1887

Journal of General Virology header logo

Volume 95, Issue 12

An assay to study chikungunya virus RNA synthesis and the mode of action of inhibitors Free

Abstract

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay P-CTP was incorporated into the CHIKV genome, subgenomic (sg) RNA and into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5′ end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15 000 . Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated replication/transcription complexes (RTCs). The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the RTC assay. In contrast to 3′dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity . In conclusion, this assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the RTC and mode of action studies on compounds with anti-CHIKV activity.

  • Received:
  • Accepted:
  • Published Online:
© 2014 The Authors
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.069690-0
2014-12-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/95/12/2683.html?itemId=/content/journal/jgv/10.1099/vir.0.069690-0&mimeType=html&fmt=ahah

References

  1. Ali N., Tardif K. D., Siddiqui A. 2002; Cell-free replication of the hepatitis C virus subgenomic replicon. J Virol 76:12001–12007 [View Article][PubMed]
     [Google Scholar]
  2. Baltimore D., Eggers H. J., Franklin R. M., Tamm I. 1963; Poliovirus-induced RNA polymerase and the effects of virus-specific inhibitors on its production. Proc Natl Acad Sci U S A 49:843–849 [View Article][PubMed]
     [Google Scholar]
  3. Barton D. J., Sawicki S. G., Sawicki D. L. 1991; Solubilization and immunoprecipitation of alphavirus replication complexes. J Virol 65:1496–1506[PubMed]
     [Google Scholar]
  4. Beaucourt S., Vignuzzi M. 2014; Ribavirin: a drug active against many viruses with multiple effects on virus replication and propagation. Molecular basis of ribavirin resistance. Curr Opin Virol 8:10–15 [View Article][PubMed]
     [Google Scholar]
  5. Bruton C. J., Kennedy S. I. 1975; Semliki Forest virus intracellular RNA: properties of the multi-stranded RNA species and kinetics of positive and negative strand synthesis. J Gen Virol 28:111–127 [View Article][PubMed]
     [Google Scholar]
  6. Burt F. J., Rolph M. S., Rulli N. E., Mahalingam S., Heise M. T. 2012; Chikungunya: a re-emerging virus. Lancet 379:662–671 [View Article][PubMed]
     [Google Scholar]
  7. Chu P. W., Westaway E. G. 1985; Replication strategy of Kunjin virus: evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication. Virology 140:68–79 [View Article][PubMed]
     [Google Scholar]
  8. Crotty S., Cameron C., Andino R. 2002; Ribavirin’s antiviral mechanism of action: lethal mutagenesis?. J Mol Med (Berl) 80:86–95 [View Article][PubMed]
     [Google Scholar]
  9. Fischer M., Staples J. E. 2014; Notes from the field: chikungunya virus spreads in the Americas - Caribbean and South America, 2013-2014. MMWR Morb Mortal Wkly Rep 63:500–501[PubMed]
     [Google Scholar]
  10. Frolova E. I., Gorchakov R., Pereboeva L., Atasheva S., Frolov I. 2010; Functional Sindbis virus replicative complexes are formed at the plasma membrane. J Virol 84:11679–11695 [View Article][PubMed]
     [Google Scholar]
  11. 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]
  12. Gibney K. B., Fischer M., Prince H. E., Kramer L. D., St George K., Kosoy O. L., Laven J. J., Staples J. E. 2011; Chikungunya fever in the United States: a fifteen year review of cases. Clin Infect Dis 52:e121–e126 [View Article][PubMed]
     [Google Scholar]
  13. Grandadam M., Caro V., Plumet S., Thiberge J. M., Souarès Y., Failloux A. B., Tolou H. J., Budelot M., Cosserat D.other authors 2011; Chikungunya virus, southeastern France. Emerg Infect Dis 17:910–913 [View Article][PubMed]
     [Google Scholar]
  14. Grun J. B., Brinton M. A. 1986; Characterization of West Nile virus RNA-dependent RNA polymerase and cellular terminal adenylyl and uridylyl transferases in cell-free extracts. J Virol 60:1113–1124[PubMed]
     [Google Scholar]
  15. Hardy R. W., Marcotrigiano J., Blight K. J., Majors J. E., Rice C. M. 2003; Hepatitis C virus RNA synthesis in a cell-free system isolated from replicon-containing hepatoma cells. J Virol 77:2029–2037 [View Article][PubMed]
     [Google Scholar]
  16. 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 [View Article][PubMed]
     [Google Scholar]
  17. Lai V. C., Dempsey S., Lau J. Y., Hong Z., Zhong W. 2003; In vitro RNA replication directed by replicase complexes isolated from the subgenomic replicon cells of hepatitis C virus. J Virol 77:2295–2300 [View Article][PubMed]
     [Google Scholar]
  18. Leparc-Goffart I., Nougairede A., Cassadou S., Prat C., de Lamballerie X. 2014; Chikungunya in the Americas. Lancet 383:514 [View Article][PubMed]
     [Google Scholar]
  19. Levin J. G., Friedman R. M. 1971; Analysis of arbovirus ribonucleic acid forms by polyacrylamide gel electrophoresis. J Virol 7:504–514[PubMed]
     [Google Scholar]
  20. Metz S. W., Gardner J., Geertsema C., Le T. T., Goh L., Vlak J. M., Suhrbier A., Pijlman G. P. 2013; Effective chikungunya virus-like particle vaccine produced in insect cells. PLoS Negl Trop Dis 7:e2124 [View Article][PubMed]
     [Google Scholar]
  21. Rezza G., Nicoletti L., Angelini R., Romi R., Finarelli A. C., Panning M., Cordioli P., Fortuna C., Boros S.other authors 2007; Infection with chikungunya virus in Italy: an outbreak in a temperate region. Lancet 370:1840–1846 [View Article][PubMed]
     [Google Scholar]
  22. Scholte F. E., Tas A., Martina B. E., Cordioli P., Narayanan K., Makino S., Snijder E. J., van Hemert M. J. 2013; Characterization of synthetic Chikungunya viruses based on the consensus sequence of recent E1-226V isolates. PLoS ONE 8:e71047 [View Article][PubMed]
     [Google Scholar]
  23. 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]
  24. Traut T. W. 1994; Physiological concentrations of purines and pyrimidines. Mol Cell Biochem 140:1–22 [View Article][PubMed]
     [Google Scholar]
  25. van Hemert M. J., de Wilde A. H., Gorbalenya A. E., Snijder E. J. 2008a; The in vitro RNA synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor. J Biol Chem 283:16525–16536 [View Article][PubMed]
     [Google Scholar]
  26. van Hemert M. J., van den Worm S. H., Knoops K., Mommaas A. M., Gorbalenya A. E., Snijder E. J. 2008b; SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro. PLoS Pathog 4:e1000054 [View Article][PubMed]
     [Google Scholar]
  27. Weaver S. C. 2014; Arrival of chikungunya virus in the new world: prospects for spread and impact on public health. PLoS Negl Trop Dis 8:e2921 [View Article][PubMed]
     [Google Scholar]
  28. Wielgosz M. M., Huang H. V. 1997; A novel viral RNA species in Sindbis virus-infected cells. J Virol 71:9108–9117[PubMed]
     [Google Scholar]
  29. Yang W., Huang M. 2009; Studying HCV RNA synthesis in vitro with replication complexes. Methods Mol Biol 510:177–184 [View Article][PubMed]
     [Google Scholar]
  30. You S., Padmanabhan R. 1999; A novel in vitro replication system for Dengue virus. Initiation of RNA synthesis at the 3′-end of exogenous viral RNA templates requires 5′- and 3′-terminal complementary sequence motifs of the viral RNA. J Biol Chem 274:33714–33722 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.069690-0
Loading
An in vitro assay to study chikungunya virus RNA synthesis and the mode of action of inhibitors
J. Gen. Virol. 95, 2683 (2014); https://doi.org/10.1099/vir.0.069690-0
/content/journal/jgv/10.1099/vir.0.069690-0
/content/journal/jgv/10.1099/vir.0.069690-0
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