siRNA inhibition of mono- and dual-infections with HIV-1 v120-A and v126-D. (A) Schematic illustration of the monoinfections or dual infections with or without siRNA treatment for a first round on U87.CD4.CXCR4 cells. The supernatant of this round was monitored at days 4, 5, 6, 7, and 8 for virus production using a radiolabelled RT assay. The RT activity (cpm/ml) over this time course with or without siRNA treatment was plotted in panel B (error bars were removed for better viewing). For the second round infection of panel A, virus-containing supernatants from the day 5 dual infections with or without dual siRNA treatment were equalized for RT activity and then added to fresh U87.CD4.CXCR4 cells. RT activity from the supernatant at day 4 from this second round infection and the first round dual infection was plotted in panel C. In the first round, a 96% inhibition was observed between dual virus production without (bar 1) versus with dual siRNA treatment (bar 2). When virus from the first round in the absence of siRNA treatment was added to the second round infection, dual siRNA treatment mediated a 61% inhibition (bar 4 versus 3). Finally, infection with siRNA-treated virus from the first round resulted in only a 21% inhibition by siRNAs in the second round (bar 6 versus 5).