Gaie Brown

Respiratory syncytial virus assembly occurs in GM1-rich regions of the host-cell membrane and alters the cellular distribution of tyrosine phosphorylated caveolin-1.

We have previously shown that respiratory syncytial virus (RSV) assembly occurs within regions of the host-cell surface membrane that are enriched in the protein caveolin-1 (cav-1). In this report, we have employed immunofluorescence microscopy to further examine the RSV assembly process. Our results show that RSV matures at regions of the cell surface that, in addition to cav-1, are enriched in the lipid-raft ganglioside GM1. Furthermore, a comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the cellular distribution of phosphocaveolin-1 (pcav-1). In mock-infected cells, pcav-1 was located at regions of the cell that interact with the extracellular matrix, termed focal adhesions (FA). In contrast, RSV-infected cells showed both a decrease in the levels of pcav-1 associated with FA and the appearance of pcav-1-containing cytoplasmic vesicles, the latter being absent in mock-infected cells. These cytoplasmic vesicles were clearly visible between 9 and 18 h post-infection and coincided with the formation of RSV filaments, although we did not observe a direct association of pcav-1 with mature virus. In addition, we noted a strong colocalization between pcav-1 and growth hormone receptor binding protein-7 (Grb7), within these cytoplasmic vesicles, which was not observed in mock-infected cells. Collectively, these findings show that the RSV assembly process occurs within specialized lipid-raft structures on the host-cell plasma membrane, induces the cellular redistribution of pcav-1 and results in the formation of cytoplasmic vesicles that contain both pcav-1 and Grb7.

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

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.

Protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein 90 in virus particle assembly

In this study, we used imaging and proteomics to identify the presence of virus-associated cellular proteins that may play a role in respiratory syncytial virus (RSV) maturation. Fluorescence microscopy of virus-infected cells revealed the presence of virus-induced cytoplasmic inclusion bodies and mature virus particles, the latter appearing as virus filaments. In situ electron tomography suggested that the virus filaments were complex structures that were able to package multiple copies of the virus genome. The virus particles were purified, and the protein content was analyzed by one-dimensional nano-LC MS/MS. In addition to all the major virus structural proteins, 25 cellular proteins were also detected, including proteins associated with the cortical actin network, energy pathways, and heat shock proteins (HSP70, HSC70, and HSP90). Representative actin-associated proteins, HSC70, and HSP90 were selected for further biological validation. The presence of β-actin, filamin-1, cofilin-1, HSC70, and HSP90 in the virus preparation was confirmed by immunoblotting using relevant antibodies. Immunofluorescence microscopy of infected cells stained with antibodies against relevant virus and cellular proteins confirmed the presence of these cellular proteins in the virus filaments and inclusion bodies. The relevance of HSP90 to virus infection was examined using the specific inhibitors 17-N-Allylamino-17-demethoxygeldanamycin. Although virus protein expression was largely unaffected by these drugs, we noted that the formation of virus particles was inhibited, and virus transmission was impaired, suggesting an important role for HSP90 in virus maturation. This study highlights the utility of proteomics in facilitating both our understanding of the role that cellular proteins play during RSV maturation and, by extrapolation, the identification of new potential targets for antiviral therapy.

Distribution of the attachment (G) glycoprotein and GM1 within the envelope of mature respiratory syncytial virus filaments revealed using field emission scanning electron microscopy

Field emission scanning electron microscopy (FE SEM) was used to visualize the distribution of virus-associated components, the virus-attachment (G) protein, and the host-cell-derived lipid, GM1, in respiratory syncytial virus (RSV) filaments. RSV-infected cells were labeled in situ with a G protein antibody (MAb30) whose presence was detected using a second antibody conjugated to colloidal gold. No bound MAb30 was detected in mock-infected cells, whereas significant quantities bound to viral filaments revealing G protein clusters throughout the filaments. GM1 was detected using cholera toxin B subunit conjugated to colloidal gold. Mock-infected cells revealed numerous GM1 clusters on the cell surface. In RSV-infected cells, these gold clusters were detected on the filaments in low, but significant, amounts, indicating the incorporation of GM1 within the viral envelope. This report describes the first use of FE SEM to map the distribution of specific structural components within the envelope of a Paramyxovirus.

Evidence that selective changes in the lipid composition of raft-membranes occur during respiratory syncytial virus infection.

We examined the structure of lipid-raft membranes in respiratory syncytial virus infected cells. Cholesterol depletion studies using methyl-beta-cyclodextrin suggested that membrane cholesterol was required for virus filament formation, but not inclusion bodies. In addition, virus filament formation coincided with elevated 3-hydroxy-3-methylglutaryl-coenzyme A reductase expression, suggesting an increase in requirement for endogenous cholesterol synthesis during virus assembly. Lipid raft membranes were examined by mass spectrometry, which suggested that virus infection induced subtle changes in the lipid composition of these membrane structures. This analysis revealed increased levels of raft-associated phosphatidylinositol (PI) and phosphorylated PI during RSV infection, which correlated with the appearance of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate (PIP(3)) within virus inclusion bodies, and inhibiting the synthesis of PIP(3) impaired the formation of progeny virus. Collectively, our analysis suggests that RSV infection induces specific changes in the composition of raft-associated lipids, and that these changes play an important role in virus maturation.

Structural analysis of hepatitis C virus core-e1 signal Peptide and requirements for cleavage of the genotype 3a signal sequence by signal Peptide peptidase.

ABSTRACT The maturation of the hepatitis C virus (HCV) core protein requires proteolytic processing by two host proteases: signal peptidase (SP) and the intramembrane-cleaving protease signal peptide peptidase (SPP). Previous work on HCV genotype 1a (GT1a) and GT2a has identified crucial residues required for efficient signal peptide processing by SPP, which in turn has an effect on the production of infectious virus particles. Here we demonstrate that the JFH1 GT2a core-E1 signal peptide can be adapted to the GT3a sequence without affecting the production of infectious HCV. Through mutagenesis studies, we identified crucial residues required for core-E1 signal peptide processing, including a GT3a sequence-specific histidine (His) at position 187. In addition, the stable knockdown of intracellular SPP levels in HuH-7 cells significantly affects HCV virus titers, further demonstrating the requirement for SPP for the maturation of core and the production of infectious HCV particles. Finally, our nuclear magnetic resonance (NMR) structural analysis of a synthetic HCV JFH1 GT2a core-E1 signal peptide provides an essential structural template for a further understanding of core processing as well as the first model for an SPP substrate within its membrane environment. Our findings give deeper insights into the mechanisms of intramembrane-cleaving proteases and the impact on viral infections.

Multiple glycosylated forms of the respiratory syncytial virus fusion protein are expressed in virus-infected cells

Analysis of the respiratory syncytial virus (RSV) fusion (F) protein in RSV-infected Vero cells showed the presence of a single F1 subunit and at least two different forms of the F2 subunit, designated F2a (21 kDa) and F2b (16 kDa), which were collectively referred to as [F2](a/b). Enzymatic deglycosylation of [F2](a/b) produced a single 10 kDa product suggesting that [F2](a/b) arises from differences in the glycosylation pattern of F2a and F2b. The detection of [F2](a/b) was dependent upon the post-translational cleavage of the F protein by furin, since its appearance was prevented in RSV-infected Vero cells treated with the furin inhibitor dec-RVKR-cmk. Analysis by protein cross-linking revealed that the F1 subunit interacted with [F2](a/b), via disulphide bonding, to produce equivalent F protein trimers, which were expressed on the surface of infected cells. Collectively, these data show that multiple F protein species are expressed in RSV-infected cells.

Increased hydroxymethylglutaryl coenzyme A reductase activity during respiratory syncytial virus infection mediates actin dependent inter-cellular virus transmission

We have examined the role that hydroxymethylglutaryl coenzyme A reductase (HMGCR) plays during respiratory syncytial virus (RSV) maturation. Imaging analysis indicated that virus-induced changes in F-actin structure correlated with the formation of virus filaments, and that these virus filaments played a direct role in virus cell-to-cell transmission. Treatment with cytochalasin D (CYD) prevented virus filament formation and virus transmission, but this could be reversed by removal of CYD. This observation, together with the presence of F-actin within the virus filaments suggested that newly polymerised F-actin was required for virus transmission. The virus-induced change in F-actin was inhibited by the HMGCR inhibitor lovastatin, and this correlated with the inhibition of both virus filament formation and the incorporation of F-actin in these virus structures. Furthermore, this inhibitory effect on virus filament formation correlated with a significant reduction in RSV transmission. Collectively these data suggested that HMGCR-mediated changes in F-actin structure play an important role in the inter-cellular transmission of mature RSV particles. These data also highlighted the interplay between cellular metabolism and RSV transmission, and demonstrate that this interaction can be targeted using anti-virus strategies.

Functional Analysis of the N -Linked Glycans Within the Fusion Protein of Respiratory Syncytial Virus

The respiratory syncytial virus fusion (F) protein is initially expressed as a single polypeptide chain (F0). The F0 subsequently undergoes posttranslational cleavage-by-cell protease activity to produce the F1 and F2 subunits. Each of the two subunits within the mature F protein is modified by the addition of N-linked glycans. The individual N-linked glycans on the F protein were selectively removed by using site-directed mutagenesis to mutate the individual glycan-acceptor sites. In this way the role of these individual glycans in targeting of the F protein to the cell surface, and on the ability of the F protein to induce membrane fusion, was examined.

Secretion of the Respiratory Syncytial Virus Fusion Protein From Insect Cells Using the Baculovirus Expression System

Sequences derived from the respiratory syncytial virus (RSV) fusion (F) protein were expressed in insect cells as recombinant glutathione-S-transferase (GST)-tagged proteins. The sequence covering the F2 subunit (GST-F2), and a truncated form of the F protein in which the transmembrane domain was removed (GST-F2/F1), were cloned into the baculovirus pAcSecG2T secretory vector. These virus sequences also had the endogenous virus signal sequence removed and replaced with a signal sequence derived from the baculovirus gp67 glycoprotein, which was present in pAcSecG2T. The recombinant RSV glycoproteins were successfully detected in expressing cells by immunofluorescence assay and in the tissue culture medium by western blot analysis. The secreted recombinant GST-F2/F1 protein was further analysed using glycosidases. Our results showed that the GST-F2/F1 protein were sensitive to peptide:N-glycosidase F (PNGase F) treatment, but not to Endoglycosidase H (EndoH) treatment. This indicates that the secreted recombinant proteins were modified by the addition of mature N-linked glycan chains.