Caveolin-1 is incorporated into mature respiratory syncytial virus particles during virus assembly on the surface of virus-infected cells

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.

Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells

“…Lastly, EM analyses of HEp-2 cells inoculated for 1 h with hRSV GFP showed that most cell-attached virus particles displayed well-defined electron-dense envelopes. These data are consistent with previous studies (33) and further support the notion that N signal on the surface of infected cells at 1 hpi is due to adsorbed, structurally intact, viral particles (Fig. 2H).…”

The Human Respiratory Syncytial Virus Matrix Protein Is Required for Maturation of Viral Filaments

“…There is ample evidence of a role for polymerized actin and actinregulatory proteins in assembly (4,6,11,22,30,32,49,53,59). However, the role of polymerized actin seems to be limited to the actual budding/release event (11,30,32), which was shown previously to occur at sites of lipid rafts (8,9,22,27,31,36,42,49,50). In contrast to polymerized actin, microtubules were reported previously to be involved in virus production at a step prior to budding (32).…”

“…Several reports have implicated cytoskeletal elements in the processes that lead to virus egress, in particular microtubules, myosin V, actin, and actin-regulatory proteins such as profilin and RhoA (30,32,59). In addition, a role for an actin/myosinbased motility system in HRSV exit has been proposed (6,53,59), and there is strong evidence for the involvement of lipid rafts (8,9,22,27,31,36,49,50,53).…”

Structural and Functional Aspects of the Small Hydrophobic (SH) Protein in the Human Respiratory Syncytial Virus

“…Lipid rafts are enriched in cholesterol and sphingolipids and form a platform for various protein-protein interactions necessary during signal transduction events (Dykstra et al, 2003), protein trafficking (Helms & Zurzolo, 2004), and also virus entry, assembly, and budding (Suzuki & Suzuki, 2006). Indeed, hRSV has been shown to utilize lipid rafts, and in particular caveolae, a caveolin-1 enriched subdomain (Werling et al, 1999;Brown et al, 2002), to gain entry and in the assembly of virus particles. Only a very low amount of SH protein is associated with the viral envelope (Rixon et al, 2004).…”