1887

Abstract

Biochemical studies revealed that nonstructural proteins of hepatitis C virus (HCV) interacted with each other and were associated with intracellular membranes. The goals of this study were to determine whether nonstructural viral proteins are colocalized at specific intracellular sites where HCV RNA is replicated and to identify the virus components of the HCV replication complex (RC). Immunofluorescence and subcellular fractionation studies were performed to determine the intracellular colocalization of nonstructural HCV proteins and the replicating RNA in a human hepatoma cell line, Huh7, in which a subgenomic HCV RNA was replicated persistently. The replicating HCV RNA was labelled with 5-bromouridine 5′-triphosphate (BrUTP). Results show that each of the nonstructural HCV proteins was colocalized predominantly with the newly synthesized HCV RNA labelled with BrUTP and an endoplasmic reticulum (ER) protein, calnexin. Consistent with these findings, subcellular fractionation and Western blot analyses revealed that the nonstructural HCV proteins were colocalized with HCV RNA mainly in the membrane fractions. Conversely, the viral nonstructural proteins and RNA remained in the soluble fractions upon treatment with detergent, confirming the membrane association of the HCV RC. HCV RNA in the membrane-bound RC was resistant to RNase treatment, whereas it became sensitive to RNases once the membranes were disrupted by treatment with detergent, suggesting that the HCV RC is assembled within membrane structures. Collectively, these findings demonstrate that HCV RNA replication occurs in the perinuclear ER membrane-bound HCV RC, containing nonstructural viral proteins and RNA.

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2003-10-01
2019-10-17
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References

  1. Banerjee, R. & Dasgupta, A. ( 2001; ). Specific interaction of hepatitis C virus protease/helicase NS3 with the 3′-terminal sequences of viral positive- and negative-strand RNA. J Virol 75, 1708–1721.[CrossRef]
    [Google Scholar]
  2. Bartenschlager, R., Ahlborn-Laake, L., Mous, J. & Jacobsen, H. ( 1993; ). Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol 67, 3835–3844.
    [Google Scholar]
  3. Bartenschlager, R., Ahlborn-Laake, L., Mous, J. & Jacobsen, H. ( 1994; ). Kinetic and structural analyses of hepatitis C virus polyprotein processing. J Virol 68, 5045–5055.
    [Google Scholar]
  4. Bartenschlager, R., Lohmann, V., Wilkinson, T. & Koch, J. O. ( 1995; ). Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation. J Virol 69, 7519–7528.
    [Google Scholar]
  5. Behrens, S. E., Tomei, L. & De Francesco, R. ( 1996; ). Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J 15, 12–22.
    [Google Scholar]
  6. 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]
  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. Bukh, J., Pietschmann, T., Lohmann, V. & 7 other authors ( 2002; ). Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc Natl Acad Sci U S A 99, 14416–14421.[CrossRef]
    [Google Scholar]
  9. 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]
  10. De Francesco, R. & Steinkuhler, C. ( 2000; ). Structure and function of the hepatitis C virus NS3–NS4A serine proteinase. Curr Top Microbiol Immunol 242, 149–169.
    [Google Scholar]
  11. Egger, D., Teterina, N., Ehrenfeld, E. & Bienz, K. ( 2000; ). Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis. J Virol 74, 6570–6580.[CrossRef]
    [Google Scholar]
  12. Egger, D., Wolk, B., Gosert, R., Bianchi, L., Blum, H. E., Moradpour, D. & Bienz, K. ( 2002; ). Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76, 5974–5984.[CrossRef]
    [Google Scholar]
  13. Froshauer, S., Kartenbeck, J. & Helenius, A. ( 1988; ). Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes. J Cell Biol 107, 2075–2086.[CrossRef]
    [Google Scholar]
  14. Gontarek, R. R., Gutshall, L. L., Herold, K. M., Tsai, J., Sathe, G. M., Mao, J., Prescott, C. & Del Vecchio, A. M. ( 1999; ). hnRNP C and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3′NTR of the HCV RNA genome. Nucleic Acids Res 27, 1457–1463.[CrossRef]
    [Google Scholar]
  15. Gosert, R., Egger, D., Lohmann, V., Bartenschlager, R., Blum, H. E., Bienz, K. & Moradpour, D. ( 2003; ). Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol 77, 5487–5492.[CrossRef]
    [Google Scholar]
  16. 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]
  17. Guo, J. T., Bichko, V. V. & Seeger, C. ( 2001; ). Effect of alpha interferon on the hepatitis C virus replicon. J Virol 75, 8516–8523.[CrossRef]
    [Google Scholar]
  18. Hardy, R. W., Marcotrigiano, J., Blight, K. J., Majors, J. E. & Rice, C. M. ( 2003; ). Hepatitis C virus RNA synthesis in a cell-free system isolated from replicon-containing hepatoma cells. J Virol 77, 2029–2037.[CrossRef]
    [Google Scholar]
  19. Haukenes, G. & Kalland, H.-H. ( 1998; ). Visualisation of ribosomal RNA (rRNA) synthesis in eukaryotic cells in culture. Methods Cell Sci 19, 295–302.[CrossRef]
    [Google Scholar]
  20. Hijikata, M., Kato, N., Ootsuyama, Y., Nakagawa, M. & Shimotohno, K. ( 1991; ). Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc Natl Acad Sci U S A 88, 5547–5551.[CrossRef]
    [Google Scholar]
  21. Hugle, T., Fehrmann, F., Bieck, E., Kohara, M., Krausslich, H. G., Rice, C. M., Blum, H. E. & Moradpour, D. ( 2001; ). The hepatitis C virus nonstructural protein 4B is an integral endoplasmic reticulum membrane protein. Virology 284, 70–81.[CrossRef]
    [Google Scholar]
  22. Hwang, S. B., Park, K. J., Kim, Y. S., Sung, Y. C. & Lai, M. M. ( 1997; ). Hepatitis C virus NS5B protein is a membrane-associated phosphoprotein with a predominantly perinuclear localization. Virology 227, 439–446.[CrossRef]
    [Google Scholar]
  23. Ikeda, M., Yi, M., Li, K. & Lemon, S. M. ( 2002; ). Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol 76, 2997–3006.[CrossRef]
    [Google Scholar]
  24. Ishido, S., Fujita, T. & Hotta, H. ( 1998; ). Complex formation of NS5B with NS3 and NS4A proteins of hepatitis C virus. Biochem Biophys Res Commun 244, 35–40.[CrossRef]
    [Google Scholar]
  25. Ito, T. & Lai, M. M. ( 1997; ). Determination of the secondary structure of and cellular protein binding to the 3′-untranslated region of the hepatitis C virus RNA genome. J Virol 71, 8698–8706.
    [Google Scholar]
  26. Lai, V. C., Dempsey, S., Lau, J. Y., Hong, Z. & Zhong, W. ( 2003; ). In vitro RNA replication directed by replicase complexes isolated from the subgenomic replicon cells of hepatitis C virus. J Virol 77, 2295–2300.[CrossRef]
    [Google Scholar]
  27. Lin, C., Lindenbach, B. D., Pragai, B. M., McCourt, D. W. & Rice, C. M. ( 1994; ). Processing in the hepatitis C virus E2–NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J Virol 68, 5063–5073.
    [Google Scholar]
  28. Lin, C., Wu, J. W., Hsiao, K. & Su, M. S. ( 1997; ). The hepatitis C virus NS4A protein: interactions with the NS4B and NS5A proteins. J Virol 71, 6465–6471.
    [Google Scholar]
  29. Lohmann, V., Korner, F., Herian, U. & Bartenschlager, R. ( 1997; ). Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. J Virol 71, 8416–8428.
    [Google Scholar]
  30. Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L. & Bartenschlager, R. ( 1999a; ). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113.[CrossRef]
    [Google Scholar]
  31. Lohmann, V., Overton, H. & Bartenschlager, R. ( 1999b; ). Selective stimulation of hepatitis C virus and pestivirus NS5B RNA polymerase activity by GTP. J Biol Chem 274, 10807–10815.[CrossRef]
    [Google Scholar]
  32. 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]
  33. Luo, G. ( 1999; ). Cellular proteins bind to the poly(U) tract of the 3′ untranslated region of hepatitis C virus RNA genome. Virology 256, 105–118.[CrossRef]
    [Google Scholar]
  34. Luo, G., Hamatake, R. K., Mathis, D. M., Racela, J., Rigat, K. L., Lemm, J. & Colonno, R. J. ( 2000; ). De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus. J Virol 74, 851–863.[CrossRef]
    [Google Scholar]
  35. Luo, G., Xin, S. & Cai, Z. ( 2003; ). Role of the 5′-proximal stem–loop structure of the 5′ untranslated region in replication and translation of hepatitis C virus RNA. J Virol 77, 3312–3318.[CrossRef]
    [Google Scholar]
  36. Mackenzie, J. M., Jones, M. K. & Young, P. R. ( 1996; ). Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication. Virology 220, 232–240.[CrossRef]
    [Google Scholar]
  37. Mottola, G., Cardinali, G., Ceccacci, A., Trozzi, C., Bartholomew, L., Torrisi, M. R., Pedrazzini, E., Bonatti, S. & Migliaccio, G. ( 2002; ). Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons. Virology 293, 31–43.[CrossRef]
    [Google Scholar]
  38. Oh, J. W., Ito, T. & Lai, M. M. ( 1999; ). A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA. J Virol 73, 7694–7702.
    [Google Scholar]
  39. Petrik, J., Parker, H. & Alexander, G. J. ( 1999; ). Human hepatic glyceraldehyde-3-phosphate dehydrogenase binds to the poly(U) tract of the 3′ non-coding region of hepatitis C virus genomic RNA. J Gen Virol 80, 3109–3113.
    [Google Scholar]
  40. Reed, K. E. & Rice, C. M. ( 2000; ). Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol Immunol 242, 55–84.
    [Google Scholar]
  41. Restrepo-Hartwig, M. A. & Ahlquist, P. ( 1996; ). Brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA synthesis. J Virol 70, 8908–8916.
    [Google Scholar]
  42. Restrepo-Hartwig, M. & Ahlquist, P. ( 1999; ). Brome mosaic virus RNA replication proteins 1a and 2a colocalize and 1a independently localizes on the yeast endoplasmic reticulum. J Virol 73, 10303–10309.
    [Google Scholar]
  43. Rice, C. M. ( 1996; ). Flaviviridae: the viruses and their replication. In Fields Virology, 3rd edn, pp. 931–956. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.
  44. Schmidt-Mende, J., Bieck, E., Hugle, T., Penin, F., Rice, C. M., Blum, H. E. & Moradpour, D. ( 2001; ). Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem 276, 44052–44063.[CrossRef]
    [Google Scholar]
  45. Schwartz, M., Chen, J., Janda, M., Sullivan, M., den Boon, J. & Ahlquist, P. ( 2002; ). A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids. Mol Cell 9, 505–514.[CrossRef]
    [Google Scholar]
  46. Selby, M. J., Glazer, E., Masiarz, F. & Houghton, M. ( 1994; ). Complex processing and protein : protein interactions in the E2 : NS2 region of HCV. Virology 204, 114–122.[CrossRef]
    [Google Scholar]
  47. Shi, S. T., Lee, K. J., Aizaki, H., Hwang, S. B. & Lai, M. M. ( 2003; ). Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2. J Virol 77, 4160–4168.[CrossRef]
    [Google Scholar]
  48. Suhy, D. A., Giddings, T. H., Jr & Kirkegaard, K. ( 2000; ). Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles. J Virol 74, 8953–8965.[CrossRef]
    [Google Scholar]
  49. Tsuchihara, K., Tanaka, T., Hijikata, M., Kuge, S., Toyoda, H., Nomoto, A., Yamamoto, N. & Shimotohno, K. ( 1997; ). Specific interaction of polypyrimidine tract-binding protein with the extreme 3′-terminal structure of the hepatitis C virus genome, the 3′X. J Virol 71, 6720–6726.
    [Google Scholar]
  50. Tu, H., Gao, L., Shi, S. T. & 7 other authors ( 1999; ). Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein. Virology 263, 30–41.[CrossRef]
    [Google Scholar]
  51. Wessel, D. & Flugge, U. I. ( 1984; ). A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138, 141–143.[CrossRef]
    [Google Scholar]
  52. Westaway, E. G., Mackenzie, J. M., Kenney, M. T., Jones, M. K. & Khromykh, A. A. ( 1997; ). Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures. J Virol 71, 6650–6661.
    [Google Scholar]
  53. Westaway, E. G., Khromykh, A. A. & Mackenzie, J. M. ( 1999; ). Nascent flavivirus RNA colocalized in situ with double-stranded RNA in stable replication complexes. Virology 258, 108–117.[CrossRef]
    [Google Scholar]
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