REAF inhibits HIV and SIV infection and occurs at an early post-entry stage of replication. (A) Western blot of HeLa-CD4 cell lysate following REAF siRNA knockdown compared with CB control. REAF is shown as three bands at 80, 130 (faint) and 220 kDa, but not all bands are detectable in all experiments. Tubulin (57 kDa) is added as a loading control. (B) 72 hr post siRNA knockdown of REAF, HeLa-CD4 cells are challenged with HIV-189.6, HIV-2CBL-23, SIVAGM (African Green Monkey; TYO-1), SIVMAC (Macaque; 32H) and SIVSM (Sooty Mangabey; B670). p24 immunostaining 48 hr post infection (pi) indicates that REAF rescues viral replication. Results are shown as fold change compared with a non-targeting control siRNA (CB). (C) siRNA knockdown of REAF results in enhanced HIV-1 RT products – early and late (0–8 hr pi) and (D) proviral DNA (Alu-gag, 2–48 hr pi). HIV-1 DNA copies are normalised to genomic GAPDH and presented per million cells. Results are mean ± s.d. of a representative experiment performed in duplicate.