1887

Abstract

We have employed immunofluorescence microscopy and transmission electron microscopy to examine the assembly and maturation of respiratory syncytial virus (RSV) in the Vero cell line C1008. RSV matures at the apical cell surface in a filamentous form that extends from the plasma membrane. We observed that inclusion bodies containing viral ribonucleoprotein (RNP) cores predominantly appeared immediately below the plasma membrane, from where RSV filaments form during maturation at the cell surface. A comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the pattern of caveolin-1 (cav-1) fluorescence staining. Analysis by immuno-electron microscopy showed that RSV filaments formed in close proximity to cav-1 clusters at the cell surface membrane. In addition, immuno-electron microscopy showed that cav-1 was closely associated with early budding RSV. Further analysis by confocal microscopy showed that cav-1 was subsequently incorporated into the envelope of RSV filaments maturing on the host cell membrane, but was not associated with other virus structures such as the viral RNPs. Although cav-1 was incorporated into the mature virus, it was localized in clusters rather than being uniformly distributed along the length of the viral filaments. Furthermore, when RSV particles in the tissue culture medium from infected cells were examined by immuno-negative staining, the presence of cav-1 on the viral envelope was clearly demonstrated. Collectively, these findings show that cav-1 is incorporated into the envelope of mature RSV particles during egress.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-83-3-611
2002-03-01
2019-10-23
Loading full text...

Full text loading...

/deliver/fulltext/jgv/83/3/0830611a.html?itemId=/content/journal/jgv/10.1099/0022-1317-83-3-611&mimeType=html&fmt=ahah

References

  1. Ali, A. & Nayak, D. P. ( 2000; ). Assembly of Sendai virus: M protein interacts with F and HN proteins and with the cytoplasmic tail and transmembrane domain of F protein. Virology 276, 289-303.[CrossRef]
    [Google Scholar]
  2. Bohn, W., Rutter, G., Hohenberg, H., Mannweiler, K. & Nobis, P. ( 1986; ). Involvement of actin filaments in budding of measles virus: studies on cytoskeletons of infected cells. Virology 149, 91-106.[CrossRef]
    [Google Scholar]
  3. Burke, E., Dupuy, L., Wall, C. & Barik, S. ( 1998; ). Role of cellular actin in the gene expression and morphogenesis of human respiratory syncytial virus. Virology 252, 137-148.[CrossRef]
    [Google Scholar]
  4. Calafat, J., Janssen, H., Démant, P., Hilgers, J. & Závada, J. ( 1983; ). Specific selection of host cell glycoproteins during assembly of murine leukaemia virus and vesicular stomatitis virus: presence of Thy-1 glycoprotein and absence of H-2, Pgp-1 and T-200 glycoproteins on the envelopes of these virus particles. Journal of General Virology 64, 1241-1253.[CrossRef]
    [Google Scholar]
  5. Cudmore, S., Cossart, P., Griffiths, G. & Way, M. ( 1995; ). Actin-based motility of vaccinia virus. Nature 378, 636-638.[CrossRef]
    [Google Scholar]
  6. Damsky, C. H., Sheffield, J. B., Tuszynski, G. P. & Warren, L. ( 1977; ). Is there a role for actin in virus budding? Journal of Cell Biology 75, 593-605.[CrossRef]
    [Google Scholar]
  7. Engelman, J. A., Chu, C., Lin, A., Jo, H., Ikezu, T., Okamoto, T., Kohtz, D. S. & Lisanti, M. P. ( 1998; ). Caveolin-mediated regulation of signaling along the p42/44 MAP kinase cascade in vivo. A role for the caveolin-scaffolding domain. FEBS Letters 428, 205-211.[CrossRef]
    [Google Scholar]
  8. Galbiati, F., Volonte, D., Engelman, J. A., Watanabe, G., Burk, R., Pestell, R. G. & Lisanti, M. P. ( 1998; ). Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade. EMBO Journal 17, 6633-6648.[CrossRef]
    [Google Scholar]
  9. Garcia, J., Garcia-Barreno, B., Vivo, A. & Melero, J. A. ( 1993; ). Cytoplasmic inclusions of respiratory syncytial virus-infected cells: formation of inclusion bodies in transfected cells that coexpress the nucleoprotein, the phosphoprotein, and the 22K protein. Virology 195, 243-247.[CrossRef]
    [Google Scholar]
  10. Garcia-Barreno, B., Delgado, T. & Melero, J. A. ( 1996; ). Identification of protein regions involved in the interaction of human respiratory syncytial virus phosphoprotein and nucleoprotein: significance for nucleocapsid assembly and formation of cytoplasmic inclusions. Journal of Virology 70, 801-808.
    [Google Scholar]
  11. Garcia-Cardena, G., Martasek, P., Masters, B. S., Skidd, P. M., Couet, J., Li, S., Lisanti, M. P. & Sessa, W. C. ( 1997; ). Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo. Journal of Biological Chemistry 272, 2543-2540.
    [Google Scholar]
  12. Ghosh, S., Gachhui, R., Crooks, C., Wu, C., Lisanti, M. P. & Stuehr, D. J. ( 1998; ). Interaction between caveolin-1 and the reductase domain of endothelial nitric-oxide synthase. Consequences for catalysis. Journal of Biological Chemistry 273, 22267-22271.[CrossRef]
    [Google Scholar]
  13. Gingras, D., Gauthier, F., Lamy, S., Desrosiers, R. R. & Beliveau, R. ( 1998; ). Localization of RhoA GTPase to endothelial caveolae-enriched membrane domains. Biochemical and Biophysical Research Communications 247, 888-893.[CrossRef]
    [Google Scholar]
  14. Gower, T. L., Peeples, M. E., Collins, P. L. & Graham, B. S. ( 2001; ). RhoA is activated during respiratory syncytial virus infection. Virology 283, 188-196.[CrossRef]
    [Google Scholar]
  15. Ju, H., Zou, R., Venema, V. J. & Venema, R. C. ( 1997; ). Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. Journal of Biological Chemistry 272, 18522-18525.[CrossRef]
    [Google Scholar]
  16. Kurzchalia, T. V., Dupree, P. & Monier, S. ( 1994; ). VIP21-caveolin, a protein of the trans-Golgi network and caveolae. FEBS Letters 346, 88-91.[CrossRef]
    [Google Scholar]
  17. Lodish, H. F. & Porter, M. ( 1980; ). Specific incorporation of host cell surface proteins into budding vesicular stomatitis virus particles. Cell 19, 161-169.[CrossRef]
    [Google Scholar]
  18. Manie, S. N., Debreyne, S., Vincent, S. & Gerlier, D. ( 2000; ). Measles virus structural components are enriched into lipid raft microdomains: a potential cellular location for virus assembly. Journal of Virology 74, 305-311.[CrossRef]
    [Google Scholar]
  19. Marschang, P., Sodroski, J., Wurzner, R. & Dierich, M. P. ( 1995; ). Decay-accelerating factor (CD55) protects human immunodeficiency virus type 1 from inactivation by human complement. European Journal of Immunology 25, 285-290.[CrossRef]
    [Google Scholar]
  20. Parry, J. E., Shirodaria, P. V. & Pringle, C. R. ( 1979; ). Pneumoviruses: the cell surface of lytically and persistently infected cells. Journal of General Virology 44, 479-491.[CrossRef]
    [Google Scholar]
  21. Pastey, M. K., Crowe, J. E.Jr & Graham, B. S. ( 1999; ). RhoA interacts with the fusion glycoprotein of respiratory syncytial virus and facilitates virus-induced syncytium formation. Journal of Virology 73, 7262-7270.
    [Google Scholar]
  22. Pickl, W. F., Pimentel-Muinos, F. X. & Seed, B. ( 2001; ). Lipid rafts and pseudotyping. Journal of Virology 75, 7175-7183.[CrossRef]
    [Google Scholar]
  23. Rey, O., Canon, J. & Krogstad, P. ( 1996; ). HIV-1 Gag protein associates with F-actin present in microfilaments. Virology 220, 530-534.[CrossRef]
    [Google Scholar]
  24. Roberts, S. R., Compans, R. W. & Wertz, G. W. ( 1995; ). Respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells. Journal of Virology 69, 2667-2673.
    [Google Scholar]
  25. Saifuddin, M., Ghassemi, M., Patki, C., Parker, C. J. & Spear, G. T. ( 1994; ). Host cell components affect the sensitivity of HIV type 1 to complement-mediated virolysis. AIDS Research and Human Retroviruses 10, 829-837.
    [Google Scholar]
  26. Sasaki, H., Nakamura, M., Ohno, T., Matsuda, Y., Yuda, Y. & Nonomura, Y. ( 1995; ). Myosin–actin interaction plays an important role in human immunodeficiency virus type 1 release from host cells. Proceedings of the National Academy of Sciences, USA 92, 2026-2030.[CrossRef]
    [Google Scholar]
  27. Smart, E. J., Graf, G. A., McNiven, M. A., Sessa, W. C., Engelman, J. A., Scherer, P. E., Okamoto, T. & Lisanti, M. P. ( 1999; ). Caveolins, liquid-ordered domains, and signal transduction. Molecular and Cellular Biology 19, 7289-7304.
    [Google Scholar]
  28. Stahlhut, M. & van Deurs, B. ( 2000; ). Identification of filamin as a novel ligand for caveolin-1: evidence for the organization of caveolin-1-associated membrane domains by the actin cytoskeleton. Molecular and Cellular Biology 11, 325-337.[CrossRef]
    [Google Scholar]
  29. Sugrue, R. J., Brown, C., Brown, G., Aitken, J. & Rixon, H. W. McL. ( 2001; ). Furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells. Journal of General Virology 82, 1375-1386.
    [Google Scholar]
  30. Taylor, G., Stott, E. J., Furze, J., Ford, J. & Sopp, P. ( 1992; ). Protective epitopes on the fusion protein of respiratory syncytial virus recognized by murine and bovine monoclonal antibodies. Journal of General Virology 73, 2217-2223.[CrossRef]
    [Google Scholar]
  31. Ulloa, L., Serra, R., Asenjo, A. & Villanueva, N. ( 1998; ). Interactions between cellular actin and human respiratory syncytial virus (HRSV). Virus Research 53, 13-25.[CrossRef]
    [Google Scholar]
  32. Vincent, S., Gerlier, D. & Manie, S. N. ( 2000; ). Measles virus assembly within membrane rafts. Journal of Virology 74, 9911-9915.[CrossRef]
    [Google Scholar]
  33. Werling, D., Hop, J. C., Chaplin, P., Collins, R. A., Taylor, G. & Howard, C. J. ( 1999; ). Involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells. Journal of Leukocyte Biology 66, 50-58.
    [Google Scholar]
  34. Wright, C., Oliver, K. C., Fenwick, F. I., Smith, N. M. & Toms, G. L. ( 1997; ). A monoclonal pool for routine immunohistochemical detection of human respiratory syncytial virus antigens in formalin fixed, paraffin-embedded tissue. Journal of Pathology 182, 238-244.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-83-3-611
Loading
/content/journal/jgv/10.1099/0022-1317-83-3-611
Loading

Data & Media loading...

Most Cited This Month

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