Respiratory syncytial virus assembles into structured filamentous virion particles independently of host cytoskeleton and related proteins

Abstract

Respiratory syncytial virus (RSV) is a single-stranded RNA virus that assembles into viral filaments at the cell surface. Virus assembly often depends on the ability of a virus to use host proteins to accomplish viral tasks. Since the fusion protein cytoplasmic tail (FCT) is critical for viral filamentous assembly, we hypothesized that host proteins important for viral assembly may be recruited by the FCT. Using a yeast two-hybrid screen, we found that filamin A interacted with FCT, and mammalian cell experiments showed it localized to viral filaments but did not affect viral replication. Furthermore, we found that a number of actin-associated proteins also were excluded from viral filaments. Actin or tubulin cytoskeletal rearrangement was not necessary for F trafficking to the cell surface or for viral assembly into filaments, but was necessary for optimal viral replication and may be important for anchoring viral filaments. These findings suggest that RSV assembly into filaments occurs independently of actin polymerization and that viral proteins are the principal drivers for the mechanical tasks involved with formation of complex, structured RSV filaments at the host cell plasma membrane.

Figure 1. Cellular localization of candidate proteins from an FCT Y2H screen.

HEp-2 cells were inoculated with RSV strain A2 at than MOI = 1.0 and incubated for 24 hours. RSV F and the indicated cellular proteins were detected by indirect immunofluorescence. Column 1 (panels A, E, I, and M) shows mock-infected cells, and columns 2–4 show RSV infected cells. Column 2 (panels B, F, J, and N) shows the indicated RSV protein; column 3 (panels C, G, K, and O) shows the indicated cellular proteins; and column 4 (panels D, H, L, and P) shows the overlay with the RSV protein in green and the cellular protein in red.

Figure 2. Cellular localization of cytoskeletal proteins found in MIRE vesicles.

HEp-2 cells were inoculated with RSV stain A2 at an MOI = 1.0 and incubated for 24 hours. RSV F and the indicated cellular proteins were detected by indirect immunofluorescence. Columns 1 and 3 (panels A, C, E, G, I, and K) show mock-infected cells, while columns 2 and 4 (panels B, D, F, H, J, and L) show RSV infected cells. RSV F is shown in green and the indicated cellular protein is shown in red.

Figure 3. Viral titers in FLNA knockdown cells.

HEp-2 cells were transduced with a lentivirus encoding an shRNA directed against the indicated protein. After selection, cell lysates were harvest and immunoblotted for the presence of filamin A, GAPDH, or actin (A). The same cells then were infected with RSV stain A2 at an MOI = 0.05 for 72 hours. Both cell-associated (B) and supernatant virus (C) yields were quantified by a plaque assay.

Figure 4. Effect of cytoskeletal inhibitors on viral assembly.

HEp-2 cells were inoculated with RSV wt strain A2 at an MOI = 1.0 and incubated for 24 hours with medium containing either vehicle of the indicated inhibitor. At 24 hours cells were fixed, and RSV F, actin, and tubulin were detected by indirect immunofluorescence. Column 1 (panels A, E, I, and M) shows mock-infected cells in the presence of vehicle. Column 2 (panels B, F, J, and N) shows mock-infected cells in the presence of the indicated inhibitor. Column 3 (panels C, G, K, and O) shows RSV infected cells in the presence of vehicle. Column 4 (panels D, H, L, and P) shows RSV infected cells in the presence of the indicated inhibitor. Panel Q: HEp-2 cells were infected with RSV at an MOI = 0.1 for 72 hours. Supernatant and cell-associated fractions were collected in duplicate and quantified using a viral plaque assay. Viral yields are presented in log₁₀ scale. Panel R: total surface expression of RSV F was determined by flow cytometric analysis in mock-infected or RSV-infected cells (MOI = 3.0) treated with vehicle or the indicated inhibitor for 24 hours. Data are plotted as mean, and error bars represent standard deviation. Weighted mean fluorescence intensity (MFI) is MFI multiplied by the frequency of positive cells.