Feb 19, 1991 - Members of the genus Pestivirus, family Togaviridae, are causative agents of economically important diseases that occur worldwide, such as ...
Vol. 65, No. 9
JOURNAL OF VIROLOGY, Sept. 1991, p. 4705-4712
0022-538X/91/094705-08$02.00/0 Copyright © 1991, American Society for Microbiology
Hog Cholera Virus: Molecular Composition of Virions from a Pestivirus HEINZ-JURGEN THIEL,* ROBERT STARK, EMILIE WEILAND, TILLMANN RUMENAPF, AND GREGOR MEYERS Federal Research Centre for Virus Diseases of Animals, P.O. Box 11 49, D-7400 Tubingen, Federal Republic of Germany Received 19 February 1991/Accepted 21 May 1991
Virions from hog cholera virus (HCV), a member of the genus Pestivirus, were analyzed by using specific antibodies. The nucleocapsid protein was found to be a 14-kDa molecule (HCV p14). An equivalent protein could also be demonstrated for virions from another pestivirus, bovine viral diarrhea virus. The HCV envelope is composed of three glycoproteins, HCV gp44/48, gp33, and gp55. All three exist in the form of disulfide-linked dimers in virus-infected cells and in virions; HCV gp44/48 and gp55 each form homodimers, whereas gp55 is also found dimerized with gp33. Such complex covalent interactions between structural glycoproteins have not been described so far for any RNA virus. Members of the genus Pestivirus, family Togaviridae, are causative agents of economically important diseases that occur worldwide, such as hog cholera, bovine viral diarrhea, and border disease of sheep (32). Complete nucleotide sequences of the genomes of two pestiviruses, bovine viral diarrhea virus (BVDV) and hog cholera virus (HCV), have been published (4, 15, 17, 22). The pestivirus genome represents a single-stranded RNA of about 12.5 kb, which is of positive polarity and comprises a single large open reading frame (ORF) (4, 15, 17). Antibodies against bacterial fusion proteins encompassing defined regions of the pestivirus polyproteins were used to identify proteins encoded by the viral genome (5, 27). The data have led to important conclusions concerning the strategy of gene expression and genome organization of this virus group. The 5'-terminal part of the pestivirus ORF is assumed to code for the nucleocapsid protein which is followed by a putative signal peptide and three heavily glycosylated proteins, which are called gp44/48, gp33, and gpS5 in HCV (5, 27, 30). If these assumptions are correct, all previous reports about structural proteins from pestiviruses are wrong or at least incomplete (9). Specifically, (i) the putative core protein has so far not been demonstrated; (ii) BVDV p125/p80 actually represent nonstructural proteins and not, as described previously, parts of the virion (20, 21); (iii) proteins with apparent molecular masses of 25, 26, 36, and 34 kDa are probably identical with gp33 (HCV) and gp25 (BVDV) but have not been described as glycosylated molecules (8, 14, 20); and (iiii) it has been reported that HCV gp33 is not part of the virion (31). However, two of the three putative structural glycoproteins have been detected in virions (8, 14). In addition, BVDV gp53 and HCV gp55 have been indirectly shown to form part of the virion since they mediate neutralization by monoclonal antibodies (MAbs) (7, 30, 31). Accordingly, the molecular composition of virions from the genus Pestivirus is still a matter of controversy. So far, the material necessary for a definitive analysis was not accessible because of poor virus yields, lack of release of virions from infected cells, and close association of virus *
with other mostly cellular components (12, 16). The last factor has made it extremely difficult to purify virions by physical means. In addition, serological reagents with defined specificities were not available. In the present report HCV virions were analyzed by using an efficient cell culture system for HCV propagation as well as specific serological reagents against different components assumed to be part of the virion. The core protein was identified for the first time. In addition, the data demonstrate complex interactions of the different structural glycoproteins by disulfide bridges.
MATERIALS AND METHODS Cells and viruses. PK15 and MDBK cells were obtained from the American Type Culture Collection, Rockville, Md. W. Schafer, Max-Planck-Institut fur Virusforschung, Tubingen, kindly provided the pig lymphoma cell line 38A1D (28). Cell lines were grown in Dulbecco modified Eagle medium-10% fetal calf serum. HCV Alfort was obtained from B. Liess, Veterinary School, Hannover, Federal Republic of Germany, and BVDV NADL was obtained from the American Type Culture Collection. In the course of animal experiments, the Alfort strain was reisolated from organs of a moribund animal and used thereafter for multiplication in 38A1D cells. Cells and virus stock were tested every 3 to 6 months for the absence of Mycoplasma contamination. Infection of cells. 38A1D cells were infected in suspension in a volume of 20 to 30 ml at a cell concentration of 5 x 107/ml at 37°C for 90 min with virus at a multiplicity of infection of 0.01 to 0.001. Thereafter, the cells were incubated in bottles with gentle stirring (Tecnomara, Femwald, Switzerland). This tissue culture system for HCV propagation has been shown to be more efficient than the use of the standard cell line, PK 15 (25). MDBK cells were infected with BVDV as layer cells at 1.5 x 106 per 3.5-cm dish. Goat anti-HCV serum. Preparation of goat anti-HCV serum after establishment of a fibroblastic cell strain and immunization with autologous HCV-infected cells has been described previously (24). Metabolic labeling of cells and viruses. 38A1D cells at 1 x 107 cells/ml were labeled for 24 h with 0.5 mCi of [35S]me-
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thionine-[35S]cysteine, [3H]lysine-[3H]arginine, or [3H]glucosamine (Amersham Buchler, Braunschweig, Federal Republic of Germany) per ml. BVDV-infected MDBK cells were also labeled for 24 h with 0.5 mCi of [35S]cysteine[35S]methionine per ml. The labeling medium contained either no cysteine and 1/20 of the normal methionine content (35S-amino acids), no lysine or arginine ([3H]lysine-[3H]arginine), or 20 mM fructose instead of glucose ([3H]glucosamine). After the labeling period, the cells were stored at -70°C. Labeling was started 24 to 48 h (HCV) or 72 h (BVDV) after infection. Purification of labeled viruses. Supernatant from infected 38A1D and MDBK cells was centrifuged first at 5,400 x g for 20 min to remove cells, nuclei, and large-cell debris. After the virus had been pelleted according to a sedimentation coefficient described for pestiviruses (S20,, of about 100) (16), the pellet was resuspended in a buffer suitable for radioimmunoprecipitation and stored at -70°C. Radioimmunoprecipitation and SDS-PAGE. Cell and virus extracts were prepared as described earlier (24) and incubated with appropriate dilutions of antibodies. Precipitates were formed with cross-linked Staphylococcus aureus (11), analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10 or 12% acrylamide) under reducing or nonreducing conditions, and processed for fluorography by using EnHance (New England Nuclear, Boston, Mass.). The dried gels were exposed to Kodak XAR-5 X-ray films at -70°C. The '4C-labeled molecular weight standards were from Amersham Buchler. Two-dimensional SDS-PAGE. Two-dimensional SDSPAGE was performed essentially as described previously (10, 26). Preparation of anti-Dl serum and anti-A3 serum. HCVderived cDNA fragments coding for amino acids 102 to 292 (D1) and amino acids 1750 to 2349 (A3) of the HCV ORF into the expression vector pEX34 were subcloned by standard procedures (13). pEX34 is identical to the previously described expression vector pEX31 (29), except for a deleted PstI site in the ampicillin resistance gene; expression and enrichment of the bacterial fusion protein were performed basically as described previously (29). Fusion proteins were further purified by preparative SDS-PAGE and, after electroelution, injected subcutaneously into rabbits with Freund adjuvant (complete for basic immunization, incomplete for booster injections). Preparation of anti-Gl serum has been described previously (26). Peptide synthesis. Peptides were synthesized on a MilliGen 9050 PepSynthesizer (Millipore) by using the Fmoc-polyamide method. HCV peptide 6 (Val-Lys-Gly-Lys-Asn-Thr-GlnAsp-Gly-Leu-Tyr-His-Asn-Lys-Asn-Lys-Pro) and HCV peptide 12 (Lys-Asn-Lys-Pro-Pro-Glu-Ser-Arg-Lys-LysLeu-Glu) correspond to amino acids 224 to 240 and amino acids 237 to 248, respectively, of the HCV ORF. Preparation of antipeptide antibodies. The HCV peptides were coupled to activated CH-Sepharose 4B (Pharmacia) as specified by the manufacturer. The peptide-coupled beads were mixed with anti-Dl serum for 16 h at 4°C and packed in a column. After extensive washing with phosphate-buffered saline, antipeptide antibodies were eluted with 0.1 M glycine (pH 2.8). Fractions containing the antipeptide antibodies were neutralized with 0.25 volume of 1 M Tris-HCl (pH 7.5). Preparation of MAbs. The procedure used for preparation of MAbs against HCV gp44/48 was basically similar to the one described previously for anti-HCV gp55 MAbs (30).
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