1887

Abstract

The replicase of Semliki Forest virus (SFV) consists of four non-structural proteins, designated nsP1–4, and is bound to cellular membranes via an amphipathic peptide and palmitoylated cysteine residues of nsP1. It was found that mutations preventing nsP1 palmitoylation also attenuated virus replication. The replacement of these cysteines by alanines, or their deletion, abolished virus viability, possibly due to disruption of interactions between nsP1 and nsP4, which is the catalytic subunit of the replicase. However, during a single infection cycle, the ability of the virus to replicate was restored due to accumulation of second-site mutations in nsP1. These mutations led to the restoration of nsP1–nsP4 interaction, but did not restore the palmitoylation of nsP1. The proteins with palmitoylation-site mutations, as well as those harbouring compensatory mutations in addition to palmitoylation-site mutations, were enzymically active and localized, at least in part, on the plasma membrane of transfected cells. Interestingly, deletion of 7 aa including the palmitoylation site of nsP1 had a relatively mild effect on virus viability and no significant impact on nsP1–nsP4 interaction. Similarly, the change of cysteine to alanine at the palmitoylation site of nsP1 of Sindbis virus had only a mild effect on virus replication. Taken together, these findings indicate that nsP1 palmitoylation as such is not the factor determining the ability to bind to cellular membranes and form a functional replicase complex. Instead, these abilities may be linked to the three-dimensional structure of nsP1 and the capability of nsP1 to interact with other components of the viral replicase complex.

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2007-07-01
2024-03-28
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References

  1. Ahola T., Kaariainen 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 [CrossRef]
    [Google Scholar]
  2. Ahola T., Laakkonen P., Vihinen H., Kaariainen 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., Kaariainen L. 1999; Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity. EMBO J 18:3164–3172 [CrossRef]
    [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 [CrossRef]
    [Google Scholar]
  5. Fata C. L., Sawicki S. G., Sawicki D. L. 2002; Modification of Asn374 of nsP1 suppresses a Sindbis virus nsP4 minus-strand polymerase mutant. J Virol 76:8641–8649 [CrossRef]
    [Google Scholar]
  6. Kaldalu N., Lepik D., Kristjuhan A., Ustav M. 2000; Monitoring and purification of proteins using bovine papillomavirus E2 epitope tags. Biotechniques 28:456–460, 462
    [Google Scholar]
  7. Karlsson G. B., Liljestrom P. 2003; Live viral vectors: Semliki Forest virus. Methods Mol Med 87:69–82
    [Google Scholar]
  8. Laakkonen P., Hyvonen M., Peranen J., Kaariainen L. 1994; Expression of Semliki Forest virus nsP1-specific methyltransferase in insect cells and in Escherichia coli. . J Virol 68:7418–7425
    [Google Scholar]
  9. Laakkonen P., Ahola T., Kaariainen 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]
  10. Laakkonen P., Auvinen P., Kujala P., Kaariainen L. 1998; Alphavirus replicase protein NSP1 induces filopodia and rearrangement of actin filaments. J Virol 72:10265–10269
    [Google Scholar]
  11. Liljestrom P., Garoff H. 1991; A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 9:1356–1361 [CrossRef]
    [Google Scholar]
  12. Peranen 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]
  13. Peranen J., Rikkonen M., Kääriäinen L. 1993; A method for exposing hidden antigenic sites in paraformaldehyde-fixed cultured cells, applied to initially unreactive antibodies. J Histochem Cytochem 41:447–454 [CrossRef]
    [Google Scholar]
  14. Rice C. M., Levis R., Strauss J. H., Huang H. V. 1987; 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 61:3809–3819
    [Google Scholar]
  15. Salonen A., Vasiljeva L., Merits A., Magden J., Jokitalo E., Kaariainen 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]
  16. 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]
  17. Spuul P., Salonen A., Merits A., Jokitalo E., Kaariainen L., Ahola T. 2007; Role of the amphipathic peptide of Semliki Forest virus replicase protein nsP1 in membrane association and virus replication. J Virol 81:872–883 [CrossRef]
    [Google Scholar]
  18. Strauss J. H., Strauss E. G. 1994; The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58:491–562
    [Google Scholar]
  19. 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]
  20. 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]
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