ride, 50mM lithium citrate, 100 mM 2-mercaptoethanol, and. 0.5% Sarcosyl NL 97, and the RNA was isolated by centrif- ugation through a CsCl cushion (8).
JOURNAL
OF VIROLOGY, Jan. 1985, p. 52-57 0022-538X/85/010052-06$02.00/0 Copyright C) 1985, American Society for Microbiology
Vol. 53, No. 1
Mapping of the Structural Gene of Pseudorabies Virus Glycoprotein A and Identification of Two Non-Glycosylated Precursor Polypeptides THOMAS C. METTENLEITER, NOEMI LUKACS, AND HANNS-JOACHIM RZIHA*
Federal Research Centre for Virus Diseases of Animals, D-7400 Tubingen, Federal Republic of Germany Received 29 June 1984/Accepted 10 September 1984
Cell-free translation of pseudorabies virus RNA isolated during the late phase of the infectious cycle yielded variety of polypeptides. A monoclonal antibody directed against one of the major viral glycoproteins, gA, immunoprecipitated two polypeptides ranging in molecular weight from 78K to 83K. To localize the structural gene for gA, we used cloned BamHI fragments of the viral DNA to select specific mRNA species and immunoprecipitated their in vitro translation products with the anti-gA monoclonal antibody. This allowed us to map the genomic region encoding the mRNA for the gA within the short unique region of the viral genome on BamHI fragments 7 and 12. Additional polypeptides encoded by this region were characterized by their electrophoretic mobility. In three virus strains tested a similar, but strain-specific, pattern of the two gA precursors was found which was not dependent on the host cell or the state of infection after reaching the late phase. a
agated in bovine and hamster kidney cells (MDBK and BHK, respectively) as described previously (30) or in swine testis cells (IBRS-2) (4). Antisera and monoclonal antibodies. For the production of antisera adult pigs were inoculated with the inactivated vaccine Nobivac and challenged with the virulent PRV strain Phylaxia 4 weeks later (30). The preparation of monoclonal antibodies and their specificity is described elsewhere (17a). Plasmids. The BamHI DNA fragments of PRV cloned in pBR325 were kindly provided by T. Ben-Porat (14). Largescale amplification and gradient centrifugation of recombinant DNA were performed by standard methods. Each plasmid DNA was identified by restriction enzyme digestion and hybridization. The physical map of the PRV BamHI fragments is adopted from that of Ladin et al. (14) and adjusted to the PRV strain Phylaxia used in our laboratory (Fig. 1). RNA isolation. Confluent cell cultures were infected with PRV at a multiplicity of 20 PFU/cell, harvested usually at 5 h postinfection (p.i.) by trypsinization (0.25% trypsin, 0.125% EDTA), and washed once in ice-cold. phosphate-buffered saline. The cells were lysed in 6 M guanidinium-hydrochloride, 50 mM lithium citrate, 100 mM 2-mercaptoethanol, and 0.5% Sarcosyl NL 97, and the RNA was isolated by centrifugation through a CsCl cushion (8). The RNA pellets were suspended in 20 mM Tris-hydrochloride (pH 7.4), 1 mM EDTA, and 0.1% sodium dodecyl sulfate (SDS) and ethanol precipitated. The RNA was collected by centrifugation, suspended in sterile distilled water, and stored at -70°C. In vitro translation and gel electrophoresis. Cell-free translation was carried out in a reticulocyte lysate (New England Nuclear Corp., Boston, Mass.) as recommended by the supplier. The reaction mixture (25 ,ul) contained 0.1 mg of total RNA per ml and [135S]methionine (50 p.Ci, 1,000 Ci/mmol; New England Nuclear Corp.) to label the in vitro-synthesized polypeptides. A 2-,ul sample of the reaction mixture was used for the analysis of the total translation products. Electrophoresis was carried out in 10% SDSpolyacrylamide gels (15). After electrophoresis the gels were
Pseudorabies virus (PRV) is a member of the herpesvirus group (Suid herpesvirus 1) (24) and contains a linear, double-stranded DNA molecule of about 90 x 106 daltons that is separated by inverted repeat sequences into a long unique (U) and a short unique (Us) region. This results in the existence of two isomeric forms of the genome, and the DNA is classified as a class 1I molecule (1). The PRV transcripts can be divided into the following three classes, which are transcribed successively: immediate-early RNA transcribed shortly after infection in the absence of viral protein synthesis, early RNA synthesized before the onset of viral replication, and late RNA emerging after virus DNA synthesis has started (7). Whereas some functions of PRV are well characterized, such as the immediate-early protein (11), the localization of the structural genes for the viral glycoproteins is unknown. Similar to other herpesviruses, enveloped PRV virions contain at least 20 proteins ranging in molecular weight from 20K to 230K (27). Among the glycoproteins specified by PRV, three major glycoproteins, designated gA, gB, and gC, have been identified (17a). Recently Hampl et al. (9) described the existence of five glycoproteins in the virion envelope. In contrast to the herpes simplex viruses, where some glycoproteins are known to be involved in neutralization (20) and virus-cell interaction (25) only limited information is available about the biological significance of the PRV glycoproteins (9). In this study we determined the coding region for gA in the short unique region of the viral genome and identified two non-glycosylated precursor proteins of PRV gA by radioimmunoprecipitation with a specific monoclonal antibody. A comparison of the two pgA of three virus strains revealed three clearly distinguishable strain-specific patterns. MATERIALS AND METHODS Viruses and cells. The PRV strains Phylaxia, Ka (obtained from H. Hampl, Berlin), and NIA-1 (obtained from J. B. McFerran, Belfast) were used in this study. Virus was prop*
Corresponding author. 52
PSEUDORABIES VIRUS GLYCOPROTEINS
VOL. 53, 1985 UL 144 5E
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FIG. 1. BamHI restriction map of PRV as published by Ladin et al. (14) and adjusted to strain Phylaxia.
treated with En3Hance (New England Nuclear Corp.), dried, and exposed to Kodak XAR-5 film at -70°C. Hybrid selection and immunoprecipitation. After sonication and heat denaturation 400 ,ug of the recombinant plasmid DNA was immobilized on 40 nitrocellulose filters (3 by 3 mm; Schleicher & Schuell Co., Keene, N.H.) (22). The filter pieces were incubated in the presence of total RNA (200 Rg) in 10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 6.8), 400 mM NaCl, and 65% (vol/vol) formamide for 3 h at 56°C. After washing in 1 x SSC (0.15 M NaCl plus 0.015 M sodium citrate)-O. 1% SDS (10 times, 60°C) and subsequently in 2 mM EDTA, pH 7.4 (4 times, 60°C), the bound RNA was eluted from the filters by boiling the samples in distilled water for 2 min and immediately cooling in liquid nitrogen (6). After the addition of 50 ,ug of yeast tRNA per ml, the RNA was ethanol precipitated and used to direct a 25-,ul in vitro translation assay. Radioimmunoprecipitation was carried out as described elsewhere (17a). Briefly, the translation reaction mixtures were diluted with 200 ,ul of phosphate-buffered saline, 1% Nonidet P-40, 0.1% deoxycholate, 0.1% SDS, 2 mg of ovalbumin per ml, 0.2% NaN3, 1 mM phenylmethylsulfonyl fluoride, 1 mM methionine, and 2.5 mM KI and preadsorbed with 50 ,lI of Staphylococcus aureus suspension (13). The samples were incubated with either S ,ul of serum or 100 Ru of hybridoma supernatant for 90 min on ice, 100 RI of S. aureus suspension was added, and the probes were further incubated for 30 min. The immunoprecipitates were washed six times with phosphate-buffered saline, 1% Nonidet P-40, 0.1% deoxycholate, 0.1% SDS, 2 mg of ovalbumin per ml, 0.2% NaN3, 1 mM phenylmethylsulfonyl fluoride, 1 mM methionine, and 2.5 mM KI and eluted in 75 RI of sample buffer (0.12 M Tris-hydrochloride [pH 6.8], 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) by heating at 96°C for 2 min. Preparation of cDNA. The eluted RNA of six hybrid selection assays was pooled and used as a template for the reverse transcriptase (J. W. Beard, Life Science, Inc.). The reaction mixture (20 RI) contained 0.04 mM dCTP, 0.04 mM dTTP, 0.4 mM dATP, 0.4 mM dGTP, 15 ,uCi of [a-32P]dCTP, 15 jCi of [oa-32P]dTTP (410 Ci/mmol; Amersham-Buchler, Braunschweig), 10 mM dithiothreitol, 6 mM MgCI2, 40 mM KC1, 50 mM Tris-hydrochloride (pH 8.3), 1 jig of oligodeoxythymidylic acid (Boehringer, Mannheim) and 6 U of reverse transcriptase (12). After incubation at 40°C for 3 h the reaction was stopped by the addition of EDTA (pH 8.5) to a final concentration of 25 mM, and the mixture was extracted with an equal volume of phenol and ether. After ethanol precipitation in the presence of 0.3 mg of calf thymus DNA per ml, the nucleic acid was collected by centrifugation, the pellets were washed three times with 70% ethanol, and the RNA was hydrolyzed in 0.3 M NaOH-1 mM EDTA (200 jil) by incubation at 65°C for 20 min. This procedure resulted in an almost complete removal of unincorporated labeled nucleotides as proven by chromatography on Sephadex G 50 (Pharmacia, Uppsala, Sweden). Blot hybridi-
53
zations to the BamHI DNA fragments of the recombinant plasmids were performed essentially as described previously (26, 29) under stringent annealing conditions. Nick translation. In vitro labeling of the recombinant clones with [a-32P]dCTP and [a-32P]dTTP (410 Ci/mmol, Amersham) to a specific activity of about 5 x 107 cpm/,ug of DNA by the nick translation technique was performed as described previously (23). Gel electrophoresis of RNA and Northern blot hybridization. Polyadenylated RNA preparations were obtained by oligodeoxythymidylic acid-cellulose (PL biochemicals) chromatography (18). The RNA was cycled two times, resulting in almost complete removal of detectable rRNA species. The quality of each RNA preparation was evaluated by in vitro translation assays. The RNA was denatured with glyoxal, electrophorized in agarose gels (3), and transferred to nitrocellulose filters as described by Thomas (28). The RNA blots were preincubated overnight at 52°C in 60% (vol/vol) formamide (deionized and recrystallized; Bethesda Research Laboratories, Rockville, Md.), 5 x SSC, 1% SDS, 5 x Denhardt solution (5), 100 ,ug of yeast tRNA (Boehringer, Mannheim) per ml, and 100 jg of denatured calf thymus DNA per ml. Hybridization with nick-translated cloned DNA fragments (5 x 105 cpm/ml) was performed for 3 days at 52°C. The filters were washed two times for 10 min in 2 x SSC-0.5% SDS at room temperature, twice in 2 x SSC-0.5% SDS for 30 min, once in 0.1 x SSC-0.1% SDS for 15 min at 72°C, and finally in 0.1 x SSC at room temperature. After drying of the filters, autoradiography was performed at -70°C with Kodak XAR-5 film and Agfa Curix intensifying screens. RESULTS Immunoprecipitation of the polypeptides synthesized in vitro. To analyze the late viral proteins, RNA was isolated at 5 h p.i. and translated in vitro, and the products were immunoprecipitated with either pig hyperimmune serum or a monoclonal antibody (designated 3/6) directed against gA. The results obtained with two different PRV strains, Phylaxia and Ka, are shown in Fig. 2. Among the in vitro translation products (Fig. 2, lanes 1 and 5) many virus-specific polypeptides were present, as demonstrated by precipitation with pig hyperimmune serum (Fig. 2, lanes 2 and 6). The prominent protein band of about 142K represents the major viral capsid protein of PRV (14). The antibody 3/6, which recognized the non-glycosylated precursors for glycoprotein A, specifically precipitated two polypeptides from the in vitro translation products (Fig. 2, lanes 4 and 8). The gA precursor molecules of the strain Phylaxia revealed molecular weights of 82K and 83K, whereas in the strain Ka two pgA of 80K and 82.5K were found (Fig. 2). To demonstrate that both pgA proteins were indeed synthesized during the late phase of infection, the cells were infected at a high multiplicity (100 PFU/cell), and total RNA was isolated at different times p.i. In vitro translation and precipitation with the monoclonal antibody (Fig. 3) showed that during the first hour p.i. no pgA polypeptide could be precipitated. The gA precursors were detected by immunoprecipitation not earlier than 2 h p.i., coinciding with the onset of the synthesis of late PRV proteins (Fig. 3A), as indicated by the appearance of the major capsid protein and other late viral polypeptides. During the course of the infection the pattern of the pgA of the strain Phylaxia (Fig. 3)
METTENLEITER, LUKACS, AND RZIHA
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