FIG 6.
Analysis of Ser5 splicing isoform anti-HIV-1 activities. (A) The expression levels of Ser5 isoforms and mutants from the pCMV6 vector were normalized to similar levels by adjusting the amounts of the expression vectors during transfection of 293T cells and detected by Western blotting. (B) Similar levels of the indicated Ser5 isoforms and mutants from the pCMV6 vector were expressed in 293T cells during production of ΔNef HIV-1 or HIV-1 expressing NL4-3 Nef (Nef43), 97ZA012 Nef (Nef97), or glycoMA. (Top) Virus production was measured by p24Gag ELISA. (Bottom) Then, equal amounts of viruses were used to infect TZM-b1 cells to determine viral infectivity. (C) Similar levels of the indicated Ser5 variants from the pCMV6 vector were expressed in J-TAg-KO cells in the presence of HIV-1, which was verified by Western blotting. (D) After viral production from the Jurkat cells was measured by p24Gag ELISA (top), equal amounts of viruses were used to measure viral infectivity (bottom). (E) The indicated Ser5 isoforms and mutants were cloned into the pBJ5 vector and expressed in 293T cells. Then, viral production and infectivity were determined similarly. The error bars represent SD from three experiments.