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Analysis of the PKR-eIF2alpha phosphorylation homology domain (PePHD) of hepatitis C virus genotype 1 in HIV-coinfected patients by ultra-deep pyrosequencing and its relationship to responses to pegylated interferon-ribavirin treatment

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Abstract

Chronic coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is among the greatest challenges facing public health worldwide. In this population, the response to hepatitis C therapy by treatment with pegylated interferon plus ribavirin (PEG-IFN+RBV) is lower than in HCV-monoinfected patients, particularly in those infected by HCV genotype 1. A PKR/eIF-2α phosphorylation homology domain (PePHD) within the E2 protein has been found to interact with PKR and inhibit PKR in vitro, suggesting a possible mechanism for HCV to evade the antiviral effects of IFN. The aim of this work was to analyze the amino acid conservation in the HCV-E2-PePHD and quasispecies diversity among HCV-HIV-coinfected patients exhibiting sustained virological response, non-response, or partial response with viral relapse to PEG-IFN+RBV by ultra-deep pyrosequencing. For this purpose, HCV-E2-PePHD PCR products were generated and sequenced directly for four patients with a sustained response, seven patients with no virological response, and four patients with viral relapse before and after treatment with PEG-IFN+RBV. HCV-E2-PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment (24 h, 4 weeks, and 12 weeks). Quasispecies analysis of the HCV-E2-PePHD and flanking genomic regions was performed using 454/Roche pyrosequencing, analyzing 39,364 sequence reads in total. The HCV-E2-PePHD sequence at the amino acid and nucleotide level was highly conserved among HCV genotype 1 strains, irrespective of the PEG-IFN+RBV response. This high degree of amino acid conservation and sporadic mutations in the HCV-E2-PePHD domain do not appear to be associated with treatment outcome. The HCV-E2-PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in patients coinfected with HCV genotype 1 and HIV.

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References

  1. Thomson EC, Smith JA, Klenerman P (2011) The natural history of early hepatitis C virus evolution; lessons from a global outbreak in HIV-1 infected individuals. J Gen Virol. doi:10.1099/vir.0.033910-0

    Google Scholar 

  2. Pang PS, Planet PJ, Glenn JS (2009) The evolution of the major hepatitis C genotypes correlates with clinical response to interferon therapy. PLoS One 4(8):e6579. doi:10.1371/journal.pone.0006579

    Article  PubMed  Google Scholar 

  3. Rockstroh JK, Mocroft A, Soriano V, Tural C, Losso MH, Horban A, Kirk O, Phillips A, Ledergerber B, Lundgren J (2005) Influence of hepatitis C virus infection on HIV-1 disease progression and response to highly active antiretroviral therapy. J Infect Dis 192(6):992–1002. doi:10.1086/432762

    Article  PubMed  Google Scholar 

  4. Torriani FJ, Rodriguez-Torres M, Rockstroh JK, Lissen E, Gonzalez-Garcia J, Lazzarin A, Carosi G, Sasadeusz J, Katlama C, Montaner J, Sette H Jr, Passe S, De Pamphilis J, Duff F, Schrenk UM, Dieterich DT (2004) Peginterferon Alfa-2a plus ribavirin for chronic hepatitis C virus infection in HIV-infected patients. N Engl J Med 351(5):438–450. doi:10.1056/NEJMoa040842

    Article  PubMed  CAS  Google Scholar 

  5. Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, Goodman ZD, Koury K, Ling M, Albrecht JK (2001) Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 358(9286):958–965 (pii:S0140673601061025)

    Article  PubMed  CAS  Google Scholar 

  6. Taylor DR, Shi ST, Romano PR, Barber GN, Lai MM (1999) Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 285(5424):107–110 (pii:7641)

    Article  PubMed  CAS  Google Scholar 

  7. Laufer N, Bolcic F, Rolon MJ, Martinez A, Reynoso R, Perez H, Salomon H, Cahn P, Quarleri J (2011) HCV RNA decline in the first 24 h exhibits high negative predictive value of sustained virologic response in HIV/HCV genotype 1 co-infected patients treated with peginterferon and ribavirin. Antiviral Res 90(1):92–97. doi:10.1016/j.antiviral.2011.02.013

    Article  PubMed  CAS  Google Scholar 

  8. Laufer N, Quarleri J, Bouzas MB, Juncos G, Cabrini M, Moretti F, Bolcic F, Fernandez-Giuliano S, Mammana L, Salomon H, Cahn P (2010) Hepatitis B virus, hepatitis C virus and HIV coinfection among people living with HIV/AIDS in Buenos Aires, Argentina. Sex Transm Dis 37(5):342–343. doi:10.1097/OLQ.0b013e3181d73c0d

    PubMed  Google Scholar 

  9. Bolcic F, Bull L, Martinez L, Reynoso R, Salomon H, Arduino R, Barnett B, Quarleri J (2008) Analysis of sequence configurations of the PKR-interacting HCV proteins from plasma and PBMC as predictors of response to interferon-alpha and ribavirin therapy in HIV-coinfected patients. Intervirology 51(4):261–264. doi:10.1159/000158523

    Article  PubMed  CAS  Google Scholar 

  10. Eriksson N, Pachter L, Mitsuya Y, Rhee SY, Wang C, Gharizadeh B, Ronaghi M, Shafer RW, Beerenwinkel N (2008) Viral population estimation using pyrosequencing. PLoS Comput Biol 4(4):e1000074. doi:10.1371/journal.pcbi.1000074

    Article  PubMed  Google Scholar 

  11. Kuiken C, Hraber P, Thurmond J, Yusim K (2008) The hepatitis C sequence database in Los Alamos. Nucleic Acids Res 36(Database issue):D512–D516. doi:10.1093/nar/gkm962

    PubMed  CAS  Google Scholar 

  12. Kuiken C, Korber B, Shafer RW (2003) HIV sequence databases. AIDS Rev 5(1):52–61

    PubMed  Google Scholar 

  13. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30(14):3059–3066

    Article  PubMed  CAS  Google Scholar 

  14. Katoh K, Asimenos G, Toh H (2009) Multiple alignment of DNA sequences with MAFFT. Methods Mol Biol 537:39–64. doi:10.1007/978-1-59745-251-9_3

    Article  PubMed  CAS  Google Scholar 

  15. Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  16. Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25(7):1253–1256. doi:10.1093/molbev/msn083

    Article  PubMed  CAS  Google Scholar 

  17. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52(5):696–704 (pii:54QHX07WB5K5XCX4)

    Article  PubMed  Google Scholar 

  18. Huson DH, Richter DC, Rausch C, Dezulian T, Franz M, Rupp R (2007) Dendroscope: an interactive viewer for large phylogenetic trees. BMC Bioinformatics 8:460. doi:10.1186/1471-2105-8-460

    Article  PubMed  Google Scholar 

  19. Huse SM, Huber JA, Morrison HG, Sogin ML, Welch DM (2007) Accuracy and quality of massively parallel DNA pyrosequencing. Genome Biol 8(7):R143. doi:10.1186/gb-2007-8-7-r143

    Article  PubMed  Google Scholar 

  20. Ogata N, Alter HJ, Miller RH, Purcell RH (1991) Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proc Natl Acad Sci USA 88(8):3392–3396

    Article  PubMed  CAS  Google Scholar 

  21. Okamoto H, Kojima M, Okada S, Yoshizawa H, Iizuka H, Tanaka T, Muchmore EE, Peterson DA, Ito Y, Mishiro S (1992) Genetic drift of hepatitis C virus during an 8.2-year infection in a chimpanzee: variability and stability. Virology 190(2):894–899

    Article  PubMed  CAS  Google Scholar 

  22. Smith DB, Pathirana S, Davidson F, Lawlor E, Power J, Yap PL, Simmonds P (1997) The origin of hepatitis C virus genotypes. J Gen Virol 78(Pt 2):321–328

    PubMed  CAS  Google Scholar 

  23. Ukai K, Ishigami M, Yoshioka K, Kawabe N, Katano Y, Hayashi K, Honda T, Yano M, Goto H (2006) Mutations in carboxy-terminal part of E2 including PKR/eIF2alpha phosphorylation homology domain and interferon sensitivity determining region of nonstructural 5A of hepatitis C virus 1b: their correlation with response to interferon monotherapy and viral load. World J Gastroenterol 12(23):3722–3728

    PubMed  CAS  Google Scholar 

  24. Afzal S, Idrees M, Akram M, Awan Z, Khubaib B, Aftab M, Fatima Z, Badar S, Hussain A (2010) Mutations in the E2-PePHD region of hepatitis C virus genotype-3a and correlation with response to interferon and ribavirin combination therapy in Pakistani patients. Virol J 7:377. doi:10.1186/1743-422X-7-377

    Article  PubMed  CAS  Google Scholar 

  25. Munoz de Rueda P, Casado J, Paton R, Quintero D, Palacios A, Gila A, Quiles R, Leon J, Ruiz-Extremera A, Salmeron J (2008) Mutations in E2-PePHD, NS5A-PKRBD, NS5A-ISDR, and NS5A-V3 of hepatitis C virus genotype 1 and their relationships to pegylated interferon-ribavirin treatment responses. J Virol 82(13):6644–6653. doi:10.1128/JVI.02231-07

    Article  PubMed  CAS  Google Scholar 

  26. Gaudy C, Lambele M, Moreau A, Veillon P, Lunel F, Goudeau A (2005) Mutations within the hepatitis C virus genotype 1b E2-PePHD domain do not correlate with treatment outcome. J Clin Microbiol 43(2):750–754. doi:10.1128/JCM.43.2.750-754.2005

    Article  PubMed  CAS  Google Scholar 

  27. Berg T, Mas Marques A, Hohne M, Wiedenmann B, Hopf U, Schreier E (2000) Mutations in the E2-PePHD and NS5A region of hepatitis C virus type 1 and the dynamics of hepatitis C viremia decline during interferon alfa treatment. Hepatology 32(6):1386–1395. doi:10.1053/jhep.2000.20527

    Article  PubMed  CAS  Google Scholar 

  28. Chayama K, Suzuki F, Tsubota A, Kobayashi M, Arase Y, Saitoh S, Suzuki Y, Murashima N, Ikeda K, Takahashi N, Kinoshita M, Kumada H (2000) Association of amino acid sequence in the PKR-eIF2 phosphorylation homology domain and response to interferon therapy. Hepatology 32(5):1138–1144. doi:10.1053/jhep.2000.19364

    Article  PubMed  CAS  Google Scholar 

  29. Gerotto M, Dal Pero F, Pontisso P, Noventa F, Gatta A, Alberti A (2000) Two PKR inhibitor HCV proteins correlate with early but not sustained response to interferon. Gastroenterology 119(6):1649–1655 (pii:S0016508500186731)

    Article  PubMed  CAS  Google Scholar 

  30. Hung CH, Lee CM, Lu SN, Lee JF, Wang JH, Tung HD, Chen TM, Hu TH, Chen WJ, Changchien CS (2003) 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 10(2):87–94 (pii:414)

    Article  PubMed  Google Scholar 

  31. Sarrazin C, Bruckner M, Herrmann E, Ruster B, Bruch K, Roth WK, Zeuzem S (2001) 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 289(1):150–163. doi:10.1006/viro.2001.1092

    Article  PubMed  CAS  Google Scholar 

  32. Jenke AC, Moser S, Orth V, Zilbauer M, Gerner P, Wirth S (2009) Mutation frequency of NS5A in patients vertically infected with HCV genotype 1 predicts sustained virological response to peginterferon alfa-2b and ribavirin combination therapy. J Viral Hepat 16(12):853–859. doi:10.1111/j.1365-2893.2009.01140.x

    Article  PubMed  CAS  Google Scholar 

  33. Yokozaki S, Katano Y, Hayashi K, Ishigami M, Itoh A, Hirooka Y, Nakano I, Goto H (2011) Mutations in two PKR-binding domains in chronic hepatitis C of genotype 3a and correlation with viral loads and interferon responsiveness. J Med Virol 83(10):1727–1732. doi:10.1002/jmv.21959

    Article  PubMed  CAS  Google Scholar 

  34. Yahoo N, Sabahi F, Shahzamani K, Malboobi MA, Jabbari H, Sharifi H, Mousavi-Fard SH, Merat S (2011) Mutations in the E2 and NS5A regions in patients infected with hepatitis C virus genotype 1a and their correlation with response to treatment. J Med Virol 83(8):1332–1337. doi:10.1002/jmv.22144

    Article  PubMed  CAS  Google Scholar 

  35. Bagaglio S, De Mitri MS, Lodrini S, Paties C, Cassini R, Bianchi G, Bernardi M, Lazzarin A, Morsica G (2005) Mutations in the E2-PePHD region of hepatitis C virus type 1b in patients with hepatocellular carcinoma. J Viral Hepat 12(3):243–250. doi:10.1111/j.1365-2893.2005.00589.x

    Article  PubMed  CAS  Google Scholar 

  36. Massanella M, Tural C, Papagno L, Garcia E, Jou A, Bofill M, Autran B, Clotet B, Blanco J (2010) Changes in T-cell subsets in HIV-HCV-coinfected patients during pegylated interferon-alpha2a plus ribavirin treatment. Antivir Ther 15(3):333–342. doi:10.3851/IMP1531

    Article  PubMed  CAS  Google Scholar 

  37. Berenguer J, von Wichmann MA, Quereda C, Miralles P, Mallolas J, Lopez-Aldeguer J, Alvarez-Pellicer J, De Miguel J, Crespo M, Guardiola JM, Tellez MJ, Galindo MJ, Arponen S, Barquilla E, Bellon JM, Gonzalez-Garcia J (2011) Effect of accompanying antiretroviral drugs on virological response to pegylated interferon and ribavirin in patients co-infected with HIV and hepatitis C virus. J Antimicrob Chemother 66(12):2843–2849. doi:10.1093/jac/dkr362

    Article  PubMed  CAS  Google Scholar 

  38. Bagaglio S, Bruno R, Lodrini S, De Mitri MS, Andreone P, Loggi E, Galli L, Lazzarin A, Morsica G (2003) Genetic heterogeneity of hepatitis C virus (HCV) in clinical strains of HIV positive and HIV negative patients chronically infected with HCV genotype 3a. J Biol Regul Homeost Agents 17(2):153–161

    PubMed  CAS  Google Scholar 

  39. Lee SH, Kim YK, Kim CS, Seol SK, Kim J, Cho S, Song YL, Bartenschlager R, Jang SK (2005) E2 of hepatitis C virus inhibits apoptosis. J Immunol 175(12):8226–8235 (pii:175/12/8226)

    PubMed  CAS  Google Scholar 

  40. Owsianka AM, Timms JM, Tarr AW, Brown RJ, Hickling TP, Szwejk A, Bienkowska-Szewczyk K, Thomson BJ, Patel AH, Ball JK (2006) Identification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding. J Virol 80(17):8695–8704. doi:10.1128/JVI.00271-06

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This study was supported partially by grants from the University of Buenos Aires (SECYT-UBA 2010-2012) and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-PIP112 200801 01773). The authors thank Sergio Mazzini (Centro Nacional de Referencia para el SIDA, Departamento de Microbiología, Facultad de Medicina, Universidad de Buenos Aires, Argentina) for his assistance during the manuscript preparation.

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The authors declare that there is no conflict of interest.

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Authors and Affiliations

  1. Departamento de Microbiología, Facultad de Medicina (UBA), Centro Nacional de Referencia para el SIDA, Paraguay 2155, Piso 11, C1121ABG, Buenos Aires, Argentina

    F. Bolcic, M. Sede, N. Laufer & J. Quarleri

  2. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina

    F. Bolcic, M. Sede, F. Moretti, M. Vazquez, N. Laufer & J. Quarleri

  3. Instituto de Agrobiotecnología Rosario (INDEAR), Buenos Aires, Argentina

    G. Westergaard & M. Vazquez

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  1. F. Bolcic
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  2. M. Sede
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Correspondence to J. Quarleri.

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Bolcic, F., Sede, M., Moretti, F. et al. Analysis of the PKR-eIF2alpha phosphorylation homology domain (PePHD) of hepatitis C virus genotype 1 in HIV-coinfected patients by ultra-deep pyrosequencing and its relationship to responses to pegylated interferon-ribavirin treatment. Arch Virol 157, 703–711 (2012). https://doi.org/10.1007/s00705-012-1230-1

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