Identification and Characterization of Pseudorabies Virus Glycoprotein ...

JOURNAL OF VIROLOGY, Aug. 1996, p. 5684–5688 0022-538X/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 70, No. 8

Identification and Characterization of Pseudorabies Virus Glycoprotein gM as a Nonessential Virion Component JOHANNES M. DIJKSTRA,1 NICO VISSER,2 THOMAS C. METTENLEITER,1

AND

BARBARA G. KLUPP1*

Institute of Molecular and Cellular Virology, Friedrich Loeffler Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany,1 and Intervet International B.V., NL-5830AA Boxmeer, The Netherlands2 Received 18 March 1996/Accepted 3 May 1996

Sequence analysis within BamHI fragment 3 of the pseudorabies virus (PrV) genome revealed an open reading frame homologous to the UL10 gene of herpes simplex virus. A rabbit antiserum directed against a synthetic oligopeptide representing the carboxy-terminal 18 amino acids of the predicted UL10 product recognized a major 45-kDa protein in lysates of purified Pr virions. In addition, a second protein of 90 kDa which could represent a dimeric form was observed. Enzymatic deglycosylation showed that the PrV UL10 protein is N glycosylated. Therefore, it was designated PrV gM according to its homolog in herpes simplex virus. A PrV mutant lacking ca. 60% of UL10 coding sequences was able to productively replicate on noncomplementing cells, demonstrating that PrV gM is not required for viral replication in cell culture. However, infectivity of the mutant virus was reduced and penetration was delayed, indicating a modulatory role of PrV gM in the initiation of infection. UL43, and UL53, which are predicted to encode hydrophobic proteins with the potential to pass the membrane several times (29). The product of the UL53 gene is the essential glycoprotein K (16). So far, no UL43 protein product has been identified in any herpesvirus. HSV-1 UL20 encodes a polypeptide of 222 amino acids (aa) with two to four potential transmembrane segments which is involved in viral egress (5). The protein encoded by HSV-1 UL10 is predicted to contain eight transmembrane domains and has recently been described as an N-glycosylated component of the virion designated gM (4, 28). HSV-1 gM is dispensable for viral replication in cell culture (3, 28). Interestingly, gM appears to be conserved throughout the Herpesviridae. A gM homolog was first identified in human cytomegalovirus as integral membrane protein, a 45-kDa structural component of the virion (27). In equine herpesvirus 1 (EHV-1), gM has been shown to represent a major structural component of the virion (33). Given the general collinearity of alphaherpesvirus genomes, we determined the nucleotide sequence (36) of a portion of BamHI fragment 3 of PrV strain Ka (18) which was suspected to contain the PrV homolog of the HSV-1 gene UL10, encoding glycoprotein M (Fig. 1A). An open reading frame whose translation product exhibited significant homologies to HSV-1 gM and respective proteins of varicella-zoster virus (11) and EHV-1 (39) of 32, 36, and 40%, respectively, was identified (12). For PrV UL10, two possible start codons were found (data not shown). Comparison with UL10 homologs in other herpesviruses favors translational start at the second start codon (24). No TATA consensus sequence was found upstream of the first putative start codon, but the sequence 59TATA-39 present 84 bp upstream of the second start codon might constitute part of the UL10 promoter (10). Assuming that translation starts at the second ATG, the gene encodes a 393-aa protein with a predicted molecular mass of 42 kDa. Homologous genes have been identified in every herpesvirus analyzed so far (1, 2, 8, 11, 14, 26, 27, 29, 37, 39, 40). The deduced PrV UL10 protein (Fig. 1B) contains eight hydrophobic domains with sufficient length and hydrophobicity to span the lipid bilayer (9, 25). In the region located between the first and second hydrophobic domains, a consensus sequence for

Pseudorabies virus (PrV), a member of the Alphaherpesvirinae (35), is an important pathogen of swine, causing Aujeszky’s disease (30). The PrV genome consists of a linear doublestranded DNA of approximately 150 kbp. As a typical class II herpesvirus, genome it is divided into a unique long (UL) and a unique short (US) region, the latter being bracketed by inverted repeats (6). Approximately 60% of the PrV genome has been sequenced. With increasing numbers of completely sequenced herpesvirus genomes, information about the identity and diversity of herpesviruses is accumulating. Within the alphaherpesviruses, gene arrangement appears to be largely collinear with the exception of a large inversion in the UL region of the PrV genome (7). Of the ca. 70 proteins specified by alphaherpesvirus genomes, we are particularly interested in viral glycoproteins. Herpesvirus envelope glycoproteins perform a number of important functions. They are involved in virus entry into and egress from cells, and they constitute important targets for the host immune response. Several glycoproteins are conserved throughout the herpesvirus family, including homologs of herpes simplex virus type 1 (HSV-1) gB, gH, and gL. Others, such as gG, gK, and possibly gE and gI, are found only in alphaherpesviruses (38). In PrV, 10 glycoproteins, designated gB, gC, gD, gE, gG, gI, gH, gK, gL, and gN, have been found (17, 30). All except gG and possibly gK are structural components of the virion. Amino acid sequence analysis characterizes most of these proteins as type I membrane proteins, comprising an amino-terminal hydrophobic stretch as a putative signal sequence and a second carboxy-terminal hydrophobic region as a transmembrane domain resulting in location of most of the amino-terminal part of the protein on the outside of the membrane. Another class of herpesvirus membrane proteins, designated type III, are multiply inserted proteins characterized by several stretches of hydrophobic amino acids. The HSV-1 genome contains four open reading frames, UL10, UL20,

* Corresponding author. Mailing address: Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany. Phone: 49-38351-7269. Fax: 49-38351-7151. 5684

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FIG. 1. Map location and deduced amino acid sequence of PrV UL10. (A) At the top is a schematic diagram of the PrV genome. Shaded boxes represent inverted repeat regions (TR, terminal repeat; IR, internal repeat) which bracket the US region and separate it from the UL part. Below is a BamHI restriction fragment map. The region encompassing the UL10 gene is enlarged, and relevant restriction sites are shown. The deletion introduced into the UL10 gene is marked, as is insertion of the BHV-1 gB expression cassette (not drawn to scale). (B) Deduced amino acid sequence of the PrV UL10 protein. Regions of the predicted polypeptide which contain a stretch of at least 16 mainly hydrophobic amino acids not interrupted by charged amino acids and therefore predicted to be membrane spanning are underlined and numbered. 1 and 2 mark charged residues. The consensus sequence for addition of N-glycans is boxed. Peptides UL10.1 (aa 376 to 393) and UL10.2 (aa 346 to 361) are in boldface letters.

addition of N-linked glycans is present (23). The position of this consensus sequence is conserved in all UL10 homologs. Of particular interest is the highly charged carboxy terminus. Of the carboxy-terminal 68 aa, 24 possess charged side chains, 12 of which are acidic and 12 of which are basic in character. Of the last 18 aa, 9 carry negative charges (Fig. 1B). The function of this highly charged domain is unclear at present. Comparison of deduced gM homologs shows conservation of (i) a short hydrophilic region at the amino terminus; (ii) eight stretches of hydrophobic amino acids suitable to span the lipid bilayer; and (iii) a hydrophilic and charged carboxy terminus varying in length between 29 (human herpesvirus 6) and 134 (HSV-1) aa. In the deduced PrV UL10 protein, all of these features are also present. To identify and characterize the PrV UL10 protein, rabbit antipeptide sera were generated. To this end, two peptides corresponding to aa 376 to 393 (UL10.1) and 346 to 361 (UL10.2) (Fig. 1B) of the predicted primary translation product were synthesized, coupled to keyhole limpet hemocyanin, and used for immunization of three rabbits each as described previously (21). Sera obtained after the third boost were further analyzed by Western blotting (immunoblotting) on lysates of purified Pr virions separated in sodium dodecyl sulfate (SDS)–13% polyacrylamide gels (21). Following transfer to nitrocellulose, filters were incubated with rabbit antisera at dilutions of 1:5,000 for the UL10-specific sera and 1:500 for the anti-gH serum 1193 (21). After incubation with peroxidaseconjugated second antibody, bound antibody was visualized by luminescence recorded on X-ray films (ECL; Amersham, Braunschweig, Germany). While serum raised against peptide UL10.2 did not show any specific reactivity (data not shown), serum directed against peptide UL10.1, corresponding to the carboxy-terminal 18 aa of the predicted translation product, specifically reacted with a prominent protein of ca. 45 kDa and a minor product of ca. 90 kDa (Fig. 2B, lane 1), as is evident by comparison with the matching preimmune serum (Fig. 2A, lane 1). Sera of all three rabbits immunized with peptide UL10.1 reacted similarly (data not shown). Preincubation of anti-UL10.1 serum with 10 mg of peptide UL10.1 inhibited detection of the 45-kDa and the 90-kDa proteins (Fig. 2C, lane 1), while incubation with peptide UL10.2 had no effect (data

not shown), demonstrating specificity of the reaction with both proteins. To analyze whether the UL10 translation product is glycosylated as predicted from the amino acid sequence, N-glycosidase F digestions of purified virions were performed. Twenty micrograms of purified virions was incubated in 50 mM potassium phosphate (pH 7.4)–20 mM EDTA–0.5% Nonidet P-40– 200 mU of N-glycosidase F (Boehringer GmbH, Mannheim, Germany) for 18 h at 378C prior to electrophoresis and Western blotting. While incubation in N-glycosidase F buffer alone had no effect on the detected protein products (Fig. 2, lanes 3),

FIG. 2. Identification and analysis of the PrV UL10 protein by Western blotting. Purified Pr virions were lysed, and proteins were separated in SDS–13% polyacrylamide gels and electrotransferred onto nitrocellulose membranes. Filters were probed with preimmune serum (A), antiserum directed against peptide UL10.1 (B; for location of the peptide, see Fig. 1B), anti-UL10.1 serum after preincubation with 10 mg of peptide UL10.1 (C), and gH-specific serum 1193 (21) (D). In lanes 1, untreated PrV virions were loaded. Lanes 2 contain proteins of mutant PrV-DgMgB. PrV virions shown in lanes 3 and 4 were incubated in N-glycosidase buffer without (lanes 3) or with (lanes 4) N-glycosidase F prior to polyacrylamide gel electrophoresis. Prior to loading onto the gel, proteins were denatured for 5 min at 958C.

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FIG. 3. In vitro translation of the UL10 protein. Whole-cell RNA isolated at 5 h p.i. was translated in vitro in a rabbit reticulocyte lysate (lanes 2 and 3), as was in vitro-transcribed UL10 mRNA (lanes 1 and 5 to 8). Translation products were separated in SDS–10% polyacrylamide gels either directly (lanes 1 and 2), after precipitation with antiserum directed against oligopeptide UL10.1 (lanes 3 and 6 to 8), or after precipitation with a negative control serum (lane 5). Lane 4 shows products of in vitro translation without exogenous RNA. Samples were loaded onto the gel either directly (lanes 1 and 2), after 1 h of incubation at 378C in loading buffer (lanes 3 to 7), or after 2 min of incubation at 958C in loading buffer (lane 8).

enzymatic digestion decreased the size of the 45-kDa form to ca. 33 kDa and that of the 90-kDa protein to ca. 70 kDa (Fig. 2, lanes 4). As a control, parallel blots were also probed with a monospecific antiserum directed against PrV gH (Fig. 2D) (21). These results demonstrate that the PrV UL10 protein is N glycosylated. Following the HSV-1 glycoprotein nomenclature, we designated it PrV gM. The higher-molecular-weight form with a size approximately double that of the 45-kDa protein is also modified by N-linked glycans and might represent a dimeric form of gM. In EHV-1, gM represents a major constituent of the virus particle (33). Although exact quantitation has not been attempted, as judged from the signal in Western blot analysis, gM appears to be a prominent virion glycoprotein in PrV, similar to the situation in EHV-1 (33) but unlike that in HSV-1 (4, 28). For in vitro transcription-translation, the UL10 open reading frame was cloned into vector pRc/CMV (InVitrogen, Leek, The Netherlands), transcribed in sense orientation by T7 polymerase, translated in a rabbit reticulocyte lysate (TNT coupled reticulocyte lysate system; Promega, Heidelberg, Germany) in the presence of [35S]methionine (ICN Biochemicals, Meckenheim, Germany), and analyzed either directly or after immunoprecipitation with anti-peptide UL10.1 serum by SDS–10% polyacrylamide gel electrophoresis as described previously (31). In addition, 2 mg of whole-cell RNA isolated 5 h after infection (20) was translated in vitro in a 25-ml reaction, and translation products were analyzed either directly (2 ml) or after immunoprecipitation of the remaining assay mixture (31). In vitro translation of in vitro-transcribed UL10 mRNA resulted in two products of 33 and 30 kDa (Fig. 3, lane 1) which are recognized by the anti-UL10.1 serum in immunoprecipitation (Fig. 3, lane 6) but not by a negative control serum (Fig. 3, lane 5). The smaller protein is most likely due to internal translation initiation at a downstream in-frame ATG at aa 41 (Fig. 1B). A 33-kDa protein is also recognized by the antiserum (Fig. 3, lane 3) among in vitro translation products of wholecell RNA isolated 5 h after infection (Fig. 3, lane 2). In Fig. 3, lane 4, translation products without addition of exogenous RNA are shown. The apparent size of the larger primary translation product matches the size of the de-N-glycosylated form but is smaller than the predicted molecular mass of 42 kDa. This difference might be due to the high hydrophobicity of the protein. Figure 3, lane 8, shows aggregation of the nonglycosylated gM precursor after immunoprecipitation of in vitro

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transcription-translation products with the anti-UL10 serum followed by 2 min of incubation at 958C compared with a parallel sample incubated for 1 h at 378C in gel loading buffer (Fig. 3, lane 7) before electrophoresis. Since the protein samples shown in Fig. 2 were boiled at 958C prior to loading on the gel, it appears as if PrV gM does not aggregate as easily as HSV-1 gM (4). In fact, boiling did not substantially alter the appearance of the gM products in Western blots compared with samples incubated at 56 or 378C (data not shown). In contrast, in vitro-translated gM precursor readily aggregated upon heating (Fig. 3, lanes 7 and 8). Although gM-homologous proteins are conserved in all herpesvirus subfamilies, gM was shown to be dispensable for replication in cell culture in HSV-1 and EHV-1 (3, 28, 32). Since the function of gM is still unclear, we sought to inactivate the PrV UL10 open reading frame by deletion/insertion mutagenesis. By a mutagenesis procedure called heterologous cis complementation (13), a 0.7-kb KpnI-SalI fragment (Fig. 1) comprising codons 126 to 353 of the UL10 open reading frame was deleted and substituted in parallel transcriptional orientation by a 3.3-kb KpnI-SalI fragment containing the bovine herpesvirus 1 (BHV-1) gB gene under control of its own promoter, resulting in plasmid pUL10DBHVgB. Complementation by BHV-1 gB restores replication competence in noncomplementing cells to a PrV gB2 mutant (34), which results in an easily selectable phenotype. After cotransfection (15) of PrV gB2 DNA with pUL10DBHVgB into Vero cells, infectious progeny appeared which was further plaque purified to obtain a homogeneous virus population. One plaque isolate, designated PrV-DgMgB, was further analyzed. Genotypic characterization of PrV-DgMgB by Southern blot analysis showed correct deletion of UL10-specific sequences and insertion of the foreign DNA as expected (data not shown). Western blot analysis of purified PrV-DgMgB virions (Fig. 2, lanes 2) confirmed the absence of the 45- and 90-kDa proteins (Fig. 2B), while gH was present in amounts similar to that in wild-type PrV (Fig. 2D). The fact that PrV-DgMgB could be isolated on noncomplementing cells indicated that UL10 is not essential for productive replication of PrV in cell culture. To analyze replication of the mutant in more detail, one-step growth kinetics were established. For comparison, PrV recombinant 9112C2, which carries the BHV-1 gB gene in the partially deleted PrV gB locus, was used to identify effects solely due to the insertion of the heterologous gene (22). As shown in Fig. 4A, PrV-9112C2 exhibits an increase in virus titer starting 8 h postinfection (p.i.), and the titer reaches near plateau levels at 24 h p.i. PrV-DgMgB, in contrast, shows a prolonged eclipse phase with lower titers at 24 h p.i. Differences in the ratio between extraand intracellular infectious virus were not observed between PrV-9112C2 and PrV-DgMgB. Compared with PrV-9112C2, final titers were reduced 10- to 50-fold in different experiments, indicating an impairment in virus growth in the absence of gM. Since the prolonged lag phase might be due to a delay in entry, penetration kinetics were investigated by low-pH inactivation of extracellular virus (22). As demonstrated in Fig. 4B, PrV9112C2 entered cells with a half-time of ca. 10 min, whereas PrV-DgMgB exhibited a longer lag phase, and it took ca. 30 min for 50% of the adsorbed virions to be internalized. In contrast to the phenotype seen in penetration, plaque sizes of PrVDgMgB were only slightly (ca. 15%) reduced compared with those of PrV-9112C2 (data not shown). Similar results were obtained with an independently isolated lacZ insertion mutant in PrV UL10 (data not shown). These data indicate a modulatory role of PrV gM in membrane fusion during initiation of infection by free virions. In HCMV, the gM-homologous pro-

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FIG. 4. One-step growth curve and penetration kinetics of gM2 PrV. (A) To assay one-step growth, Vero cells were infected at a multiplicity of infection of 2 with either PrV-9112C2 or PrV-DgMgB for 1 h at 48C. The inoculum was then removed, and the cells were overlaid with prewarmed medium to initiate penetration. After 2 h of incubation at 378C, extracellular virus was inactivated by low-pH treatment. Immediately thereafter and at the times indicated, cells and supernatant were harvested and titrated. Values for intra- and extracellular virus titers were added and plotted. Data from a representative experiment are shown. (B) For analysis of penetration kinetics, Vero cells in six-well culture dishes were infected with approximately 500 PFU of PrV-9112C2 or PrV-DgMgB per well for 1 h at 48C. Then the inoculum was removed, and cells were overlaid with prewarmed medium to initiate penetration. Immediately thereafter and at the time points indicated, extracellular virus was inactivated by low-pH treatment. After 2 days of incubation in minimal essential medium–0.5% methylcellulose, plaques were counted. The percentage of PFU surviving the low-pH treatment was calculated with reference to the values for phosphate-buffered saline-treated control plates. Values represent the averages of two independent experiments. Standard deviations are indicated.

tein is part of the gC-II glycoprotein complex, which has been shown to bind heparin and therefore probably functions in attachment of virions (19). However, in PrV, gC represents the only virion protein able to productively interact with heparan sulfate on the cell surface (30), and it therefore appears unlikely that PrV gM plays a role in this process. In HSV-1, EHV-1, and PrV, absence of functional gM resulted in only relatively slight phenotypic alterations (references 28 and 32 and this study). Therefore, the conservation of gM-homologous proteins throughout the herpesviruses is puzzling. Among the alphaherpesviruses analyzed, gM proteins exhibit homologies of between 32 and 40%, which indicates a degree of conservation similar to that for the essential gH and gK homologs. Whether a more prominent phenotype can be observed after infection of the natural host is under investigation. Nucleotide sequence accession number. The PrV UL10 sequence is contained in GenBank under accession number X97257. This work was supported by grant ERBCHTX-CT 920029 from the EC. The technical assistance of Uta Hartwig and Wim Verbruggen is greatly appreciated. REFERENCES 1. Albrecht, J. C., J. Nicholas, D. Biller, K. Cameron, B. Biesinger, C. Newman, S. Wittmann, M. Craxton, H. Coleman, B. Fleckenstein, and R. Honess. 1992. Primary structure of the herpesvirus saimiri genome. J. Virol. 66:5047– 5058. 2. Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J. Farrell, T. J. Gibson, T. Hatfull, G. S. Hudson, S. C. Satchwell, C. Seguin, P. S. Tuffnell, and B. G. Barrell. 1984. DNA sequence and expression of the B95-8 EpsteinBarr virus genome. Nature (London) 310:207–211. 3. Baines, J. D., and B. Roizman. 1991. The open reading frames UL3, UL4, UL10, and UL16 are dispensable for the replication of herpes simplex virus 1 in cell culture. J. Virol. 65:938–944.

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