Figure 3. Cholesterol facilitates viral RNA synthesis and attenuates viral 3CD^pro processing.

(A) Acute free cholesterol extraction from PM inhibits PV replication. Mean data +/− SEM of PV replication and cholesterol levels is plotted (n=50 cells for each condition).

(B) Cholesterol rescues CVB3 replicon replication. Mean data +/− SEM, from 6 replicates for each condition is plotted.

(C) Inhibition of replication organelle biogenesis after acute MβCD treatment. Antibodies against dsRNA and 3A proteins detect replication organelles. Scale bar 5um.

(D) Cell-free PV RNA translation and synthesis assay. Viral RNA synthesis assayed by ³²P-CTP (top) and translation assayed by ³⁵S-methionine labeling (bottom) on filipin treated membranes.

(E) Immunoblot analysis of 3CD^pro, 2BC and 3AB processing after mock; MβCD (1 hr pre-treatment); or filipin treatment (at 3 hr pi). Viral proteins were harvested at 5 hr pi.

(F) 3CD^pro/3D^pol protein ratio after cholesterol perturbation. Mean data +/− SEM from 3 independent experiments is plotted.

Figure 3 related to Figure S3.