1887

Abstract

Hepatitis C virus (HCV) genotype 3 is widely distributed, and genotype 3-infected patients achieve a lower cure rate in direct-acting antiviral (DAA) therapy and are associated with a higher risk of hepatic steatosis than patients with other genotypes. Thus, the study of the virology and pathogenesis of genotype 3 HCV is increasingly relevant. Here, we developed a full-length infectious clone and a subgenomic replicon for the genotype 3a isolate, CH3a. From an infected serum, we constructed a full-length CH3a clone, however, it was nonviable in Huh7.5.1 cells. Next, we systematically adapted several intergenotypic recombinants containing Core-NS2 and 5′UTR-NS5A from CH3a, and other sequences from a replication-competent genotype 2 a clone JFH1. Adaptive mutations were identified, of which several combinations facilitated the replication of CH3a-JFH1 recombinants; however, they failed to adapt to the full-length CH3a and the recombinants containing CH3a NS5B. Thus, we attempted to separately adapt CH3a NS5B-3′UTR by constructing an intragenotypic recombinant using 5′UTR-NS5A from an infectious genotype 3a clone, DBN3acc, from which L3004P/M in NS5B and a deletion of 11 nucleotides (Δ11nt) downstream of the polyU/UC tract of the 3′UTR were identified and demonstrated to efficiently improve virus production. Finally, we combined functional 5′UTR-NS5A and NS5B-3′UTR sequences that carried the selected mutations to generate full-length CH3a with 26 or 27 substitutions (CH3acc), and both revealed efficient replication and virus spread in transfected and infected cells, releasing HCV of 10 f.f.u. ml. CH3acc was inhibited by DAAs targeting NS3/4A, NS5A and NS5B in a dose-dependent manner. The selected mutations permitted the development of subgenomic replicon CH3a-SGRep, by which L3004P, L3004M and Δ11nt were proven, together with a single-cycle virus production assay, to facilitate virus assembly, release, and RNA replication. CH3acc clones and CH3a-SGRep replicon provide new tools for the study of HCV genotype 3.

Funding
This study was supported by the:
  • Entrepreneurial Talent Team Award of Guangdong Province (Award 2016ZT06S252)
    • Principle Award Recipient: Yi-PingLi
  • National Basic Research Program of China (973 Program) (Award 2015CB554301)
    • Principle Award Recipient: Yi-PingLi
  • National Natural Science Foundation of China (Award 81901557)
    • Principle Award Recipient: PingYin
  • National Natural Science Foundation of China (Award 81971938)
    • Principle Award Recipient: Yi-PingLi
  • The Innovation Research Team for Basic and Clinical Studies on Chronic Liver Diseases of 2018 High-Level Health Teams of Zhuhai
    • Principle Award Recipient: Yi-PingLi
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/content/journal/jgv/10.1099/jgv.0.001704
2021-12-24
2024-03-28
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References

  1. Pawlotsky JM, Feld JJ, Zeuzem S, Hoofnagle JH. From non-A, non-B hepatitis to hepatitis C virus cure. J Hepatol 2015; 62:S87–99 [View Article] [PubMed]
    [Google Scholar]
  2. Borgia SM, Hedskog C, Parhy B, Hyland RH, Stamm LM et al. Identification of a novel hepatitis C virus genotype from Punjab, India: Expanding classification of hepatitis C virus into 8 genotypes. J Infect Dis 2018; 218:1722–1729 [View Article] [PubMed]
    [Google Scholar]
  3. Webster DP, Klenerman P, Dusheiko GM. Hepatitis C. Lancet 2015; 385:1124–1135 [View Article] [PubMed]
    [Google Scholar]
  4. Roudot-Thoraval F. Epidemiology of hepatitis C virus infection. Clin Res Hepatol Gastroenterol 2021; 45:101596 [View Article] [PubMed]
    [Google Scholar]
  5. 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 [View Article] [PubMed]
    [Google Scholar]
  6. Thrift AP, El-Serag HB, Kanwal F. Global epidemiology and burden of HCV infection and HCV-related disease. Nat Rev Gastroenterol Hepatol 2017; 14:122–132 [View Article] [PubMed]
    [Google Scholar]
  7. Kanwal F, Kramer JR, Ilyas J, Duan Z, El-Serag HB. HCV genotype 3 is associated with an increased risk of cirrhosis and hepatocellular cancer in a national sample of U.S. Veterans with HCV. Hepatology 2014; 60:98–105 [View Article] [PubMed]
    [Google Scholar]
  8. 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 [View Article] [PubMed]
    [Google Scholar]
  9. Wu N, Rao HY, Yang WB, Gao ZL, Yang RF et al. Impact of hepatitis C virus genotype 3 on liver disease progression in a Chinese national cohort. Chin Med J (Engl) 2020; 133:253–261 [View Article] [PubMed]
    [Google Scholar]
  10. Stanciu C, Muzica CM, Girleanu I, Cojocariu C, Sfarti C et al. An update on direct antiviral agents for the treatment of hepatitis C. Expert Opin Pharmacother 2021; 22:1729–1741 [View Article] [PubMed]
    [Google Scholar]
  11. Fathi H, Clark A, Hill NR, Dusheiko G. Effectiveness of current and future regimens for treating genotype 3 hepatitis C virus infection: a large-scale systematic review. BMC Infect Dis 2017; 17:722. [View Article] [PubMed]
    [Google Scholar]
  12. Zhuang L, Li J, Zhang Y, Ji S, Li Y et al. Real-world effectiveness of direct-acting antiviral regimens against Hepatitis C Virus (HCV) genotype 3 infection: a systematic review and meta-analysis. Ann Hepatol 2021; 23:100268 [View Article] [PubMed]
    [Google Scholar]
  13. European Association for the Study of the Liver. Electronic address: [email protected] Clinical Practice Guidelines Panel: Chair EASL Governing Board representative Panel members EASL recommendations on treatment of hepatitis C: Final update of the series☆. J Hepatol 2020; 73:1170–1218 [View Article] [PubMed]
    [Google Scholar]
  14. Huang J-F, Huang C-F, Yeh M-L, Dai C-Y, Yu M-L et al. Updates in the management and treatment of HCV genotype 3, what are the remaining challenges?. Expert Rev Anti Infect Ther 2018; 16:907–912 [View Article] [PubMed]
    [Google Scholar]
  15. Spearman CW, Dusheiko GM, Hellard M, Sonderup M. Hepatitis C. Lancet 2019; 394:1451–1466 [View Article] [PubMed]
    [Google Scholar]
  16. Ju W, Yang S, Feng S, Wang Q, Liu S et al. Hepatitis C virus genotype and subtype distribution in Chinese chronic hepatitis C patients: nationwide spread of HCV genotypes 3 and 6. Virol J 2015; 12:109. [View Article] [PubMed]
    [Google Scholar]
  17. Zhang Y, Chen LM, He M. Hepatitis C Virus in mainland China with an emphasis on genotype and subtype distribution. Virol J 2017; 14:41. [View Article] [PubMed]
    [Google Scholar]
  18. 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 [View Article] [PubMed]
    [Google Scholar]
  19. Lu L, Wu T, Xiong L, Li C, Nguyen MH et al. Analysis of HCV-6 isolates among Asian-born immigrants in North America reveals their high genetic diversity and a new subtype. Virology 2016; 492:25–31 [View Article] [PubMed]
    [Google Scholar]
  20. Li Y-P, 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 [View Article]
    [Google Scholar]
  21. 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 [View Article]
    [Google Scholar]
  22. Gottwein JM, Jensen TB, Mathiesen CK, Meuleman P, Serre SBN et al. Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A. J Virol 2011; 85:8913–8928 [View Article] [PubMed]
    [Google Scholar]
  23. Zhao F, Zhao T, Deng L, Lv D, Zhang X et al. Visualizing the essential role of complete virion assembly machinery in efficient hepatitis C Virus cell-to-cell transmission by a viral infection-activated split-intein-mediated reporter system. J Virol 2017; 91:e01720-16. [View Article] [PubMed]
    [Google Scholar]
  24. Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T et al. Robust hepatitis C virus infection in vitro . Proc Natl Acad Sci U S A 2005; 102:9294–9299 [View Article] [PubMed]
    [Google Scholar]
  25. Li Y-P, Ramirez S, Jensen SB, Purcell RH, Gottwein JM et al. Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system. Proc Natl Acad Sci U S A 2012; 109:19757–19762 [View Article] [PubMed]
    [Google Scholar]
  26. Li Y-P, Ramirez S, Gottwein JM, Scheel TKH, Mikkelsen L et al. Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains. Proc Natl Acad Sci U S A 2012; 109:E1101–10 [View Article] [PubMed]
    [Google Scholar]
  27. Yu M, Peng B, Chan K, Gong R, Yang H et al. Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture. Antimicrob Agents Chemother 2014; 58:2638–2646 [View Article] [PubMed]
    [Google Scholar]
  28. Chen M, Zheng F, Yuan G, Duan X, Rong L et al. Development of an infectious cell culture system for hepatitis C virus genotype 6a clinical isolate using a novel strategy and its sensitivity to direct-acting antivirals. Front Microbiol 2018; 9:2950 [View Article] [PubMed]
    [Google Scholar]
  29. Lanford RE, Sureau C, Jacob JR, White R, Fuerst TR. Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand-specific RT/PCR. Virology 1994; 202:606–614 [View Article] [PubMed]
    [Google Scholar]
  30. Russell RS, Meunier JC, Takikawa S, Faulk K, Engle RE et al. Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus. Proc Natl Acad Sci U S A 2008; 105:4370–4375 [View Article] [PubMed]
    [Google Scholar]
  31. Li Y-P, Ramirez S, Mikkelsen L, Bukh J. Efficient infectious cell culture systems of the hepatitis c virus (hcv) prototype strains hcv-1 and h77. J Virol 2015; 89:811–823 [View Article] [PubMed]
    [Google Scholar]
  32. Ramirez S, Fernandez-Antunez C, Mikkelsen LS, Pedersen J, Li Y-P et al. Cell Culture Studies of the Efficacy and Barrier to Resistance of Sofosbuvir-Velpatasvir and Glecaprevir-Pibrentasvir against Hepatitis C Virus Genotypes 2a, 2b, and 2c. Antimicrob Agents Chemother 2020; 64:e01888-19. [View Article] [PubMed]
    [Google Scholar]
  33. Ramirez S, Li Y-P, Jensen SB, Pedersen J, Gottwein JM et al. Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors. Hepatology 2014; 59:395–407 [View Article] [PubMed]
    [Google Scholar]
  34. Kim S, Date T, Yokokawa H, Kono T, Aizaki H et al. Development of hepatitis C virus genotype 3a cell culture system. Hepatology 2014; 60:1838–1850 [View Article] [PubMed]
    [Google Scholar]
  35. Li Y-P, Gottwein JM, Scheel TK, Jensen TB, Bukh J. MicroRNA-122 antagonism against hepatitis C virus genotypes 1-6 and reduced efficacy by host RNA insertion or mutations in the HCV 5’ UTR. Proc Natl Acad Sci U S A 2011; 108:4991–4996 [View Article] [PubMed]
    [Google Scholar]
  36. Friebe P, Boudet J, Simorre JP, Bartenschlager R. Kissing-loop interaction in the 3’ end of the hepatitis C virus genome essential for RNA replication. J Virol 2005; 79:380–392 [View Article] [PubMed]
    [Google Scholar]
  37. Niepmann M, Shalamova LA, Gerresheim GK, Rossbach O. Signals Involved in Regulation of Hepatitis C Virus RNA Genome Translation and Replication. Front Microbiol 2018; 9:395. [View Article] [PubMed]
    [Google Scholar]
  38. You S, Stump DD, Branch AD, Rice CM. A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication. J Virol 2004; 78:1352–1366 [View Article] [PubMed]
    [Google Scholar]
  39. You S, Rice CM. 3’ RNA elements in hepatitis C virus replication: kissing partners and long poly(U). J Virol 2008; 82:184–195 [View Article] [PubMed]
    [Google Scholar]
  40. Sagan SM, Chahal J, Sarnow P. cis-Acting RNA elements in the hepatitis C virus RNA genome. Virus Res 2015; 206:90–98 [View Article] [PubMed]
    [Google Scholar]
  41. Diviney S, Tuplin A, Struthers M, Armstrong V, Elliott RM et al. A hepatitis C virus cis-acting replication element forms a long-range RNA-RNA interaction with upstream RNA sequences in NS5B. J Virol 2008; 82:9008–9022 [View Article] [PubMed]
    [Google Scholar]
  42. Murayama A, Weng L, Date T, Akazawa D, Tian X et al. RNA polymerase activity and specific RNA structure are required for efficient HCV replication in cultured cells. PLoS Pathog 2010; 6:e1000885 [View Article] [PubMed]
    [Google Scholar]
  43. 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 [View Article]
    [Google Scholar]
  44. Scheel TKH, Gottwein JM, Carlsen THR, Li Y-P, Jensen TB et al. Efficient culture adaptation of hepatitis C virus recombinants with genotype-specific core-NS2 by using previously identified mutations. J Virol 2011; 85:2891–2906 [View Article] [PubMed]
    [Google Scholar]
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