Identification of Cytoplasmic Chaperone Networks Relevant for Respiratory Syncytial Virus Replication

FIGURE 1. An RNAi screen for chaperones and co-chaperones involved in RSV replication. (A) Overview of the experimental approach. (B) Results of the primary screening, showing the strictly standardized mean deviation (SSMD) and the statistical significance (q-value). n = 6. (C) Results of the secondary screening, showing the SSMD and statistical significance. n = 6.

FIGURE 2. VCP/p97 inhibition reduces RSV and VSV production. (A) The effect of the VCP/p97 inhibitor CB-5083 on A549 cell viability (n = 6). (B) The effect of CB-5083 on RSV virus production in A549 (n = 6). (C) The effect of the VCP/p97 inhibitor CB-5083 on Hela-H1 cell viability (n = 6). (D,E) The effect of CB-5083 on RSV (n = 7) (D), vesicular stomatitis virus (VSV) (n = 7) (E), or coxsackievirus B3 (CVB3) production (n = 4). (F) Virus production in Hela-H1 cells. Results indicate the mean and SEM. nsp > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 by a two-tail, unpaired Mann-Whitney test/Wilcoxon rank sum exact test.

FIGURE 3. VCP/p97 is not involved in the early steps of infection. The VCP/p97 inhibitor CB-5083 was added at the indicated time point relative to infection (hours post-infection, hpi) with RSV (A) or VSV (B) and virus production was quantified. Data represent the mean and SEM of three replicates. ns, p > 0.05 by t-test on log-transformed data vs. drug treatment 1 h prior to infection (–1 hpi).

FIGURE 4. VCP/p97 is involved in multiple steps of the RSV and VSV infection cycle. Cells were treated with the VCP/p97 inhibitor CB-5083 or the Hsp90 inhibitor 17-DMAG and infected with RSV (top row) or VSV (bottom row). Subsequently, virus production was measured by plaque assay (A,D) and intracellular replication was measured by the expression of a reporter protein (B,E). The ratio of the antiviral effect observed for virus replication compared to virus production (C,F). Plotted are the means and SEM for three replicates. nsp > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by t-test on log-transformed data.

FIGURE 5. VCP/p97 treatment does not block the trafficking of viral glycoproteins to the surface. Cells were transfected with a plasmid encoding the VSV glycoprotein (VSV-G). Following 24 h, cells were either treated or mock-treated with the VCP/p97 inhibitor CB-5083. Following an additional 24 h, VSV-G expression was assessed by flow cytometry. Data represent the mean and SEM of three replicates. **p < 0.01 by t-test.