Skip to content
1887

Journal of General Virology header logo Journal of General Virology

Volume 84, Issue 6

Amino acid mutations in the replicase protein nsP3 of Semliki Forest virus cumulatively affect neurovirulence No Access

Abstract

It has been shown previously that an avirulent Semliki Forest virus (SFV) clone, rA774, engineered to carry the nsP3 gene of the virulent clone SFV4 becomes highly neurovirulent and is lethal for adult BALB/c mice. rA774, like several other alphaviruses, has an opal termination codon close to the 5′ end of nsP3 (aa 469), while SFV4 has an arginine residue at this position. Mutation of the opal codon to an arginine residue increases the virulence of rA774 but does not reconstruct the severe neurovirulence of SFV4. Additionally, nsP3 amino acid sequences differ between these two strains by eight amino acids and by a deletion of seven amino acids in the C-terminal third of rA774 nsP3. This study shows that neurovirulence can be reconstituted gradually by exchanging individual amino acids and is fully retained when combinations of two nsP3 mutations, V→I and L→F, V→I and D→N, A→E and G→A or T→A and F→L, are introduced into an rA774 derivative carrying R. The critical role of the arginine codon for neurovirulence was confirmed further by the acquisition of a fully lethal phenotype following the introduction of R into a moderately virulent rA774 recombinant carrying the SFV4 nsP1 and nsP2 genes. In conclusion, virulence determinants in SFV are distributed over a wide region of the nonstructural genes.

  • Received:
  • Accepted:
  • Published Online:
SGM
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.18936-0
2003-06-01
2025-06-19
Loading full text...

Full text loading...

References

  1. Ahola T., Kääriäinen L. 1995; Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP. Proc Natl Acad Sci U S A 92:507–511
     [Google Scholar]
  2. Ahola T., Laakkonen P., Vihinen H., Kääriäinen L. 1997; Critical residues of Semliki Forest virus RNA capping enzyme involved in methyltransferase and guanylyltransferase-like activities. J Virol 71:392–397
     [Google Scholar]
  3. 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
     [Google Scholar]
  4. 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
     [Google Scholar]
  5. Amor S., Scallan M. F., Morris M. M., Dyson H., Fazakerley J. K. 1996; Role of immune responses in protection and pathogenesis during Semliki Forest virus encephalitis. J Gen Virol 77:281–291
     [Google Scholar]
  6. Costanzi C., Pehrson J. R. 1998; Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals. Nature 393:599–601
     [Google Scholar]
  7. Csankovszki G., Panning B., Bates B., Pehrson J. R., Jaenisch R. 1999; Conditional deletion of Xist disrupts histone macroH2A localization but not maintenance of X inactivation. Nat Genet 22:323–324
     [Google Scholar]
  8. Dryga S. A., Dryga O. A., Schlesinger S. 1997; Identification of mutations in a Sindbis virus variant able to establish persistent infection in BHK cells: the importance of a mutation in the nsP2 gene. Virology 228:74–83
     [Google Scholar]
  9. Fazakerley J. K., Pathak S., Scallan M., Amor S., Dyson H. 1993; Replication of the A7(74) strain of Semliki Forest virus is restricted in neurons. Virology 195:627–637
     [Google Scholar]
  10. Froshauer S., Kartenbeck J., Helenius A. 1988; Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes. J Cell Biol 107:2075–2086
     [Google Scholar]
  11. 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
     [Google Scholar]
  12. Gomez de Cedron M., Ehsani N., Mikkola M. L., Garcia J. A., Kääriäinen L. 1999; RNA helicase activity of Semliki Forest virus replicase protein NSP2. FEBS Lett 448:19–22
     [Google Scholar]
  13. Gorbalenya A. E., Koonin E. V., Lai M. M. 1991; Putative papain-related thiol proteases of positive-strand RNA viruses. Identification of rubi- and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha- and coronaviruses. FEBS Lett 288:201–205
     [Google Scholar]
  14. Hardy W. R., Strauss J. H. 1989; Processing the nonstructural polyproteins of Sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans . J Virol 63:4653–4664
     [Google Scholar]
  15. Haseloff J., Goelet P., Zimmern D., Ahlquist R., Dasgupta R., Kaesberg P. 1984; Striking similarities in amino acid sequence among nonstructural proteins encoded by RNA viruses that have dissimilar genomic organization. Proc Natl Acad Sci U S A 81:4358–4362
     [Google Scholar]
  16. Heise M. T., Simpson D. A., Johnston R. E. 2000; Sindbis-group alphavirus replication in periosteum and endosteum of long bones in adult mice. J Virol 74:9294–9299
     [Google Scholar]
  17. Kamer G., Argos P. 1984; Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res 12:7269–7282
     [Google Scholar]
  18. Kujala P., Ikäheimonen A., Ehsani N., Vihinen H., Auvinen P., Kääriäinen L. 2001; Biogenesis of the Semliki Forest virus RNA replication complex. J Virol 75:3873–3884
     [Google Scholar]
  19. Laakkonen P., Hyvönen M., Peränen J., Kääriäinen L. 1994; Expression of Semliki Forest virus nsP1-specific methyltransferase in insect cells and in Escherichia coli . J Virol 68:7418–7425
     [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
     [Google Scholar]
  21. Lampio A., Kilpeläinen 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
     [Google Scholar]
  22. Lastarza M. W., Grakoui A., Rice C. M. 1994a; 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
     [Google Scholar]
  23. LaStarza M. W., Lemm J. A., Rice C. M. 1994b; 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]
  24. Lemm J. A., Rümenapf 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]
  25. Liang Y., Yao J., Gillam S. 2000; Rubella virus nonstructural protein protease domains involved in trans - and cis -cleavage activities. J Virol 74:5412–5423
     [Google Scholar]
  26. 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]
  27. Mehta S., Pathak S., Webb H. E. 1990; Induction of membrane proliferation in mouse CNS by gold sodium thiomalate with reference to increased virulence of the avirulent Semliki Forest virus. Biosci Rep 10:271–279
     [Google Scholar]
  28. Oliver K. R., Fazakerley J. K. 1998; Transneuronal spread of Semliki Forest virus in the developing mouse olfactory system is determined by neuronal maturity. Neuroscience 82:867–877
     [Google Scholar]
  29. Oliver K. R., Scallan M. F., Dyson H., Fazakerley J. K. 1997; Susceptibility to a neurotropic virus and its changing distribution in the developing brain is a function of CNS maturity. J Neurovirol 3:38–48
     [Google Scholar]
  30. Pathak S., Illavia S. J., Webb H. E. 1983; The identification and role of cells involved in CNS demyelination in mice after Semliki Forest virus infection: an ultrastructural study. Prog Brain Res 59:237–254
     [Google Scholar]
  31. Pehrson J. R., Fuji R. N. 1998; Evolutionary conservation of histone macroH2A subtypes and domains. Nucleic Acids Res 26:2837–2842
     [Google Scholar]
  32. 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]
  33. 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
     [Google Scholar]
  34. Rikkonen M., Peränen J., Kääriäinen L. 1992; Nuclear and nucleolar targeting signals of Semliki Forest virus nonstructural protein nsP2. Virology 189:462–473
     [Google Scholar]
  35. Rikkonen M., Peränen J., Kääriäinen L. 1994; ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2. J Virol 68:5804–5810
     [Google Scholar]
  36. 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
     [Google Scholar]
  37. 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
     [Google Scholar]
  38. 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
     [Google Scholar]
  39. Scallan M. F., Fazakerley J. K. 1999; Aurothiolates enhance the replication of Semliki Forest virus in the CNS and the exocrine pancreas. J Neurovirol 5:392–400
     [Google Scholar]
  40. Simpson D. A., Davis N. L., Lin S. C., Russell D., Johnston R. E. 1996; Complete nucleotide sequence and full-length cDNA clone of S.A.AR86 a South African alphavirus related to Sindbis. Virology 222:464–469
     [Google Scholar]
  41. Strauss J. H., Strauss E. G. 1994; The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58:491–562
     [Google Scholar]
  42. 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
     [Google Scholar]
  43. Takkinen K. 1986; Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus. Nucleic Acids Res 14:5667–5682
     [Google Scholar]
  44. Takkinen K., Peränen J., Kääriäinen L. 1991; Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein. J Gen Virol 72:1627–1633
     [Google Scholar]
  45. 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]
  46. 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
     [Google Scholar]
  47. van der Heijden M. W., Bol J. F. 2002; Composition of alphavirus-like replication complexes: involvement of virus and host encoded proteins. Arch Virol 147:875–898
     [Google Scholar]
  48. 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
     [Google Scholar]
  49. 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
     [Google Scholar]
  50. Vihinen H., Saarinen J. 2000; Phosphorylation site analysis of Semliki Forest virus nonstructural protein 3. J Biol Chem 275:27775–27783
     [Google Scholar]
  51. 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
     [Google Scholar]
  52. 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]
/content/journal/jgv/10.1099/vir.0.18936-0
Loading
Amino acid mutations in the replicase protein nsP3 of Semliki Forest virus cumulatively affect neurovirulence
J. Gen. Virol. 84, 1525 (2003); https://doi.org/10.1099/vir.0.18936-0
/content/journal/jgv/10.1099/vir.0.18936-0
/content/journal/jgv/10.1099/vir.0.18936-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error