Mutations that reduce CA binding to CPSF6 rescue T54A.

<p>(A) HeLa cells stably transduced with the control LPCX vector or one overexpressing CPSF6-358 were infected with VSV-G-pseudotyped GFP reporter viruses. Infectivity of T54A was significantly lower in CPSF6-358-expressing cells than in control cells (p<0.001). (B) T54A and T54A-A105T CA tubes (5 µM) were incubated with 500, 250, or 125 µg of HeLa cell extracts for 1 hr with gentle mixing. The tubes were pelleted, and proteins analyzed by non-reducing SDS-PAGE and immunoblotting for CPSF6 and CA. Input represents 10% of initial reaction. The results are representative of three independent experiments. (C) Quantification of CPSF6 association with CA tubes relative to amount of pelleted CA. (D) HeLa cells were infected with RT-normalized VSV-G-pseudotyped GFP reporter viruses in the presence or absence of aphidicolin (Aph). Reduction of T54A infectivity in aphidicolin-treated cells was statistically significant (p<0.02???). Results are one representative experiment of at least three experiments. Standard deviations of a single triplicate experiment are indicated with the error bars (A and D).