1887

Abstract

During virus multiplication, the viral genome is recognized and recruited for replication based on specific -acting elements. Here, we dissected the important -acting sequence elements in Semliki Forest virus RNA by using a -replication system. As the viral replicase is expressed from a separate plasmid, the template RNA can be freely modified in this system. We show that the -acting element at the beginning of the non-structural protein 1 (nsP1) coding region together with the end of the 3′ UTR are the minimal requirements for minus-strand synthesis. To achieve a high level of replication, the native 5′ UTR was also needed. The virus-induced membranous replication compartments (spherules) were only detected when a replication-competent template was present with an active replicase and minus strands were produced. No translation could be detected from the minus strands, suggesting that they are segregated from the cytoplasm. Minus strands could not be recruited directly to initiate the replication process. Thus, there is only one defined pathway for replication, starting with plus-strand recognition followed by concomitant spherule formation and minus-strand synthesis.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000448
2016-06-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/97/6/1395.html?itemId=/content/journal/jgv/10.1099/jgv.0.000448&mimeType=html&fmt=ahah

References

  1. Buchholz U. J., Finke S., Conzelmann K. K. 1999; Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J Virol 73:251–259[PubMed]
    [Google Scholar]
  2. den Boon J. A., Ahlquist P. 2010; Organelle-like membrane compartmentalization of positive-strand RNA virus replication factories. Annu Rev Microbiol 64:241–256 [View Article][PubMed]
    [Google Scholar]
  3. Fayzulin R., Frolov I. 2004; Changes of the secondary structure of the 5' end of the Sindbis virus genome inhibit virus growth in mosquito cells and lead to accumulation of adaptive mutations. J Virol 78:4953–4964 [View Article][PubMed]
    [Google Scholar]
  4. Frolov I., Hardy R., Rice C. M. 2001; Cis-acting RNA elements at the 5' end of Sindbis virus genome RNA regulate minus- and plus-strand RNA synthesis. RNA 7:1638–1651 [View Article][PubMed]
    [Google Scholar]
  5. 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 [View Article][PubMed]
    [Google Scholar]
  6. George J., Raju R. 2000; Alphavirus RNA genome repair and evolution: molecular characterization of infectious sindbis virus isolates lacking a known conserved motif at the 3' end of the genome. J Virol 74:9776–9785 [View Article][PubMed]
    [Google Scholar]
  7. Gorchakov R., Hardy R., Rice C. M., Frolov I. 2004; Selection of functional 5' cis-acting elements promoting efficient sindbis virus genome replication. J Virol 78:61–75 [View Article][PubMed]
    [Google Scholar]
  8. Gorchakov R., Garmashova N., Frolova E., Frolov I. 2008; Different types of nsP3-containing protein complexes in Sindbis virus-infected cells. J Virol 82:10088–10101 [View Article][PubMed]
    [Google Scholar]
  9. Grimley P. M., Berezesky I. K., Friedman R. M. 1968; Cytoplasmic structures associated with an arbovirus infection: loci of viral ribonucleic acid synthesis. J Virol 2:1326–1338[PubMed]
    [Google Scholar]
  10. Hardy R. W. 2006; The role of the 3' terminus of the Sindbis virus genome in minus-strand initiation site selection. Virology 345:520–531 [View Article][PubMed]
    [Google Scholar]
  11. Hardy R. W., Rice C. M. 2005; Requirements at the 3' end of the sindbis virus genome for efficient synthesis of minus-strand RNA. J Virol 79:4630–4639 [View Article][PubMed]
    [Google Scholar]
  12. Hellström K., Vihinen H., Kallio K., Jokitalo E., Ahola T. 2015; Correlative light and electron microscopy enables viral replication studies at the ultrastructural level. Methods 90:49–56 [View Article][PubMed]
    [Google Scholar]
  13. Hyde J. L., Chen R., Trobaugh D. W., Diamond M. S., Weaver S. C., Klimstra W. B., Wilusz J. 2015; The 5' and 3' ends of alphavirus RNAs--Non-coding is not non-functional. Virus Res 206:99–107 [View Article][PubMed]
    [Google Scholar]
  14. Kääriäinen L., Ahola T. 2002; Functions of alphavirus nonstructural proteins in RNA replication. Prog Nucleic Acid Res Mol Biol 71:187–222 [View Article][PubMed]
    [Google Scholar]
  15. Kallio K., Hellström K., Balistreri G., Spuul P., Jokitalo E., Ahola T. 2013; Template RNA length determines the size of replication complex spherules for Semliki Forest virus. J Virol 87:9125–9134 [View Article][PubMed]
    [Google Scholar]
  16. Kallio K., Hellström K., Jokitalo E., Ahola T. 2016; RNA replication and membrane modification require the same functions of alphavirus nonstructural proteins. J Virol 90:1687–1692 [View Article][PubMed]
    [Google Scholar]
  17. Kato H., Takeuchi O., Mikamo-Satoh E., Hirai R., Kawai T., Matsushita K., Hiiragi A., Dermody T. S., Fujita T., Akira S. 2008; Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5. J Exp Med 205:1601–1610 [View Article][PubMed]
    [Google Scholar]
  18. Kuhn R. J., Hong Z., Strauss J. H. 1990; Mutagenesis of the 3' nontranslated region of Sindbis virus RNA. J Virol 64:1465–1476[PubMed]
    [Google Scholar]
  19. Kulasegaran-Shylini R., Atasheva S., Gorenstein D. G., Frolov I. 2009; Structural and functional elements of the promoter encoded by the 5' untranslated region of the Venezuelan equine encephalitis virus genome. J Virol 83:8327–8339 [View Article][PubMed]
    [Google Scholar]
  20. 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[PubMed]
    [Google Scholar]
  21. Levis R., Schlesinger S., Huang H. V. 1990; Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription. J Virol 64:1726–1733[PubMed]
    [Google Scholar]
  22. Li M. L., Stollar V. 2004; Identification of the amino acid sequence in Sindbis virus nsP4 that binds to the promoter for the synthesis of the subgenomic RNA. Proc Natl Acad Sci U S A 101:9429–9434 [View Article][PubMed]
    [Google Scholar]
  23. Li M. L., Stollar V. 2007; Distinct sites on the Sindbis virus RNA-dependent RNA polymerase for binding to the promoters for the synthesis of genomic and subgenomic RNA. J Virol 81:4371–4373 [View Article][PubMed]
    [Google Scholar]
  24. Liu Y., Wimmer E., Paul A. V. 2009; Cis-acting RNA elements in human and animal plus-strand RNA viruses. Biochim Biophys Acta 1789:495–517 [View Article][PubMed]
    [Google Scholar]
  25. Mai J., Sawicki S. G., Sawicki D. L. 2009; Fate of minus-strand templates and replication complexes produced by a p23-cleavage-defective mutant of Sindbis virus. J Virol 83:8553–8564 [View Article][PubMed]
    [Google Scholar]
  26. Michel G., Petrakova O., Atasheva S., Frolov I. 2007; Adaptation of Venezuelan equine encephalitis virus lacking 51-nt conserved sequence element to replication in mammalian and mosquito cells. Virology 362:475–487 [View Article][PubMed]
    [Google Scholar]
  27. Miller S., Krijnse-Locker J. 2008; Modification of intracellular membrane structures for virus replication. Nat Rev Microbiol 6:363–374 [View Article][PubMed]
    [Google Scholar]
  28. Neuvonen M., Ahola T. 2009; Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites. J Mol Biol 385:212–225 [View Article][PubMed]
    [Google Scholar]
  29. Nickens D. G., Hardy R. W. 2008; Structural and functional analyses of stem-loop 1 of the Sindbis virus genome. Virology 370:158–172 [View Article][PubMed]
    [Google Scholar]
  30. Niesters H. G., Strauss J. H. 1990a; Defined mutations in the 5′ non translated sequence of Sindbis virus RNA. J Virol 64:4162–4168
    [Google Scholar]
  31. Niesters H. G., Strauss J. H. 1990b; Mutagenesis of the conserved 51-nucleotide region of Sindbis virus. J Virol 64:1639–1647
    [Google Scholar]
  32. Nikonov A., Mölder T., Sikut R., Kiiver K., Männik A., Toots U., Lulla A., Lulla V., Utt A., other authors . 2013; RIG-I and MDA-5 detection of viral RNA-dependent RNA polymerase activity restricts positive-strand RNA virus replication. PLoS Pathog 9:e1003610 [View Article][PubMed]
    [Google Scholar]
  33. Rupp J. C., Sokoloski K. J., Gebhart N. N., Hardy R. W. 2015; Alphavirus RNA synthesis and non-structural protein functions. J Gen Virol 96:2483–2500 [View Article][PubMed]
    [Google Scholar]
  34. Salonen A., Ahola T., Kääriäinen L. 2005; Viral RNA replication in association with cellular membranes. Curr Top Microbiol Immunol 285:139–173[PubMed]
    [Google Scholar]
  35. Spuul P., Balistreri G., Kääriäinen L., Ahola T. 2010; Phosphatidylinositol 3-kinase, actin, and microtubule-dependent transport of Semliki Forest Virus replication complexes from the plasma membrane to modified lysosomes. J Virol 84:7543–7557 [View Article][PubMed]
    [Google Scholar]
  36. Spuul P., Balistreri G., Hellström K., Golubtsov A. V., Jokitalo E., Ahola T. 2011; Assembly of alphavirus replication complexes from RNA and protein components in a novel trans-replication system in mammalian cells. J Virol 85:4739–4751 [View Article][PubMed]
    [Google Scholar]
  37. Strauss J. H., Strauss E. G. 1994; The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58:491–562[PubMed]
    [Google Scholar]
  38. Thal M. A., Wasik B. R., Posto J., Hardy R. W. 2007; Template requirements for recognition and copying by Sindbis virus RNA-dependent RNA polymerase. Virology 358:221–232 [View Article][PubMed]
    [Google Scholar]
  39. Wengler G., Wengler G., Gross H. S. 1979; Replicative form of Semliki Forest virus RNA contains an unpaired guanosine. Nature 282:754–756 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000448
Loading
/content/journal/jgv/10.1099/jgv.0.000448
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
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