siRNA-Based Inhibition of Autophagy in Herpes Simplex Virus-Infected Immature Dendritic Cells
Transcription
Take two electroporation cuvettes. One cuvette contains a small interfering RNA, or siRNA, designed to inhibit the production of FIP200, an autophagy-inducing protein.
The second cuvette contains a scrambled siRNA as a control that does not inhibit FIP200 production.
Add immature dendritic cells, or iDCs; electroporate for the cellular entry of siRNA and then plate the cells.
The internalized siRNA binds to an RNA-inducing silencing complex.
The complex with the FIP200-specific siRNA cleaves its target mRNA, inhibiting FIP200 production, while the control siRNA-treated iDCs produce FIP200.
Harvest the cells and add the herpes simplex virus. Upon viral envelope-cell membrane fusion, the released DNA enters the nucleus and leads to the production of viral proteins and nucleocapsids.
Viral proteins disrupt nuclear lamins. FIP200 forms a complex that triggers a signaling cascade, inducing autophagy of the disrupted lamins to target them for degradation and facilitating the escape of viral nucleocapsids into the cytoplasm.
siRNA-mediated inhibition of FIP200 production restricts lamin autophagy, preventing nucleocapsid escape.
The cells are ready to assess siRNA-induced autophagy inhibition.
