siRNA-Based Inhibition of Autophagy in Herpes Simplex Virus-Infected Immature Dendritic Cells

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.

On day 3.5 post adherence, transfer 12 million iDCs into a 50-milliliter tube. Centrifuge them at 300 times g for 5 minutes, and then discard the supernatant. In parallel, perform flow cytometric analysis to monitor the maturation status.

Gently wash the iDCs in 5 milliliters of OptiMEM without phenol red, and centrifuge them at 300 times g for 5 minutes. Discard the supernatant and resuspend the cells in 200 microliters of OptiMEM, adjusting the cell concentration to 6 million cells per 100 microliters.

Add 75 picamoles of either FIP200-specific or scrambled siRNA to 4-millimeter electrode cuvettes and transfer 100 microliters of the cell suspension into the respective cuvette. Directly pulse the iDCs using an electroporation apparatus.

After electroporation, transfer the iDCs into six-well plates with fresh pre-warmed DC medium, seeding the cells at a final concentration of 1 million cells per milliliter, and place them in the incubator. After 48 hours, examine the morphology of the electroporated iDCs microscopically, then harvest the cells with a cell scraper, and transfer them into 15-milliliter tubes.

Rinse the wells with 1 milliliter of PBS supplemented with 0.01% EDTA and transfer the solution to the respective tubes. Then, split the cells for each siRNA condition as described in the manuscript.

Use 500,000 cells to assess maturation status and cell viability. Use 1 million cells for western blot analysis to verify 200 specific knockdown efficiency, and use the remaining cells for HSV-1 infection experiments.