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. 1997 Jul;71(7):5415–5422. doi: 10.1128/jvi.71.7.5415-5422.1997

Serine protease of pestiviruses: determination of cleavage sites.

N Tautz 1, K Elbers 1, D Stoll 1, G Meyers 1, H J Thiel 1
PMCID: PMC191781  PMID: 9188613

Abstract

The single-stranded genomic RNA of pestiviruses is of positive polarity and encompasses one large open reading frame of about 4,000 codons. The resulting polyprotein is processed co- and posttranslationally by virus-encoded and host cell proteases to give rise to the mature viral proteins. A serine protease residing in the nonstructural (NS) protein NS3 (p80) has been shown to be essential for the release of the NS proteins located downstream of NS3. In this report the NS3 serine protease-dependent cleavage sites for bovine viral diarrhea virus (BVDV) strain CP7 are described. Proteins used for analysis were generated in Escherichia coli or in eukaryotic cells by the use of the T7 vaccinia virus system. The N termini of NS4A, NS4B, NS5A, and NS5B were determined by protein sequencing. Analysis of the data obtained showed that leucine at P1 is the only position conserved for all cleavage sites. At P1' alanine is found at the NS4A-NS4B site, whereas serine resides at this position at the NS3-NS4A, NS4B-NS5A, and NS5A-NS5B cleavage sites. For all cleavage sites the amino acids found at P1 and P1' are conserved for different genotypes of pestiviruses, despite the high degree of sequence variation found between these viruses. It is therefore assumed that the cleavage sites determined for BVDV CP7 are representative of those for all pestiviruses.

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Selected References

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  1. Bartenschlager R., Ahlborn-Laake L., Mous J., Jacobsen H. Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol. 1993 Jul;67(7):3835–3844. doi: 10.1128/jvi.67.7.3835-3844.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bartenschlager R., Ahlborn-Laake L., Yasargil K., Mous J., Jacobsen H. Substrate determinants for cleavage in cis and in trans by the hepatitis C virus NS3 proteinase. J Virol. 1995 Jan;69(1):198–205. doi: 10.1128/jvi.69.1.198-205.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bazan J. F., Fletterick R. J. Detection of a trypsin-like serine protease domain in flaviviruses and pestiviruses. Virology. 1989 Aug;171(2):637–639. doi: 10.1016/0042-6822(89)90639-9. [DOI] [PubMed] [Google Scholar]
  4. Becher P., Shannon A. D., Tautz N., Thiel H. J. Molecular characterization of border disease virus, a pestivirus from sheep. Virology. 1994 Feb;198(2):542–551. doi: 10.1006/viro.1994.1065. [DOI] [PubMed] [Google Scholar]
  5. Colett M. S., Larson R., Gold C., Strick D., Anderson D. K., Purchio A. F. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology. 1988 Jul;165(1):191–199. doi: 10.1016/0042-6822(88)90672-1. [DOI] [PubMed] [Google Scholar]
  6. Collett M. S., Larson R., Belzer S. K., Retzel E. Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus. Virology. 1988 Jul;165(1):200–208. doi: 10.1016/0042-6822(88)90673-3. [DOI] [PubMed] [Google Scholar]
  7. Collett M. S., Moennig V., Horzinek M. C. Recent advances in pestivirus research. J Gen Virol. 1989 Feb;70(Pt 2):253–266. doi: 10.1099/0022-1317-70-2-253. [DOI] [PubMed] [Google Scholar]
  8. Collett M. S., Wiskerchen M., Welniak E., Belzer S. K. Bovine viral diarrhea virus genomic organization. Arch Virol Suppl. 1991;3:19–27. doi: 10.1007/978-3-7091-9153-8_3. [DOI] [PubMed] [Google Scholar]
  9. Conzelmann K. K., Schnell M. Rescue of synthetic genomic RNA analogs of rabies virus by plasmid-encoded proteins. J Virol. 1994 Feb;68(2):713–719. doi: 10.1128/jvi.68.2.713-719.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Corapi W. V., Donis R. O., Dubovi E. J. Characterization of a panel of monoclonal antibodies and their use in the study of the antigenic diversity of bovine viral diarrhea virus. Am J Vet Res. 1990 Sep;51(9):1388–1394. [PubMed] [Google Scholar]
  11. Corapi W. V., Donis R. O., Dubovi E. J. Monoclonal antibody analyses of cytopathic and noncytopathic viruses from fatal bovine viral diarrhea virus infections. J Virol. 1988 Aug;62(8):2823–2827. doi: 10.1128/jvi.62.8.2823-2827.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. De Moerlooze L., Lecomte C., Brown-Shimmer S., Schmetz D., Guiot C., Vandenbergh D., Allaer D., Rossius M., Chappuis G., Dina D. Nucleotide sequence of the bovine viral diarrhoea virus Osloss strain: comparison with related viruses and identification of specific DNA probes in the 5' untranslated region. J Gen Virol. 1993 Jul;74(Pt 7):1433–1438. doi: 10.1099/0022-1317-74-7-1433. [DOI] [PubMed] [Google Scholar]
  13. De Moerlooze L., Struman I., Renard A., Martial J. A. Stabilization of T7-promoter-based pARHS expression vectors using the parB locus. Gene. 1992 Sep 21;119(1):91–93. doi: 10.1016/0378-1119(92)90070-6. [DOI] [PubMed] [Google Scholar]
  14. Deng R., Brock K. V. Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1. Virology. 1992 Dec;191(2):867–869. doi: 10.1016/0042-6822(92)90262-n. [DOI] [PubMed] [Google Scholar]
  15. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Doucet J. P., Trifaró J. M. A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution. Anal Biochem. 1988 Feb 1;168(2):265–271. doi: 10.1016/0003-2697(88)90317-x. [DOI] [PubMed] [Google Scholar]
  17. Elbers K., Tautz N., Becher P., Stoll D., Rümenapf T., Thiel H. J. Processing in the pestivirus E2-NS2 region: identification of proteins p7 and E2p7. J Virol. 1996 Jun;70(6):4131–4135. doi: 10.1128/jvi.70.6.4131-4135.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Failla C., Tomei L., De Francesco R. Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins. J Virol. 1994 Jun;68(6):3753–3760. doi: 10.1128/jvi.68.6.3753-3760.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Graf L., Craik C. S., Patthy A., Roczniak S., Fletterick R. J., Rutter W. J. Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin. Biochemistry. 1987 May 5;26(9):2616–2623. doi: 10.1021/bi00383a031. [DOI] [PubMed] [Google Scholar]
  22. Grakoui A., McCourt D. W., Wychowski C., Feinstone S. M., Rice C. M. Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites. J Virol. 1993 May;67(5):2832–2843. doi: 10.1128/jvi.67.5.2832-2843.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hedstrom L., Szilagyi L., Rutter W. J. Converting trypsin to chymotrypsin: the role of surface loops. Science. 1992 Mar 6;255(5049):1249–1253. doi: 10.1126/science.1546324. [DOI] [PubMed] [Google Scholar]
  24. Hijikata M., Mizushima H., Akagi T., Mori S., Kakiuchi N., Kato N., Tanaka T., Kimura K., Shimotohno K. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol. 1993 Aug;67(8):4665–4675. doi: 10.1128/jvi.67.8.4665-4675.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kessler S. W. Use of protein A-bearing staphylococci for the immunoprecipitation and isolation of antigens from cells. Methods Enzymol. 1981;73(Pt B):442–459. doi: 10.1016/0076-6879(81)73084-2. [DOI] [PubMed] [Google Scholar]
  26. Kim J. L., Morgenstern K. A., Lin C., Fox T., Dwyer M. D., Landro J. A., Chambers S. P., Markland W., Lepre C. A., O'Malley E. T. Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell. 1996 Oct 18;87(2):343–355. doi: 10.1016/s0092-8674(00)81351-3. [DOI] [PubMed] [Google Scholar]
  27. Kolykhalov A. A., Agapov E. V., Rice C. M. Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing. J Virol. 1994 Nov;68(11):7525–7533. doi: 10.1128/jvi.68.11.7525-7533.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lin C., Prágai B. M., Grakoui A., Xu J., Rice C. M. Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics. J Virol. 1994 Dec;68(12):8147–8157. doi: 10.1128/jvi.68.12.8147-8157.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Love R. A., Parge H. E., Wickersham J. A., Hostomsky Z., Habuka N., Moomaw E. W., Adachi T., Hostomska Z. The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site. Cell. 1996 Oct 18;87(2):331–342. doi: 10.1016/s0092-8674(00)81350-1. [DOI] [PubMed] [Google Scholar]
  30. Meyers G., Rümenapf T., Thiel H. J. Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology. 1989 Aug;171(2):555–567. doi: 10.1016/0042-6822(89)90625-9. [DOI] [PubMed] [Google Scholar]
  31. Meyers G., Tautz N., Becher P., Thiel H. J., Kümmerer B. M. Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs. J Virol. 1996 Dec;70(12):8606–8613. doi: 10.1128/jvi.70.12.8606-8613.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Meyers G., Tautz N., Dubovi E. J., Thiel H. J. Viral cytopathogenicity correlated with integration of ubiquitin-coding sequences. Virology. 1991 Feb;180(2):602–616. doi: 10.1016/0042-6822(91)90074-L. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Meyers G., Tautz N., Stark R., Brownlie J., Dubovi E. J., Collett M. S., Thiel H. J. Rearrangement of viral sequences in cytopathogenic pestiviruses. Virology. 1992 Nov;191(1):368–386. doi: 10.1016/0042-6822(92)90199-Y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meyers G., Thiel H. J. Cytopathogenicity of classical swine fever virus caused by defective interfering particles. J Virol. 1995 Jun;69(6):3683–3689. doi: 10.1128/jvi.69.6.3683-3689.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Meyers G., Thiel H. J., Rümenapf T. Classical swine fever virus: recovery of infectious viruses from cDNA constructs and generation of recombinant cytopathogenic defective interfering particles. J Virol. 1996 Mar;70(3):1588–1595. doi: 10.1128/jvi.70.3.1588-1595.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Moormann R. J., Warmerdam P. A., van der Meer B., Schaaper W. M., Wensvoort G., Hulst M. M. Molecular cloning and nucleotide sequence of hog cholera virus strain Brescia and mapping of the genomic region encoding envelope protein E1. Virology. 1990 Jul;177(1):184–198. doi: 10.1016/0042-6822(90)90472-4. [DOI] [PubMed] [Google Scholar]
  37. Moormann R. J., van Gennip H. G., Miedema G. K., Hulst M. M., van Rijn P. A. Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus. J Virol. 1996 Feb;70(2):763–770. doi: 10.1128/jvi.70.2.763-770.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pocock D. H., Howard C. J., Clarke M. C., Brownlie J. Variation in the intracellular polypeptide profiles from different isolates of bovine virus diarrhoea virus. Arch Virol. 1987;94(1-2):43–53. doi: 10.1007/BF01313724. [DOI] [PubMed] [Google Scholar]
  39. Purchio A. F., Larson R., Collett M. S. Characterization of bovine viral diarrhea virus proteins. J Virol. 1984 May;50(2):666–669. doi: 10.1128/jvi.50.2.666-669.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ridpath J. F., Bolin S. R. The genomic sequence of a virulent bovine viral diarrhea virus (BVDV) from the type 2 genotype: detection of a large genomic insertion in a noncytopathic BVDV. Virology. 1995 Sep 10;212(1):39–46. doi: 10.1006/viro.1995.1451. [DOI] [PubMed] [Google Scholar]
  41. Ruggli N., Tratschin J. D., Mittelholzer C., Hofmann M. A. Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA. J Virol. 1996 Jun;70(6):3478–3487. doi: 10.1128/jvi.70.6.3478-3487.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rümenapf T., Unger G., Strauss J. H., Thiel H. J. Processing of the envelope glycoproteins of pestiviruses. J Virol. 1993 Jun;67(6):3288–3294. doi: 10.1128/jvi.67.6.3288-3294.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  44. Stark R., Meyers G., Rümenapf T., Thiel H. J. Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus. J Virol. 1993 Dec;67(12):7088–7095. doi: 10.1128/jvi.67.12.7088-7095.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  46. Suzich J. A., Tamura J. K., Palmer-Hill F., Warrener P., Grakoui A., Rice C. M., Feinstone S. M., Collett M. S. Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes. J Virol. 1993 Oct;67(10):6152–6158. doi: 10.1128/jvi.67.10.6152-6158.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tamura J. K., Warrener P., Collett M. S. RNA-stimulated NTPase activity associated with the p80 protein of the pestivirus bovine viral diarrhea virus. Virology. 1993 Mar;193(1):1–10. doi: 10.1006/viro.1993.1097. [DOI] [PubMed] [Google Scholar]
  48. Tautz N., Meyers G., Stark R., Dubovi E. J., Thiel H. J. Cytopathogenicity of a pestivirus correlates with a 27-nucleotide insertion. J Virol. 1996 Nov;70(11):7851–7858. doi: 10.1128/jvi.70.11.7851-7858.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tautz N., Meyers G., Thiel H. J. Processing of poly-ubiquitin in the polyprotein of an RNA virus. Virology. 1993 Nov;197(1):74–85. doi: 10.1006/viro.1993.1568. [DOI] [PubMed] [Google Scholar]
  50. Tautz N., Thiel H. J., Dubovi E. J., Meyers G. Pathogenesis of mucosal disease: a cytopathogenic pestivirus generated by an internal deletion. J Virol. 1994 May;68(5):3289–3297. doi: 10.1128/jvi.68.5.3289-3297.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Thiel H. J., Stark R., Weiland E., Rümenapf T., Meyers G. Hog cholera virus: molecular composition of virions from a pestivirus. J Virol. 1991 Sep;65(9):4705–4712. doi: 10.1128/jvi.65.9.4705-4712.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vanderheijden N., De Moerlooze L., Vandenbergh D., Chappuis G., Renard A., Lecomte C. Expression of the bovine viral diarrhoea virus Osloss p80 protein: its use as ELISA antigen for cattle serum antibody detection. J Gen Virol. 1993 Jul;74(Pt 7):1427–1431. doi: 10.1099/0022-1317-74-7-1427. [DOI] [PubMed] [Google Scholar]
  53. Venekei I., Szilágyi L., Gráf L., Rutter W. J. Attempts to convert chymotrypsin to trypsin. FEBS Lett. 1996 Jan 29;379(2):143–147. [PubMed] [Google Scholar]
  54. Warrener P., Collett M. S. Pestivirus NS3 (p80) protein possesses RNA helicase activity. J Virol. 1995 Mar;69(3):1720–1726. doi: 10.1128/jvi.69.3.1720-1726.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wiskerchen M., Collett M. S. Pestivirus gene expression: protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing. Virology. 1991 Sep;184(1):341–350. doi: 10.1016/0042-6822(91)90850-b. [DOI] [PubMed] [Google Scholar]
  56. Xu J., Mendez E., Caron P. R., Lin C., Murcko M. A., Collett M. S., Rice C. M. Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication. J Virol. 1997 Jul;71(7):5312–5322. doi: 10.1128/jvi.71.7.5312-5322.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]

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