1887

Abstract

Hepatitis C virus (HCV) genotype (GT) 3 is the second most prevalent of the seven HCV genotypes and exhibits the greatest resistance to the highly potent, direct-acting antivirals (DAAs) that are currently in use. Previously a stable cell line harbouring the S52 GT3 sub-genomic replicon (SGR) was established, but this SGR was unable to robustly replicate transiently. As transient SGRs are a critical tool in the development of DAAs, and in the study of viral resistance, we sought to establish a transient SGR system based on S52. Next-generation sequencing was used to identify putative culture-adaptive substitutions that had arisen during long-term selection of the S52 SGR. A subset of these substitutions was built back into the S52 SGR in the context of a CpG/UpA-low luciferase reporter, with a single point mutation in NS4A conferring the greatest replication capability upon S52. Modification of the innate immune-sensing pathways of Huh7.5 hepatoma cells by expression of the parainfluenza virus type 5 V protein and SEC14L2 resulted in a further enhancement of S52 replication. Furthermore, this transiently replicating SGR showed genotype-specific differences in sensitivity to two clinically relevant NS5A DAAs. In conclusion, we report that a single substitution in NS4A, coupled with host cell modifications, enabled robust levels of transient replication by the GT3 S52 SGR. This system will have beneficial uses in both basic research into the unique aspects of GT3 biology and drug discovery.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000932
2017-10-01
2020-12-03
Loading full text...

Full text loading...

/deliver/fulltext/jgv/98/10/2495.html?itemId=/content/journal/jgv/10.1099/jgv.0.000932&mimeType=html&fmt=ahah

References

  1. Messina JP, Humphreys I, Flaxman A, Brown A, Cooke GS et al. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology 2015;61:77–87 [CrossRef][PubMed]
    [Google Scholar]
  2. Perz JF, Armstrong GL, Farrington LA, Hutin YJ, Bell BP. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J Hepatol 2006;45:529–538 [CrossRef][PubMed]
    [Google Scholar]
  3. Smith DB, Bukh J, Kuiken C, Muerhoff AS, Rice CM et al. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource. Hepatology 2014;59:318–327 [CrossRef][PubMed]
    [Google Scholar]
  4. FakhriRavari A, Malakouti M, Brady R. Interferon-free treatments for chronic hepatitis C genotype 1 infection. J Clin Transl Hepatol 2016;4:97–112 [CrossRef][PubMed]
    [Google Scholar]
  5. Jacobson IM, Gordon SC, Kowdley KV, Yoshida EM, Rodriguez-Torres M et al. Sofosbuvir for hepatitis C genotype 2 or 3 in patients without treatment options. N Engl J Med 2013;368:1867–1877 [CrossRef][PubMed]
    [Google Scholar]
  6. Sulkowski MS, Gardiner DF, Rodriguez-Torres M, Reddy KR, Hassanein T et al. Daclatasvir plus sofosbuvir for previously treated or untreated chronic HCV infection. N Engl J Med 2014;370:211–221 [CrossRef][PubMed]
    [Google Scholar]
  7. Sulkowski MS, Jacobson IM, Nelson DR. Daclatasvir plus sofosbuvir for HCV infection. N Engl J Med 2014;370:1560–1561 [CrossRef][PubMed]
    [Google Scholar]
  8. Dore GJ, Lawitz E, Hézode C, Shafran SD, Ramji A et al. Daclatasvir plus peginterferon and ribavirin is noninferior to peginterferon and ribavirin alone, and reduces the duration of treatment for HCV genotype 2 or 3 infection. Gastroenterology 2015;148:355–366 [CrossRef][PubMed]
    [Google Scholar]
  9. Molina JM, Orkin C, Iser DM, Zamora FX, Nelson M et al. Sofosbuvir plus ribavirin for treatment of hepatitis C virus in patients co-infected with HIV (PHOTON-2): a multicentre, open-label, non-randomised, phase 3 study. Lancet 2015;385:1098–1106 [CrossRef][PubMed]
    [Google Scholar]
  10. Wyles DL, Ruane PJ, Sulkowski MS, Dieterich D, Luetkemeyer A et al. Daclatasvir plus sofosbuvir for HCV in patients coinfected with HIV-1. N Engl J Med 2015;373:714–725 [CrossRef][PubMed]
    [Google Scholar]
  11. Foster GR, Irving WL, Cheung MC, Walker AJ, Hudson BE et al. Impact of direct acting antiviral therapy in patients with chronic hepatitis C and decompensated cirrhosis. J Hepatol 2016;64:1224–1231 [CrossRef][PubMed]
    [Google Scholar]
  12. Nkontchou G, Ziol M, Aout M, Lhabadie M, Baazia Y et al. HCV genotype 3 is associated with a higher hepatocellular carcinoma incidence in patients with ongoing viral C cirrhosis. J Viral Hepat 2011;18:e516–22 [CrossRef][PubMed]
    [Google Scholar]
  13. Gimeno-Ballester V, Buti M, San Miguel R, Riveiro M, Esteban R. Interferon-free therapies for patients with chronic hepatitis C genotype 3 infection: a systematic review. J Viral Hepat 2017; [CrossRef][PubMed]
    [Google Scholar]
  14. Lohmann V, Körner F, Koch J, Herian U, Theilmann L et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 1999;285:110–113 [CrossRef][PubMed]
    [Google Scholar]
  15. Kato T, Date T, Miyamoto M, Furusaka A, Tokushige K et al. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 2003;125:1808–1817 [CrossRef][PubMed]
    [Google Scholar]
  16. Saeed M, Scheel TK, Gottwein JM, Marukian S, Dustin LB et al. Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells. Antimicrob Agents Chemother 2012;56:5365–5373 [CrossRef][PubMed]
    [Google Scholar]
  17. Saeed M, Gondeau C, Hmwe S, Yokokawa H, Date T et al. Replication of hepatitis C virus genotype 3a in cultured cells. Gastroenterology 2013;144:56–58 [CrossRef][PubMed]
    [Google Scholar]
  18. Yu M, Corsa AC, Xu S, Peng B, Gong R et al. In vitro efficacy of approved and experimental antivirals against novel genotype 3 hepatitis C virus subgenomic replicons. Antiviral Res 2013;100:439–445 [CrossRef][PubMed]
    [Google Scholar]
  19. Lanford RE, Guerra B, Lee H. Hepatitis C virus genotype 1b chimeric replicon containing genotype 3 NS5A domain. Virology 2006;355:192–202 [CrossRef][PubMed]
    [Google Scholar]
  20. Wang C, Valera L, Jia L, Kirk MJ, Gao M et al. In vitro activity of daclatasvir on hepatitis C virus genotype 3 NS5A. Antimicrob Agents Chemother 2013;57:611–613 [CrossRef][PubMed]
    [Google Scholar]
  21. Kylefjord H, Danielsson A, Sedig S, Belda O, Wiktelius D et al. Transient replication of a hepatitis C virus genotype 1b replicon chimera encoding NS5A-5B from genotype 3a. J Virol Methods 2014;195:156–163 [CrossRef][PubMed]
    [Google Scholar]
  22. Gottwein JM, Scheel TK, Callendret B, Li YP, Eccleston HB et al. Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies. J Virol 2010;84:5277–5293 [CrossRef][PubMed]
    [Google Scholar]
  23. Witteveldt J, Martin-Gans M, Simmonds P. Enhancement of the replication of hepatitis C virus replicons of genotypes 1 to 4 by manipulation of CpG and UpA dinucleotide frequencies and use of cell lines expressing SECL14L2 for antiviral resistance testing. Antimicrob Agents Chemother 2016;60:2981–2992 [CrossRef][PubMed]
    [Google Scholar]
  24. Lin Y, Sun M, Fuentes SM, Keim CD, Rothermel T et al. Inhibition of interleukin-6 expression by the V protein of parainfluenza virus 5. Virology 2007;368:262–272 [CrossRef][PubMed]
    [Google Scholar]
  25. Lu LL, Puri M, Horvath CM, Sen GC. Select paramyxoviral V proteins inhibit IRF3 activation by acting as alternative substrates for inhibitor of κB kinase ε (IKKe)/TBK1. J Biol Chem 2008;283:14269–14276 [CrossRef][PubMed]
    [Google Scholar]
  26. Saeed M, Andreo U, Chung HY, Espiritu C, Branch AD et al. SEC14L2 enables pan-genotype HCV replication in cell culture. Nature 2015;524:471–475 [CrossRef][PubMed]
    [Google Scholar]
  27. Hernandez D, Zhou N, Ueland J, Monikowski A, Mcphee F. Natural prevalence of NS5A polymorphisms in subjects infected with hepatitis C virus genotype 3 and their effects on the antiviral activity of NS5A inhibitors. J Clin Virol 2013;57:13–18 [CrossRef][PubMed]
    [Google Scholar]
  28. 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 [CrossRef][PubMed]
    [Google Scholar]
  29. Li YP, Ramirez S, Humes D, Jensen SB, Gottwein JM et al. Differential sensitivity of 5'UTR-NS5A recombinants of hepatitis C virus genotypes 1-6 to protease and NS5A inhibitors. Gastroenterology 2014;146:812–821 [CrossRef][PubMed]
    [Google Scholar]
  30. 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 [CrossRef][PubMed]
    [Google Scholar]
  31. Tulloch F, Atkinson NJ, Evans DJ, Ryan MD, Simmonds P. RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. Elife 2014;3:e04531 [CrossRef][PubMed]
    [Google Scholar]
  32. Atkinson NJ, Witteveldt J, Evans DJ, Simmonds P. The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication. Nucleic Acids Res 2014;42:4527–4545 [CrossRef][PubMed]
    [Google Scholar]
  33. He B, Paterson RG, Stock N, Durbin JE, Durbin RK et al. Recovery of paramyxovirus simian virus 5 with a V protein lacking the conserved cysteine-rich domain: the multifunctional V protein blocks both interferon-β induction and interferon signaling. Virology 2002;303:15–32 [CrossRef][PubMed]
    [Google Scholar]
  34. Poole E, He B, Lamb RA, Randall RE, Goodbourn S. The V proteins of simian virus 5 and other paramyxoviruses inhibit induction of interferon-β. Virology 2002;303:33–46 [CrossRef][PubMed]
    [Google Scholar]
  35. Andrus L, Marukian S, Jones CT, Catanese MT, Sheahan TP et al. Expression of paramyxovirus V proteins promotes replication and spread of hepatitis C virus in cultures of primary human fetal liver cells. Hepatology 2011;54:1901–1912 [CrossRef][PubMed]
    [Google Scholar]
  36. 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 [CrossRef][PubMed]
    [Google Scholar]
  37. Thomson E, Ip CL, Badhan A, Christiansen MT, Adamson W et al. Comparison of next-generation sequencing technologies for comprehensive assessment of full-length hepatitis C viral genomes. J Clin Microbiol 2016;54:2470–2484 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000932
Loading
/content/journal/jgv/10.1099/jgv.0.000932
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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