Hsp70 Isoforms Are Essential for the Formation of Kaposi’s Sarcoma-Associated Herpesvirus Replication and Transcription Compartments
Fig 10
Specific depletion of Hsc70 significantly reduced KSHV lytic transcription in HEK-293T rKSHV.219 cells.
Cells were transfected twice with 100 nM Hsc70-specific siRNA or 100 nM scramble siRNA. Following four days post-siRNA transfection, cells were either reactivated for 24 h or remained unreactivated and total RNA and protein were extracted from the same sample. Despite achieving ~ 85% Hsc70 knockdown at the mRNA level (A), significant amounts of Hsc70 protein remained in Hsc70 siRNA-treated samples (B). Despite this small knockdown at the protein level, in Hsc70-depleted cells there was a significant decrease in the amount of multiple viral transcripts from various temporal classes as quantified by RT-PCR analysis (C). Viral DNA replication was assessed by qPCR following Hsc70-knockdown. Cells were reactivated for 72 h after four days post-siRNA transfection. No significant differences were observed between scramble and Hsc70-treated cells (D). Similar virion production was detected in scramble and Hsc70-treated cells. Cells were reactivated for 72 h after four days post-siRNA transfection, culture medium was centrifuged and immediately incubated with HEK-293T cells for 24 h. Total RNA was then isolated and qRT-PCR carried out (E). Incomplete Hsc70-knockdown at the protein level was observed by Western blotting even after seven days post-siRNA transfection in HEK-293T rKSHV.219 cells. This demonstrates the remarkable stability of Hsc70 in this cell line (F). The average of three independent transfections is shown with error bars as standard deviation (A, C-E).