. 6. Chou, P. Y., and G. Fasman. 1978. Prediction of the secondary structure of proteins from their amino acid sequence. Adv. Enzymol. Relat. Areas Mol. Biol. 47:145-148. 7. Cranage, M. P., G. Smith, S. Bell, H. Hart, C. Brown, A. Bankier, T. Tomlinson, B. Barell, and A. Minson. 1988. Identification and expression of a human cytomegalovirus glycoprotein with homology to the Epstein-Barr virus BXLF2 product, varicella-zoster virus gpIII, and herpes simplex virus type 1 glycoprotein H. J. Virol. 62:1416-1422. 8. Desai, P., P. Schaffer, and A. Minson. 1988. Excretion of noninfectious virus particles lacking glycoprotein H by a temperature sensitive mutant of herpes simplex virus type 1: evidence that gH is essential for virion infectivity. J. Gen. Virol. 69:1147-1156. 9. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis for the VAX. Nucleic Acids Res. 12:387-395. 10. Edson, C. M., and D. Thorley-Lawson. 1983. Synthesis and processing of three major envelope glycoproteins of EpsteinBarr virus. J. Virol. 46:547-556. 11. Foa-Tomasi, L., E. Avitabile, A. Boscaro, R. Brandimarti, R. Gualandri, R. Manservigi, F. Dall'Olio, F. Serafini-Cessi, and G. Campadelli-Fiume. 1991. Herpes simplex virus (HSV) glycoprotein H is partially processed in a cell line that expresses the glycoprotein and fully processed in cells infected with deletion or ts mutants in the known HSV glycoproteins. Virology 180:474-482. 12. Forrester, A. J., V. Sullivan, A. Simmons, B. Blacklaws, G. Smith, A. Nash, and A. Minson. 1991. Induction of protective immunity with antibody to herpes simplex virus type 1 glycoprotein H (gH) and analysis of the immune response to gH expressed in recombinant vaccinia virus. J. Gen. Virol. 72:369375. 13. Fuller, A. O., R. Santos, and P. Spear. 1989. Neutralizing antibodies specific for glycoprotein gH of herpes simplex virus permit viral attachment to cells but prevent penetration. J. Virol. 63:3435-3443. 14. Ghiasi, H., A. Nesburn, and S. Wechsler. 1991. Cell surface expression of herpes simplex virus type 1 glycoprotein H in recombinant baculovirus-infected cells. Virology 185:187-194. 15. Gompels, U., A. Carss, C. Saxby, D. Hancock, A. Forrester, and

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