1887

Abstract

The hepatitis C virus (HCV) p7 ion channel and non-structural protein 2 (NS2) are both required for efficient assembly and release of nascent virions, yet precisely how these proteins are able to influence this process is unclear. Here, we provide both biochemical and cell biological evidence for a functional interaction between p7 and NS2. We demonstrate that in the context of a genotype 1b subgenomic replicon the localization of NS2 is affected by the presence of an upstream p7 with its cognate signal peptide derived from the C terminus of E2 (SPp7). Immunofluorescence analysis revealed that the presence of SPp7 resulted in the targeting of NS2 to sites closely associated with viral replication complexes. In addition, biochemical analysis demonstrated that, in the presence of SPp7, a significant proportion of NS2 was found in a detergent (Triton X-100)-insoluble fraction, which also contained a marker of detergent resistant rafts. In contrast, in replicons lacking p7, NS2 was entirely detergent soluble and the altered localization was lost. Furthermore, we found that serine 168 within NS2 was required for its localization adjacent to replication complexes, but not for its accumulation in the detergent-insoluble fraction. NS2 physically interacted with NS5A and this interaction was dependent on both p7 and serine 168 within NS2. Mutational and pharmacological analyses demonstrated that these effects were not a consequence of p7 ion channel function, suggesting that p7 possesses an alternative function that may influence the coordination of virus genome replication and particle assembly.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.027441-0
2011-04-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/92/4/819.html?itemId=/content/journal/jgv/10.1099/vir.0.027441-0&mimeType=html&fmt=ahah

References

  1. Aizaki, H., Lee, K. J., Sung, V. M., Ishiko, H. & Lai, M. M.(2004). Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. Virology 324, 450–461.[CrossRef] [Google Scholar]
  2. Aoubala, M., Holt, J., Clegg, R. A., Rowlands, D. J. & Harris, M.(2001). The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis. J Gen Virol 82, 1637–1646. [Google Scholar]
  3. Brohm, C., Steinmann, E., Friesland, M., Lorenz, I. C., Patel, A., Penin, F., Bartenschlager, R. & Pietschmann, T.(2009). Characterization of determinants important for hepatitis C virus p7 function in morphogenesis by using trans-complementation. J Virol 83, 11682–11693.[CrossRef] [Google Scholar]
  4. Carrère-Kremer, S., Montpellier-Pala, C., Cocquerel, L., Wychowski, C., Penin, F. & Dubuisson, J.(2002). Subcellular localization and topology of the p7 polypeptide of hepatitis C virus. J Virol 76, 3720–3730.[CrossRef] [Google Scholar]
  5. Carrère-Kremer, S., Montpellier, C., Lorenzo, L., Brulin, B., Cocquerel, L., Belouzard, S., Penin, F. & Dubuisson, J.(2004). Regulation of hepatitis C virus polyprotein processing by signal peptidase involves structural determinants at the p7 sequence junctions. J Biol Chem 279, 41384–41392.[CrossRef] [Google Scholar]
  6. Clarke, D., Griffin, S., Beales, L., Gelais, C. S., Burgess, S., Harris, M. & Rowlands, D.(2006). Evidence for the formation of a heptameric ion channel complex by the hepatitis C virus p7 protein in vitro. J Biol Chem 281, 37057–37068.[CrossRef] [Google Scholar]
  7. 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]
  8. Flajolet, M., Rotondo, G., Daviet, L., Bergametti, F., Inchauspé, G., Tiollais, P., Transy, C. & Legrain, P.(2000). A genomic approach of the hepatitis C virus generates a protein interaction map. Gene 242, 369–379.[CrossRef] [Google Scholar]
  9. Franck, N., Le Seyec, J., Guguen-Guillouzo, C. & Erdtmann, L.(2005). Hepatitis C virus NS2 protein is phosphorylated by the protein kinase CK2 and targeted for degradation to the proteasome. J Virol 79, 2700–2708.[CrossRef] [Google Scholar]
  10. Gao, L., Aizaki, H., He, J. W. & Lai, M. M.(2004). Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft. J Virol 78, 3480–3488.[CrossRef] [Google Scholar]
  11. 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]
  12. Griffin, S.(2010). Inhibition of HCV p7 as a therapeutic target. Curr Opin Investig Drugs 11, 175–181. [Google Scholar]
  13. Griffin, S. D. C., Beales, L. P., Clarke, D. S., Worsfold, O., Evans, S. D., Jaeger, J., Harris, M. P. G. & Rowlands, D. J.(2003). The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine. FEBS Lett 535, 34–38.[CrossRef] [Google Scholar]
  14. Griffin, S. D., Harvey, R., Clarke, D. S., Barclay, W. S., Harris, M. & Rowlands, D. J.(2004). A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria. J Gen Virol 85, 451–461.[CrossRef] [Google Scholar]
  15. Griffin, S., Clarke, D., McCormick, C., Rowlands, D. & Harris, M.(2005). Signal peptide cleavage and internal targeting signals direct the hepatitis C virus p7 protein to distinct intracellular membranes. J Virol 79, 15525–15536.[CrossRef] [Google Scholar]
  16. 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] [Google Scholar]
  17. Hughes, M., Gretton, S., Shelton, H., Brown, D. D., McCormick, C. J., Angus, A. G., Patel, A. H., Griffin, S. & Harris, M.(2009). A conserved proline between domains II and III of hepatitis C virus NS5A influences both RNA replication and virus assembly. J Virol 83, 10788–10796.[CrossRef] [Google Scholar]
  18. 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] [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. Kato, T., Date, T., Miyamoto, M., Furusaka, A., Tokushige, K., Mizokami, M. & Wakita, T.(2003). Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125, 1808–1817.[CrossRef] [Google Scholar]
  21. Krieger, N., Lohmann, V. & Bartenschlager, R.(2001). Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75, 4614–4624.[CrossRef] [Google Scholar]
  22. Lavanchy, D.(1999). Global surveillance and control of hepatitis C. Report of a WHO Consultation organized in collaboration with the Viral Hepatitis Prevention Board, Antwerp, Belgium. J Viral Hepat 6, 35–47.[CrossRef] [Google Scholar]
  23. Lin, C., Lindenbach, B. D., Prágai, 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]
  24. 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]
  25. Luik, P., Chew, C., Aittoniemi, J., Chang, J., Wentworth, P., Jr, Dwek, R. A., Biggin, P. C., Vénien-Bryan, C. & Zitzmann, N.(2009). The 3-dimensional structure of a hepatitis C virus p7 ion channel by electron microscopy. Proc Natl Acad Sci U S A 106, 12712–12716.[CrossRef] [Google Scholar]
  26. 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] [Google Scholar]
  27. Macdonald, A., Crowder, K., Street, A., McCormick, C., Saksela, K. & Harris, M.(2003). The hepatitis C virus NS5A protein inhibits activating protein-1 function by perturbing Ras-ERK pathway signalling. J Biol Chem 278, 17775–17784.[CrossRef] [Google Scholar]
  28. 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] [Google Scholar]
  29. McCormick, C. J., Challinor, L., Macdonald, A., Rowlands, D. J. & Harris, M.(2004). Introduction of replication-competent hepatitis C virus transcripts using a tetracycline-regulable baculovirus delivery system. J Gen Virol 85, 429–439.[CrossRef] [Google Scholar]
  30. Moradpour, D., Penin, F. & Rice, C. M.(2007). Replication of hepatitis C virus. Nat Rev Microbiol 5, 453–463.[CrossRef] [Google Scholar]
  31. Pavlović, D., Neville, D. C., Argaud, O., Blumberg, B., Dwek, R. A., Fischer, W. B. & Zitzmann, N.(2003). The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc Natl Acad Sci U S A 100, 6104–6108.[CrossRef] [Google Scholar]
  32. 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] [Google Scholar]
  33. Premkumar, A., Wilson, L., Ewart, G. D. & Gage, P. W.(2004). Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride. FEBS Lett 557, 99–103.[CrossRef] [Google Scholar]
  34. 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]
  35. Sakai, A., Claire, M. S., Faulk, K., Govindarajan, S., Emerson, S. U., Purcell, R. H. & Bukh, J.(2003). The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences. Proc Natl Acad Sci U S A 100, 11646–11651.[CrossRef] [Google Scholar]
  36. Santolini, E., Pacini, L., Fipaldini, C., Migliaccio, G. & Monica, N.(1995). The NS2 protein of hepatitis C virus is a transmembrane polypeptide. J Virol 69, 7461–7471. [Google Scholar]
  37. 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]
  38. Steinmann, E., Penin, F., Kallis, S., Patel, A. H., Bartenschlager, R. & Pietschmann, T.(2007a). Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions. PLoS Pathog 3, e103.[CrossRef] [Google Scholar]
  39. Steinmann, E., Whitfield, T., Kallis, S., Dwek, R. A., Zitzmann, N., Pietschmann, T. & Bartenschlager, R.(2007b). Antiviral effects of amantadine and iminosugar derivatives against hepatitis C virus. Hepatology 46, 330–338. [Google Scholar]
  40. StGelais, C., Tuthill, T. J., Clarke, D. S., Rowlands, D. J., Harris, M. & Griffin, S.(2007). Inhibition of hepatitis C virus p7 membrane channels in a liposome-based assay system. Antiviral Res 76, 48–58.[CrossRef] [Google Scholar]
  41. StGelais, C., Foster, T. L., Verow, M., Atkins, E., Fishwick, C. W., Rowlands, D., Harris, M. & Griffin, S.(2009). Determinants of hepatitis C virus p7 ion channel function and drug sensitivity identified in vitro. J Virol 83, 7970–7981.[CrossRef] [Google Scholar]
  42. Targett-Adams, P., Boulant, S. & McLauchlan, J.(2008). Visualization of double-stranded RNA in cells supporting hepatitis C virus RNA replication. J Virol 82, 2182–2195.[CrossRef] [Google Scholar]
  43. Tellinghuisen, T. L., Foss, K. L. & Treadaway, J.(2008). Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. PLoS Pathog 4, e1000032.[CrossRef] [Google Scholar]
  44. 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] [Google Scholar]
  45. Welbourn, S., Green, R., Gamache, I., Dandache, S., Lohmann, V., Bartenschlager, R., Meerovitch, K. & Pause, A.(2005). Hepatitis C virus NS2/3 processing is required for NS3 stability and viral RNA replication. J Biol Chem 280, 29604–29611.[CrossRef] [Google Scholar]
  46. Wozniak, A. L., Griffin, S., Rowlands, D. J., Harris, M., Yi, M., Lemon, S. M. & Weinman, S. A.(2010). Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production. PLoS Pathog 6, e1001087.[CrossRef] [Google Scholar]
  47. Yang, G., Pevear, D. C., Collett, M. S., Chunduru, S., Young, D. C., Benetatos, C. & Jordan, R.(2004). Newly synthesized hepatitis C virus replicon RNA is protected from nuclease activity by a protease-sensitive factor(s). J Virol 78, 10202–10205.[CrossRef] [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] [Google Scholar]
  49. Yi, M., Ma, Y., Yates, J. & Lemon, S. M.(2009). Trans-complementation of an NS2 defect in a late step in hepatitis C virus (HCV) particle assembly and maturation. PLoS Pathog 5, e1000403.[CrossRef] [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.027441-0
Loading
/content/journal/jgv/10.1099/vir.0.027441-0
Loading

Data & Media loading...

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