1887

Abstract

Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core–NS2 and NS5A from genotypes 1–7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core–NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core–NS2/NS5A recombinants produced infectivity titres of 10–10 f.f.u. ml. Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core and NS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ER associated at later stages. Finally, we found that different genotypes showed varying levels of core/cLD co-localization, with a possible effect on viral assembly/release. In summary, we developed a panel of HCV genotype 1–7 core–NS2/NS5A recombinants producing infectious virus, and an immunostaining protocol detecting the core and NS5A proteins from seven different genotypes. These systems will allow, for the first time, investigation of core/NS5A interactions during assembly and release of HCV particles of all major genotypes.

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2013-10-01
2019-10-23
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References

  1. Alter H. J., Seeff L. B.. ( 2000;). Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome. . Semin Liver Dis 20:, 17–35. [CrossRef][PubMed]
    [Google Scholar]
  2. Appel N., Zayas M., Miller S., Krijnse-Locker J., Schaller T., Friebe P., Kallis S., Engel U., Bartenschlager R.. ( 2008;). Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. . PLoS Pathog 4:, e1000035. [CrossRef][PubMed]
    [Google Scholar]
  3. Bartenschlager R., Penin F., Lohmann V., André P.. ( 2011;). Assembly of infectious hepatitis C virus particles. . Trends Microbiol 19:, 95–103. [CrossRef][PubMed]
    [Google Scholar]
  4. Boson B., Granio O., Bartenschlager R., Cosset F. L.. ( 2011;). A concerted action of hepatitis C virus p7 and nonstructural protein 2 regulates core localization at the endoplasmic reticulum and virus assembly. . PLoS Pathog 7:, e1002144. [CrossRef][PubMed]
    [Google Scholar]
  5. Boulant S., Targett-Adams P., McLauchlan J.. ( 2007;). Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus. . J Gen Virol 88:, 2204–2213. [CrossRef][PubMed]
    [Google Scholar]
  6. Bukh J., Purcell R. H., Miller R. H.. ( 1993;). At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide. . Proc Natl Acad Sci U S A 90:, 8234–8238. [CrossRef][PubMed]
    [Google Scholar]
  7. Bukh J., Meuleman P., Tellier R., Engle R. E., Feinstone S. M., Eder G., Satterfield W. C., Govindarajan S., Krawczynski K.. & other authors ( 2010;). Challenge pools of hepatitis C virus genotypes 1-6 prototype strains: replication fitness and pathogenicity in chimpanzees and human liver-chimeric mouse models. . J Infect Dis 201:, 1381–1389. [CrossRef][PubMed]
    [Google Scholar]
  8. Camus G., Herker E., Modi A. A., Haas J. T., Ramage H. R., Farese R. V. Jr, Ott M.. ( 2013;). Diacylglycerol acyltransferase-1 localizes hepatitis C virus NS5A protein to lipid droplets and enhances NS5A interaction with the viral capsid core. . J Biol Chem 288:, 9915–9923. [CrossRef][PubMed]
    [Google Scholar]
  9. Gastaminza P., Cheng G., Wieland S., Zhong J., Liao W., Chisari F. V.. ( 2008;). Cellular determinants of hepatitis C virus assembly, maturation, degradation, and secretion. . J Virol 82:, 2120–2129. [CrossRef][PubMed]
    [Google Scholar]
  10. Gottwein J. M., Bukh J.. ( 2008;). Cutting the gordian knot-development and biological relevance of hepatitis C virus cell culture systems. . Adv Virus Res 71:, 51–133. [CrossRef][PubMed]
    [Google Scholar]
  11. Gottwein J. M., Scheel T. K., Hoegh A. M., Lademann J. B., Eugen-Olsen J., Lisby G., Bukh J.. ( 2007;). Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. . Gastroenterology 133:, 1614–1626. [CrossRef][PubMed]
    [Google Scholar]
  12. Gottwein J. M., Scheel T. K., Jensen T. B., Lademann J. B., Prentoe J. C., Knudsen M. L., Hoegh A. M., Bukh J.. ( 2009;). Development and characterization of hepatitis C virus genotype 1-7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs. . Hepatology 49:, 364–377. [CrossRef][PubMed]
    [Google Scholar]
  13. Gottwein J. M., Scheel T. K., Callendret B., Li Y. P., Eccleston H. B., Engle R. E., Govindarajan S., Satterfield W., Purcell R. H.. & other authors ( 2010;). 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 84:, 5277–5293. [CrossRef][PubMed]
    [Google Scholar]
  14. Gottwein J. M., Scheel T. K., Jensen T. B., Ghanem L., Bukh J.. ( 2011;). Differential efficacy of protease inhibitors against HCV genotypes 2a, 3a, 5a, and 6a NS3/4A protease recombinant viruses. . Gastroenterology 141:, 1067–1079. [CrossRef][PubMed]
    [Google Scholar]
  15. Griffin S., Stgelais C., Owsianka A. M., Patel A. H., Rowlands D., Harris M.. ( 2008;). Genotype-dependent sensitivity of hepatitis C virus to inhibitors of the p7 ion channel. . Hepatology 48:, 1779–1790. [CrossRef][PubMed]
    [Google Scholar]
  16. Herker E., Harris C., Hernandez C., Carpentier A., Kaehlcke K., Rosenberg A. R., Farese R. V. Jr, Ott M.. ( 2010;). Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1. . Nat Med 16:, 1295–1298. [CrossRef][PubMed]
    [Google Scholar]
  17. Huang L., Hwang J., Sharma S. D., Hargittai M. R., Chen Y., Arnold J. J., Raney K. D., Cameron C. E.. ( 2005;). Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein. . J Biol Chem 280:, 36417–36428. [CrossRef][PubMed]
    [Google Scholar]
  18. Huang H., Sun F., Owen D. M., Li W., Chen Y., Gale M. Jr, Ye J.. ( 2007;). Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins. . Proc Natl Acad Sci U S A 104:, 5848–5853. [CrossRef][PubMed]
    [Google Scholar]
  19. Jensen T. B., Gottwein J. M., Scheel T. K., Hoegh A. M., Eugen-Olsen J., Bukh J.. ( 2008;). Highly efficient JFH1-based cell-culture system for hepatitis C virus genotype 5a: failure of homologous neutralizing-antibody treatment to control infection. . J Infect Dis 198:, 1756–1765. [CrossRef][PubMed]
    [Google Scholar]
  20. Jirasko V., Montserret R., Appel N., Janvier A., Eustachi L., Brohm C., Steinmann E., Pietschmann T., Penin F., Bartenschlager R.. ( 2008;). Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly. . J Biol Chem 283:, 28546–28562. [CrossRef][PubMed]
    [Google Scholar]
  21. Jirasko V., Montserret R., Lee J. Y., Gouttenoire J., Moradpour D., Penin F., Bartenschlager R.. ( 2010;). Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly. . PLoS Pathog 6:, e1001233. [CrossRef][PubMed]
    [Google Scholar]
  22. Kaul A., Woerz I., Meuleman P., Leroux-Roels G., Bartenschlager R.. ( 2007;). Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant. . J Virol 81:, 13168–13179. [CrossRef][PubMed]
    [Google Scholar]
  23. Kaul A., Stauffer S., Berger C., Pertel T., Schmitt J., Kallis S., Zayas M., Lohmann V., Luban J., Bartenschlager R.. ( 2009;). Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics. . PLoS Pathog 5:, e1000546. [CrossRef][PubMed]
    [Google Scholar]
  24. Kim J. L., Morgenstern K. A., Lin C., Fox T., Dwyer M. D., Landro J. A., Chambers S. P., Markland W., Lepre C. A.. & other authors ( 1996;). Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. . Cell 87:, 343–355. [CrossRef][PubMed]
    [Google Scholar]
  25. Lindenbach B. D.. ( 2013;). Virion assembly and release. . Curr Top Microbiol Immunol 369:, 199–218. [CrossRef][PubMed]
    [Google Scholar]
  26. Lindenbach B. D., Evans M. J., Syder A. J., Wölk B., Tellinghuisen T. L., Liu C. C., Maruyama T., Hynes R. O., Burton D. R.. & other authors ( 2005;). Complete replication of hepatitis C virus in cell culture. . Science 309:, 623–626. [CrossRef][PubMed]
    [Google Scholar]
  27. Lindenbach B. D., Prágai B. M., Montserret R., Beran R. K., Pyle A. M., Penin F., Rice C. M.. ( 2007;). The C terminus of hepatitis C virus NS4A encodes an electrostatic switch that regulates NS5A hyperphosphorylation and viral replication. . J Virol 81:, 8905–8918. [CrossRef][PubMed]
    [Google Scholar]
  28. Love R. A., Parge H. E., Wickersham J. A., Hostomsky Z., Habuka N., Moomaw E. W., Adachi T., Hostomska Z.. ( 1996;). The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site. . Cell 87:, 331–342. [CrossRef][PubMed]
    [Google Scholar]
  29. Ma Y., Anantpadma M., Timpe J. M., Shanmugam S., Singh S. M., Lemon S. M., Yi M.. ( 2011;). Hepatitis C virus NS2 protein serves as a scaffold for virus assembly by interacting with both structural and nonstructural proteins. . J Virol 85:, 86–97. [CrossRef][PubMed]
    [Google Scholar]
  30. Masaki T., Suzuki R., Murakami K., Aizaki H., Ishii K., Murayama A., Date T., Matsuura Y., Miyamura T.. & other authors ( 2008;). Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles. . J Virol 82:, 7964–7976. [CrossRef][PubMed]
    [Google Scholar]
  31. McLauchlan J., Lemberg M. K., Hope G., Martoglio B.. ( 2002;). Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. . EMBO J 21:, 3980–3988. [CrossRef][PubMed]
    [Google Scholar]
  32. Miyanari Y., Atsuzawa K., Usuda N., Watashi K., Hishiki T., Zayas M., Bartenschlager R., Wakita T., Hijikata M., Shimotohno K.. ( 2007;). The lipid droplet is an important organelle for hepatitis C virus production. . Nat Cell Biol 9:, 1089–1097. [CrossRef][PubMed]
    [Google Scholar]
  33. Pedersen J., Carlsen T. H., Prentoe J., Ramirez S., Jensen T. B., Forns X., Alter H., Foung S. K., Law M.. & other authors ( 2013;). Neutralization resistance of hepatitis C virus can be overcome by recombinant human monoclonal antibodies. . Hepatology doi: 10.1002/hep.26524 [Epub ahead of print]. [CrossRef][PubMed]
    [Google Scholar]
  34. Pietschmann T., Kaul A., Koutsoudakis G., Shavinskaya A., Kallis S., Steinmann E., Abid K., Negro F., Dreux M.. & other authors ( 2006;). Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. . Proc Natl Acad Sci U S A 103:, 7408–7413. [CrossRef][PubMed]
    [Google Scholar]
  35. Popescu C. I., Callens N., Trinel D., Roingeard P., Moradpour D., Descamps V., Duverlie G., Penin F., Héliot L.. & other authors ( 2011;). NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly. . PLoS Pathog 7:, e1001278. [CrossRef][PubMed]
    [Google Scholar]
  36. Prentoe J., Jensen T. B., Meuleman P., Serre S. B., Scheel T. K., Leroux-Roels G., Gottwein J. M., Bukh J.. ( 2011;). Hypervariable region 1 differentially impacts viability of hepatitis C virus strains of genotypes 1 to 6 and impairs virus neutralization. . J Virol 85:, 2224–2234. [CrossRef][PubMed]
    [Google Scholar]
  37. Sakai A., Takikawa S., Thimme R., Meunier J. C., Spangenberg H. C., Govindarajan S., Farci P., Emerson S. U., Chisari F. V.. & other authors ( 2007;). In vivo study of the HC-TN strain of hepatitis C virus recovered from a patient with fulminant hepatitis: RNA transcripts of a molecular clone (pHC-TN) are infectious in chimpanzees but not in Huh7.5 cells. . J Virol 81:, 7208–7219. [CrossRef][PubMed]
    [Google Scholar]
  38. Scheel T. K., Gottwein J. M., Jensen T. B., Prentoe J. C., Hoegh A. M., Alter H. J., Eugen-Olsen J., Bukh J.. ( 2008;). Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization. . Proc Natl Acad Sci U S A 105:, 997–1002. [CrossRef][PubMed]
    [Google Scholar]
  39. Scheel T. K., Gottwein J. M., Carlsen T. H., Li Y. P., Jensen T. B., Spengler U., Weis N., Bukh J.. ( 2011a;). Efficient culture adaptation of hepatitis C virus recombinants with genotype-specific core-NS2 by using previously identified mutations. . J Virol 85:, 2891–2906. [CrossRef][PubMed]
    [Google Scholar]
  40. Scheel T. K., Gottwein J. M., Mikkelsen L. S., Jensen T. B., Bukh J.. ( 2011b;). Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferon-α. . Gastroenterology 140:, 1032–1042, 1042.e6. [CrossRef][PubMed]
    [Google Scholar]
  41. Scheel T. K., Prentoe J., Carlsen T. H., Mikkelsen L. S., Gottwein J. M., Bukh J.. ( 2012;). Analysis of functional differences between hepatitis C virus NS5A of genotypes 1-7 in infectious cell culture systems. . PLoS Pathog 8:, e1002696. [CrossRef][PubMed]
    [Google Scholar]
  42. Shavinskaya A., Boulant S., Penin F., McLauchlan J., Bartenschlager R.. ( 2007;). The lipid droplet binding domain of hepatitis C virus core protein is a major determinant for efficient virus assembly. . J Biol Chem 282:, 37158–37169. [CrossRef][PubMed]
    [Google Scholar]
  43. Simmonds P., Holmes E. C., Cha T. A., Chan S. W., McOmish F., Irvine B., Beall E., Yap P. L., Kolberg J., Urdea M. S.. ( 1993;). Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. . J Gen Virol 74:, 2391–2399. [CrossRef][PubMed]
    [Google Scholar]
  44. Simmonds P., Bukh J., Combet C., Deléage G., Enomoto N., Feinstone S., Halfon P., Inchauspé G., Kuiken C.. & other authors ( 2005;). Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. . Hepatology 42:, 962–973. [CrossRef][PubMed]
    [Google Scholar]
  45. Tellinghuisen T. L., Marcotrigiano J., Rice C. M.. ( 2005;). Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. . Nature 435:, 374–379. [CrossRef][PubMed]
    [Google Scholar]
  46. Wakita T., Pietschmann T., Kato T., Date T., Miyamoto M., Zhao Z., Murthy K., Habermann A., Kräusslich H.-G.. & other authors ( 2005;). Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. . Nat Med 11:, 791–796. [CrossRef][PubMed]
    [Google Scholar]
  47. Yanagi M., Purcell R. H., Emerson S. U., Bukh J.. ( 1999;). Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras. . Virology 262:, 250–263. [CrossRef][PubMed]
    [Google Scholar]
  48. Yi M., Ma Y., Yates J., Lemon S. M.. ( 2007;). Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus. . J Virol 81:, 629–638. [CrossRef][PubMed]
    [Google Scholar]
  49. Zhong J., Gastaminza P., Cheng G., Kapadia S., Kato T., Burton D. R., Wieland S. F., Uprichard S. L., Wakita T., Chisari F. V.. ( 2005;). Robust hepatitis C virus infection in vitro. . Proc Natl Acad Sci U S A 102:, 9294–9299. [CrossRef][PubMed]
    [Google Scholar]
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