Host RAB11FIP5 protein inhibits the release of Kaposi’s sarcoma-associated herpesvirus particles by promoting lysosomal degradation of ORF45
Fig 5
Knockdown of endogenous RAB11FIP5 promotes the release of progeny virus.
(A) iSLK.RGB cells were transfected with control siRNA and two RAB11FIP5-specific siRNAs (#1 and #2). The knockdown efficiency was determined by western blotting. (B) iSLK.RGB cells were transfected with control siRNA and siRAB11FIP5-#2. Twenty-four hours after transfection, cells were induced with dox for different time points as indicated. Extracellular virions were collected from the culture medium and treated with DNase I. Viral DNA was extracted, and KSHV genomic DNA copy numbers were estimated by qPCR by comparison with external standards containing known concentrations of the viral K9 plasmid. (C) Supernatants (500 μl) collected from dox-induced cells at 72 hpi were incubated with HEK293T cells. The infection rate of HEK293T cells was examined by fluorescence microscopy. (D) Flow cytometry analysis and quantitation of the percentage of RFP+ cells from (C). (E) Intracellular KSHV genomic DNA was extracted from harvested cells and quantified by qPCR with normalization to GAPDH. (F) The transcription levels of KSHV genes were measured at 72 hpi. (G) The expression levels of several KSHV proteins were determined by immunoblotting at the indicated time post induction.