286 Effect of PRA1 Suppression on SIV Virion Release, Infectivity, and Envelope Content P Blancou*, DT Evans, RC Desrosiers Harvard Univ, Southborough, MA
Background: Prenylated Rab acceptor (PRA1) was identified as a potential cellular binding partner of the SIV gp41 cytoplasmic domain (gp41CD) in a 2 yeast hybrid screen of a human, phytohemagglutinin-activated T-cell cDNA library. This interaction was confirmed by mammalian 2-hybrid assays. PRA1 is a 21kDa protein that binds prenylated Rab proteins in their GTP-bound state and appears to participate in intracellular vesicular trafficking. To determine if PRA1 has an effect on the assembly and release of infectious virus particles, we produced SIV by transient transfection under conditions of PRA1 suppression.
Methods: Suppression of PRA-1 expression was achieved by RNAi. RNAi-mediated inhibition of PRA1 expression was confirmed by flow cytometry in experiments that utilized a PRA1-GFP fusion construct. Viral release, infectivity and envelope virion content were assessed in transient transfection assays under conditions of maximal PRA1 suppression. The assembly and release of SIV was assessed by p27 ELISA. To assess infectivity, CEMx174 cells were infected with different amounts of SIV expressing GFP and analyzed by flow cytometry. Envelope incorporation into virions was quantitated using virions obtained from clarified culture cell supernatants by ultracentrifugation; identical quantities of p27 were analyzed by Western blotting with anti-gp41 and gp120 antibodies.
Results: PRA1 expression was decreased more than 5-fold by the presence of RNAi. However, no change in virion release, infectivity, or envelope content was observed.
Conclusions: These data suggest that the PRA1/gp41CD interaction does not effect SIVmac239 virion production, infectivity or envelope incorporation. These results are consistent with the dispensability of the SIV gp41 cytoplasmic domain for replication in cultured cells.
