Fig 6.

Effect of host autophagy response on virus replication. (A) SNV infection induces autophagy in infected HUVEC. HUVEC were infected with SNV at an MOI of 10, and then cells were collected at 12-h time intervals, lysed in RIPA buffer, and stored at −20°C. The total proteins in cell lysates were quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). Equal amounts of proteins of each sample (10 μg) were loaded on an SDS-PAGE gel, and the LC3-II/LC3-I ratios were assessed by Western blotting. Autophagy was triggered as early as 12 h postinfection. (B) Induction of autophagy is essential for virus replication. HUVEC were transfected with either control siRNA or specific siRNA targeting the autophagy-essential genes BCLN-1 and Atg-7. At 48 h posttransfection, cells were infected with SNV at an MOI of 10. Virus production was monitored by quantifying S-segment RNA using real-time PCR. A similar experiment was performed with cells treated with lysosome protease inhibitors E64d and pepstatin A, as described in Materials and Methods. Virus replication in untreated cells resembled that of cells treated with a control siRNA and is not shown.