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. 1994 Sep;68(9):5755–5764. doi: 10.1128/jvi.68.9.5755-5764.1994

Proteolytic processing of the replicase ORF1a protein of equine arteritis virus.

E J Snijder 1, A L Wassenaar 1, W J Spaan 1
PMCID: PMC236979  PMID: 8057457

Abstract

To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire ORF1a protein, results in cleavage products of approximately 29, 61, 22, 31, 41, and 3 kDa, which were named nonstructural proteins (nsps) 1 through 6, respectively. Pulse-chase experiments revealed that the cleavages at the nsp1/2 and nsp2/3 junctions are the most rapid processing steps. The remaining nsp3456 precursor is first cleaved at the nsp4/5 site. Final processing of the nsp34 and nsp56 intermediates is extremely slow. As predicted from previous in vitro translation experiments (E. J. Snijder, A. L. M. Wassenaar, and W. J. M. Spaan, J. Virol. 66:7040-7048, 1992), a cysteine protease domain in nsp1 was shown to be responsible for the nsp1/2 cleavage. The other processing steps are carried out by the putative EAV serine protease in nsp4 and by a third protease, which remains to be identified.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BRYANS J. T., CROWE M. E., DOLL E. R., MCCOLLUM W. H. Isolation of a filterable agent causing arteritis of horses and abortion by mares; its differentiation from the equine abortion (influenza) virus. Cornell Vet. 1957 Jan;47(1):3–41. [PubMed] [Google Scholar]
  2. Baker S. C., Yokomori K., Dong S., Carlisle R., Gorbalenya A. E., Koonin E. V., Lai M. M. Identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus. J Virol. 1993 Oct;67(10):6056–6063. doi: 10.1128/jvi.67.10.6056-6063.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baric R. S., Fu K., Schaad M. C., Stohlman S. A. Establishing a genetic recombination map for murine coronavirus strain A59 complementation groups. Virology. 1990 Aug;177(2):646–656. doi: 10.1016/0042-6822(90)90530-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barton D. J., Sawicki S. G., Sawicki D. L. Solubilization and immunoprecipitation of alphavirus replication complexes. J Virol. 1991 Mar;65(3):1496–1506. doi: 10.1128/jvi.65.3.1496-1506.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bazan J. F., Fletterick R. J. Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7872–7876. doi: 10.1073/pnas.85.21.7872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bonilla P. J., Gorbalenya A. E., Weiss S. R. Mouse hepatitis virus strain A59 RNA polymerase gene ORF 1a: heterogeneity among MHV strains. Virology. 1994 Feb;198(2):736–740. doi: 10.1006/viro.1994.1088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boursnell M. E., Brown T. D., Foulds I. J., Green P. F., Tomley F. M., Binns M. M. Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus. J Gen Virol. 1987 Jan;68(Pt 1):57–77. doi: 10.1099/0022-1317-68-1-57. [DOI] [PubMed] [Google Scholar]
  8. Bredenbeek P. J., Pachuk C. J., Noten A. F., Charité J., Luytjes W., Weiss S. R., Spaan W. J. The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism. Nucleic Acids Res. 1990 Apr 11;18(7):1825–1832. doi: 10.1093/nar/18.7.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen Z., Kuo L., Rowland R. R., Even C., Faaberg K. S., Plagemann P. G. Sequences of 3' end of genome and of 5' end of open reading frame 1a of lactate dehydrogenase-elevating virus and common junction motifs between 5' leader and bodies of seven subgenomic mRNAs. J Gen Virol. 1993 Apr;74(Pt 4):643–659. doi: 10.1099/0022-1317-74-4-643. [DOI] [PubMed] [Google Scholar]
  10. Denison M. R., Zoltick P. W., Hughes S. A., Giangreco B., Olson A. L., Perlman S., Leibowitz J. L., Weiss S. R. Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events. Virology. 1992 Jul;189(1):274–284. doi: 10.1016/0042-6822(92)90703-R. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Denison M. R., Zoltick P. W., Leibowitz J. L., Pachuk C. J., Weiss S. R. Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59. J Virol. 1991 Jun;65(6):3076–3082. doi: 10.1128/jvi.65.6.3076-3082.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Dougherty W. G., Semler B. L. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes. Microbiol Rev. 1993 Dec;57(4):781–822. doi: 10.1128/mr.57.4.781-822.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Elroy-Stein O., Fuerst T. R., Moss B. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6126–6130. doi: 10.1073/pnas.86.16.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Godeny E. K., Chen L., Kumar S. N., Methven S. L., Koonin E. V., Brinton M. A. Complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-elevating virus (LDV). Virology. 1993 Jun;194(2):585–596. doi: 10.1006/viro.1993.1298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gorbalenya A. E., Donchenko A. P., Blinov V. M., Koonin E. V. Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold. FEBS Lett. 1989 Jan 30;243(2):103–114. doi: 10.1016/0014-5793(89)80109-7. [DOI] [PubMed] [Google Scholar]
  18. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis. Nucleic Acids Res. 1989 Jun 26;17(12):4847–4861. doi: 10.1093/nar/17.12.4847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Herold J., Raabe T., Schelle-Prinz B., Siddell S. G. Nucleotide sequence of the human coronavirus 229E RNA polymerase locus. Virology. 1993 Aug;195(2):680–691. doi: 10.1006/viro.1993.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jang S. K., Kräusslich H. G., Nicklin M. J., Duke G. M., Palmenberg A. C., Wimmer E. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol. 1988 Aug;62(8):2636–2643. doi: 10.1128/jvi.62.8.2636-2643.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jore J., De Geus B., Jackson R. J., Pouwels P. H., Enger-Valk B. E. Poliovirus protein 3CD is the active protease for processing of the precursor protein P1 in vitro. J Gen Virol. 1988 Jul;69(Pt 7):1627–1636. doi: 10.1099/0022-1317-69-7-1627. [DOI] [PubMed] [Google Scholar]
  22. Kao C. C., Quadt R., Hershberger R. P., Ahlquist P. Brome mosaic virus RNA replication proteins 1a and 2a from a complex in vitro. J Virol. 1992 Nov;66(11):6322–6329. doi: 10.1128/jvi.66.11.6322-6329.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  24. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Lee H. J., Shieh C. K., Gorbalenya A. E., Koonin E. V., La Monica N., Tuler J., Bagdzhadzhyan A., Lai M. M. The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology. 1991 Feb;180(2):567–582. doi: 10.1016/0042-6822(91)90071-I. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lemm J. A., Rice C. M. Roles of nonstructural polyproteins and cleavage products in regulating Sindbis virus RNA replication and transcription. J Virol. 1993 Apr;67(4):1916–1926. doi: 10.1128/jvi.67.4.1916-1926.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Meulenberg J. J., Hulst M. M., de Meijer E. J., Moonen P. L., den Besten A., de Kluyver E. P., Wensvoort G., Moormann R. J. Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virology. 1993 Jan;192(1):62–72. doi: 10.1006/viro.1993.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Plagemann P. G., Moennig V. Lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand RNA viruses. Adv Virus Res. 1992;41:99–192. doi: 10.1016/S0065-3527(08)60036-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Reichlin M. Use of glutaraldehyde as a coupling agent for proteins and peptides. Methods Enzymol. 1980;70(A):159–165. doi: 10.1016/s0076-6879(80)70047-2. [DOI] [PubMed] [Google Scholar]
  31. Schaad M. C., Stohlman S. A., Egbert J., Lum K., Fu K., Wei T., Jr, Baric R. S. Genetics of mouse hepatitis virus transcription: identification of cistrons which may function in positive and negative strand RNA synthesis. Virology. 1990 Aug;177(2):634–645. doi: 10.1016/0042-6822(90)90529-Z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shirako Y., Strauss J. H. Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis. J Virol. 1994 Mar;68(3):1874–1885. doi: 10.1128/jvi.68.3.1874-1885.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  34. Snijder E. J., Horzinek M. C. Toroviruses: replication, evolution and comparison with other members of the coronavirus-like superfamily. J Gen Virol. 1993 Nov;74(Pt 11):2305–2316. doi: 10.1099/0022-1317-74-11-2305. [DOI] [PubMed] [Google Scholar]
  35. Snijder E. J., Wassenaar A. L., Spaan W. J. The 5' end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease. J Virol. 1992 Dec;66(12):7040–7048. doi: 10.1128/jvi.66.12.7040-7048.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Snijder E. J., den Boon J. A., Bredenbeek P. J., Horzinek M. C., Rijnbrand R., Spaan W. J. The carboxyl-terminal part of the putative Berne virus polymerase is expressed by ribosomal frameshifting and contains sequence motifs which indicate that toro- and coronaviruses are evolutionarily related. Nucleic Acids Res. 1990 Aug 11;18(15):4535–4542. doi: 10.1093/nar/18.15.4535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Troxler M., Egger D., Pfister T., Bienz K. Intracellular localization of poliovirus RNA by in situ hybridization at the ultrastructural level using single-stranded riboprobes. Virology. 1992 Dec;191(2):687–697. doi: 10.1016/0042-6822(92)90244-j. [DOI] [PubMed] [Google Scholar]
  38. Vennema H., Rijnbrand R., Heijnen L., Horzinek M. C., Spaan W. J. Enhancement of the vaccinia virus/phage T7 RNA polymerase expression system using encephalomyocarditis virus 5'-untranslated region sequences. Gene. 1991 Dec 15;108(2):201–209. doi: 10.1016/0378-1119(91)90435-E. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ypma-Wong M. F., Dewalt P. G., Johnson V. H., Lamb J. G., Semler B. L. Protein 3CD is the major poliovirus proteinase responsible for cleavage of the P1 capsid precursor. Virology. 1988 Sep;166(1):265–270. doi: 10.1016/0042-6822(88)90172-9. [DOI] [PubMed] [Google Scholar]
  40. de Groot R. J., Hardy W. R., Shirako Y., Strauss J. H. Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo. EMBO J. 1990 Aug;9(8):2631–2638. doi: 10.1002/j.1460-2075.1990.tb07445.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. de Vries A. A., Chirnside E. D., Bredenbeek P. J., Gravestein L. A., Horzinek M. C., Spaan W. J. All subgenomic mRNAs of equine arteritis virus contain a common leader sequence. Nucleic Acids Res. 1990 Jun 11;18(11):3241–3247. doi: 10.1093/nar/18.11.3241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. de Vries A. A., Chirnside E. D., Horzinek M. C., Rottier P. J. Structural proteins of equine arteritis virus. J Virol. 1992 Nov;66(11):6294–6303. doi: 10.1128/jvi.66.11.6294-6303.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. den Boon J. A., Snijder E. J., Chirnside E. D., de Vries A. A., Horzinek M. C., Spaan W. J. Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily. J Virol. 1991 Jun;65(6):2910–2920. doi: 10.1128/jvi.65.6.2910-2920.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. van Berlo M. F., Zeegers J. J., Horzinek M. C., van der Zeijst B. A. Antigenic comparison of equine arteritis virus (EAV) and lactic dehydrogenase virus (LDV); binding of staphylococcal protein A to the nucleocapsid protein of EAV. Zentralbl Veterinarmed B. 1983 May;30(4):297–304. doi: 10.1111/j.1439-0450.1983.tb01846.x. [DOI] [PubMed] [Google Scholar]

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