1887

Abstract

Among the least-known hepatitis C virus proteins is the non-structural protein 4B (NS4B). It localizes to the endoplasmic reticulum (ER) membrane and induces membrane changes, resulting in a membranous web that is reported to be the locale for virus replication. A model was presented previously for the topology of recombinant HCV NS4B of the 1a genotype based on data. In this model, the N-terminal tail of a considerable fraction of the NS4B molecules was translocated into the ER lumen via a post-translational process, giving the protein a dual transmembrane topology. It is now reported that translocation of the N terminus also occurs for processed NS4B expressed in cells in the context of the polyprotein. In the presence of NS5A, however, a lower degree of translocation was observed, which may indicate that NS5A influences the topology of NS4B. expression studies of NS4B from all major genotypes demonstrated that translocation of the N terminus to the ER lumen is conserved across genotypes. This clearly suggests an important function for this feature. Furthermore, when disrupting a previously reported amphipathic helix (AH) in the N terminus of NS4B, translocation was inhibited. As a disrupted AH also abolished the ability of NS4B to rearrange membranes, these data indicate for the first time an association between translocation of the N terminus and membrane rearrangement. Finally, the present experiments also confirm the predicted location of the first luminal loop to be around aa 112.

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2006-11-01
2019-11-19
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References

  1. Adams, N. J., Chamberlain, R. W., Taylor, L. A., Davidson, F., Lin, C. K., Elliott, R. M. & Simmonds, P. ( 1997; ). Complete coding sequence of hepatitis C virus genotype 6a. Biochem Biophys Res Commun 234, 393–396.[CrossRef]
    [Google Scholar]
  2. Appel, N., Herian, U. & Bartenschlager, R. ( 2005; ). Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A. J Virol 79, 896–909.[CrossRef]
    [Google Scholar]
  3. 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]
  4. 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]
  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. Bordier, C. ( 1981; ). Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 256, 1604–1607.
    [Google Scholar]
  7. Bruss, V. & Vieluf, K. ( 1995; ). Functions of the internal pre-S domain of the large surface protein in hepatitis B virus particle morphogenesis. J Virol 69, 6652–6657.
    [Google Scholar]
  8. Bruss, V., Lu, X., Thomssen, R. & Gerlich, W. H. ( 1994; ). Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein. EMBO J 13, 2273–2279.
    [Google Scholar]
  9. Chamberlain, R. W., Adams, N., Saeed, A. A., Simmonds, P. & Elliott, R. M. ( 1997a; ). Complete nucleotide sequence of a type 4 hepatitis C virus variant, the predominant genotype in the Middle East. J Gen Virol 78, 1341–1347.
    [Google Scholar]
  10. Chamberlain, R. W., Adams, N. J., Taylor, L. A., Simmonds, P. & Elliott, R. M. ( 1997b; ). The complete coding sequence of hepatitis C virus genotype 5a, the predominant genotype in South Africa. Biochem Biophys Res Commun 236, 44–49.[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. Egger, D., Wölk, B., Gosert, R., Bianchi, L., Blum, H. E., Moradpour, D. & Bienz, K. ( 2002; ). Expression of hepatitits C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76, 5974–5984.[CrossRef]
    [Google Scholar]
  13. Einav, S., Elazar, M., Danieli, T. & Glenn, J. S. ( 2004; ). A nucleotide binding motif in hepatitis C virus (HCV) NS4B mediates HCV RNA replication. J Virol 78, 11288–11295.[CrossRef]
    [Google Scholar]
  14. Elazar, M., Liu, P., Rice, C. M. & Glenn, J. S. ( 2004; ). An N-terminal amphipathic helix in hepatitis C virus (HCV) NS4B mediates membrane association, correct localization of replication complex proteins, and HCV RNA replication. J Virol 78, 11393–11400.[CrossRef]
    [Google Scholar]
  15. El-Hage, N. & Luo, G. ( 2003; ). Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA. J Gen Virol 84, 2761–2769.[CrossRef]
    [Google Scholar]
  16. Evans, M. J., Rice, C. M. & Goff, S. P. ( 2004; ). Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication. Proc Natl Acad Sci U S A 101, 13038–13043.[CrossRef]
    [Google Scholar]
  17. Gallinari, P., Brennan, D., Nardi, C., Brunetti, M., Tomei, L., Steinkühler, C. & De Francesco, R. ( 1998; ). Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus. J Virol 72, 6758–6769.
    [Google Scholar]
  18. 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]
  19. Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M. & Rice, C. M. ( 1993; ). Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites. J Virol 67, 2832–2843.
    [Google Scholar]
  20. Gretton, S. N., Taylor, A. I. & McLauchlan, J. ( 2005; ). Mobility of the hepatitis C virus NS4B protein on the endoplasmic reticulum membrane and membrane-associated foci. J Gen Virol 86, 1415–1421.[CrossRef]
    [Google Scholar]
  21. 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]
  22. Hegde, R. S. & Lingappa, V. R. ( 1999; ). Regulation of protein biogenesis at the endoplasmic reticulum membrane. Trends Cell Biol 9, 132–137.[CrossRef]
    [Google Scholar]
  23. Hijikata, M., Mizushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., Tanaka, T., Kimura, K. & Shimotohno, K. ( 1993; ). 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]
  24. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. ( 1989; ). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.[CrossRef]
    [Google Scholar]
  25. Isherwood, B. J. & Patel, A. H. ( 2005; ). Analysis of the processing and transmembrane topology of the E2p7 protein of hepatitis C virus. J Gen Virol 86, 667–676.[CrossRef]
    [Google Scholar]
  26. Kim, D. W., Gwack, Y., Han, J. H. & Choe, J. ( 1995; ). C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem Biophys Res Commun 215, 160–166.[CrossRef]
    [Google Scholar]
  27. Kim, J.-E., Song, W. K., Chung, K. M., Back, S. H. & Jang, S. K. ( 1999; ). Subcellular localization of hepatitis C viral proteins in mammalian cells. Arch Virol 144, 329–343.[CrossRef]
    [Google Scholar]
  28. Koch, J. O. & Bartenschlager, R. ( 1999; ). Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A, and NS4B. J Virol 73, 7138–7146.
    [Google Scholar]
  29. Lambert, C. & Prange, R. ( 2001; ). Dual topology of the hepatitis B virus large envelope protein: determinants influencing post-translational pre-S translocation. J Biol Chem 276, 22265–22272.[CrossRef]
    [Google Scholar]
  30. Le Seyec, J., Chouteau, P., Cannie, I., Guguen-Guillouzo, C. & Gripon, P. ( 1998; ). Role of the pre-S2 domain of the large envelope protein in hepatitis B virus assembly and infectivity. J Virol 72, 5573–5578.
    [Google Scholar]
  31. Le Seyec, J., Chouteau, P., Cannie, I., Guguen-Guillouzo, C. & Gripon, P. ( 1999; ). Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain. J Virol 73, 2052–2057.
    [Google Scholar]
  32. Levy, D. ( 1996; ). Membrane proteins which exhibit multiple topological orientations. Essays Biochem 31, 49–60.
    [Google Scholar]
  33. Lindström, H., Lundin, M., Häggström, S. & Persson, M. A. A. ( 2006; ). Mutations of the Hepatitis C virus protein NS4B on either side of the ER membrane affect the efficiency of subgenomic replicons. Virus Res (in press). doi:10.1016/j.virusres.2006.05.008
    [Google Scholar]
  34. Lohmann, V., Körner, F., Koch, J.-O., 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]
  35. Lohmann, V., Körner, F., Dobierzewska, A. & Bartenschlager, R. ( 2001; ). Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol 75, 1437–1449.[CrossRef]
    [Google Scholar]
  36. Lohmann, V., Hoffmann, S., Herian, U., Penin, F. & Bartenschlager, R. ( 2003; ). Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J Virol 77, 3007–3019.[CrossRef]
    [Google Scholar]
  37. Lundin, M., Monné, M., Widell, A., von Heijne, G. & Persson, M. A. A. ( 2003; ). Topology of the membrane-associated hepatitis C virus protein NS4B. J Virol 77, 5428–5438.[CrossRef]
    [Google Scholar]
  38. Major, M. E., Rehermann, B. & Feinstone, S. M. ( 2001; ). Hepatitis C viruses. In Fields Virology, pp. 1127–1161. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
  39. Moradpour, D., Bieck, E., Hügle, T., Wels, W., Wu, J. Z., Hong, Z., Blum, H. E. & Bartenschlager, R. ( 2002; ). Functional properties of a monoclonal antibody inhibiting the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem 277, 593–601.[CrossRef]
    [Google Scholar]
  40. Moradpour, D., Gosert, R., Egger, D., Penin, F., Blum, H. E. & Bienz, K. ( 2003; ). Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex. Antiviral Res 60, 103–109.[CrossRef]
    [Google Scholar]
  41. Mottola, G., Cardinal, G., Cessacci, 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]
  42. Namba, K., Naka, K., Dansako, H., Nozaki, A., Ikeda, M., Shiratori, Y., Shimotohno, K. & Kato, N. ( 2004; ). Establishment of hepatitis C virus replicon cell lines possessing interferon-resistant phenotype. Biochem Biophys Res Commun 323, 299–309.[CrossRef]
    [Google Scholar]
  43. Neurath, A. R., Kent, S. B. H., Strick, N. & Parker, K. ( 1986; ). Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus. Cell 46, 429–436.[CrossRef]
    [Google Scholar]
  44. Nilsson, I. & von Heijne, G. ( 1993; ). Determination of the distance between the oligosaccharyltransferase active site and the endoplasmatic reticulum membrane. J Biol Chem 268, 5798–5801.
    [Google Scholar]
  45. Ostapchuk, P., Hearing, P. & Ganem, D. ( 1994; ). A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis. EMBO J 13, 1048–1057.
    [Google Scholar]
  46. Popov, M., Tam, L. Y., Li, J. & Reithmeier, R. A. F. ( 1997; ). Mapping of the ends of transmembrane segments in a polytopic membrane protein. J Biol Chem 272, 18325–18332.[CrossRef]
    [Google Scholar]
  47. Prange, R. & Streeck, R. E. ( 1995; ). Novel transmembrane topology of the hepatitis B virus envelope proteins. EMBO J 14, 247–256.
    [Google Scholar]
  48. Yanagi, M., Purcell, R. H., Emerson, S. U. & Bukh, J. ( 1997; ). Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci U S A 94, 8738–8743.[CrossRef]
    [Google Scholar]
  49. Zhu, Q., Guo, J.-T. & Seeger, C. ( 2003; ). Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells. J Virol 77, 9204–9210.[CrossRef]
    [Google Scholar]
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vol. , part 11, pp. 3263 – 3272

Sequences of the oligonucleotides used for mutagenesis, transcriptions and construction of the different vectors [ PDF] (116 KB)



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