Endogenous 90K lowers the infectivity of HIV-1. (A) Confocal microscopy of TZM-bl cells immunostained for endogenous 90K and DAPI. Scale bar: 10 μm. (B) Western Blotting analysis of TZM-bl cells using an anti-90K antibody. Values indicate the relative 90K protein expression, as measured by Infrared imaging-based quantification. (C) Normalized 90K mRNA levels of the cells shown in (A). (D) Following siRNA transfection, TZM-bl cells were infected with VSV-G-pseudotyped HIV-1NL4.3 over-night, followed by thorough washing of the cells in order to remove excess virus. Three days post infection, supernatants were analyzed for released p24 capsid antigen and (E) particle infectivity, defined as infectivity per ng p24. Bars show the arithmetic means ± S.E.M. of four experiments. (F) 90K knockdown efficiencies of the three experiments shown in (D and E) were validated by quantitative RT-PCR. Bars show the arithmetic means ± S.E.M. (G) Lysates of infected cells and (H) of sucrose cushion purified viruses from one representative experiment were analyzed by Western Blotting. Percentages indicate the efficiency of gp120 processing, calculated as the signal ratio of gp120 relative to (gp120 + gp160), the relative 90K expression, calculated as the signal ratio of 90K relative to MAPK, and the relative gp120 incorporation, calculated as the amount of gp120 per p24, respectively. **: p < 0.02 (Student’s T-Test).