Virion-associated Vpr up-regulates ULBP-2 expression in noninfected target cells and triggers NK cell–mediated killing. (A) Human primary CD4⁺ T lymphocytes were exposed to indinavir-treated noninfectious viral particles that were trans-packaged with VprWT or the VprQ65R mutant, as indicated, and cell-surface expression of ULBP-2 was monitored 24 hours after exposure using specific mAbs directed against ULBP-2 and appropriate fluorochrome-conjugated secondary reagents. (B) HIV-1ΔVpr and HIV-1ΔVpr LF/PS viruses (P6-mutated Gag-encoding virus that does not incorporate Vpr) trans-packaged with HA-tagged VprWT or VprQ65R were produced as described in “Production of lentiviral vectors and HIV-1 viruses.” Virion-associated HA-tagged VprWT and VprQ65R were detected by Western blotting using anti-HA mAbs. (C) Primary CD4⁺ T lymphocytes were exposed to reverse transcriptase- and integrase-defective (HIVΔVprΔRTΔIN) viral particles that were trans-packaged with VprWT or the VprQ65R mutant as indicated, and cell-surface expression of ULBP-2 was monitored 24 hours after exposure. MFI values were calculated by subtracting the corresponding isotype-control values (dashed line). (D) Primary CD4⁺ T lymphocytes exposed to indinavir-treated noninfectious viral particles containing VprWT or VprQ65R were added 24 hours after exposure to autologous primary NK cells in a 4-hour ⁵¹Cr release assay, as indicated. Error bars represent SEM. Results shown are representative of the data obtained from 2 independent donors.