1887

Abstract

The structural part of the hepatitis C virus (HCV) genome encodes a capsid protein, C, and two envelope glycoproteins, E1 and E2, released from the virus polyprotein precursor by signalase(s) cleavage(s). The processing of E1 was investigated by infecting simian cells with recombinant vaccinia viruses expressing parts of the HCV structural proteins. When the predicted E1 sequence was expressed alone (amino acid residues 174–370 of the polyprotein) or with the capsid protein gene (residues 1–370), it showed an apparent molecular mass of 35 kDa as measured by SDS-PAGE analysis. However, when E1 was expressed as part of a truncated C-E1-truncated E2 polypeptide (residues 132–383), the processed E1 product had the expected apparent molecular mass of 31 kDa, suggesting that flanking sequences are necessary for the generation of the mature 31 kDa E1 form. The N-terminal sequence of the two E1 forms was found to be the same. Analysis of the glycosylation pattern showed that, in both species, only four of the five potential -linked glycosylation sites were recognized, indicating that glycosylation was not involved in the molecular mass difference. We showed that expression of E1 with or without the hydrophobic stretch of amino acids residues 371–383, defined as the E2 signal sequence, may be responsible for the difference in electrophoretic mobility of the two E1 species. translation assays and site-directed mutagenesis experiments suggest that this sequence remains part of the 31 kDa E1 mature protein.

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1996-05-01
2024-03-29
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References

  1. Baron M. D., Ebel T., Suomalainen M. 1992; Intracellular transport of rubella virus structural proteins expressed from cloned cDNA. Journal of General Virology 73:1073–1086
    [Google Scholar]
  2. Bartenschlager R., Ahlbornlaake L., Mous J., Jacobsen H. 1993; Nonstructural protein-3 of the hepatitis-C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. Journal of Virology 67:3835–3844
    [Google Scholar]
  3. Cahour A., Falgout B., Lai C. J. 1992; Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease. Journal of Virology 66:1535–1542
    [Google Scholar]
  4. Chen P. J., Lin M. H., Tai K. F., Liu P. C., Lin C. J., Chen D. S. 1992; The Taiwanese hepatitis-C virus genome - sequence determination and mapping of the 5′ termini of viral genomic and antigenomic RNA. Virology 188:102–113
    [Google Scholar]
  5. Choo Q.-L., Kuo G., Weiner A. J., Overby L. R., Bradley D. W., Houghton M. 1989; Isolation of a cDNA clone derived from a blood-borne non-A non-B viral hepatitis genome. Science 244:359–362
    [Google Scholar]
  6. Choo Q.-L., Richman K. H., Han J. H., Berger K., Lee C., Dong C., Gallegos C., Coit D., Medina-Selby A., Barr P. J., Weiner A. J., Bradley D. W., Kuo G., Houghton M. 1991; Genetic organization and diversity of the hepatitis C virus. Proceedings of the National Academy of Sciences, USA 88:2451–2455
    [Google Scholar]
  7. Drillien R., Spehner D., Kirn A. 1982; Complementation and genetic linkage between vaccinia virus temperature-sensitive mutants. Virology 119:372–381
    [Google Scholar]
  8. Dubuisson J., Hsu H. H., Cheung R. C., Greenberg H. B., Russell D. G., Rice C. M. 1994; Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses. Journal of Virology 68:6147–6160
    [Google Scholar]
  9. Feinstone S. M., Alter H. J., Dienes H. P., Shimizu Y., Popper H., Blackmore D., Sly D., London W. T., Purcell R. H. 1981; Non-A, non-B hepatitis in chimpanzees and marmosets. Journal of Infectious Diseases 144:588–598
    [Google Scholar]
  10. Francki R. I., Fauquet C. M., Knudson D. L., Brown F. (editors) 1991 Classification and Nomenclature of Viruses. Fifth Report of the International Committee on Taxonomy of Viruses pp 223–233 Wein and New York: Springer-Verlag;
    [Google Scholar]
  11. Fuerst T. R., Niles E. G., Studier F. W., Moss B. 1986; Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proceedings of the National Academy of Sciences, USA 83:8122–8126
    [Google Scholar]
  12. Grakoui A., McCourt D. W., Wychowski C., Feinstone S. M., Rice C. M. 1993a; A second hepatitis-C virus-encoded proteinase. Proceedings of the National Academy of Sciences, USA 90:10583–10587
    [Google Scholar]
  13. Grakoui A., McCourt D. W., Wychowski C., Feinstone S. M., Rice C. M. 1993b; Characterization of the hepatitis-C virus-encoded serine proteinase–determination of proteinase-dependent polyprotein cleavage sites. Journal of Virology 67:2832–2843
    [Google Scholar]
  14. Grakoui A., Wychowski C., Lin C., Feinstone S. M., Rice C. M. 1993c; Expression and identification of hepatitis C virus polyprotein cleavage products. Journal of Virology 67:1385–1395
    [Google Scholar]
  15. Gübler U., Hoffman B. J. 1983; A simple and very efficient method for generating cDNA libraries. Gene 25:263–269
    [Google Scholar]
  16. Hijikata M., Kato N., Ootsuyama Y., Nakagawa M., Shimotohno K. 1991; Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proceedings of the National Academy of Sciences, USA 88:5547–5551
    [Google Scholar]
  17. Hijikata M., Mizushima H., Tanji Y., Komoda Y., Hirowatari Y., Akagi T., Kato N., Kimura K., Shimotohno K. 1993; Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis-C virus. Proceedings of the National Academy of Sciences, USA 90:10773–10777
    [Google Scholar]
  18. Inchauspe G., Zebedee S., Lee D.-H., Sugitani M., Nasoff M., Prince A. M. 1991; Genomic structure of the human prototype strain H of hepatitis C virus: comparison with the American and Japanese isolates. Proceedings of the National Academy of Sciences, USA 88:10292–10296
    [Google Scholar]
  19. Kato N., Hijikata M., Ootsuyama Y., Nakagawa M., Ohkoshi S., Sugimura T., Shimotohno K. 1990; Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proceedings of the National Academy of Sciences, USA 87:9524–9528
    [Google Scholar]
  20. Kieny M. P., Lathe R., Drillien R., Spehner D., Skory S., Schmitt D., Wiktor T., Koprowski H., Lecocq J. P. 1984; Expression of rabies virus glycoprotein from a recombinant vaccinia virus. Nature 312:163–166
    [Google Scholar]
  21. Konishi E., Pincus S., Fonseca B. A. L., Shope R. E., Paoletti E., Mason P. W. 1991; Comparison of protective immunity elicited by recombinant vaccinia viruses that synthesize E or NS1 of Japanese encephalitis virus. Virology 185:401–410
    [Google Scholar]
  22. Kornfeld R., Kornfeld S. 1985; Assembly of asparagine-linked oligosaccharides. Annual Review of Biochemistry 54:631–664
    [Google Scholar]
  23. Kuo G., Choo Q.-L., Alter H. J., Gitnick G. L., Redecker A. G., Purcell R. H., Miyamura T., Dienstag J. L., Alter M. J., Stevens C. E., Tegtmeier G. E., Bonino F., Colombo M., Lee W. S., Kuo C., Berger K., Shuster J. R., Overby L. R., Bradley D. W., Houghton M. 1989; An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 244:362–364
    [Google Scholar]
  24. Lanford R. E., Notv all L., Chavez D., White R., Frenzel G., Simonsen C., Kim J. 1993; Analysis of hepatitis-C virus capsid, E1, and E2/NS1 proteins expressed in insect cells. Virology 197:225–235
    [Google Scholar]
  25. Lin C., Lindenbach B. D., Pragai B., McCourt D. W., Rice C. M. 1994; Processing of the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. Journal of Virology 68:5063–5073
    [Google Scholar]
  26. Maley F., Trimble R. B., Tarentino A. L., Plummer T. H. 1989; Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Analytical Biochemistry 180:195–204
    [Google Scholar]
  27. Manabe S., Fuke I., Tanishita O., Kaji C., Gomi Y., Yoshida S., Mori C., Takamizawa A., Yoshida I., Okayama H. 1994; Production of non-structural proteins of hepatitis C virus requires a putative viral protease encoded by NS3. Virology 198:636–644
    [Google Scholar]
  28. Matsuura Y., Harada S., Suzuki R., Watanabe Y., Inoue Y., Saito I., Miyamura T. 1992; Expression of processed envelope protein of hepatitis-C virus in mammalian and insect cells. Journal of Virology 66:1425–1431
    [Google Scholar]
  29. Matsuura Y., Suzuki T., Suzuki R., Sato M., Aizaki H., Saito I., Miyamura T. 1994; Processing of E1 and E2 glycoproteins of hepatitis C virus expressed in mammalian and insect cells. Virology 205:141–150
    [Google Scholar]
  30. Mizushima H., Hijikata M., Asabe S. I., Hirota M., Kimura K., Shimotohno K. 1994; Two hepatitis C virus glycoprotein E2 products with different C termini. Journal of Virology 68:6215–6222
    [Google Scholar]
  31. Moss B., Elroy-Stein O., Mizukami T., Alexander W. A., Fuerst T. R. 1990; New mammalian expression vectors. Nature 348:91–92
    [Google Scholar]
  32. Okamoto H., Okada S., Sugiyama Y., Kurai K., Iizuka H., Machida A., Miyakama Y., Mayumi M. 1991; Nucleotide sequence of the genomic RNA of hepatitis C virus isolated from a human carrier: comparison with reported isolates for conserved and divergent regions. Journal of General Virology 72:2697–2704
    [Google Scholar]
  33. Ralston R., Thudium K., Berger K., Kuo C., Gervase B., Hall Selby M., Kuo G., Houghton M., Choo Q.-L. 1993; Characterization of hepatitis-C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses. Journal of Virology 67:6753–6761
    [Google Scholar]
  34. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. 1988; Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491
    [Google Scholar]
  35. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual 2nd edn New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  36. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, USA 74:5463–5467
    [Google Scholar]
  37. Selby M. J., Glazer E., Masiarz F., Houghton M. 1994; Complex processing and protein:protein interactions in the E2:NS2 region of HCV. Virology 204:114–122
    [Google Scholar]
  38. Takamisawa A., Mori C., Fuke I., Manabe S., Murakami S., Jujita J., Onishi E., Andoh T., Yoshida I., Okayama H. 1991; Structure and organization of the hepatitis C virus genome isolated from human carriers. Journal of Virology 65:1105–1113
    [Google Scholar]
  39. Tarentino A. L., Plummer T. H., Maley F. 1974; The release of intact oligosaccharides from specific glycoproteins by endo-β-N- acetylglucosaminidase H. Journal of Biological Chemistry 249:818–824
    [Google Scholar]
  40. Tomei L., Failla C., Santolini E., DeFrancesco R., Lamonica N. 1993; NS3 is a serine protease required for processing of hepatitis-C virus polyprotein. Journal of Virology 67:4017–4026
    [Google Scholar]
  41. Wychowski C., van der Were S., Girard M. 1985; Nuclear localization of poliovirus capsid polypeptide VP1 expressed as a fusion protein with SV40-VP1. Gene 37:63–71
    [Google Scholar]
  42. Zarlenga D. S., Gamble H. R. 1987; Simultaneous isolation of preparative amounts of RNA and DNA from Trichinella spiralis by cesium trifluoroacetate isopycnic centrifugation. Analytical Biochemistry 162:569–574
    [Google Scholar]
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