1887

Abstract

Hijacking host membranes to assemble a membrane-associated viral replicase is a hallmark of almost all positive-strand RNA viruses. However, how the virus co-opts host factors to facilitate this energy-unfavourable process is incompletely understood. In a previous study, using hepatitis C virus (HCV) as a model and employing affinity purification of the viral replicase, we identified a valosin-containing protein (p97/VCP), a member of the ATPases associated with diverse cellular activities (AAA+ ATPase family) as a viral replicase-associated host factor. It is required for viral replication, depending on its ATPase activity. In this study, we used VCP pharmacological inhibitors and short hairpin (sh) RNA-mediated knockdown to ablate VCP function and then dissected the roles of VCP in viral replicase assembly in an HCV subgenomic replicon system and a viral replicase assembly surrogate system. Ablation of VCP specifically resulted in the pronounced formation of an SDS-resistant aggregation of HCV NS5A and the reduction of hyperphosphorylation of NS5A. The NS5A dimerization domain was indispensable for aggregation and the NS5A disordered regions also contributed to a lesser extent. The reduction of the hyperphosphorylation of NS5A coincided with the aggregation of NS5A. We propose that HCV may co-opt VCP to disaggregate an aggregation-prone replicase module to facilitate its replicase assembly.

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2017-07-01
2024-12-03
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References

  1. 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]
  2. Binder M, Sulaimanov N, Clausznitzer D, Schulze M, Hüber CM et al. Replication vesicles are load- and choke-points in the hepatitis C virus lifecycle. PLoS Pathog 2013; 9:e1003561 [View Article][PubMed]
    [Google Scholar]
  3. Belema M, Nguyen VN, Bachand C, Deon DH, Goodrich JT et al. Hepatitis C virus NS5A replication complex inhibitors: the discovery of daclatasvir. J Med Chem 2014; 57:2013–2032 [View Article][PubMed]
    [Google Scholar]
  4. 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]
  5. O'Boyle, 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]
  6. Lavanchy D. Evolving epidemiology of hepatitis C virus. Clin Microbiol Infect 2011; 17:107–115 [View Article][PubMed]
    [Google Scholar]
  7. Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat Rev Microbiol 2007; 5:453–463 [View Article][PubMed]
    [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. Verdegem D, Badillo A, Wieruszeski JM, Landrieu I, Leroy A et al. Domain 3 of NS5A protein from the hepatitis C virus has intrinsic α-helical propensity and is a substrate of cyclophilin A. J Biol Chem 2011; 286:20441–20454 [View Article][PubMed]
    [Google Scholar]
  16. 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]
  17. Tellinghuisen TL, Foss KL, Treadaway J. Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. PLoS Pathog 2008; 4:e1000032 [View Article][PubMed]
    [Google Scholar]
  18. Tellinghuisen TL, Foss KL, Treadaway JC, Rice CM. Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein. J Virol 2008; 82:1073–1083 [View Article][PubMed]
    [Google Scholar]
  19. Nagy PD, Pogany J. The dependence of viral RNA replication on co-opted host factors. Nat Rev Microbiol 2011; 10:137–149 [View Article][PubMed]
    [Google Scholar]
  20. 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]
  21. 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]
  22. Hanson PI, Whiteheart SW. AAA+ proteins: have engine, will work. Nat Rev Mol Cell Biol 2005; 6:519–529 [View Article][PubMed]
    [Google Scholar]
  23. Marukian S, Jones CT, Andrus L, Evans MJ, Ritola KD et al. Cell culture-produced hepatitis C virus does not infect peripheral blood mononuclear cells. Hepatology 2008; 48:1843–1850 [View Article][PubMed]
    [Google Scholar]
  24. 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]
  25. Chou TF, Brown SJ, Minond D, Nordin BE, Li K et al. Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways. Proc Natl Acad Sci USA 2011; 108:4834–4839 [View Article][PubMed]
    [Google Scholar]
  26. Murray CL, Rice CM. Turning hepatitis C into a real virus. Annu Rev Microbiol 2011; 65:307–327 [View Article][PubMed]
    [Google Scholar]
  27. 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]
  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. Hou F, Sun L, Zheng H, Skaug B, Jiang QX et al. MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response. Cell 2011; 146:448–461 [View Article][PubMed]
    [Google Scholar]
  30. 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]
  31. 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]
  32. Tyedmers J, Mogk A, Bukau B. Cellular strategies for controlling protein aggregation. Nat Rev Mol Cell Biol 2010; 11:777–788 [View Article][PubMed]
    [Google Scholar]
  33. 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]
  34. Watts GD, Wymer J, Kovach MJ, Mehta SG, Mumm S et al. Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nat Genet 2004; 36:377–381 [View Article][PubMed]
    [Google Scholar]
  35. Kobayashi T, Manno A, Kakizuka A. Involvement of valosin-containing protein (VCP)/p97 in the formation and clearance of abnormal protein aggregates. Genes Cells 2007; 12:889–901 [View Article][PubMed]
    [Google Scholar]
  36. Eyre NS, Hampton-Smith RJ, Aloia AL, Eddes JS, Simpson KJ et al. Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation. Virology 2016; 491:27–44 [View Article][PubMed]
    [Google Scholar]
  37. Evans MJ, Rice CM, Goff SP. Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication. Proc Natl Acad Sci USA 2004; 101:13038–13043 [View Article][PubMed]
    [Google Scholar]
  38. Blight KJ, Kolykhalov AA, Rice CM. Efficient initiation of HCV RNA replication in cell culture. Science 2000; 290:1972–1974 [View Article][PubMed]
    [Google Scholar]
  39. Koch JO, Bartenschlager R. Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A, and NS4B. J Virol 1999; 73:7138–7146[PubMed]
    [Google Scholar]
  40. Arita M, Wakita T, Shimizu H. Valosin-containing protein (VCP/p97) is required for poliovirus replication and is involved in cellular protein secretion pathway in poliovirus infection. J Virol 2012; 86:5541–5553 [View Article][PubMed]
    [Google Scholar]
  41. Wu KX, Phuektes P, Kumar P, Goh GY, Moreau D et al. Human genome-wide RNAi screen reveals host factors required for enterovirus 71 replication. Nat Commun 2016; 7:13150 [View Article][PubMed]
    [Google Scholar]
  42. Barajas D, Martín IF, Pogany J, Risco C, Nagy PD. Noncanonical role for the host Vps4 AAA+ ATPase ESCRT protein in the formation of Tomato bushy stunt virus replicase. PLoS Pathog 2014; 10:e1004087 [View Article][PubMed]
    [Google Scholar]
  43. Diaz A, Zhang J, Ollwerther A, Wang X, Ahlquist P. Host ESCRT proteins are required for bromovirus RNA replication compartment assembly and function. PLoS Pathog 2015; 11:e1004742 [View Article][PubMed]
    [Google Scholar]
  44. Buchberger A, Schindelin H, Hänzelmann P. Control of p97 function by cofactor binding. FEBS Lett 2015; 589:2578–2589 [View Article][PubMed]
    [Google Scholar]
  45. Lindenbach BD, Evans MJ, Syder AJ, Wölk B, Tellinghuisen TL et al. Complete replication of hepatitis C virus in cell culture. Science 2005; 309:623–626 [View Article][PubMed]
    [Google Scholar]
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