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. 2004 Feb 10;191(2):932–940. doi: 10.1016/0042-6822(92)90268-T

Identification of the active site residues in the nsP2 proteinase of sindbis virus

Ellen G Strauss 1,1, Raoul J De Groot 1,2, Randy Levinson 1,3, James H Strauss 1
PMCID: PMC7131396  PMID: 1448929

Abstract

The nonstructural polyproteins of Sindbis virus are processed by a virus-encoded proteinase which is located in the C-terminal domain of nsP2. Here we have performed a mutagenic analysis to identify the active site residues of this proteinase. Substitution of other amino acids for either Cys-481 or His-558 completely abolished proteolytic processing of Sindbis virus polyproteins in vitro. Substitutions within this domain for a second cysteine conserved among alphaviruses, for four other conserved histidines, or for a conserved serine did not affect the activity of the enzyme. These results suggest that nsP2 is a papain-like proteinase whose catalytic dyad is composed of Cys-481 and His-558. Since an asparagine residue has been implicated in the active site of papain, we changed the four conserved asparagine residues in the C-terminal half of nsP2 and found that all could be substituted without total loss of activity. Among papain-like proteinases, the residue following the catalytic histidine is alanine or glycine in the plant and animal enzymes, and the presence of Trp-559 in alphaviruses is unusual. A mutant enzyme containing Ala-559 was completely inactive, implying that Trp-559 is essential for a functional proteinase. All of these mutations were introduced into a full-length clone of Sindbis virus from which infectious RNA could be transcribed in vitro, and the effects of these changes on viability were tested. In all cases it was found that mutations which abolished proteolytic activity were lethal, whether or not these mutations were in the catalytic residues, indicating that proteolysis of the nonstructural polyprotein is essential for Sindbis replication.

References

  1. Baker E.N., Drenth J. The thiol proteases: Structure and mechanism. In: Jurnak F.A., McPherson A., editors. Vol. 3. Wiley; New York: 1987. pp. 314–367. (Biological Macromolecules and Assemblies: The Active Sites of Enzymes). [Google Scholar]
  2. Baker S.C., Shieh C.-K., Soe L.H., Chang M.-F., Vannier D.M., Lai M.M.C. Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein. J. Virol. 1991;63:3693–3699. doi: 10.1128/jvi.63.9.3693-3699.1989. [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;171:637–639. doi: 10.1016/0042-6822(89)90639-9. [DOI] [PubMed] [Google Scholar]
  4. Bazan J.F., Fletterick R.J. Structural and catalytic models of trypsin-like viral proteases. Sem. Virol. 1990;1:311–322. [Google Scholar]
  5. Boege U., Wengler G., Wengler G., Wittman-Liebold B. Primary structure of the core proteins of the alphaviruses Semliki Forest virus and Sindbis virus. Virology. 1981;113:293–303. doi: 10.1016/0042-6822(81)90156-2. [DOI] [PubMed] [Google Scholar]
  6. Boursnell M.E.G., Brown T.D.K., 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;68:57–77. doi: 10.1099/0022-1317-68-1-57. [DOI] [PubMed] [Google Scholar]
  7. Brocklehurst K. Acyl group transfer-cysteine proteinases. In: Page M.I., Williams A., editors. Enzyme Mechanisms. The Royal Society of Chemistry; London: 1987. pp. 140–158. [Google Scholar]
  8. Carne A., Moore C.H. The amino acid sequence of the tryptic peptides of Actinidin, a proteolytic enzyme from the fruit of Actinidia chinensis. Biochem. J. 1978;173:73–83. doi: 10.1042/bj1730073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chambers T.J., Weir R.C., Grakoui A., McCourt D.W., Bazan J.F., Fletterick R.J., Rice C.M. Vol. 87. 1990. Evidence that the N-terminal domain of nonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein; pp. 8898–8902. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Choi G.H., Pawlyk D.M., Nuss D.L. The autocatalytic protease p29 encoded by a hypovirulence-associated virus of the chestnut blight fungus resembles the potyvirus-encoded protease HC-Pro. Virology. 1991;183:747–752. doi: 10.1016/0042-6822(91)91004-z. [DOI] [PubMed] [Google Scholar]
  11. Choi H.-K., Tong L., Minor W., Dumas P., Boege U., Rossmann M.G., Wengler G. Structure of Sindbis virus core protein reveals a chymotrypsin-like serine proteinase and the organization of the virion. Nature. 1991;354:37–43. doi: 10.1038/354037a0. [DOI] [PubMed] [Google Scholar]
  12. De Groot R.J., Hardy W.R., Shirako Y., Strauss J.H. Cleavage-site preferences of Sindbis virus polyproteins containing the nonstructural proteinase: Evidence for temporal regulation of polyprotein processing in vivo. EMBO J. 1990;9:2631–2638. doi: 10.1002/j.1460-2075.1990.tb07445.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. De Groot R.J., Rümenapf T., Kuhn R.J., Strauss E.G., Strauss J.H. Vol. 88. 1991. Sindbis virus RNA polymerase is degraded by the N-end rule pathway; pp. 8967–8971. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dominguez G., Wang C.-Y., Frey T.K. Sequence of the genome RNA of rubella virus: Evidence for genetic rearrangement during togavirus evolution. Virology. 1990;177:225–238. doi: 10.1016/0042-6822(90)90476-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Faragher S.G., Meek A.D.J., Rice C.M., Dalgarno L. Genome sequences of a mouse-avirulent and a mouse-virulent strain of Ross River virus. Virology. 1988;163:509–526. doi: 10.1016/0042-6822(88)90292-9. [DOI] [PubMed] [Google Scholar]
  16. Garavito R.M., Rossmann M.G., Argos P., Eventoff W. Convergence of active site geometries. Biochemistry. 1977;16:5065–5071. doi: 10.1021/bi00642a019. [DOI] [PubMed] [Google Scholar]
  17. Goldbach R. Plant viral proteinases. Sem. Virol. 1990;1:335–346. [Google Scholar]
  18. 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;243:103–114. doi: 10.1016/0014-5793(89)80109-7. [DOI] [PubMed] [Google Scholar]
  19. Gorbalenya A.E., Koonin E.V., Lai M.M.C. Putative papain-related thiol proteases of positive-strand RNA viruses. FEBS Lett. 1991;288:201–205. doi: 10.1016/0014-5793(91)81034-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hahn C.S., Strauss E.G., Strauss J.H. Vol. 82. 1985. Sequence analysis of three Sindbis virus mutants temperature-sensitive in the capsid autoprotease; pp. 4648–4652. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hahn C.S., Strauss J.H. Site-directed mutagenesis of the proposed catalytic amino acids of the Sindbis virus capsid protein autoprotease. J. Virol. 1990;64:3069–3073. doi: 10.1128/jvi.64.6.3069-3073.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hahn Y.S., Strauss E.G., Strauss J.H. Mapping of RNA- temperature-sensitive mutants of Sindbis virus: Assignment of complementation groups A, B, and G to nonstructural proteins. J. Virol. 1989;63:3142–3150. doi: 10.1128/jvi.63.7.3142-3150.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hardy W.R., Hahn Y., S. De Groot R.J., Strauss E.G., Strauss J.H. Synthesis and processing of the nonstructural polyproteins of several temperature-sensitive mutants of Sindbis virus. Virology. 1990;177:199–208. doi: 10.1016/0042-6822(90)90473-5. [DOI] [PubMed] [Google Scholar]
  24. Hardy W.R., Strauss J.H. Processing the nonstructural proteins of Sindbis virus: Nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and trans. J. Virol. 1989;63:4653–4664. doi: 10.1128/jvi.63.11.4653-4664.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Harris K.S., Hellen C.U.T., Wimmer E. Proteolytlc processing in the replication of picornaviruses. Sem. Virol. 1990;1:323–333. [Google Scholar]
  26. Higaki J.N., Gibson B.W., Craik C.S. Evolution of catalysis in the serine proteases. CSHSQB. 1987;52:615–621. doi: 10.1101/sqb.1987.052.01.070. [DOI] [PubMed] [Google Scholar]
  27. Kamphuis I.G., Drenth J., Baker E.N. Thiol proteases: Comparative studies based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain. J. Mol. Biol. 1985;182:317–329. doi: 10.1016/0022-2836(85)90348-1. [DOI] [PubMed] [Google Scholar]
  28. Kamphuis I.G., Kalk K.H., Swarte M.B.A., Drenth J. Structure of papain refined at 1.65 Å resolution. J. Mol. Biol. 1984;179:233–256. doi: 10.1016/0022-2836(84)90467-4. [DOI] [PubMed] [Google Scholar]
  29. Kashiwazaki S., Minobe Y., Hibino H. Nucleotide sequence of barley yellow mosaic virus RNA 2. J. Gen. Virol. 1991;72:995–999. doi: 10.1099/0022-1317-72-4-995. [DOI] [PubMed] [Google Scholar]
  30. Kunkel T.A. Vol. 82. 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection; pp. 488–492. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  32. Lee H.A., Shieh C.-K., Gorbalenya A.E., Koonin E.V., La Monica N., Tuler J., Bagdzhadzhyan A., Lai M.M.C. The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology. 1991;180:567–582. doi: 10.1016/0042-6822(91)90071-I. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Levinson R., Strauss J.H., Strauss E.G. Determination of the complete nucleotide sequence of the genomic RNA of O'Nyong-nyong virus and its use in the construction of phylogenetic trees. Virology. 1990;175:110–123. doi: 10.1016/0042-6822(90)90191-s. [DOI] [PubMed] [Google Scholar]
  34. Loeb D.D., Hutchinson C.A.I., Edgell N.H., Farmerie W.G., Swanstrom R. Mutational analysis of human immunodeficiency virus type I protease suggests functional homology with aspartic proteinases. J. Virol. 1989;63:111–121. doi: 10.1128/jvi.63.1.111-121.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mitchel R.E.J., Chaiken I.M., Smith E.L. The complete amino acid sequence of papain. J. Biol. Chem. 1970;245:3485–3492. [PubMed] [Google Scholar]
  36. Navia M.A., Fitzgerald P.M.D., McKeever B.M., Leu C.-T., Heimbach J.C., Herber W.K., Sigal I.S., Darke P.L., Springer J.P. Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1. Nature. 1989;337:615–620. doi: 10.1038/337615a0. [DOI] [PubMed] [Google Scholar]
  37. Oh C.-S., Carrington J.C. Identification of essential residues in potyvirus proteinase HC-Pro by site-directed mutagenesis. Virology. 1989;173:692–699. doi: 10.1016/0042-6822(89)90582-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ohno S., Emori Y., Imaioh S., Kawasaki H., Kisaragi M., Suzuki K. Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein. Nature. 1984;312:566–570. doi: 10.1038/312566a0. [DOI] [PubMed] [Google Scholar]
  39. Pears C.J., Mahbubani H.M., Williams J.G. Characterization of two highly diverged but developmentally co-regulated cysteine proteinase genes in Dictyostelium discoideum. Nucleic Acids Res. 1985;13:8853–8866. doi: 10.1093/nar/13.24.8853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Preugschat F., Lenches E.M., Strauss J.H. Flavivirus enzyme-substrate interactions studied with chimeric proteins: Identification of an intragenic locus important for substrate recognition. J. Virol. 1991;65:4749–4758. doi: 10.1128/jvi.65.9.4749-4758.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rice C.M., Levis R., Strauss J.H., Huang H.V. Production of infectious RNA transcripts from Sindbis virus cDNA clones: Mapping of lethal mutations, rescue of a temperature sensitive marker, and in vitro mutagenesis to generate defined mutants. J. Virol. 1987;61:3809–3819. doi: 10.1128/jvi.61.12.3809-3819.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ritonja A., Popovic T., Turk V., Wiedenmann K., Machleidt W. Amino acid sequence of human liver cathepsin B. FEBS Lett. 1985;181:169–172. doi: 10.1016/0014-5793(85)81136-4. [DOI] [PubMed] [Google Scholar]
  43. Ritonja A., Rowan A.D., Buttle D.J., Rawlings N.D., Turk V., Barrett A.J. Stem bromeam Amino acid sequence and implications for weak binding of cystatin. FEBS Lett. 1989;247:419–424. doi: 10.1016/0014-5793(89)81383-3. [DOI] [PubMed] [Google Scholar]
  44. Sambrook J., Fritsch E.F., Maniatis T. 2nd ed. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY: 1989. (Molecular Cloning: A Laboratory Manual). [Google Scholar]
  45. Shirako Y., Niklasson B., Dalrymple J.M., Strauss E.G., Strauss J.H. Structure of the Ockelbo virus genome and its relationship to other Sindbis viruses. Virology. 1991;182:753–764. doi: 10.1016/0042-6822(91)90616-j. [DOI] [PubMed] [Google Scholar]
  46. Shirako Y., Strauss J.H. Cleavage between nsP1 and nsP2 initiates the processing pathway of Sindbis virus nonstructural polyprotein P123. Virology. 1990;177:54–64. doi: 10.1016/0042-6822(90)90459-5. [DOI] [PubMed] [Google Scholar]
  47. Strauss E.G., Rice C.M., Strauss J.H. Complete nucleotide sequence of the genomic RNA of Sindbis virus. Virology. 1984;133:92–110. doi: 10.1016/0042-6822(84)90428-8. [DOI] [PubMed] [Google Scholar]
  48. Strauss E.G., Strauss J.H. Structure and replication of the alphavirus genome. In: Schlesinger S., Schlesinger M.J., editors. The Togaviridae and Flaviviridae. Plenum; New York: 1986. pp. 35–90. [Google Scholar]
  49. Strauss J.H., editor. Viral proteinases. Vol. 1. 1990. pp. 307–384. (Sem. Virol.). [Google Scholar]
  50. Strauss J.H., Strauss E.G. Alphavirus proteinases. Sem. Virol. 1990;1:347–356. [Google Scholar]
  51. Takkinen K. Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus. Nucleic Acids Res. 1986;14:5667–5682. doi: 10.1093/nar/14.14.5667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wiederanders B., Broemma D., Kirschke H., Kalkinnen N., Rinne A., Paquette T., Toothman P. Primary structure of bovine cathepsin S. FEBS Lett. 1991;286:189–192. doi: 10.1016/0014-5793(91)80971-5. [DOI] [PubMed] [Google Scholar]
  53. Wiskerchen M., Belzer S.K., Collett M.S. Pestivlrus gene expression: The first protein product of the bovine viral diarrhea virus large open reading frame, P20, possesses proteolytic activity. J. Virol. 1991;65:4508–4514. doi: 10.1128/jvi.65.8.4508-4514.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zoller M.J., Smith M. Laboratory Methods: Oligonucleotide-directed mutagenesis: A simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984;3:477–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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