FIP1C depletion alters HIV-1 Env incorporation, cell surface levels, and viral growth in the H9 T cell line.
(A) FIP1C depletion was achieved using shRNA in HeLa cells (left) and the H9 T cell line (right). Populations of FIP1C-depleted HeLa and H9 cells were then divided and infected with VSV-pseudotyped NL4-3 or NL4-3delCT144. Released viruses were harvested 2 days after infection (HeLa) or 4 days after infection (H9) and evaluated for protein content by Western blotting. Note that results using specific antiserum used for detection of gp120 and gp160 in cells and particles are shown; gp41 particle blots were probed with a gp41-specific monoclonal antibody. (B) Infectivity of released viral particles from the experiment shown in (A) was measured in the TZM-bl reporter cell line, expressed as infectious units/ng p24. (C) Plasma membrane (left) and total cellular (permeabilized) levels of HIV-1 Env (right) were detected using flow cytometry 4 days after infection with VSV-G- pseudotyped NL4-3 WT or NL4-3delCT144 viruses. The relative level of Env on plasma membrane compared to total Env was calculated and plotted in panel (D) for control and FIP1C-depleted H9 cells. (E) FIP1C-depleted (open circles) and control shRNA-treated H9 cells (filled squares) were infected with VSV-pseudotyped viruses indicated at MOI 0.5. Viral growth was monitored over time by detection of p24 in the culture supernatant. CT144 growth (triangles) shown in control H9 cells.