In this report, the interaction between respiratory syncytial virus (RSV) and heat shock protein 70 (HSP70) was examined. Although no significant increase in total HSP70 protein levels was observed during virus infection, analysis of the HSP70 content in lipid-raft membranes from mock- and virus-infected cells revealed an increase in the levels of raft-associated HSP70 during virus infection. Fluorescence microscopy demonstrated that this transport of HSP70 into lipid-raft membranes correlated with the appearance of HSP70 within virus-induced inclusion bodies. Furthermore, co-localisation of HSP70 with the virus N protein and the raft lipid GM1 was observed within these structures. Immunoprecipitation experiments demonstrated the ability of HSP70 to interact with the virus polymerase complex in lipid-rafts in an ATP-dependent manner. Collectively, these data suggest that RSV may induce cellular changes which allow the recruitment of specific host-cell factors, via lipid-raft membranes, to the polymerase complex.

The cellular distribution of the small hydrophobic (SH) protein in respiratory syncytial virus (RSV)-infected cells was examined. Although the SH protein was distributed throughout the cytoplasm, it appeared to accumulate in the Golgi complex within membrane structures that were enriched in the raft lipid, GM1. The ability of the SH protein to interact with lipid-raft membranes was further confirmed by examining its detergent-solubility properties in Triton X-100 at 4 6C. This analysis showed that a large proportion of the SH protein exhibited detergent-solubility characteristics that were consistent with an association with lipid-raft membranes. Analysis of virus-infected cells by immuno-transmission electron microscopy revealed SH protein clusters on the cell surface, but only very low levels of the protein appeared to be associated with mature virus filaments and inclusion bodies. These data suggest that during virus infection, the compartments in the secretory pathway, such as the endoplasmic reticulum (ER) and Golgi complex, are major sites of accumulation of the SH protein. Furthermore, although a significant amount of this protein interacts with lipid-raft membranes within the Golgi complex, its presence within mature virus filaments is minimal.

The assembly of respiratory syncytial virus (RSV) in lipid-rafts was examined in Hep2 cells. Confocal and electron microscopy showed that during RSV assembly, the cellular distribution of the complement regulatory proteins, decay accelerating factor (CD55) and CD59, changes and high levels of these cellular proteins are incorporated into mature virus filaments. The detergent-solubility properties of CD55, CD59, and the RSV fusion (F) protein were found to be consistent with each protein being located predominantly within lipid-raft structures. The levels of these proteins in cell-released virus were examined by immunoelectronmicroscopy and found to account for between 5% and 15% of the virus attachment (G) glycoprotein levels. Collectively, our findings suggest that an intimate association exists between RSV and lipid-raft membranes and that significant levels of these host-derived raft proteins, such as those regulating complement activation, are subsequently incorporated into the envelope of mature virus particles.

The interaction between the respiratory syncytial virus (RSV) polymerase complex and lipid rafts was examined in HEp2 cells. Lipid-raft membranes were prepared from virus-infected cells and their protein content was analysed by Western blotting and mass spectrometry. This analysis revealed the presence of the N, P, L, M2-1 and M proteins. However, these proteins appeared to differ from one another in their association with these structures, with the M2-1 protein showing a greater partitioning into raft membranes compared to that of the N, P or M proteins. Determination of the polymerase activity profile of the gradient fractions revealed that 95% of the detectable viral enzyme activity was associated with lipid-raft membranes. Furthermore, analysis of virus-infected cells by confocal microscopy suggested an association between these proteins and the raft-lipid, GM1. Together, these results provide evidence that the RSV polymerase complex is able to associate with lipid rafts in virus-infected cells.

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