The cell-associated, but not the secreted species of 90K, exerts the antiviral activity. (A) 293T cells were either transfected with pBR.HIV-1 NL4.3 IRES GFP and inoculated with culture supernatant originating from vector-transfected or 90K-myc-transfected 293T cells (left half), or cotransfected with HIV-1 wt and pcDNA6.90K-myc or empty vector (right half). Two days post transfection, the amount of infectious HIV-1 in the culture supernatant was determined. Shown are the arithmetic means ± S.D. of triplicates originating from one representative experiment out of two. Supernatants of the cultures from (A) were analyzed for 90K-myc content. (B) A constant amount of HIV-1 IRES GFP virions was incubated for 15 min at 37°C with decreasing amounts of supernatant derived from 293T cells that had been transfected with empty vector or pcDNA6.90K-myc. TZM-bl were inoculated with the pretreated virus and analyzed for GFP expression three days later. Shown are the values of one representative experiment out of two. An aliquot of the pretreated inoculum that was given to the TZM-bl cells was analyzed by Western Blotting. (C) 293T cells were cotransfected with pBR.HIV-1 NL4.3 IRES GFP and indicated expression plasmids. Two days post transfection, the amount of infectious HIV-1 in the culture supernatant was determined. Values are normalized to the empty vector-cotransfected control. Shown are the arithmetic means ± S.E.M. of three independent experiments. Cell lysates and supernatants from one representive experiment were analyzed by Western Blotting. * : p < 0.05 (Student’s T-Test).