Ectopic expression of Nedd4 augments supernatant HIV-1 p24 levels Independent of HECT Domain Function.

<p>293 cells were transfected with HXB2 and a plasmid encoding either rat Nedd4 (rNedd4) or a rat Nedd4 catalytic domain mutant (rNedd4CSmut) and at 48 hr post-transfection, cell supernatants were collected and p24 assays were performed as described (Material and Methods). Schematics highlighting the Ca²⁺/Calmodulin-regulated phospholipid binding domain (C2), WW protein binding domains (WW1–3) and E3 catalytic domain (HECT) for rNedd4 and rNedd4CSmut are shown (A). Mean p24± standard error values, calculated from data obtained from transfections conducted with rNedd4 (n = 7) and rNedd4CSmut (n = 5), are graphically represented (B). Statistical significance was shown for the increased p24 levels by rNedd4 (rNedd4 vs control at 48 hr: p<0.01; rNedd4CSmut vs control at 48 hr; p<0.01) using a one way ANOVA followed by Tukey’s multiple comparison test. No statistical difference was shown for rNedd4CSmut compared to rNedd4 at 48 hr (p>0.05) using this analysis. Immunoblot analysis, using a rabbit anti-mouse Nedd4 antibody, was performed on detergent soluble cell fractions as described (Materials and Methods). Nedd4 and Nedd4CSmut protein bands are indicated (arrows) (C).