1887

Abstract

Daclatasvir (DCV) is a highly potent direct-acting antiviral that targets the non-structural protein 5A (NS5A) of hepatitis C virus (HCV) and has been used with great clinical success. Previous studies have demonstrated its impact on viral replication complex assembly. However, the precise mechanisms by which DCV impairs the replication complex assembly remains elusive. In this study, by using HCV subgenomic replicons and a viral replicase assembly surrogate system in which the HCV NS3-5B polyprotein is expressed to mimic the viral replicase assembly, we assessed the impact of DCV on the aggregation and tertiary structure of NS5A, the protein–protein interactions within the viral replicase and the quaternary structure of the viral replicase. We found that DCV did not affect aggregation and tertiary structure of NS5A. DCV induced a quaternary structural change of the viral replicase, as evidenced by selective increase of NS4B’s sensitivity to proteinase K digestion. Mechanically, DCV impaired the NS4B-involved protein–protein interactions within the viral replicase. These phenotypes were consistent with the phenotypes of several reported NS4B mutants that abolish the viral replicase assembly. The DCV-resistant mutant Y93H was refractory to the DCV-induced reduction of the NS4B-involved protein interactions and the quaternary structural change of the viral replicase. In addition, Y93H reduced NS4B-involved protein–protein interactions within the viral replicase and attenuated viral replication. We propose that DCV may induce a positional change of NS5A, which allosterically affects protein interactions within the replicase components and disrupts replicase assembly.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001180
2018-12-05
2024-12-05
Loading full text...

Full text loading...

/deliver/fulltext/jgv/100/1/69.html?itemId=/content/journal/jgv/10.1099/jgv.0.001180&mimeType=html&fmt=ahah

References

  1. Lavanchy D. Evolving epidemiology of hepatitis C virus. Clin Microbiol Infect 2011; 17:107–115 [View Article][PubMed]
    [Google Scholar]
  2. Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat Rev Microbiol 2007; 5:453–463 [View Article][PubMed]
    [Google Scholar]
  3. Gu M, Rice CM. Structures of hepatitis C virus nonstructural proteins required for replicase assembly and function. Curr Opin Virol 2013; 3:129–136 [View Article][PubMed]
    [Google Scholar]
  4. Romero-Brey I, Merz A, Chiramel A, Lee JY, Chlanda P et al. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS Pathog 2012; 8:e1003056 [View Article][PubMed]
    [Google Scholar]
  5. Kohli A, Shaffer A, Sherman A, Kottilil S. Treatment of hepatitis C: a systematic review. JAMA 2014; 312:631–640 [View Article][PubMed]
    [Google Scholar]
  6. Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN et al. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 2010; 465:96–100 [View Article][PubMed]
    [Google Scholar]
  7. Elazar M, Cheong KH, Liu P, Greenberg HB, Rice CM et al. Amphipathic helix-dependent localization of NS5A mediates hepatitis C virus RNA replication. J Virol 2003; 77:6055–6061 [View Article][PubMed]
    [Google Scholar]
  8. Love RA, Brodsky O, Hickey MJ, Wells PA, Cronin CN. Crystal structure of a novel dimeric form of NS5A domain I protein from hepatitis C virus. J Virol 2009; 83:4395–4403 [View Article][PubMed]
    [Google Scholar]
  9. Tellinghuisen TL, Marcotrigiano J, Rice CM. Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 2005; 435:374–379 [View Article][PubMed]
    [Google Scholar]
  10. Dujardin M, Madan V, Montserret R, Ahuja P, Huvent I et al. A Proline-tryptophan turn in the intrinsically disordered domain 2 of ns5a protein is essential for hepatitis c virus rna replication. J Biol Chem 2015; 290:19104–19120 [View Article][PubMed]
    [Google Scholar]
  11. Ross-Thriepland D, Amako Y, Harris M. The C terminus of NS5A domain II is a key determinant of hepatitis C virus genome replication, but is not required for virion assembly and release. J Gen Virol 2013; 94:1009–1018 [View Article][PubMed]
    [Google Scholar]
  12. Appel N, Zayas M, Miller S, Krijnse-Locker J, Schaller T et al. Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathog 2008; 4:e1000035 [View Article][PubMed]
    [Google Scholar]
  13. Verdegem D, Badillo A, Wieruszeski JM, Landrieu I, Leroy A et al. Domain 3 of NS5A protein from the hepatitis C virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin A. J Biol Chem 2011; 286:20441–20454 [View Article][PubMed]
    [Google Scholar]
  14. Ross-Thriepland D, Harris M. Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!. J Gen Virol 2015; 96:727–738 [View Article][PubMed]
    [Google Scholar]
  15. Lee C, Ma H, Hang JQ, Leveque V, Sklan EH et al. The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein. Virology 2011; 414:10–18 [View Article][PubMed]
    [Google Scholar]
  16. Qiu D, Lemm JA, O'Boyle DR, Sun JH, Nower PT et al. The effects of NS5A inhibitors on NS5A phosphorylation, polyprotein processing and localization. J Gen Virol 2011; 92:2502–2511 [View Article][PubMed]
    [Google Scholar]
  17. Chukkapalli V, Berger KL, Kelly SM, Thomas M, Deiters A et al. Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides. Virology 2015; 476:168–179 [View Article][PubMed]
    [Google Scholar]
  18. Targett-Adams P, Graham EJ, Middleton J, Palmer A, Shaw SM et al. Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action. J Virol 2011; 85:6353–6368 [View Article][PubMed]
    [Google Scholar]
  19. Berger C, Romero-Brey I, Radujkovic D, Terreux R, Zayas M et al. Daclatasvir-like inhibitors of NS5A block early biogenesis of hepatitis C virus-induced membranous replication factories, independent of RNA replication. Gastroenterology 2014; 147:1094–1105 [View Article][PubMed]
    [Google Scholar]
  20. Boson B, Denolly S, Turlure F, Chamot C, Dreux M et al. Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites. Gastroenterology 2017; 152:895–907 [View Article][PubMed]
    [Google Scholar]
  21. den Boon JA, Ahlquist P. Organelle-like membrane compartmentalization of positive-strand RNA virus replication factories. Annu Rev Microbiol 2010; 64:241–256 [View Article][PubMed]
    [Google Scholar]
  22. Yi Z, Fang C, Zou J, Xu J, Song W et al. Affinity purification of the Hepatitis C Virus Replicase identifies valosin-containing protein, a member of the ATPases associated with diverse cellular activities family, as an active virus replication modulator. J Virol 2016; 90:9953–9966 [View Article][PubMed]
    [Google Scholar]
  23. Yi Z, Pan T, Wu X, Song W, Wang S et al. Hepatitis C virus co-opts Ras-GTPase-activating protein-binding protein 1 for its genome replication. J Virol 2011; 85:6996–7004 [View Article][PubMed]
    [Google Scholar]
  24. Kato T, Date T, Miyamoto M, Zhao Z, Mizokami M et al. Nonhepatic cell lines HeLa and 293 support efficient replication of the hepatitis C virus genotype 2a subgenomic replicon. J Virol 2005; 79:592–596 [View Article][PubMed]
    [Google Scholar]
  25. Sun JH, O'Boyle DR, Fridell RA, Langley DR, Wang C et al. Resensitizing daclatasvir-resistant hepatitis C variants by allosteric modulation of NS5A. Nature 2015; 527:245–248 [View Article][PubMed]
    [Google Scholar]
  26. Yi Z, Yuan Z. Aggregation of a hepatitis C virus replicase module induced by ablation of p97/VCP. J Gen Virol 2017; 98:1667–1678 [View Article][PubMed]
    [Google Scholar]
  27. Magnaghi P, D'Alessio R, Valsasina B, Avanzi N, Rizzi S et al. Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death. Nat Chem Biol 2013; 9:548–556 [View Article][PubMed]
    [Google Scholar]
  28. Alberti S, Halfmann R, Lindquist S. Biochemical, cell biological, and genetic assays to analyze amyloid and prion aggregation in yeast. Methods Enzymol 2010; 470:709–734 [View Article][PubMed]
    [Google Scholar]
  29. Jiang F, Zhou K, Ma L, Gressel S, Doudna JA. Structural biology. A Cas9-guide RNA complex preorganized for target DNA recognition. Science 2015; 348:1477–1481 [View Article][PubMed]
    [Google Scholar]
  30. Feng Y, de Franceschi G, Kahraman A, Soste M, Melnik A et al. Global analysis of protein structural changes in complex proteomes. Nat Biotechnol 2014; 32:1036–1044 [View Article][PubMed]
    [Google Scholar]
  31. Bozzacco L, Yi Z, Andreo U, Conklin CR, Li MM et al. Chaperone-assisted protein folding is critical for yellow fever Virus NS3/4A cleavage and replication. J Virol 2016; 90:3212–3228 [View Article][PubMed]
    [Google Scholar]
  32. Miyanari Y, Hijikata M, Yamaji M, Hosaka M, Takahashi H et al. Hepatitis C virus non-structural proteins in the probable membranous compartment function in viral genome replication. J Biol Chem 2003; 278:50301–50308 [View Article][PubMed]
    [Google Scholar]
  33. Romero-Brey I, Berger C, Kallis S, Kolovou A, Paul D et al. NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication. MBio 2015; 6:e00759 [View Article][PubMed]
    [Google Scholar]
  34. Paul D, Hoppe S, Saher G, Krijnse-Locker J, Bartenschlager R. Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment. J Virol 2013; 87:10612–10627 [View Article][PubMed]
    [Google Scholar]
  35. Paul D, Madan V, Bartenschlager R. Hepatitis C virus RNA replication and assembly: living on the fat of the land. Cell Host Microbe 2014; 16:569–579 [View Article][PubMed]
    [Google Scholar]
  36. Lundin M, Lindström H, Grönwall C, Persson MA. Dual topology of the processed hepatitis C virus protein NS4B is influenced by the NS5A protein. J Gen Virol 2006; 87:3263–3272 [View Article][PubMed]
    [Google Scholar]
  37. Lundin M, Monné M, Widell A, von Heijne G, Persson MA. Topology of the membrane-associated hepatitis C virus protein NS4B. J Virol 2003; 77:5428–5438 [View Article][PubMed]
    [Google Scholar]
  38. Paul D, Madan V, Ramirez O, Bencun M, Stoeck IK et al. Glycine zipper motifs in Hepatitis C Virus Nonstructural Protein 4B Are required for the establishment of viral replication organelles. J Virol 2018; 92: [View Article][PubMed]
    [Google Scholar]
  39. Paul D, Romero-Brey I, Gouttenoire J, Stoitsova S, Krijnse-Locker J et al. NS4B self-interaction through conserved C-terminal elements is required for the establishment of functional hepatitis C virus replication complexes. J Virol 2011; 85:6963–6976 [View Article][PubMed]
    [Google Scholar]
  40. Fiskum G, Craig SW, Decker GL, Lehninger AL. The cytoskeleton of digitonin-treated rat hepatocytes. Proc Natl Acad Sci USA 1980; 77:3430–3434 [View Article][PubMed]
    [Google Scholar]
  41. Du X, Pan T, Xu J, Zhang Y, Song W et al. Hepatitis C virus replicative double-stranded RNA is a potent interferon inducer that triggers interferon production through MDA5. J Gen Virol 2016; 97:2868–2882 [View Article][PubMed]
    [Google Scholar]
  42. Lambert SM, Langley DR, Garnett JA, Angell R, Hedgethorne K et al. The crystal structure of NS5A domain 1 from genotype 1a reveals new clues to the mechanism of action for dimeric HCV inhibitors. Protein Sci 2014; 23:723–734 [View Article][PubMed]
    [Google Scholar]
  43. O'Boyle Ii DR, Sun JH, Nower PT, Lemm JA, Fridell RA et al. Characterizations of HCV NS5A replication complex inhibitors. Virology 2013; 444:343–354 [View Article][PubMed]
    [Google Scholar]
  44. Biswas A, Treadaway J, Tellinghuisen TL. Interaction between Nonstructural Proteins NS4B and NS5A Is essential for proper NS5A localization and Hepatitis C Virus RNA Replication. J Virol 2016; 90:7205–7218 [View Article][PubMed]
    [Google Scholar]
  45. Fridell RA, Qiu D, Valera L, Wang C, Rose RE et al. Distinct functions of NS5A in hepatitis C virus RNA replication uncovered by studies with the NS5A inhibitor BMS-790052. J Virol 2011; 85:7312–7320 [View Article][PubMed]
    [Google Scholar]
  46. Lindenbach BD. Virion assembly and release. Curr Top Microbiol Immunol 2013; 369:199–218 [View Article][PubMed]
    [Google Scholar]
/content/journal/jgv/10.1099/jgv.0.001180
Loading
/content/journal/jgv/10.1099/jgv.0.001180
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