1887

Abstract

Virus-encoded proteinase activity of hepatitis A virus (HAV) was studied . Genomic regions coding for segments of the viral polyprotein were expressed by transcription and translation in rabbit reticulocyte lysates. Polyproteins translated from synthetic transcripts encoding P1–P2 or ΔVP1–P2 were not processed indicating that no proteolytic activity is encoded within P2 of HAV, in contrast to other picornaviruses. Proteinase activity was, however, detected in the genomic region encoding 3C. Mutant transcripts (µ) which encode an alanine in place of the cysteine residue at amino acid position 172 of 3C did not yield proteolytic activity, consistent with the hypothesis that proteinase 3C is a cysteine-containing trypsin-like proteinase. Processing products 3ABC and P3 were identified by immunoprecipitation, providing evidence that proteolytic cleavage occurs at the 2C/3A and less frequently at the 3C/3D junction. For cleavages at either site, the complete 3D moiety was not required. In general, analysis of cleavage products was made difficult by the presence of polypeptides which were translated from internal start sites, predominantly within the P3 region. Since only small amounts of the full-length products P1–P2–P3 or P2–P3 were translated, possible cleavage of P1 and P2 by 3C could not be resolved in this system. Furthermore, no intermolecular cleavage could be detected when translated polypeptides of the P3 region were incubated with P1, P1–P2 or P2–P3μ as substrates.

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1993-04-01
2021-10-25
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References

  1. Bazan J. F., Fletterick R. J. 1988; Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications. Proceedings of the National Academy of Sciences, U.S.A. 85:7872–7876
    [Google Scholar]
  2. Brown E. A., Day S. P., Jansen R. W., Lemon S. M. 1991; The 5′ nontranslated region of hepatitis A virus RNA: secondary structure and elements required for translation in vitro. Journal of Virology 85:5828–5838
    [Google Scholar]
  3. Cohen J. I., Ticehurst J. R., Purcell R., Buckler-White A., Baroudy R. M. 1987; Complete nucleotide sequences of hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses. Journal of Virology 61:50–59
    [Google Scholar]
  4. Dorner A. J., Semler B. L., Jackson R., Hanecak R., Duprey E., Wimmer E. 1984; In vitro translation of poliovirus RNA: utilization of internal sites in reticulocyte lysate. Journal of Virology 50:507–514
    [Google Scholar]
  5. Gauss-Müller V. 1990; Studies on hepatitis A virus 3C proteinase. In Viral Hepatitis 1990 pp 65–68 Edited by Hollinger B. Baltimore: Williams & Wilkins;
    [Google Scholar]
  6. Gauss-Müller V., Jurgensen D., Deutzmann R. 1991; Autoproteolytic cleavage of recombinant 3C proteinase of hepatitis A virus. Virology 182:861–864
    [Google Scholar]
  7. Gorbalenya A., Donchenko A. P., Blinov V. M., Koonin E. V. 1989; Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases: a distinct protein superfamily with common structural fold. FEBS Letters 234:103–114
    [Google Scholar]
  8. Hämmerle T., Hellen C. T., Wimmer E. 1991; Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase. Journal of Biological Chemistry 266:5412–5416
    [Google Scholar]
  9. Harmon S. A., Updike W., Summers D. F., Ehrenfeld E. 1992; Polyprotein processing in cis and in trans by hepatitis A virus 3C protease cloned and expressed in E. coli. Journal of Virology 66:5242–5247
    [Google Scholar]
  10. Jackson R. J. 1986; A detailed kinetic analysis of the in vitro synthesis and processing of encephalomyocarditis virus products. Virology 149:114–127
    [Google Scholar]
  11. Jackson R. J. 1989; Comparison of encephalomyocarditis virus and poliovirus with respect to translation initiation and processing in vitro. In Molecular Aspects of Picornavirus Infection and Detection pp 51–71 Edited by Semler B. L., Ehrenfeld E. Washington: American Society for Microbiology;
    [Google Scholar]
  12. Jia X. Y., Ehrenfeld E., Summers D. F. 1991 a; Proteolytic activity of hepatitis A virus 3C protein. Journal of Virology 65:2595–2600
    [Google Scholar]
  13. Jia X. Y., Scheper G., Brown D., Updike W., Harmon S., Richards S., Summers D. F., Ehrenfeld E. 1991 b; Translation of hepatitis A virus RNA in vitro: aberrant internal initiations influenced by 5′ noncoding region. Virology 182:712–722
    [Google Scholar]
  14. Jore J., DeGeus B., Jackson R. J., Pouwels P. H., Enger-Valk B. E. 1988; Poliovirus protein 3CD is the active protease for processing of the precursor protein P1 in vitro. Journal of General Virology 69:1627–1636
    [Google Scholar]
  15. Kräusslich H. G., Wimmer E. 1988; Viral proteinases.. Annual Review of Biochemistry 57:701–754
    [Google Scholar]
  16. Kunkel T. 1985; Rapid and efficient site-specific mutagenesis without phenotypic selection. Proceedings of the National Academy of Sciences, U.S.A. 82:488–192
    [Google Scholar]
  17. Kusov Y. Y., SOMMERGRUBER W., SCHREIBER M., GAUSS-MULLER V. 1992; Intermolecular cleavage of hepatitis A virus (HAV) precursor protein P1–P2 by recombinant HAV proteinase 3C. Journal of Virology 66:6794–6796
    [Google Scholar]
  18. Malcolm B. A., Chin S. M., Jewell D. A., Stratton-Thomas J. R., Thudium K. B., Ralston R., Rosenberg S. 1992; Expression and characterization of recombinant hepatitis A virus 3C proteinase. Biochemistry 31:3358–3363
    [Google Scholar]
  19. Molla A., Paul A. V., Wimmer E. 1991; Cell-free, de-novo synthesis of poliovirus. Science 254:1647–1651
    [Google Scholar]
  20. Nicklin M. JH., Kräusslich H. G., Toyoda H., Dunn J. J., Wimmer E. 1987; Poliovirus polypeptide precursor: expression in vitro and processing by exogenous 3C and 2A proteinases. Proceedings of the National Academy of Sciences, U.S.A. 84:4002–4006
    [Google Scholar]
  21. Palmenberg A. C. 1990; Proteolytic processing of picornaviral polyproteins. Annual Review of Microbiology 44:603–624
    [Google Scholar]
  22. Paul A. V., Tada H., von der Helm K., Wissel T., Kiehn R., Wimmer E., Deinhardt F. 1987; The entire nucleotide sequence of the genome of human hepatitis A virus (isolate MBB). Virus Research 8:153–171
    [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A. 74:5463–5467
    [Google Scholar]
  24. Ticehurst J., Cohen J. I., Feinstone S. M., Purcell R. H., Jansen W., Lemon S. M. 1989; Replication of hepatitis A virus: new ideas from studies with cloned cDNA. In Molecular Aspects of Picornavirus Infection and Detection pp 27–50 Edited by Semler B. L., Ehrenfeld E. Washington: American Society for Microbiology;
    [Google Scholar]
  25. Updike W. S., Tesar M., Ehrenfeld E. 1991; Detection of hepatitis A virus protein in infected BS-C-1 cells. Virology 185:411–418
    [Google Scholar]
  26. Ypma-Wong M. F., Semler B. L. 1987; Processing determinant required for in vitro cleavage of the poliovirus P1 precursor to capsid proteins. Journal of Virology 61:3199–3207
    [Google Scholar]
  27. Ypma-Wong M. F., Dew alt P. G., Johnson V. H., Lamb J. G., Semler B. L. 1988; Protein 3CD is the major poliovirus proteinase responsible for cleavage of the P1 capsid precursor. Virology 166:265–270
    [Google Scholar]
  28. Yu S. F., Lloyd R. E. 1992; Characterization of the roles of conserved cysteine and histidine residues in poliovirus 2A protease. Virology 186:725–735
    [Google Scholar]
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