Downregulation of viral RNA translation by hepatitis C virus non-structural protein NS5A requires the poly(U/UC) sequence in the 3′ UTR Free

Abstract

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3′ UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5′ and 3′ UTRs of HCV because substitution of either sequence with the 5′ and 3′ UTRs of enterovirus 71 or a cap structure at the 5′ end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3′ UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3′ UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3′ UTR.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.000141
2015-08-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/96/8/2114.html?itemId=/content/journal/jgv/10.1099/vir.0.000141&mimeType=html&fmt=ahah

References

  1. Bai Y., Zhou K., Doudna J.A. 2013; Hepatitis C virus 3′ UTR regulates viral translation through direct interactions with the host translation machinery. Nucleic Acids Res 41:7861–7874 [View Article][PubMed]
    [Google Scholar]
  2. Bradrick S.S., Walters R.W., Gromeier M. 2006; The hepatitis C virus 3′-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase. Nucleic Acids Res 34:1293–1303 [View Article][PubMed]
    [Google Scholar]
  3. Bung C., Bochkaeva Z., Terenin I., Zinovkin R., Shatsky I.N., Niepmann M. 2010; Influence of the hepatitis C virus 3′-untranslated region on IRES-dependent and cap-dependent translation initiation. FEBS Lett 584:837–842 [View Article][PubMed]
    [Google Scholar]
  4. Chang G.H., Lin L., Luo Y.J., Cai L.J., Wu X.Y., Xu H.M., Zhu Q.Y. 2010; Sequence analysis of six enterovirus 71 strains with different virulences in humans. Virus Res 151:66–73 [View Article][PubMed]
    [Google Scholar]
  5. Dustin L.B., Rice C.M. 2007; Flying under the radar: the immunobiology of hepatitis C. Annu Rev Immunol 25:71–99 [View Article][PubMed]
    [Google Scholar]
  6. Foster T.L., Belyaeva T., Stonehouse N.J., Pearson A.R., Harris M. 2010; All three domains of the hepatitis C virus nonstructural NS5A protein contribute to RNA binding. J Virol 84:9267–9277 [View Article][PubMed]
    [Google Scholar]
  7. Gonzalez O., Fontanes V., Raychaudhuri S., Loo R., Loo J., Arumugaswami V., Sun R., Dasgupta A., French S.W. 2009; The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production. Hepatology 50:1756–1764 [View Article][PubMed]
    [Google Scholar]
  8. He Y., Yan W., Coito C., Li Y., Gale M. Jr, Katze M.G. 2003; The regulation of hepatitis C virus (HCV) internal ribosome-entry site-mediated translation by HCV replicons and nonstructural proteins. J Gen Virol 84:535–543 [View Article][PubMed]
    [Google Scholar]
  9. Hoffman B., Liu Q. 2011; Hepatitis C viral protein translation: mechanisms and implications in developing antivirals. Liver Int 31:1449–1467 [View Article][PubMed]
    [Google Scholar]
  10. Huang L., Sineva E.V., Hargittai M.R., Sharma S.D., Suthar M., Raney K.D., Cameron C.E. 2004; Purification and characterization of hepatitis C virus non-structural protein 5A expressed in Escherichia coli . Protein Expr Purif 37:144–153 [View Article][PubMed]
    [Google Scholar]
  11. Huang L., Hwang J., Sharma S.D., Hargittai M.R., Chen Y., Arnold J.J., Raney K.D., Cameron C.E. 2005; Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein. J Biol Chem 280:36417–36428 [View Article][PubMed]
    [Google Scholar]
  12. Hughes M., Griffin S., Harris M. 2009; Domain III of NS5A contributes to both RNA replication and assembly of hepatitis C virus particles. J Gen Virol 90:1329–1334 [View Article][PubMed]
    [Google Scholar]
  13. Ikeda M., Yi M., Li K., Lemon S.M. 2002; Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol 76:2997–3006 [View Article][PubMed]
    [Google Scholar]
  14. Imbert I., Dimitrova M., Kien F., Kieny M.P., Schuster C. 2003; Hepatitis C virus IRES efficiency is unaffected by the genomic RNA 3′NTR even in the presence of viral structural or non-structural proteins. J Gen Virol 84:1549–1557 [View Article][PubMed]
    [Google Scholar]
  15. Isken O., Baroth M., Grassmann C.W., Weinlich S., Ostareck D.H., Ostareck-Lederer A., Behrens S.E. 2007; Nuclear factors are involved in hepatitis C virus RNA replication. RNA 13:1675–1692 [View Article][PubMed]
    [Google Scholar]
  16. Jackel-Cram C., Qiao L., Xiang Z., Brownlie R., Zhou Y., Babiuk L., Liu Q. 2010; Hepatitis C virus genotype-3a core protein enhances sterol regulatory element-binding protein-1 activity through the phosphoinositide 3-kinase–Akt-2 pathway. J Gen Virol 91:1388–1395 [View Article][PubMed]
    [Google Scholar]
  17. Jones C.T., Murray C.L., Eastman D.K., Tassello J., Rice C.M. 2007; Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol 81:8374–8383 [View Article][PubMed]
    [Google Scholar]
  18. Kalliampakou K.I., Kalamvoki M., Mavromara P. 2005; Hepatitis C virus (HCV) NS5A protein downregulates HCV IRES-dependent translation. J Gen Virol 86:1015–1025 [View Article][PubMed]
    [Google Scholar]
  19. Kohler J.J., Nettles J.H., Amblard F., Hurwitz S.J., Bassit L., Stanton R.A., Ehteshami M., Schinazi R.F. 2014; Approaches to hepatitis C treatment and cure using NS5A inhibitors. Infect Drug Resist 7:41–56[PubMed]
    [Google Scholar]
  20. Lohmann V. 2009; HCV replicons: overview and basic protocols. Methods Mol Biol 510:145–163 [View Article][PubMed]
    [Google Scholar]
  21. Lourenço S., Costa F., Débarges B., Andrieu T., Cahour A. 2008; Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by cis-acting RNA elements and trans-acting viral factors. FEBS J 275:4179–4197 [View Article][PubMed]
    [Google Scholar]
  22. Macdonald A., Harris M. 2004; Hepatitis C virus NS5A: tales of a promiscuous protein. J Gen Virol 85:2485–2502 [View Article][PubMed]
    [Google Scholar]
  23. Niepmann M. 2013; Hepatitis C virus RNA translation. Curr Top Microbiol Immunol 369:143–166[PubMed]
    [Google Scholar]
  24. Qiao L., Wu Q., Lu X., Zhou Y., Fernández-Alvarez A., Ye L., Zhang X., Han J., Casado M., Liu Q. 2013; SREBP-1a activation by HBx and the effect on hepatitis B virus enhancer II/core promoter. Biochem Biophys Res Commun 432:643–649 [View Article][PubMed]
    [Google Scholar]
  25. Ross-Thriepland D., Harris M. 2015; Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!. J Gen Virol727–738 [View Article][PubMed]
    [Google Scholar]
  26. Shetty S., Stefanovic S., Mihailescu M.R. 2013; Hepatitis C virus RNA: molecular switches mediated by long-range RNA–RNA interactions?. Nucleic Acids Res 41:2526–2540 [View Article][PubMed]
    [Google Scholar]
  27. Song Y., Friebe P., Tzima E., Jünemann C., Bartenschlager R., Niepmann M. 2006; The hepatitis C virus RNA 3′-untranslated region strongly enhances translation directed by the internal ribosome entry site. J Virol 80:11579–11588 [View Article][PubMed]
    [Google Scholar]
  28. Suzuki T., Ishii K., Aizaki H., Wakita T. 2007; Hepatitis C viral life cycle. Adv Drug Deliv Rev 59:1200–1212 [View Article][PubMed]
    [Google Scholar]
  29. Toroney R., Nallagatla S.R., Boyer J.A., Cameron C.E., Bevilacqua P.C. 2010; Regulation of PKR by HCV IRES RNA: importance of domain II and NS5A. J Mol Biol 400:393–412 [View Article][PubMed]
    [Google Scholar]
  30. Tuplin A., Struthers M., Simmonds P., Evans D.J. 2012; A twist in the tail: SHAPE mapping of long-range interactions and structural rearrangements of RNA elements involved in HCV replication. Nucleic Acids Res 40:6908–6921 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.000141
Loading
/content/journal/jgv/10.1099/vir.0.000141
Loading

Data & Media loading...

Most cited Most Cited RSS feed