1887

Abstract

The hepatitis C virus (HCV) non-structural protein NS4B induces morphological changes in the endoplasmic reticulum (ER) membrane that may have a direct role in viral RNA replication. A chimeric GFP–NS4B fusion protein located to the ER membrane and to foci that were attached to the ER. These embrane-ssociated oci (MAFs) could be related to the membrane alterations observed in cells that replicate HCV RNA. The relationship of MAFs to pre-existing cellular structures is not known. Indirect immunofluorescence analysis demonstrated that they did not contain a cellular marker for vesicles, which have been implicated in the replication of other viruses. From photobleaching studies to examine diffusion of NS4B, the GFP-tagged protein had reduced mobility on MAFs compared with on the ER membrane. This slower mobility suggested that NS4B is likely to form different interactions on MAFs and the ER.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.80768-0
2005-05-01
2020-01-21
Loading full text...

Full text loading...

/deliver/fulltext/jgv/86/5/vir861415.html?itemId=/content/journal/jgv/10.1099/vir.0.80768-0&mimeType=html&fmt=ahah

References

  1. 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]
  2. 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]
  3. Gao, L., Aizaki, H., He, J.-W. & Lai, M. M. C. ( 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]
  4. 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]
  5. Hope, R. G. & McLauchlan, J. ( 2000; ). Sequence motifs required for lipid droplet association and protein stability are unique to the hepatitis C virus core protein. J Gen Virol 81, 1913–1925.
    [Google Scholar]
  6. 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]
  7. Ikeda, M., Yi, M.-K., 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]
  8. Konan, K. V., Giddings, T. H., Jr, Ikeda, M., Li, K., Lemon, S. M. & Kirkegaard, K. ( 2003; ). Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus. J Virol 77, 7843–7855.[CrossRef]
    [Google Scholar]
  9. Lohmann, V., Korner, 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]
  10. Lundin, M., Monne, 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]
  11. Marguet, D., Spiliotis, E. T., Pentcheva, T., Lebowitz, M., Schneck, J. & Edidin, M. ( 1999; ). Lateral diffusion of GFP-tagged H2Ld molecules and of GFP–TAP1 reports on the assembly and retention of these molecules in the endoplasmic reticulum. Immunity 11, 231–240.[CrossRef]
    [Google Scholar]
  12. Moradpour, D., Evans, M. J., Gosert, R., Yuan, Z.-H., Blum, H. E., Goff, S. P., Lindenbach, B. D. & Rice, C. M. ( 2004; ). Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes. J Virol 78, 7400–7409.[CrossRef]
    [Google Scholar]
  13. Niv, H., Gutman, O., Kloog, Y. & Henis, Y. I. ( 2002; ). Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells. J Cell Biol 157, 865–872.[CrossRef]
    [Google Scholar]
  14. Patel, J., Patel, A. H. & McLauchlan, J. ( 1999; ). Covalent interactions are not required to permit or stabilize the non-covalent association of hepatitis C virus glycoproteins E1 and E2. J Gen Virol 80, 1681–1690.
    [Google Scholar]
  15. Reits, E. A. J. & Neefjes, J. J. ( 2001; ). From fixed to FRAP: measuring protein mobility and activity in living cells. Nat Cell Biol 3, E145–E147.[CrossRef]
    [Google Scholar]
  16. Rust, R. C., Landmann, L., Gosert, R., Tang, B. L., Hong, W.-J., Hauri, H.-P., Egger, D. & Bienz, K. ( 2001; ). Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex. J Virol 75, 9808–9818.[CrossRef]
    [Google Scholar]
  17. Saffman, P. G. & Delbruck, M. ( 1975; ). Brownian motion in biological membranes. Proc Natl Acad Sci U S A 72, 3111–3113.[CrossRef]
    [Google Scholar]
  18. Stephens, D. J. ( 2003; ). De novo formation, fusion and fission of mammalian COPII-coated endoplasmic reticulum exit sites. EMBO Rep 4, 210–217.[CrossRef]
    [Google Scholar]
  19. Stephens, D. J., Lin-Marq, N., Pagano, A., Pepperkok, R. & Paccaud, J. P. ( 2000; ). COPI-coated ER-to-Golgi transport complexes segregate from COPII in close proximity to ER exit sites. J Cell Sci 113, 2177–2185.
    [Google Scholar]
  20. Targett-Adams, P., Chambers, D., Gledhill, S., Hope, R. G., Coy, J. F., Girod, A. & McLauchlan, J. ( 2003; ). Live cell analysis and targeting of the lipid droplet-binding adipocyte differentiation-related protein. J Biol Chem 278, 15998–16007.[CrossRef]
    [Google Scholar]
  21. Vaz, W. L. C., Criado, M., Madeira, V. M. C., Schoellmann, G. & Jovin, T. M. ( 1982; ). Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching. Biochemistry 21, 5608–5612.[CrossRef]
    [Google Scholar]
  22. 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]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.80768-0
Loading
/content/journal/jgv/10.1099/vir.0.80768-0
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