Fig 5. SMPD4 knockdown facilitates PRV Spread.

A: Tri-chamber Anterograde Spread Assay workflow–Dissociated SCG neurons are seeded in the soma-S-compartment (left), growing axons penetrate through the middle-M-compartment into the neurite-N-compartment (right). siRNA are administered in the S-compartment for 3 days, followed by infection in the S-compartment. The spread of virus particles into the N-compartment can be detected by fluorescent expression of GFP-Us9 or mRFP-VP26 in the N-compartment. B: SMPD4 siRNA knockdown. Dissociated SCG neurons were transfected with 50uM of siRNA against SMPD4 (+) or Non-Target controls (-). At 3 days post siRNA transfection (labeled pre-infection), samples were collected and assayed on SDS-PAGE western blot to confirm protein knockdown. After the anterograde sorting assay, Soma form the S-compartment were collected again to measure knockdown for the duration of the assay. Each lane represents a different chamber. C: Robust spread detected after SMPD4 knockdown. At 48 hpi, the N-compartment of chamber treated with siRNA-SMPD4 (left) displayed greater GFP-Us9 (top) and mRFP-VP26 (bottom) signal, in comparison to the si:NonTarget negative-control (right chamber). D: Virus titers after anterograde sorting assay. N-sup represents virus particles that have sorted into the N-compartment and released into the supernatant. N-cells represents particles sorted into the N-compartment but confined inside the axons or PK-15 cells. S-Sup represent particles released into the supernatant of the S-compartment. Titer was measured by counting plaques on a monolayer of PK-15 cells. Statistics were performed using 2way-ANOVA test. E: SMPD4 localization after PRV infection. Confocal microscopy of siRNA transduced SCG cell body infected with virus expressing GFP-Us9^WT and mRFP-VP26 capsids (PRV 341). After 12 hpi, cells were fixed for immunofluorescence staining of SMPD4. White arrow indicates foci of mRFP-VP26 and SMPD4 colocalization. Arrowhead indicates foci of mRFP-VP26 and GFP-Us9 colocalization. F: Quantification of mRFP-VP26 capsid distribution. All cytoplasmic mRFP-VP26 capsid foci were quantified for colocalization with GFP-Us9 and/or SMPD4 foci.