1887

Journal of General Virology header logo

Volume 87, Issue 11

Dual topology of the processed hepatitis C virus protein NS4B is influenced by the NS5A protein Free

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.

  • Received:
  • Accepted:
  • Published Online:
SGM
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.82211-0
2006-11-01
2024-04-16
Loading full text...

Full text loading...

/deliver/fulltext/jgv/87/11/3263.html?itemId=/content/journal/jgv/10.1099/vir.0.82211-0&mimeType=html&fmt=ahah

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 Knipe D. M., Howley P. M. Philadelphia, PA: Lippincott Williams & Wilkins;
     [Google Scholar]
  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]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.82211-0
Loading
Dual topology of the processed hepatitis C virus protein NS4B is influenced by the NS5A protein
J. Gen. Virol. 87, 3263 (2006); https://doi.org/10.1099/vir.0.82211-0
/content/journal/jgv/10.1099/vir.0.82211-0
/content/journal/jgv/10.1099/vir.0.82211-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

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