1887

Abstract

Major virulence determinants of (SFV) lie within the non-structural genes that form the replicase complex proteins. Gene exchange between virulent and avirulent viruses has shown that the nsP3 gene, which has essential 5′ conserved domains and a non-essential hypervariable 3′ domain, is one of the virulence determinants. This protein plays a role in subgenomic 26S and negative-strand RNA synthesis and is thought to function with nsP1 to anchor replication complexes to cell membrane structures. Studies to date have focused on analysing the effect of mutational changes spread over the whole gene on virulence of the virus. The virulent SFV4 virus, derived from an infectious clone, was utilized to analyse the effect on virulence of large deletions in the hypervariable domain of nsP3. Two viruses with different in-frame deletions that spanned this domain showed reduced rates of RNA synthesis and multiplication in cell culture. In adult BALB/c mice, these viruses were avirulent after intramuscular and intraperitoneal inoculation, and brains sampled from infected mice showed minimal or no evidence of pathology. These deleted viruses had greatly reduced virulence when administered by the intranasal route and brains from infected mice showed lesions that were much less severe than those seen in SFV4 infection. Mice surviving infection with the deleted viruses resisted challenge with the virulent L10 strain, indicating induction of protective immunity. This work establishes that deletions in the nsP3 hypervariable domain attenuate virulence after peripheral inoculation and also reduce virulence after intranasal inoculation.

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2006-04-01
2024-10-03
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References

  1. Ahola T., Lampio A., Auvinen P., Kääriäinen L. 1999; Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity. EMBO J 18:3164–3172 [CrossRef]
    [Google Scholar]
  2. Ahola T., Kujala P., Tuittila M., Blom T., Laakkonen P., Hinkkanen A., Auvinen P. 2000; Effects of palmitoylation of replicase protein nsP1 on alphavirus infection. J Virol 74:6725–6733 [CrossRef]
    [Google Scholar]
  3. Atkins G. J., Sheahan B. J., Liljeström P. 1999; The molecular pathogenesis of Semliki Forest virus: a model virus made useful?. J Gen Virol 80:2287–2297
    [Google Scholar]
  4. Balluz I. M., Glasgow G. M., Killen H. M., Mabruk M. J., Sheahan B. J., Atkins G. J. 1993; Virulent and avirulent strains of Semliki Forest virus show similar cell tropism for the murine central nervous system but differ in the severity and rate of induction of cytolytic damage. Neuropathol Appl Neurobiol 19:233–239 [CrossRef]
    [Google Scholar]
  5. De I., Fata-Hartley C., Sawicki S. G., Sawicki D. L. 2003; Functional analysis of nsP3 phosphoprotein mutants of Sindbis virus. J Virol 77:13106–13116 [CrossRef]
    [Google Scholar]
  6. de Groot R. J., Hardy W. R., Shirako Y., Strauss J. H. 1990; Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo. EMBO J 9:2631–2638
    [Google Scholar]
  7. Fata C. L., Sawicki S. G., Sawicki D. L. 2002a; Modification of Asn374 of nsP1 suppresses a Sindbis virus nsP4 minus-strand polymerase mutant. J Virol 76:8641–8649 [CrossRef]
    [Google Scholar]
  8. Fata C. L., Sawicki S. G., Sawicki D. L. 2002b; Alphavirus minus-strand RNA synthesis: identification of a role for Arg183 of the nsP4 polymerase. J Virol 76:8632–8640 [CrossRef]
    [Google Scholar]
  9. Fazakerley J. K. 2002; Pathogenesis of Semliki Forest virus encephalitis. J Neurovirol 8:66–74 [CrossRef]
    [Google Scholar]
  10. Fazakerley J. K. 2004; Semliki Forest virus infection of laboratory mice: a model to study the pathogenesis of viral encephalitis. Arch Virol Suppl 18:179–190
    [Google Scholar]
  11. Fazakerley J. K., Boyd A., Mikkola M. L., Kääriäinen L. 2002; A single amino acid change in the nuclear localization sequence of the nsP2 protein affects the neurovirulence of Semliki Forest virus. J Virol 76:392–396 [CrossRef]
    [Google Scholar]
  12. Glasgow G. M., Sheahan B. J., Atkins G. J., Wahlberg J. M., Salminen A., Liljeström P. 1991; Two mutations in the envelope glycoprotein E2 of Semliki Forest virus affecting the maturation and entry patterns of the virus alter pathogenicity for mice. Virology 185:741–748 [CrossRef]
    [Google Scholar]
  13. Glasgow G. M., Killen H. M., Liljeström P., Sheahan B. J., Atkins G. J. 1994; A single amino acid change in the E2 spike protein of a virulent strain of Semliki Forest virus attenuates pathogenicity. J Gen Virol 75:663–668 [CrossRef]
    [Google Scholar]
  14. Hahn Y. S., Strauss E. G., Strauss J. H. 1989; Mapping of RNA- temperature-sensitive mutants of Sindbis virus: assignment of complementation groups A, B, and G to nonstructural proteins. J Virol 63:3142–3150
    [Google Scholar]
  15. Kääriäinen L., Söderlund H. 1978; Structure and replication of alphaviruses. Curr Top Microbiol Immunol 82:15–69
    [Google Scholar]
  16. Kääriäinen L., Ahola T. 2002; Functions of alphavirus nonstructural proteins in RNA replication. Prog Nucleic Acid Res Mol Biol 71:187–222
    [Google Scholar]
  17. Kim K. H., Rumenapf T., Strauss E. G., Strauss J. H. 2004; Regulation of Semliki Forest virus RNA replication: a model for the control of alphavirus pathogenesis in invertebrate hosts. Virology 323:153–163 [CrossRef]
    [Google Scholar]
  18. Koonin E. V., Dolja V. V. 1993; Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol 28:375–430 [CrossRef]
    [Google Scholar]
  19. Kujala P., Ikaheimonen A., Ehsani N., Vihinen H., Auvinen P., Kääriäinen L. 2001; Biogenesis of the Semliki Forest virus RNA replication complex. J Virol 75:3873–3884 [CrossRef]
    [Google Scholar]
  20. Laakkonen P., Ahola T., Kääriäinen L. 1996; The effects of palmitoylation on membrane association of Semliki forest virus RNA capping enzyme. J Biol Chem 271:28567–28571 [CrossRef]
    [Google Scholar]
  21. Lampio A., Kilpelainen I., Pesonen S., Karhi K., Auvinen P., Somerharju P., Kääriäinen L. 2000; Membrane binding mechanism of an RNA virus-capping enzyme. J Biol Chem 275:37853–37859 [CrossRef]
    [Google Scholar]
  22. LaStarza M. W., Lemm J. A., Rice C. M. 1994a; Genetic analysis of the nsP3 region of Sindbis virus: evidence for roles in minus-strand and subgenomic RNA synthesis. J Virol 68:5781–5791
    [Google Scholar]
  23. LaStarza M. W., Grakoui A., Rice C. M. 1994b; Deletion and duplication mutations in the C-terminal nonconserved region of Sindbis virus nsP3: effects on phosphorylation and on virus replication in vertebrate and invertebrate cells. Virology 202:224–232 [CrossRef]
    [Google Scholar]
  24. Lemm J. A., Rice C. M. 1993; Roles of nonstructural polyproteins and cleavage products in regulating Sindbis virus RNA replication and transcription. J Virol 67:1916–1926
    [Google Scholar]
  25. Lemm J. A., Rumenapf T., Strauss E. G., Strauss J. H., Rice C. M. 1994; Polypeptide requirements for assembly of functional Sindbis virus replication complexes: a model for the temporal regulation of minus- and plus-strand RNA synthesis. EMBO J 13:2925–2934
    [Google Scholar]
  26. Lemm J. A., Bergqvist A., Read C. M., Rice C. M. 1998; Template-dependent initiation of Sindbis virus RNA replication in vitro. J Virol 72:6546–6553
    [Google Scholar]
  27. Li G. P., La Starza M. W., Hardy W. R., Strauss J. H., Rice C. M. 1990; Phosphorylation of Sindbis virus nsP3 in vivo and in vitro. Virology 179:416–427 [CrossRef]
    [Google Scholar]
  28. Liljeström P., Lusa S., Huylebroeck D., Garoff H. 1991; In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release. J Virol 65:4107–4113
    [Google Scholar]
  29. Meissner J. D., Huang C. Y., Pfeffer M., Kinney R. M. 1999; Sequencing of prototype viruses in the Venezuelan equine encephalitis antigenic complex. Virus Res 64:43–59 [CrossRef]
    [Google Scholar]
  30. Merits A., Vasiljeva L., Ahola T., Kääriäinen L., Auvinen P. 2001; Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease. J Gen Virol 82:765–773
    [Google Scholar]
  31. Oberste M. S., Parker M. D., Smith J. F. 1996; Complete sequence of Venezuelan equine encephalitis virus subtype IE reveals conserved and hypervariable domains within the C terminus of nsP3. Virology 219:314–320 [CrossRef]
    [Google Scholar]
  32. Pathak S., Webb H. E. 1974; Possible mechanisms for the transport of Semliki forest virus into and within mouse brain. An electron-microscopic study. J Neurol Sci 23:175–184 [CrossRef]
    [Google Scholar]
  33. Pehrson J. R. L., Fuji R. N. 1998; Evolutionary conservation of histone macroH2A subtypes and domains. Nucleic Acids Res 26:2837–2842 [CrossRef]
    [Google Scholar]
  34. Peränen J. 1991; Localization and phosphorylation of Semliki Forest virus non-structural protein nsP3 expressed in COS cells from a cloned cDNA. J Gen Virol 72:195–199 [CrossRef]
    [Google Scholar]
  35. Peränen J., Kääriäinen L. 1991; Biogenesis of type I cytopathic vacuoles in Semliki Forest virus-infected BHK cells. J Virol 65:1623–1627
    [Google Scholar]
  36. Peränen J., Takkinen K., Kalkkinen N., Kääriäinen L. 1988; Semliki Forest virus-specific non-structural protein nsp3 is a phosphoprotein. J Gen Virol 69:2165–2178 [CrossRef]
    [Google Scholar]
  37. Peränen J., Laakkonen P., Hyvönen M., Kääriäinen L. 1995; The alphavirus replicase protein nsp1 is membrane-associated and has affinity to endocytic organelles. Virology 208:610–620 [CrossRef]
    [Google Scholar]
  38. Rikkonen M. 1996; Functional significance of the nuclear-targeting and NTP-binding motifs of Semliki Forest virus nonstructural protein nsP2. Virology 218:352–361 [CrossRef]
    [Google Scholar]
  39. Saleh S. M., Poidinger M., Mackenzie J. S., Broom A. K., Lindsay M. D., Hall R. A. 2003; Complete genomic sequence of the Australian south-west genotype of Sindbis virus: comparisons with other Sindbis strains and identification of a unique deletion in the 3′-untranslated region. Virus Genes 26:317–327 [CrossRef]
    [Google Scholar]
  40. Salonen A., Vasiljeva L., Merits A., Magden J., Jokitalo E., Kääriäinen L. 2003; Properly folded nonstructural polyprotein directs the Semliki Forest virus replication complex to the endosomal compartment. J Virol 77:1691–1702 [CrossRef]
    [Google Scholar]
  41. Sammin D. J., Butler D., Atkins G. J., Sheahan B. J. 1999; Cell death mechanisms in the olfactory bulb of rats infected intranasally with Semliki Forest virus. Neuropathol Appl Neurobiol 25:236–243 [CrossRef]
    [Google Scholar]
  42. Santagati M. G., Määttä J. A., Itäranta P. V., Salmi A. A., Hinkkanen A. E. 1995; The Semliki Forest virus E2 gene as a virulence determinant. J Gen Virol 76:47–52 [CrossRef]
    [Google Scholar]
  43. Santagati M. G., Määttä J. A., Röyttä M., Salmi A. A., Hinkkanen A. E. 1998; The significance of the 3′-nontranslated region and E2 amino acid mutations in the virulence of Semliki Forest virus in mice. Virology 243:66–77 [CrossRef]
    [Google Scholar]
  44. Shirako Y., Strauss J. H. 1994; 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 68:1874–1885
    [Google Scholar]
  45. Shirako Y., Strauss E. G., Strauss J. H. 2000; Suppressor mutations that allow sindbis virus RNA polymerase to function with nonaromatic amino acids at the N-terminus: evidence for interaction between nsP1 and nsP4 in minus-strand RNA synthesis. Virology 276:148–160 [CrossRef]
    [Google Scholar]
  46. Smyth J. W., Fleeton M. N., Sheahan B. J., Atkins G. J. 2005; Treatment of rapidly growing K-BALB and CT26 mouse tumours using Semliki Forest virus and its derived vector. Gene Ther 12:147–159 [CrossRef]
    [Google Scholar]
  47. Soilu-Hänninen M., Erälinna J. P., Hukkanen V., Röyttä M., Salmi A. A., Salonen R. 1994; Semliki Forest virus infects mouse brain endothelial cells and causes blood-brain barrier damage. J Virol 68:6291–6298
    [Google Scholar]
  48. Strauss J. H., Strauss E. G. 1994; The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58:491–562
    [Google Scholar]
  49. Strauss E. G., Levinson R., Rice C. M., Dalrymple J., Strauss J. H. 1988; Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: sequence and comparison with those of other alphaviruses. Virology 164:265–274 [CrossRef]
    [Google Scholar]
  50. Suopanki J., Sawicki D. L., Sawicki S. G., Kääriäinen L. 1998; Regulation of alphavirus 26S mRNA transcription by replicase component nsP2. J Gen Virol 79:309–319
    [Google Scholar]
  51. Tarbatt C. J., Glasgow G. M., Mooney D. A., Sheahan B. J., Atkins G. J. 1997; Sequence analysis of the avirulent, demyelinating A7 strain of Semliki Forest virus. J Gen Virol 78:1551–1557
    [Google Scholar]
  52. Tuittila M., Hinkkanen A. E. 2003; Amino acid mutations in the replicase protein nsP3 of Semliki Forest virus cumulatively affect neurovirulence. J Gen Virol 84:1525–1533 [CrossRef]
    [Google Scholar]
  53. Tuittila M. T., Santagati M. G., Röyttä M., Määttä J. A., Hinkkanen A. E. 2000; Replicase complex genes of Semliki Forest virus confer lethal neurovirulence. J Virol 74:4579–4589 [CrossRef]
    [Google Scholar]
  54. Vasiljeva L., Merits A., Auvinen P., Kääriäinen L. 2000; Identification of a novel function of the Alphavirus capping apparatus. RNA 5′-triphosphatase activity of Nsp2. J Biol Chem 275:17281–17287 [CrossRef]
    [Google Scholar]
  55. Vasiljeva L., Valmu L., Kääriäinen L., Merits A. 2001; Site-specific protease activity of the carboxyl-terminal domain of Semliki Forest virus replicase protein nsP2. J Biol Chem 276:30786–30793 [CrossRef]
    [Google Scholar]
  56. Vasiljeva L., Merits A., Golubtsov A., Sizemskaja V., Kääriäinen L., Ahola T. 2003; Regulation of the sequential processing of Semliki Forest virus replicase polyprotein. J Biol Chem 278:41636–41645 [CrossRef]
    [Google Scholar]
  57. Vihinen H., Saarinen J. 2000; Phosphorylation site analysis of Semliki Forest virus nonstructural protein 3. J Biol Chem 275:27775–27783
    [Google Scholar]
  58. Vihinen H., Ahola T., Tuittila M., Merits A., Kääriäinen L. 2001; Elimination of phosphorylation sites of Semliki Forest virus replicase protein nsP3. J Biol Chem 276:5745–5752 [CrossRef]
    [Google Scholar]
  59. Wang Y. F., Sawicki S. G., Sawicki D. L. 1991; Sindbis virus nsP1 functions in negative-strand RNA synthesis. J Virol 65:985–988
    [Google Scholar]
  60. Wang Y. F., Sawicki S. G., Sawicki D. L. 1994; Alphavirus nsP3 functions to form replication complexes transcribing negative-strand RNA. J Virol 68:6466–6475
    [Google Scholar]
  61. Wang H. L., O'Rear J., Stollar V. 1996; Mutagenesis of the Sindbis virus nsP1 protein: effects on methyltransferase activity and viral infectivity. Virology 217:527–531 [CrossRef]
    [Google Scholar]
  62. Weston J., Villoing S., Bremont M. & 8 other authors 2002; Comparison of two aquatic alphaviruses, salmon pancreas disease virus and sleeping disease virus, by using genome sequence analysis, monoclonal reactivity, and cross-infection. J Virol 76:6155–6163 [CrossRef]
    [Google Scholar]
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