1887

Abstract

The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and the assembly of infectious virus particles. NS5A comprises three domains, separated by low-complexity sequences. Whilst the function of domain I appears to be predominantly involved with genome replication, the roles of domains II and III are less well defined. It has been reported previously that a deletion spanning the majority of domain II but retaining the C-terminal 35 residues had no effect on virus production; however, deletion of the entire domain II eliminated genome replication, pointing to a key role for the C terminus of this domain. Recent work has also highlighted this region as the potential binding site of the host factor cyclophilin A (CypA). To define this requirement for replication in more detail, and to investigate the involvement of CypA, we conducted a mutagenic study of the C-terminal 30 residues of domain II within the context of both the infectious JFH-1 virus and a JFH-1-derived subgenomic replicon. We showed that 12 of these residues were absolutely required for virus genome replication, whilst mutations of the remainder either had no phenotype or exhibited a partial reduction in genome replication. There was an absolute correlation between the datasets for virus and subgenomic replicon, indicating that this region is involved solely in the process of genome replication. Comparison of our data with a previously published analysis of the same region in genotype 1b revealed some important differences between the two genotypes of HCV.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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2013-05-01
2024-03-29
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References

  1. Appel N., Pietschmann T., Bartenschlager R. 2005; Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain. J Virol 79:3187–3194 [View Article][PubMed]
    [Google Scholar]
  2. Appel N., Zayas M., Miller S., Krijnse-Locker J., Schaller T., Friebe P., Kallis S., Engel U., Bartenschlager R. 2008; Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathog 4:e1000035 [View Article][PubMed]
    [Google Scholar]
  3. Chatterji U., Lim P., Bobardt M. D., Wieland S., Cordek D. G., Vuagniaux G., Chisari F., Cameron C. E., Targett-Adams P.other authors 2010; HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A-cyclophilin A interaction to cyclophilin inhibitors. J Hepatol 53:50–56 [View Article][PubMed]
    [Google Scholar]
  4. Coelmont L., Hanoulle X., Chatterji U., Berger C., Snoeck J., Bobardt M., Lim P., Vliegen I., Paeshuyse J.other authors 2010; DEB025 (Alisporivir) inhibits hepatitis C virus replication by preventing a cyclophilin A induced cis-trans isomerisation in domain II of NS5A. PLoS ONE 5:e13687 [View Article][PubMed]
    [Google Scholar]
  5. Evans M. J., Rice C. M., Goff S. P. 2004; Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication. Proc Natl Acad Sci U S A 101:13038–13043 [View Article][PubMed]
    [Google Scholar]
  6. Feuerstein S., Solyom Z., Aladag A., Favier A., Schwarten M., Hoffmann S., Willbold D., Brutscher B. 2012; Transient structure and SH3 interaction sites in an intrinsically disordered fragment of the hepatitis C virus protein NS5A. J Mol Biol 420:310–323 [View Article][PubMed]
    [Google Scholar]
  7. Fischer G., Gallay P., Hopkins S. 2010; Cyclophilin inhibitors for the treatment of HCV infection. Curr Opin Investig Drugs 11:911–918[PubMed]
    [Google Scholar]
  8. 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]
  9. Foster T. L., Gallay P., Stonehouse N. J., Harris M. 2011; Cyclophilin A interacts with domain II of hepatitis C virus NS5A and stimulates RNA binding in an isomerase-dependent manner. J Virol 85:7460–7464 [View Article][PubMed]
    [Google Scholar]
  10. Fridell R. A., Qiu D., Wang C., Valera L., Gao M. 2010; Resistance analysis of the hepatitis C virus NS5A inhibitor BMS-790052 in an in vitro replicon system. Antimicrob Agents Chemother 54:3641–3650 [View Article][PubMed]
    [Google Scholar]
  11. Gao M., Nettles R. E., Belema M., Snyder L. B., Nguyen V. N., Fridell R. A., Serrano-Wu M. H., Langley D. R., Sun J.-H.other authors 2010; Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 465:96–100 [View Article][PubMed]
    [Google Scholar]
  12. Goh P.-Y., Tan Y.-J., Lim S. P., Lim S. G., Tan Y. H., Hong W. J. 2001; The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage. Virology 290:224–236 [View Article][PubMed]
    [Google Scholar]
  13. Griffin S. 2010; Inhibition of HCV p7 as a therapeutic target. Curr Opin Investig Drugs 11:175–181[PubMed]
    [Google Scholar]
  14. Hanoulle X., Badillo A., Wieruszeski J. M., Verdegem D., Landrieu I., Bartenschlager R., Penin F., Lippens G. 2009a; Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B. J Biol Chem 284:13589–13601 [View Article][PubMed]
    [Google Scholar]
  15. Hanoulle X., Verdegem D., Badillo A., Wieruszeski J. M., Penin F., Lippens G. 2009b; Domain 3 of non-structural protein 5A from hepatitis C virus is natively unfolded. Biochem Biophys Res Commun 381:634–638 [View Article][PubMed]
    [Google Scholar]
  16. He Y., Staschke K. A., Tan S.-L. 2006; HCV NS5A: a multifunctional regulator of cellular pathways and virus replication. In Hepatitis C Viruses: Genomes and Molecular Biology pp. 267–292 Edited by Tan S. L. Norfolk, UK: Horizon Bioscience;
    [Google Scholar]
  17. Hughes M., Griffin S., Harris M. 2009a; 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]
  18. Hughes M., Gretton S., Shelton H., Brown D. D., McCormick C. J., Angus A. G., Patel A. H., Griffin S., Harris M. 2009b; A conserved proline between domains II and III of hepatitis C virus NS5A influences both RNA replication and virus assembly. J Virol 83:10788–10796 [View Article][PubMed]
    [Google Scholar]
  19. Hwang J., Huang L., Cordek D. G., Vaughan R., Reynolds S. L., Kihara G., Raney K. D., Kao C. C., Cameron C. E. 2010; Hepatitis C virus nonstructural protein 5A: biochemical characterization of a novel structural class of RNA-binding proteins. J Virol 84:12480–12491 [View Article][PubMed]
    [Google Scholar]
  20. Jirasko V., Montserret R., Appel N., Janvier A., Eustachi L., Brohm C., Steinmann E., Pietschmann T., Penin F., Bartenschlager R. 2008; Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly. J Biol Chem 283:28546–28562 [View Article][PubMed]
    [Google Scholar]
  21. Jones D. M., Patel A. H., Targett-Adams P., McLauchlan J. 2009; The hepatitis C virus NS4B protein can trans-complement viral RNA replication and modulates production of infectious virus. J Virol 83:2163–2177 [View Article][PubMed]
    [Google Scholar]
  22. Liang Y., Ye H., Kang C. B., Yoon H. S. 2007; Domain 2 of nonstructural protein 5A (NS5A) of hepatitis C virus is natively unfolded. Biochemistry 46:11550–11558 [View Article][PubMed]
    [Google Scholar]
  23. Love R. A., Brodsky O., Hickey M. J., Wells P. A., Cronin C. N. 2009; Crystal structure of a novel dimeric form of NS5A domain I protein from hepatitis C virus. J Virol 83:4395–4403 [View Article][PubMed]
    [Google Scholar]
  24. Ma Y., Anantpadma M., Timpe J. M., Shanmugam S., Singh S. M., Lemon S. M., Yi M. 2011; Hepatitis C virus NS2 protein serves as a scaffold for virus assembly by interacting with both structural and nonstructural proteins. J Virol 85:86–97 [View Article][PubMed]
    [Google Scholar]
  25. 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]
  26. Macdonald A., Crowder K., Street A., McCormick C., Saksela K., Harris M. 2003; The hepatitis C virus non-structural NS5A protein inhibits activating protein-1 function by perturbing Ras–ERK pathway signaling. J Biol Chem 278:17775–17784 [View Article][PubMed]
    [Google Scholar]
  27. Moradpour D., Penin F., Rice C. M. 2007; Replication of hepatitis C virus. Nat Rev Microbiol 5:453–463 [View Article][PubMed]
    [Google Scholar]
  28. Shepard C. W., Finelli L., Alter M. J. 2005; Global epidemiology of hepatitis C virus infection. Lancet Infect Dis 5:558–567 [View Article][PubMed]
    [Google Scholar]
  29. Shimakami T., Hijikata M., Luo H., Ma Y. Y., Kaneko S., Shimotohno K., Murakami S. 2004; Effect of interaction between hepatitis C virus NS5A and NS5B on hepatitis C virus RNA replication with the hepatitis C virus replicon. J Virol 78:2738–2748 [View Article][PubMed]
    [Google Scholar]
  30. Shimakami T., Yamane D., Jangra R. K., Kempf B. J., Spaniel C., Barton D. J., Lemon S. M. 2012; Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex. Proc Natl Acad Sci U S A 109:941–946 [View Article][PubMed]
    [Google Scholar]
  31. Shirota Y., Luo H., Qin W., Kaneko S., Yamashita T., Kobayashi K., Murakami S. 2002; Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity. J Biol Chem 277:11149–11155 [View Article][PubMed]
    [Google Scholar]
  32. Simmonds P., Bukh J., Combet C., Deléage G., Enomoto N., Feinstone S., Halfon P., Inchauspé G., Kuiken C.other authors 2005; Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 42:962–973 [View Article][PubMed]
    [Google Scholar]
  33. Takeuchi T., Katsume A., Tanaka T., Abe A., Inoue K., Tsukiyama-Kohara K., Kawaguchi R., Tanaka S., Kohara M. 1999; Real-time detection system for quantification of hepatitis C virus genome. Gastroenterology 116:636–642 [View Article][PubMed]
    [Google Scholar]
  34. Targett-Adams P., McLauchlan J. 2005; Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon. J Gen Virol 86:3075–3080 [View Article][PubMed]
    [Google Scholar]
  35. Tellinghuisen T. L., Marcotrigiano J., Rice C. M. 2005; Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 435:374–379 [View Article][PubMed]
    [Google Scholar]
  36. Tellinghuisen T. L., Foss K. L., Treadaway J. 2008a; Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. PLoS Pathog 4:e1000032 [View Article][PubMed]
    [Google Scholar]
  37. Tellinghuisen T. L., Foss K. L., Treadaway J. C., Rice C. M. 2008b; Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein. J Virol 82:1073–1083 [View Article][PubMed]
    [Google Scholar]
  38. Wakita T., Pietschmann T., Kato T., Date T., Miyamoto M., Zhao Z., Murthy K., Habermann A., Kräusslich H. G.other authors 2005; Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791–796 [View Article][PubMed]
    [Google Scholar]
  39. Yang F., Robotham J. M., Grise H., Frausto S., Madan V., Zayas M., Bartenschlager R., Robinson M., Greenstein A. E.other authors 2010; A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach. PLoS Pathog 6:e1001118 [View Article][PubMed]
    [Google Scholar]
  40. Yang P. L., Gao M., Lin K., Liu Q., Villareal V. A. 2011; Anti-HCV drugs in the pipeline. Curr Opin Virol 1:607–616 [View Article][PubMed]
    [Google Scholar]
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