Figure 2
Figure 2. Detection of virus after restimulation of infected cells. (A) Method used for infection and restimulation. Resting CD4+ T cells cultured for 3 days with CCL19 or PHA/IL-2 or left unactivated were subjected to a second round of activation by the addition of activated PBMCs (aPBMC) at a ratio of 1:1 together with PHA and IL-2. The PHA was removed the next day, and cultures were maintained in IL-2 alone. In some experiments, the integrase inhibitor L8 was added before the second stimulation. As a control for the activity of L8, L8 was added 24 hours before the initial infection of PHA-stimulated PBMCs. Supernatants and cells were collected for quantification of RT and integrated HIV-1 DNA, respectively. (B) RT production after restimulation of resting CD4+ T cells. Resting CD4+ T cells were cultured for 3 days with CCL19 (●), or IL2/PHA (■) or unactivated (△) before infection with HIV-1 AD8 or mock (△). Cultures were restimulated (closed symbols, solid lines) or not restimulated (open symbols, dashed lines) as described above in panel A. RT activity in culture supernatant from a representative experiment is shown (from 3 replicate experiments). (C) RT production after restimulation in the presence and absence of an integrase inhibitor. Resting CD4+ T cells were cultured for 3 days with CCL19 (●) or unactivated (◇) before infection with HIV-1 AD8 or mock (△). Integration competent virus in CCL19-activated or unactivated CD4+ T cells was identified by culture in the presence (closed symbols, solid lines) or absence (open symbols, dashed lines) of an HIV-1 integrase inhibitor (L-8; Merck) for 24 hours before the second round of activation with PBMC, PHA and IL-2. The PHA was removed the next day and the cultures were kept in IL-2 with or without L-8. As a control for activity of L8, PBMCs were stimulated with PHA/IL-2 and then infected in the presence (■) or absence (□) of L8 (added 24 hours before infection). RT activity in culture supernatant from a representative experiment is shown (from a total of 4 replicate experiments). (D) The identical experiment to panel C but infection was with pNL4.3. RT activity in culture supernatant from a representative experiment is shown (from a total of 2 replicate experiments).

Detection of virus after restimulation of infected cells. (A) Method used for infection and restimulation. Resting CD4+ T cells cultured for 3 days with CCL19 or PHA/IL-2 or left unactivated were subjected to a second round of activation by the addition of activated PBMCs (aPBMC) at a ratio of 1:1 together with PHA and IL-2. The PHA was removed the next day, and cultures were maintained in IL-2 alone. In some experiments, the integrase inhibitor L8 was added before the second stimulation. As a control for the activity of L8, L8 was added 24 hours before the initial infection of PHA-stimulated PBMCs. Supernatants and cells were collected for quantification of RT and integrated HIV-1 DNA, respectively. (B) RT production after restimulation of resting CD4+ T cells. Resting CD4+ T cells were cultured for 3 days with CCL19 (●), or IL2/PHA (■) or unactivated (△) before infection with HIV-1 AD8 or mock (△). Cultures were restimulated (closed symbols, solid lines) or not restimulated (open symbols, dashed lines) as described above in panel A. RT activity in culture supernatant from a representative experiment is shown (from 3 replicate experiments). (C) RT production after restimulation in the presence and absence of an integrase inhibitor. Resting CD4+ T cells were cultured for 3 days with CCL19 (●) or unactivated (◇) before infection with HIV-1 AD8 or mock (△). Integration competent virus in CCL19-activated or unactivated CD4+ T cells was identified by culture in the presence (closed symbols, solid lines) or absence (open symbols, dashed lines) of an HIV-1 integrase inhibitor (L-8; Merck) for 24 hours before the second round of activation with PBMC, PHA and IL-2. The PHA was removed the next day and the cultures were kept in IL-2 with or without L-8. As a control for activity of L8, PBMCs were stimulated with PHA/IL-2 and then infected in the presence (■) or absence (□) of L8 (added 24 hours before infection). RT activity in culture supernatant from a representative experiment is shown (from a total of 4 replicate experiments). (D) The identical experiment to panel C but infection was with pNL4.3. RT activity in culture supernatant from a representative experiment is shown (from a total of 2 replicate experiments).

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