Figure 7.

PI(4,5)P₂ disruption impairs FIV particle production. Feline CRFK cells were co-transfected with pFIV-Orf2rep and plasmids expressing human Arf6 in either its native or constitutively active form (Q67L) or human 5-phosphatase-IV (5ptaseIV) in its native or catalytically inactive form (5ptase Δ1) using Lipofectamine LTX PLUS reagent (Invitrogen), at a DNA ratio of 9:1 (FIV:Arf6/5ptaseIV). Cells were metabolically labeled with [³⁵S] Met/Cys and FIV proteins were immunoprecipitated from cell and viral lysates with anti-FIV p24gag (see Materials and Methods). Virus release efficiency was calculated as the proportion of virion-associated FIV capsid compared with total FIV Gag, normalized to samples containing FIV plasmid alone. Error bars indicate standard error of the mean (SEM) from at least 3 independent experiments. Reductions in virus release in the presence of Arf6/Q67L and 5ptaseIV were statistically significant (paired Student’s t-test).