Influence of α₁PI on HIV-1 infection kinetics in serum-free medium. To examine whether α₁PI might exert its influence on virus or cells, cells were cultured overnight in serum-free medium and infected with varying doses of virus in the presence of varying concentrations of α₁PI. To avoid biasing the outcome against adherent cells, U937 clones were infected directly in microplates. For comparison, cells were infected in microtubes, washed to remove free virus, and transferred to clean wells of a microplate. In vitro infectivity of HIV-1 permissive clone 10 was significantly increased in the presence of increasing concentrations of α₁PI. Clone 17 failed to be infected at any viral dose or any concentration of α₁PI. Cells were infected with HIV-1_NL4-3 using (A) 1 × 10^-2 MOI, (B) 1 × 10^-3 MOI, or (C) 1 × 10^-4 MOI. Clone 10 infected in the absence of α₁PI (•) was equivalent to infectivity in the presence of 0.3 μM α₁PI (▪) and increased in the presence of 3 μM (▴), or 30 μM (▾) active α₁PI. Clone 10 exhibited low RT activity when cells were infected in microfuge tubes and transferred to wells of a tissue-culture plate in any concentration of α₁PI (♦). Infectivity experiments were confirmed independently by the Laboratory of Immunoregulation, NIAID, NIH. A representative set of results is depicted. Infectivity outcome was determined in duplicate by measuring RT activity of cell-free supernatants. Mean values are depicted. (D) RT activity produced by clones 10 and 17 infected simultaneously in the presence (closed symbols), but not in the absence (open symbols), of α₁PI is represented. RT activity was produced by HIV-1 nonpermissive clone 17 following pretreatment with LPS and LBP (▾). Cells infected in the presence of LPS and α₁PI and the absence of LBP (▪) produced diminished peak RT activity and prolonged time to peak activity. RT activity was not produced by clone 17 in the absence of LPS and LBP even though α₁PI was present (♦). Peak RT activity produced by clone 10 infected in parallel is represented for comparison (•). To examine colocalization of HIV-1 with receptors, clone 10 was incubated with HIV-1_NL4-3 in serum as a source of α₁PI. At 15-minute intervals, cells were washed free of virus, fixed, and slides were prepared. Following incubation for 15 minutes with HIV-1_NL4-3, (E) HLE (green), CD4 (red), and HIV-1 (silver) were copatched, and (F) HLE (green), CXCR4 (red), and HIV-1 (silver) were copatched. (G) Copatching of HLE (green), CD4 (red), and HIV-1 (silver) on clone 10 was not detected in the absence of HIV-1_NL4-3. HIV-1 coreceptors were detected by CLSM using polyclonal anti-HLE, monoclonal anti-CD4, and monoclonal anti-HIV specific for epitopes proximal to the V3 loop. Bar represents 30 μm. HIV-1 infectivity and CLSM were performed at least 3 times and representative data are presented.