1887

Abstract

Robust production of infectious hepatitis C virus (HCV) in cell culture was realized by using the JFH1 strain and the homologous chimeric J6/JFH1 strain in Huh-7.5 cells, a highly HCV-permissive subclone of Huh-7 cells. In this study, we aimed to establish a more efficient HCV-production system and to gain some insight into the adaptation mechanisms of efficient HCV production. By serial passaging of J6/JFH1-infected Huh-7.5 cells, we obtained culture-adapted J6/JFH1 variants, designated P-27, P-38 and P-47. Sequence analyses revealed that the adaptive mutant viruses P-27, P-38 and P-47 possessed eight mutations [four in E2, two in NS2, one in NS5A and one in NS5B), 10 mutations [two additional mutations in the 5′-untranslated region (5′-UTR) and core] and 11 mutations (three additional mutations in 5′-UTR, core and NS5B), respectively. We introduced amino acid substitutions into the wild-type J6/JFH1 clone, generated recombinant viruses with adaptive mutations and analysed their infectivity and ability to produce infectious viruses. The viruses with the adaptive mutations exhibited higher expression of HCV proteins than did the wild type in Huh-7.5 cells. Moreover, we provide evidence suggesting that the mutation N534H in the E2 glycoprotein of the mutant viruses conferred an advantage at the entry level. We thus demonstrate that an efficient HCV-production system could be obtained by introducing adaptive mutations into the J6/JFH1 genome. The J6/JFH1-derived mutant viruses presented here would be a good tool for producing HCV particles with enhanced infectivity and for studying the molecular mechanism of HCV entry.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.010983-0
2009-07-01
2020-01-24
Loading full text...

Full text loading...

/deliver/fulltext/jgv/90/7/1681.html?itemId=/content/journal/jgv/10.1099/vir.0.010983-0&mimeType=html&fmt=ahah

References

  1. Aizaki, H., Morikawa, K., Fukasawa, M., Hara, H., Inoue, Y., Tani, H., Saito, K., Nishijima, M., Hanada, K. & other authors ( 2008; ). Critical role of virion-associated cholesterol and sphingolipid in hepatitis C virus infection. J Virol 82, 5715–5724.[CrossRef]
    [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]
    [Google Scholar]
  3. Bartenschlager, R. & Sparacio, S. ( 2007; ). Hepatitis C virus molecular clones and their replication capacity in vivo and in cell culture. Virus Res 127, 195–207.[CrossRef]
    [Google Scholar]
  4. Bartosch, B., Vitelli, A., Granier, C., Goujon, C., Dubuisson, J., Pascale, S., Scarselli, E., Cortese, R., Nicosia, A. & Cosset, F. L. ( 2003; ). Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J Biol Chem 278, 41624–41630.[CrossRef]
    [Google Scholar]
  5. Blight, K. J., Kolykhalov, A. A. & Rice, C. M. ( 2000; ). Efficient initiation of HCV RNA replication in cell culture. Science 290, 1972–1974.[CrossRef]
    [Google Scholar]
  6. Blight, K. J., McKeating, J. A. & Rice, C. M. ( 2002; ). Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 76, 13001–13014.[CrossRef]
    [Google Scholar]
  7. Brass, V., Bieck, E., Montserret, R., Wolk, B., Hellings, J. A., Blum, H. E., Penin, F. & Moradpour, D. ( 2002; ). An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J Biol Chem 277, 8130–8139.[CrossRef]
    [Google Scholar]
  8. Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W. & Houghton, M. ( 1989; ). Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359–362.[CrossRef]
    [Google Scholar]
  9. Choo, Q. L., Richman, K. H., Han, J. H., Berger, K., Lee, C., Dong, C., Gallegos, C., Coit, D., Medina-Selby, R. & other authors ( 1991; ). Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A 88, 2451–2455.[CrossRef]
    [Google Scholar]
  10. Delgrange, D., Pillez, A., Castelain, S., Cocquerel, L., Rouille, Y., Dubuisson, J., Wakita, T., Duverlie, G. & Wychowski, C. ( 2007; ). Robust production of infectious viral particles in Huh-7 cells by introducing mutations in hepatitis C virus structural proteins. J Gen Virol 88, 2495–2503.[CrossRef]
    [Google Scholar]
  11. Dimitrova, M., Imbert, I., Kieny, M. P. & Schuster, C. ( 2003; ). Protein-protein interactions between hepatitis C virus nonstructural proteins. J Virol 77, 5401–5414.[CrossRef]
    [Google Scholar]
  12. Evans, M. J., von Hahn, T., Tscherne, D. M., Syder, A. J., Panis, M., Wolk, B., Hatziioannou, T., McKeating, J. A., Bieniasz, P. D. & Rice, C. M. ( 2007; ). Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446, 801–805.[CrossRef]
    [Google Scholar]
  13. Goffard, A., Callens, N., Bartosch, B., Wychowski, C., Cosset, F. L., Montpellier, C. & Dubuisson, J. ( 2005; ). Role of N-linked glycans in the functions of hepatitis C virus envelope glycoproteins. J Virol 79, 8400–8409.[CrossRef]
    [Google Scholar]
  14. 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]
    [Google Scholar]
  15. Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M. & Rice, C. M. ( 1993; ). A second hepatitis C virus-encoded proteinase. Proc Natl Acad Sci U S A 90, 10583–10587.[CrossRef]
    [Google Scholar]
  16. Helle, F. & Dubuisson, J. ( 2008; ). Hepatitis C virus entry into host cells. Cell Mol Life Sci 65, 100–112.[CrossRef]
    [Google Scholar]
  17. Hijikata, M., Mizushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., Tanaka, T., Kimura, K. & Shimotohno, K. ( 1993a; ). Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol 67, 4665–4675.
    [Google Scholar]
  18. Hijikata, M., Mizushima, H., Tanji, Y., Komoda, Y., Hirowatari, Y., Akagi, T., Kato, N., Kimura, K. & Shimotohno, K. ( 1993b; ). Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A 90, 10773–10777.[CrossRef]
    [Google Scholar]
  19. Jones, C. T., Murray, C. L., Eastman, D. K., Tassello, J. & Rice, C. M. ( 2007; ). Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol 81, 8374–8383.[CrossRef]
    [Google Scholar]
  20. 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]
    [Google Scholar]
  21. Kuo, G., Choo, Q. L., Alter, H. J., Gitnick, G. L., Redeker, A. G., Purcell, R. H., Miyamura, T., Dienstag, J. L., Alter, M. J. & other authors ( 1989; ). An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 244, 362–364.[CrossRef]
    [Google Scholar]
  22. Lindenbach, B. D., Evans, M. J., Syder, A. J., Wolk, 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]
    [Google Scholar]
  23. Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L. & Bartenschlager, R. ( 1999; ). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113.[CrossRef]
    [Google Scholar]
  24. Lohmann, V., Korner, F., Dobierzewska, A. & Bartenschlager, R. ( 2001; ). Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol 75, 1437–1449.[CrossRef]
    [Google Scholar]
  25. Lorenz, I. C., Marcotrigiano, J., Dentzer, T. G. & Rice, C. M. ( 2006; ). Structure of the catalytic domain of the hepatitis C virus NS2–3 protease. Nature 442, 831–835.[CrossRef]
    [Google Scholar]
  26. Manns, M. P., Foster, G. R., Rockstroh, J. K., Zeuzem, S., Zoulim, F. & Houghton, M. ( 2007; ). The way forward in HCV treatment – finding the right path. Nat Rev Drug Discov 6, 991–1000.[CrossRef]
    [Google Scholar]
  27. Masaki, T., Suzuki, R., Murakami, K., Aizaki, H., Ishii, K., Murayama, A., Date, T., Matsuura, Y., Miyamura, T., Wakita, T. & Suzuki, T. ( 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]
    [Google Scholar]
  28. 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]
    [Google Scholar]
  29. 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]
    [Google Scholar]
  30. Muramatsu, S., Ishido, S., Fujita, T., Itoh, M. & Hotta, H. ( 1997; ). Nuclear localization of the NS3 protein of hepatitis C virus and factors affecting the localization. J Virol 71, 4954–4961.
    [Google Scholar]
  31. Murray, C. L., Jones, C. T., Tassello, J. & Rice, C. M. ( 2007; ). Alanine scanning of the hepatitis C virus core protein reveals numerous residues essential for production of infectious virus. J Virol 81, 10220–10231.[CrossRef]
    [Google Scholar]
  32. Owsianka, A. M., Timms, J. M., Tarr, A. W., Brown, R. J., Hickling, T. P., Szwejk, A., Bienkowska-Szewczyk, K., Thomson, B. J., Patel, A. H. & Ball, J. K. ( 2006; ). Identification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding. J Virol 80, 8695–8704.[CrossRef]
    [Google Scholar]
  33. Penin, F., Brass, V., Appel, N., Ramboarina, S., Montserret, R., Ficheux, D., Blum, H. E., Bartenschlager, R. & Moradpour, D. ( 2004; ). Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A. J Biol Chem 279, 40835–40843.[CrossRef]
    [Google Scholar]
  34. Pietschmann, T., Lohmann, V., Kaul, A., Krieger, N., Rinck, G., Rutter, G., Strand, D. & Bartenschlager, R. ( 2002; ). Persistent and transient replication of full-length hepatitis C virus genomes in cell culture. J Virol 76, 4008–4021.[CrossRef]
    [Google Scholar]
  35. Pileri, P., Uematsu, Y., Campagnoli, S., Galli, G., Falugi, F., Petracca, R., Weiner, A. J., Houghton, M., Rosa, D. & other authors ( 1998; ). Binding of hepatitis C virus to CD81. Science 282, 938–941.[CrossRef]
    [Google Scholar]
  36. Poynard, T., Yuen, M. F., Ratziu, V. & Lai, C. L. ( 2003; ). Viral hepatitis C. Lancet 362, 2095–2100.[CrossRef]
    [Google Scholar]
  37. Roccasecca, R., Ansuini, H., Vitelli, A., Meola, A., Scarselli, E., Acali, S., Pezzanera, M., Ercole, B. B., McKeating, J. & other authors ( 2003; ). Binding of the hepatitis C virus E2 glycoprotein to CD81 is strain specific and is modulated by a complex interplay between hypervariable regions 1 and 2. J Virol 77, 1856–1867.[CrossRef]
    [Google Scholar]
  38. Russell, R. S., Meunier, J. C., Takikawa, S., Faulk, K., Engle, R. E., Bukh, J., Purcell, R. H. & Emerson, S. U. ( 2008; ). Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus. Proc Natl Acad Sci U S A 105, 4370–4375.[CrossRef]
    [Google Scholar]
  39. Saito, I., Miyamura, T., Ohbayashi, A., Harada, H., Katayama, T., Kikuchi, S., Watanabe, Y., Koi, S., Onji, M. & other authors ( 1990; ). Hepatitis C virus infection is associated with the development of hepatocellular carcinoma. Proc Natl Acad Sci U S A 87, 6547–6549.[CrossRef]
    [Google Scholar]
  40. Scarselli, E., Ansuini, H., Cerino, R., Roccasecca, R. M., Acali, S., Filocamo, G., Traboni, C., Nicosia, A., Cortese, R. & Vitelli, A. ( 2002; ). The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J 21, 5017–5025.[CrossRef]
    [Google Scholar]
  41. Schaller, T., Appel, N., Koutsoudakis, G., Kallis, S., Lohmann, V., Pietschmann, T. & Bartenschlager, R. ( 2007; ). Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes. J Virol 81, 4591–4603.[CrossRef]
    [Google Scholar]
  42. Sumpter, R., Jr, Loo, Y. M., Foy, E., Li, K., Yoneyama, M., Fujita, T., Lemon, S. M. & Gale, M., Jr ( 2005; ). Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I. J Virol 79, 2689–2699.[CrossRef]
    [Google Scholar]
  43. Takigawa, Y., Nagano-Fujii, M., Deng, L., Hidajat, R., Tanaka, M., Mizuta, H. & Hotta, H. ( 2004; ). Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome. Microbiol Immunol 48, 591–598.[CrossRef]
    [Google Scholar]
  44. Tellinghuisen, T. L., Marcotrigiano, J., Gorbalenya, A. E. & Rice, C. M. ( 2004; ). The NS5A protein of hepatitis C virus is a zinc metalloprotein. J Biol Chem 279, 48576–48587.[CrossRef]
    [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]
    [Google Scholar]
  46. Tellinghuisen, T. L., Foss, K. L., Treadaway, J. C. & Rice, C. M. ( 2008; ). Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein. J Virol 82, 1073–1083.[CrossRef]
    [Google Scholar]
  47. Wakita, T., Pietschmann, T., Kato, T., Date, T., Miyamoto, M., Zhao, Z., Murthy, K., Habermann, A., Krausslich, 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]
    [Google Scholar]
  48. Yamaga, A. K. & Ou, J. H. ( 2002; ). Membrane topology of the hepatitis C virus NS2 protein. J Biol Chem 277, 33228–33234.[CrossRef]
    [Google Scholar]
  49. Yi, M., Villanueva, R. A., Thomas, D. L., Wakita, T. & Lemon, S. M. ( 2006; ). Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci U S A 103, 2310–2315.[CrossRef]
    [Google Scholar]
  50. 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]
    [Google Scholar]
  51. 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]
    [Google Scholar]
  52. Zhong, J., Gastaminza, P., Chung, J., Stamataki, Z., Isogawa, M., Cheng, G., McKeating, J. A. & Chisari, F. V. ( 2006; ). Persistent hepatitis C virus infection in vitro: coevolution of virus and host. J Virol 80, 11082–11093.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.010983-0
Loading
/content/journal/jgv/10.1099/vir.0.010983-0
Loading

Data & Media loading...

Most cited articles

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