1887

Abstract

Non-structural 5A protein (NS5A) has emerged as an important pharmacological target for hepatitis C virus (HCV). However, little is known about the conformation of NS5A intracellularly or how NS5A inhibitors achieve the picomolar (pM) inhibition of virus replication. Here, we have presented two structurally related small molecules, one that potently inhibits HCV replication and selects for resistance in NS5A, and another that is inactive. Resistance to this antiviral was greater in genotype 1a than in genotype 1b replicons and mapped to domain 1 of NS5A. Using a novel cell-based assay that measures the intracellular proximity of fluorescent tags covalently attached to NS5A, we showed that only the active antiviral specifically disrupted the close proximity of inter- and intramolecular positions of NS5A. The active antiviral, termed compound 1, caused a repositioning of both the N and C termini of NS5A, including disruption of the close approximation of the N termini of two different NS5A molecules in a multimolecular complex. These data provide the first study of how antivirals that select resistance in domain 1 of NS5A alter the cellular conformation of NS5A. This class of antiviral disrupts the close proximity of the N termini of domain 1 in a NS5A complex but also alters the conformation of domain 3, and leads to large aggregates of NS5A. Current models predict that a multicomponent cocktail of antivirals is needed to treat HCV infection, so a mechanistic understanding of what each component does to the viral machinery will be important.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.054569-0
2014-02-01
2019-09-20
Loading full text...

Full text loading...

/deliver/fulltext/jgv/95/2/363.html?itemId=/content/journal/jgv/10.1099/vir.0.054569-0&mimeType=html&fmt=ahah

References

  1. Adams S. R. , Campbell R. E. , Gross L. A. , Martin B. R. , Walkup G. K. , Yao Y. , Llopis J. , Tsien R. Y. . ( 2002; ). New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. . J Am Chem Soc 124:, 6063–6076. [CrossRef] [PubMed]
    [Google Scholar]
  2. Ansari I. U. , Striker R. T. . ( 2012; ). Subtype specific differences in NS5A domain II reveals involvement of proline at position 310 in cyclosporine susceptibility of hepatitis C virus. . Viruses 4:, 3303–3315. [CrossRef] [PubMed]
    [Google Scholar]
  3. Ansari I. U. , Allen T. , Berical A. , Stock P. G. , Barin B. , Striker R. . ( 2013; ). Phenotypic analysis of NS5A variant from liver transplant patient with increased cyclosporine susceptibility. . Virology 436:, 268–273. [CrossRef] [PubMed]
    [Google Scholar]
  4. Bechtel J. , Crosby R. , Wang A. , Woldu E. , Van Horn S. , Horton J. , Remlinger K. , Creech K. , Carballo L. H. . & other authors ( 2011; ). In vitro profiling of GSK2336805, a potent and selective inhibitor of HCV NS5A. . In EASL 46th Annual Meeting, 31 March–2 April, Berlin, Germany 2011.
    [Google Scholar]
  5. Bhattacharya D. , Ansari I. H. , Mehle A. , Striker R. . ( 2012; ). Fluorescence resonance energy transfer-based intracellular assay for the conformation of hepatitis C virus drug target NS5A. . J Virol 86:, 8277–8286. [CrossRef] [PubMed]
    [Google Scholar]
  6. Blight K. J. , McKeating J. A. , Marcotrigiano J. , Rice C. M. . ( 2003; ). Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture. . J Virol 77:, 3181–3190. [CrossRef] [PubMed]
    [Google Scholar]
  7. Chen P. , Couch R. , Duan M. , Grimes R. M. , Kazmierski W. M. , Norton B. A. , Tallant M. . ( 2010; ). Preparation of biphenyls end capped with amino acids or peptide derivatives for treating HCV infection. . Patent, Publication no. WO 2011028596 A1.
  8. Das S. C. , Nayak D. , Zhou Y. , Pattnaik A. K. . ( 2006; ). Visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells. . J Virol 80:, 6368–6377. [CrossRef] [PubMed]
    [Google Scholar]
  9. Enomoto N. , Sakuma I. , Asahina Y. , Kurosaki M. , Murakami T. , Yamamoto C. , Ogura Y. , Izumi N. , Marumo F. , Sato C. . ( 1996; ). Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. . N Engl J Med 334:, 77–82. [CrossRef] [PubMed]
    [Google Scholar]
  10. 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. [CrossRef] [PubMed]
    [Google Scholar]
  11. Fridell R. A. , Wang C. , Sun J. H. , O’Boyle D. R. II , Nower P. , Valera L. , Qiu D. , Roberts S. , Huang X. . & other authors ( 2011; ). Genotypic and phenotypic analysis of variants resistant to hepatitis C virus nonstructural protein 5A replication complex inhibitor BMS-790052 in humans: in vitro and in vivo correlations. . Hepatology 54:, 1924–1935. [CrossRef] [PubMed]
    [Google Scholar]
  12. 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. [CrossRef] [PubMed]
    [Google Scholar]
  13. Ghany M. G. , Nelson D. R. , Strader D. B. , Thomas D. L. , Seeff L. B. . American Association for Study of Liver Diseases ( 2011; ). An update on treatment of genotype 1 chronic hepatitis C virus infection: 2011 practice guideline by the American Association for the Study of Liver Diseases. . Hepatology 54:, 1433–1444. [CrossRef] [PubMed]
    [Google Scholar]
  14. Guedj J. , Dahari H. , Rong L. , Sansone N. D. , Nettles R. E. , Cotler S. J. , Layden T. J. , Uprichard S. L. , Perelson A. S. . ( 2013; ). Modeling shows that the NS5A inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis C virus half-life. . Proc Natl Acad Sci U S A 110:, 3991–3996. [CrossRef] [PubMed]
    [Google Scholar]
  15. 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. [CrossRef] [PubMed]
    [Google Scholar]
  16. Lee C. , Ma H. , Hang J. Q. , Leveque V. , Sklan E. H. , Elazar M. , Klumpp K. , Glenn J. S. . ( 2011; ). The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein. . Virology 414:, 10–18. [CrossRef] [PubMed]
    [Google Scholar]
  17. Lemm J. A. , O’Boyle D. II , Liu M. , Nower P. T. , Colonno R. , Deshpande M. S. , Snyder L. B. , Martin S. W. , St Laurent D. R. . & other authors ( 2010; ). Identification of hepatitis C virus NS5A inhibitors. . J Virol 84:, 482–491. [CrossRef] [PubMed]
    [Google Scholar]
  18. Lohmann V. , Hoffmann S. , Herian U. , Penin F. , Bartenschlager R. . ( 2003; ). Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. . J Virol 77:, 3007–3019. [CrossRef] [PubMed]
    [Google Scholar]
  19. Lok A. S. , Gardiner D. F. , Lawitz E. , Martorell C. , Everson G. T. , Ghalib R. , Reindollar R. , Rustgi V. , McPhee F. . & other authors ( 2012; ). Preliminary study of two antiviral agents for hepatitis C genotype 1. . N Engl J Med 366:, 216–224. [CrossRef] [PubMed]
    [Google Scholar]
  20. 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. [CrossRef] [PubMed]
    [Google Scholar]
  21. Membreno F. E. , Espinales J. C. , Lawitz E. J. . ( 2013; ). Cyclophilin inhibitors for hepatitis C therapy. . Clin Liver Dis 17:, 129–139. [CrossRef] [PubMed]
    [Google Scholar]
  22. Miyawaki A. , Tsien R. Y. . ( 2000; ). Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. . Methods Enzymol 327:, 472–500. [CrossRef] [PubMed]
    [Google Scholar]
  23. O’Boyle, I. D. R. , Sun, J. H. , Nower, P. T. , Lemm J. A. , Fridell R. A. , Wang C. , Romine J. L. , Belema M. , Nguyen V. N. . & other authors ( 2013; ). Characterizations of HCV NS5A replication complex inhibitors. . Virology 444:, 343–354.[CrossRef]
    [Google Scholar]
  24. Puyang X. , Poulin D. L. , Mathy J. E. , Anderson L. J. , Ma S. , Fang Z. , Zhu S. , Lin K. , Fujimoto R. . & other authors ( 2010; ). Mechanism of resistance of hepatitis C virus replicons to structurally distinct cyclophilin inhibitors. . Antimicrob Agents Chemother 54:, 1981–1987. [CrossRef] [PubMed]
    [Google Scholar]
  25. Qiu D. , Lemm J. A. , O’Boyle D. R. II , Sun J. H. , Nower P. T. , Nguyen V. , Hamann L. G. , Snyder L. B. , Deon D. H. . & other authors ( 2011; ). The effects of NS5A inhibitors on NS5A phosphorylation, polyprotein processing and localization. . J Gen Virol 92:, 2502–2511. [CrossRef] [PubMed]
    [Google Scholar]
  26. Schmitz U. , Tan S. L. . ( 2008; ). NS5A–from obscurity to new target for HCV therapy. . Recent Pat Antiinfect Drug Discov 3:, 77–92. [CrossRef] [PubMed]
    [Google Scholar]
  27. Suk-Fong Lok A. . ( 2013; ). HCV NS5A inhibitors in development. . Clin Liver Dis 17:, 111–121. [CrossRef] [PubMed]
    [Google Scholar]
  28. Targett-Adams P. , Graham E. J. , Middleton J. , Palmer A. , Shaw S. M. , Lavender H. , Brain P. , Tran T. D. , Jones L. H. . & other authors ( 2011; ). Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action. . J Virol 85:, 6353–6368. [CrossRef] [PubMed]
    [Google Scholar]
  29. 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. [CrossRef] [PubMed]
    [Google Scholar]
  30. Tellinghuisen T. L. , Foss K. L. , Treadaway J. C. . ( 2008; ). Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. . PLoS Pathog 4:, e1000032.[CrossRef]
    [Google Scholar]
  31. van den Hoff M. J. , Moorman A. F. , Lamers W. H. . ( 1992; ). Electroporation in ‘intracellular’ buffer increases cell survival. . Nucleic Acids Res 20:, 2902. [CrossRef] [PubMed]
    [Google Scholar]
  32. WHO ( 2011; ). Hepatitis C. Fact sheet no. 164. . World Health Organization;, Geneva, Switzerland:.
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.054569-0
Loading
/content/journal/jgv/10.1099/vir.0.054569-0
Loading

Data & Media loading...

Supplementary material 

PDF

Most Cited This Month

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