1887

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Volume 104, Issue 9

Mutational analysis reveals a novel role for hepatitis C virus NS5A domain I in cyclophilin-dependent genome replication Open Access

Abstract

The hepatitis C virus (HCV) NS5A protein is comprised of three domains (D1–3). Previously, we observed that two alanine substitutions in D1 (V67A, P145A) abrogated replication of a genotype 2a isolate (JFH-1) sub-genomic replicon (SGR) in Huh7 cells, but this phenotype was partially restored in Huh7.5 cells. Here we demonstrate that five additional residues, surface-exposed and proximal to V67 or P145, exhibited the same phenotype. In contrast, the analogous mutants in a genotype 3a isolate (DBN3a) SGR exhibited different phenotypes in each cell line, consistent with fundamental differences in the functions of genotypes 2 and 3 NS5A. The difference between Huh7 and Huh7.5 cells was reminiscent of the observation that cyclophilin inhibitors are more potent against HCV replication in the former and suggested a role for D1 in cyclophilin dependence. Consistent with this, all JFH-1 and DBN3a mutants exhibited increased sensitivity to cyclosporin A treatment compared to wild-type. Silencing of cyclophilin A (CypA) in Huh7 cells inhibited replication of both JFH-1 and DBN3a. However, in Huh7.5 cells CypA silencing did not inhibit JFH-1 wild-type, but abrogated replication of all the JFH-1 mutants, and both DBN3a wild-type and all mutants. CypB silencing in Huh7 cells had no effect on DBN3a, but abrogated replication of JFH-1. CypB silencing in Huh7.5 cells had no effect on either SGR. Lastly, we confirmed that JFH-1 NS5A D1 interacted with CypA . These data demonstrate both a direct involvement of NS5A D1 in cyclophilin-dependent genome replication and functional differences between genotype 2 and 3 NS5A.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): cyclophilin , cyclosporin , genome replication , genotype , hepatitis C virus , non-structural 5A protein (NS5A) and sub-genomic replicon
Funding
This study was supported by the:
  • Medical Research Council (Award MR/S001026/1)
    • Principle Award Recipient: MarkHarris
  • Wellcome Trust (Award 096670)
    • Principle Award Recipient: MarkHarris
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2023-09-06
2024-05-17
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References

  1. Younossi ZM, Wong G, Anstee QM, Henry L. The global burden of liver disease. Clin Gastroenterol Hepatol 2023; 21:1978–1991 [View Article] [PubMed]
     [Google Scholar]
  2. Chan A, Patel K, Naggie S. Genotype 3 infection: the last stand of hepatitis C virus. Drugs 2017; 77:131–144 [View Article] [PubMed]
     [Google Scholar]
  3. Smith D, Magri A, Bonsall D, Ip CLC, Trebes A et al. Resistance analysis of genotype 3 hepatitis C virus indicates subtypes inherently resistant to nonstructural protein 5A inhibitors. Hepatology 2019; 69:1861–1872 [View Article] [PubMed]
     [Google Scholar]
  4. Simmonds P, Becher P, Bukh J, Gould EA, Meyers G et al. ICTV virus taxonomy profile: Flaviviridae. J Gen Virol 2017; 98:2–3 [View Article] [PubMed]
     [Google Scholar]
  5. Yin C, Goonawardane N, Stewart H, Harris M. A role for domain I of the hepatitis C virus NS5A protein in virus assembly. PLoS Pathog 2018; 14:e1006834 [View Article] [PubMed]
     [Google Scholar]
  6. Chen S, Harris M. NS5A domain I antagonises PKR to facilitate the assembly of infectious hepatitis C virus particles. PLoS Pathog 2023; 19:e1010812 [View Article] [PubMed]
     [Google Scholar]
  7. Foster TL, Gallay P, Stonehouse NJ, Harris M. Cyclophilin A Interacts with domain II of hepatitis C virus NS5A and stimulates RNA binding in an isomerase-dependent manner. J Virol 2011; 85:7460–7464 [View Article]
     [Google Scholar]
  8. Yang F, Robotham JM, Grise H, Frausto S, Madan V et al. A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach. PLoS Pathog 2010; 6:e1001118 [View Article]
     [Google Scholar]
  9. Liu Z, Yang F, Robotham JM, Tang H. Critical role of cyclophilin A and its prolyl-peptidyl isomerase activity in the structure and function of the hepatitis C virus replication complex. J Virol 2009; 83:6554–6565 [View Article]
     [Google Scholar]
  10. 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]
  11. Watashi K, Ishii N, Hijikata M, Inoue D, Murata T et al. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell 2005; 19:111–122 [View Article]
     [Google Scholar]
  12. Harding MW, Handschumacher RE. Cyclophilin, a primary molecular target for cyclosporine. Structural and functional implications. Transplantation 1988; 46:29S–35S [View Article] [PubMed]
     [Google Scholar]
  13. Mamatis JE, Pellizzari-Delano IE, Gallardo-Flores CE, Colpitts CC. Emerging roles of cyclophilin A in regulating viral cloaking. Front Microbiol 2022; 13:828078 [View Article] [PubMed]
     [Google Scholar]
  14. Fischer G, Gallay P, Hopkins S. Cyclophilin inhibitors for the treatment of HCV infection. Curr Opin Investig Drugs 2010; 11:911–918
     [Google Scholar]
  15. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res 1982; 42:3858–3863 [PubMed]
     [Google Scholar]
  16. Sumpter R, Loo YM, Foy E, Li K, Yoneyama M et al. Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I. J Virol 2005; 79:2689–2699 [View Article]
     [Google Scholar]
  17. Blight KJ, McKeating JA, Rice CM. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 2002; 76:13001–13014 [View Article]
     [Google Scholar]
  18. Hughes M, Griffin S, Harris M. Domain III of NS5A contributes to both RNA replication and assembly of hepatitis C virus particles. JGenVirol 2009; 90:1329–1334 [View Article]
     [Google Scholar]
  19. Ramirez S, Mikkelsen LS, Gottwein JM, Bukh J. Robust HCV genotype 3a infectious cell culture system permits identification of escape variants with resistance to sofosbuvir. Gastroenterology 2016; 151:973–985 e2 [View Article] [PubMed]
     [Google Scholar]
  20. Ward JC, Bowyer S, Chen S, Fernandes Campos GR, Ramirez S et al. Insights into the unique characteristics of hepatitis C virus genotype 3 revealed by development of a robust sub-genomic DBN3a replicon. J Gen Virol 2020; 101:1182–1190 [View Article] [PubMed]
     [Google Scholar]
  21. Colpitts CC, Ridewood S, Schneiderman B, Warne J, Tabata K et al. Hepatitis C virus exploits cyclophilin A to evade PKR. Elife 2020; 9:e52237 [View Article] [PubMed]
     [Google Scholar]
  22. Macdonald A, Crowder K, Street A, McCormick C, Saksela K et al. The hepatitis C virus non-structural NS5A protein inhibits activating protein-1 function by perturbing ras-ERK pathway signaling. J Biol Chem 2003; 278:17775–17784 [View Article] [PubMed]
     [Google Scholar]
  23. Targett-Adams P, McLauchlan J. Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon. JGenVirol 2005; 86:3075–3080 [View Article]
     [Google Scholar]
  24. Ramirez S, Bukh J. Current status and future development of infectious cell-culture models for the major genotypes of hepatitis C virus: essential tools in testing of antivirals and emerging vaccine strategies. Antiviral Res 2018; 158:264–287 [View Article] [PubMed]
     [Google Scholar]
  25. Watanabe N, Suzuki T, Date T, Hussan HA, Hmwe SS et al. Establishment of infectious genotype 4 cell culture-derived hepatitis C virus. J Gen Virol 2020; 101:188–197 [View Article] [PubMed]
     [Google Scholar]
  26. Hanoulle X, Badillo A, Wieruszeski J-M, Verdegem D, Landrieu I et al. Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B. J Biol Chem 2009; 284:13589–13601 [View Article] [PubMed]
     [Google Scholar]
  27. Foster TL, Belyaeva T, Stonehouse NJ, Pearson AR, Harris M. All three domains of the hepatitis C virus nonstructural NS5A protein contribute to RNA binding. J Virol 2010; 84:9267–9277 [View Article]
     [Google Scholar]
  28. Shanmugam S, Nichols AK, Saravanabalaji D, Welsch C, Yi M. HCV NS5A dimer interface residues regulate HCV replication by controlling its self-interaction, hyperphosphorylation, subcellular localization and interaction with cyclophilin A. PLoS Pathog 2018; 14:e1007177 [View Article] [PubMed]
     [Google Scholar]
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Mutational analysis reveals a novel role for hepatitis C virus NS5A domain I in cyclophilin-dependent genome replication
J. Gen. Virol. 104, 001886 (2023); https://doi.org/10.1099/jgv.0.001886
/content/journal/jgv/10.1099/jgv.0.001886
/content/journal/jgv/10.1099/jgv.0.001886
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