Figure 3. A “G” residue at the −1 position reduces HIV-1 subtype B susceptibility to restriction by human PYHIN proteins.

(A and B) HEK293T cells were cotransfected with the indicated proviral constructs (2.5 μg) and either a vector control or expression constructs for human nuclear PYHIN proteins (1 μg). Two days post-transfection, infectious virus yield was determined by TZM-bl cells infection (A), and p24 in the supernatant was assessed by ELISA (B). The left panels show absolute values and the right panels infectious virus and p24 production in the presence of the indicated PYHIN proteins relative to the vector control (100%). Shown are average values (±SD) obtained from four independent experiments.

(C–F) HEK293T cells were cotransfected with either an empty vector or expression plasmids for Sp1 (C and D) or NF-κB (E and F) and the indicated proviral HIV-1 constructs. Infectious virus yields (C and E) and p24 levels (D and F) were determined 2 days post-transfection. The left panel shows absolute and the right panel relative levels of production of described above. Curves were derived from three to four independent experiments and show average values (±SEM).

(G) Effect of Sp1 or NF-κB overexpression on infectious virus (top) and p24 antigen (bottom) yield of the wild-type and A349G HIV-1 CH058 IMCs. Values were derived from (C)–(F) at the highest concentration of Sp1 and NF-κB expression, respectively. **p < 0.01; ***p < 0.001.

See also Figure S3.