Hepatitis C virus diversity and evolution in the full open-reading frame during antiviral therapy
Nathan A Cannon, Maureen J Donlin, Xiaofeng Fan, Rajeev Aurora, John E Tavis, Virahep-C Study Group, Nathan A Cannon, Maureen J Donlin, Xiaofeng Fan, Rajeev Aurora, John E Tavis, Virahep-C Study Group
Abstract
Background: Pegylated interferon plus ribavirin therapy for hepatitis C virus (HCV) fails in approximately half of genotype 1 patients. Treatment failure occurs either by nonresponse (minimal declines in viral titer) or relapse (robust initial responses followed by rebounds of viral titers during or after therapy). HCV is highly variable genetically. To determine if viral genetic differences contribute to the difference between response and relapse, we examined the inter-patient genetic diversity and mutation pattern in the full open reading frame HCV genotype 1a consensus sequences.
Methodology/principal findings: Pre- and post-therapy sequences were analyzed for 10 nonresponders and 10 relapsers from the Virahep-C clinical study. Pre-therapy interpatient diversity among the relapsers was higher than in the nonresponders in the viral NS2 and NS3 genes, and post-therapy diversity was higher in the relapsers for most of HCV's ten genes. Pre-therapy diversity among the relapsers was intermediate between that of the non-responders and responders to therapy. The average mutation rate was just 0.9% at the amino acid level and similar numbers of mutations occurred in the nonresponder and relapser sequences, but the mutations in NS2 of relapsers were less conservative than in nonresponders. Finally, the number and distribution of regions under positive selection was similar between the two groups, although the nonresponders had more foci of positive selection in E2.
Conclusions/significance: The HCV sequences were unexpectedly stable during failed antiviral therapy, both nonresponder and relapser sequences were under selective pressure during therapy, and variation in NS2 may have contributed to the difference in response between the nonresponder and relapser groups. These data support a role for viral genetic variability in determining the outcome of anti-HCV therapy, with those sequences that are more distant from an optimal sequence being less able to resist the pressures of interferon-based therapy.
Trial registration: ClinicalTrials.gov NCT00038974.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
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References
- Global burden of disease (GBD) for hepatitis C. J Clin Pharmacol. 2004;44:20–29.
- Alter MJ. Epidemiology of hepatitis C virus infection. World J Gastroenterol. 2007;13:2436–2441.
- Armstrong GL, Wasley A, Simard EP, McQuillan GM, Kuhnert WL, et al. The prevalence of hepatitis C virus infection in the United States, 1999 through 2002. Ann Intern Med. 2006;144:705–714.
- Kim WR, Brown RS, Jr, Terrault NA, El-Serag H. Burden of liver disease in the United States: summary of a workshop. Hepatology. 2002;36:227–242.
- Baker DE. Pegylated interferon plus ribavirin for the treatment of chronic hepatitis C. Reviews in Gastroenterological Disorders. 2003;2:93–109.
- Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. New England Journal of Medicine. 2002;347:975–982.
- McHutchison JG, Gordon SC, Schiff ER, Shiffman ML, Lee WM, et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. New England Journal of Medicine. 1998;339:1485–1492.
- Conjeevaram HS, Fried MW, Jeffers LJ, Terrault NA, Wiley-Lucas TE, et al. Peginterferon and ribavirin treatment in African American and Caucasian American patients with hepatitis C genotype 1. Gastroenterology. 2006;131:470–477.
- Branch AD, Stump DD, Gutierrez JA, Eng F, Walewski JL. The hepatitis C virus alternate reading frame (ARF) and its family of novel products: the alternate reading frame protein/F-protein, the double-frameshift protein, and others. Semin Liver Dis. 2005;25:105–117.
- Varaklioti A, Vassilaki N, Georgopoulou U, Mavromara P. Alternate translation occurs within the core coding region of the hepatitis C viral genome. J Biol Chem. 2002;277:17713–17721.
- Walewski JL, Keller TR, Stump DD, Branch AD. Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame. RNA. 2001;7:710–721.
- Bukh J, Miller R, Purcell R. Genetic heterogeneity of hepatitis c virus: quasispecies and genotypes. Seminars in Liver Disease. 1995;15:41–63.
- Robertson B, Myers G, Howard C, Brettin T, Bukh J, et al. Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization. Archives of Virology. 1998;143:2493–2503.
- Simmonds P, Holmes EC, Cha TA, Chan SW, McOmish F, et al. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. Journal of General Virology. 1993;74:2391–2399.
- Simmonds P. Genetic diversity and evolution of hepatitis C virus–15 years on. J Gen Virol. 2004;85:3173–3188.
- Simmonds P, Bukh J, Combet C, Deleage G, Enomoto N, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology. 2005;42:962–973.
- Gaudieri S, Rauch A, Park LP, Freitas E, Herrmann S, et al. Evidence of viral adaptation to HLA class I-restricted immune pressure in chronic hepatitis C virus infection. J Virol. 2006;80:11094–11104.
- Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet. 2001;358:958–965.
- Strader DB, Wright T, Thomas DL, Seeff LB. Diagnosis, management, and treatment of hepatitis C. Hepatology. 2004;39:1147–1171.
- Gale MJ, Jr, Korth MJ, Tang NM, Tan SL, Hopkins DA, et al. Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology. 1997;230:217–227.
- Dal PF, Tang KH, Gerotto M, Bortoletto G, Paulon E, et al. Impact of NS5A sequences of Hepatitis C virus genotype 1a on early viral kinetics during treatment with peginterferon- alpha 2a plus ribavirin. J Infect Dis. 2007;196:998–1005.
- Kohashi T, Maekawa S, Sakamoto N, Kurosaki M, Watanabe H, et al. Site-specific mutation of the interferon sensitivity-determining region (ISDR) modulates hepatitis C virus replication. J Viral Hepat. 2006;13:582–590.
- Veillon P, Payan C, Le Guillou-Guillemette H, Gaudy C, Lunel F. Quasispecies evolution in NS5A region of hepatitis C virus genotype 1b during interferon or combined interferon-ribavirin therapy. World J Gastroenterol. 2007;13:1195–1203.
- Chayama K, Suzuki F, Tsubota A, Kobayashi M, Arase Y, et al. Association of amino acid sequence in the PKR-eIF2 phosphorylation homology domain and response to interferon therapy. Hepatology. 2000;32:1138–1144.
- Gaudy C, Lambele M, Moreau A, Veillon P, Lunel F, et al. Mutations within the hepatitis C virus genotype 1b E2-PePHD domain do not correlate with treatment outcome. J Clin Microbiol. 2005;43:750–754.
- Gerotto M, Dal PF, Pontisso P, Noventa F, Gatta A, et al. Two PKR inhibitor HCV proteins correlate with early but not sustained response to interferon. Gastroenterology. 2000;119:1649–1655.
- Gupta R, Subramani M, Khaja MN, Madhavi C, Roy S, et al. Analysis of mutations within the 5′ untranslated region, interferon sensitivity region, and PePHD region as a function of response to interferon therapy in hepatitis C virus-infected patients in India. J Clin Microbiol. 2006;44:709–715.
- Hung CH, Lee CM, Lu SN, Lee JF, Wang JH, et al. Mutations in the NS5A and E2-PePHD region of hepatitis C virus type 1b and correlation with the response to combination therapy with interferon and ribavirin. J Viral Hepat. 2003;10:87–94.
- Puig-Basagoiti F, Saiz JC, Forns X, Ampurdanes S, Gimenez-Barcons M, et al. Influence of the genetic heterogeneity of the ISDR and PePHD regions of hepatitis C virus on the response to interferon therapy in chronic hepatitis C. J Med Virol. 2001;65:35–44.
- Sarrazin C, Bruckner M, Herrmann E, Ruster B, Bruch K, et al. Quasispecies heterogeneity of the carboxy-terminal part of the E2 gene including the PePHD and sensitivity of hepatitis C virus 1b isolates to antiviral therapy. Virology. 2001;289:150–163.
- Yang SS, Lai MY, Chen DS, Chen GH, Kao JH. Mutations in the NS5A and E2-PePHD regions of hepatitis C virus genotype 1b and response to combination therapy of interferon plus ribavirin. Liver Int. 2003;23:426–433.
- Donlin MJ, Cannon NA, Yao E, Li J, Wahed A, Taylor MW, et al. Pretreatment sequence diversity differences in the full-length hepatitis C virus open reading frame correlate with early response to therapy. J Virol. 2007;81:8211–8224.
- Hadziyannis SJ, Sette H, Jr, Morgan TR, Balan V, Diago M, et al. Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose. Ann Intern Med. 2004;140:346–355.
- Macdonald A, Crowder K, Street A, McCormick C, Harris M. The hepatitis C virus NS5A protein binds to members of the Src family of tyrosine kinases and regulates kinase activity. J Gen Virol. 2004;85:721–729.
- van Doorn LJ, Capriles I, Maertens G, DeLeys R, Murray K, et al. Sequence evolution of the hypervariable region in the putative envelope region E2/NS1 of hepatitis C virus is correlated with specific humoral immune responses. J Virol. 1995;69:773–778.
- Francois C, Duverlie G, Rebouillat D, Khorsi H, Castelain S, et al. Expression of hepatitis C virus proteins interferes with the antiviral action of interferon independently of PKR-mediated control of protein synthesis. J Virol. 2000;74:5587–5596.
- Taylor DR, Shi ST, Romano PR, Garber GN, Lai MMC. Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science. 1999;285:107–110.
- Lorenz IC, Marcotrigiano J, Dentzer TG, Rice CM. Structure of the catalytic domain of the hepatitis C virus NS2-3 protease. Nature. 2006;442:831–835.
- Yao N, Reichert P, Taremi SS, Prosise WW, Weber PC. Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase. Structure. 1999;7:1353–1363.
- 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.
- O'Farrell D, Trowbridge R, Rowlands D, Jager J. Substrate complexes of hepatitis C virus RNA polymerase (HC-J4): structural evidence for nucleotide import and de-novo initiation. J Mol Biol. 2003;326:1025–1035.
- Poon AF, Kosakovsky Pond SL, Bennett P, Richman DD, Leigh Brown AJ, et al. Adaptation to human populations is revealed by within-host polymorphisms in HIV-1 and hepatitis C virus. PLoS Pathog. 2007;3:e45.
- Abbate I, Lo IO, Di SR, Cappiello G, Girardi E, et al. HVR-1 quasispecies modifications occur early and are correlated to initial but not sustained response in HCV-infected patients treated with pegylated- or standard-interferon and ribavirin. J Hepatol. 2004;40:831–836.
- Chambers TJ, Fan X, Droll DA, Hembrador E, Slater T, et al. Quasispecies heterogeneity within the E1/E2 region as a pretreatment variable during pegylated interferon therapy of chronic hepatitis C virus infection. J Virol. 2005;79:3071–3083.
- Forns X, Purcell RH, Bukh J. Quasispecies in viral persistence and pathogenesis of hepatitis C virus. Trends Microbiol. 1999;7:402–410.
- Morishima C, Polyak SJ, Ray R, Doherty MC, Di Bisceglie AM, et al. Hepatitis C virus-specific immune responses and quasi-species variability at baseline are associated with nonresponse to antiviral therapy during advanced hepatitis C. J Infect Dis. 2006;193:931–940.
- von WM, Lee JH, Ruster B, Kronenberger B, Sarrazin C, et al. Dynamics of hepatitis C virus quasispecies turnover during interferon-alpha treatment. J Viral Hepat. 2003;10:413–422.
- Arataki K, Kumada H, Toyota K, Ohishi W, Takahashi S, et al. Evolution of hepatitis C virus quasispecies during ribavirin and interferon-alpha-2b combination therapy and interferon-alpha-2b monotherapy. Intervirology. 2006;49:352–361.
- Chen S, Wang YM. Genetic evolution of structural region of hepatitis C virus in primary infection. World J Gastroenterol. 2002;8:686–693.
- Odeberg J, Yun Z, Sonnerborg A, Weiland O, Lundeberg J. Variation in the hepatitis C virus NS5a region in relation to hypervariable region 1 heterogeneity during interferon treatment. J Med Virol. 1998;56:33–38.
- Pawlotsky JM, Germanidis G, Frainais PO, Bouvier M, Soulier A, et al. Evolution of the hepatitis C virus second envelope protein hypervariable region in chronically infected patients receiving alpha interferon therapy. J Virol. 1999;73:6490–6499.
- Polyak SJ, McArdle S, Liu SL, Sullivan DG, Chung M, et al. Evolution of hepatitis C virus quasispecies in hypervariable region 1 and the putative interferon sensitivity-determining region during interferon therapy and natural infection. J Virol. 1998;72:4288–4296.
- Kaukinen P, Sillanpaa M, Kotenko S, Lin R, Hiscott J, et al. Hepatitis C virus NS2 and NS3/4A proteins are potent inhibitors of host cell cytokine/chemokine gene expression. Virol J. 2006;3:66.
- Jones CT, Murray CL, Eastman DK, Tassello J, Rice CM. Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol. 2007;81:8374–8383.
- Brown RJ, Juttla VS, Tarr AW, Finnis R, Irving WL, et al. Evolutionary dynamics of hepatitis C virus envelope genes during chronic infection. J Gen Virol. 2005;86:1931–1942.
- Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, et al. Comparison of full-length sequences of interferon-sensitve and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J Clin Invest. 1995;96:224–230.
- Vuillermoz I, Khattab E, Sablon E, Ottevaere I, Durantel D, et al. Genetic variability of hepatitis C virus in chronically infected patients with viral breakthrough during interferon-ribavirin therapy. J Med Virol. 2004;74:41–53.
- Hamano K, Sakamoto N, Enomoto N, Izumi N, Asahina Y, et al. Mutations in the NS5B region of the hepatitis C virus genome correlate with clinical outcomes of interferon-alpha plus ribavirin combination therapy. J Gastroenterol Hepatol. 2005;20:1401–1409.
- Yao E, Tavis JE. A general method for nested RT-PCR amplification and sequencing the complete HCV genotype 1 open reading frame. Virol J. 2005;2:88.
- Fan X, Xu Y, Di Bisceglie AM. Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples. Biochem Biophys Res Commun. 2006;346:1163–1172.
- Walewski JL, Gutierrez JA, Branch-Elliman W, Stump DD, Keller TR, et al. Mutation Master: profiles of substitutions in hepatitis C virus RNA of the core, alternate reading frame, and NS2 coding regions. RNA. 2002;8:557–571.
- Kuiken C, Yusim K, Boykin L, Richardson R. The Los Alamos hepatitis C sequence database. Bioinformatics. 2005;21:379–384.
- Kumar S, Tamura K, Nei M. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform. 2004;5:150–163.
- Fares MA. SWAPSC: sliding window analysis procedure to detect selective constraints. Bioinformatics. 2004;20:2867–2868.
- Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998;8:186–194.
- Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998;8:175–185.
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