FIG 3.

HIV-1 OGH: a double reporter construct used to analyze the impact of LEDGINs on immediate latency and reactivation. (a) Schematic representation of the HIV-1 OGH construct (51, 55, 56). HIV-1 OGH is a replication-deficient vector containing an eGFP gene under the control of the viral LTR promoter. HIV-1 OGH also carries a constitutively active transcriptional unit of an mKO2 reporter driven by an EF1α promoter. (b) Timeline of the transduction and reactivation experiments. SupT1 cells were transduced in the presence of CX14442 or GS-9822. Three days postransduction, vector and compounds were washed away, and flow cytometry analysis was performed. Eight days postransduction, cells were reactivated with 10 ng/ml TNF-α for 24 h. At day 9, 24 h postreactivation, cells were analyzed by flow cytometry. (c) Representative dot plot of SupT1 cells, transduced with 120 × 10⁶ pg of HIV-1 OGH on day 9 (condition without TNF-α), showing how flow cytometry makes it possible to distinguish between different cell populations. Cells only expressing mKO2 from the constitutively active EF1α promoter have an inactive LTR and are thus considered to be latently transduced (quadrant C). If cells are productively transduced (quadrant B), the viral LTR promoter will drive eGFP expression as well, resulting in double positive cells.