Figure 4.

HIV-1 inhibition by tat­-targeting CRISPR-Cas9 was maintained after a second round of infection and co-culture of infected cells with unprotected T cells. (a) As shown by the timeline, T cells were first infected with HIV-1_NL4-3 equivalent to 200 pg of p24 and cultured for eight days. The supernatants were collected and virions equivalent to 200 pg of p24 were used on an infected to an uninfected batch of the same group of cells to represent the second round of infection. The supernatant p24 levels at 4 dpi were measured and compared to samples from the first round of infection at the same time point. The assays were performed three times with average values (±SE,) as shown. An unpaired t-test was done to identify significant differences. *, p < 0.05. (b) At 4 dpi of the first round of infection, infected cells (5x10⁴) were collected, washed two times with fresh medium, and co-cultured with unprotected, wild-type MT-4 T cells (5x10⁴). The supernatant p24 levels were measured at eight days post-co-culture. The average values (±SE) of three assays are shown. One-way ANOVA applied to all groups and Dunnett’s multiple comparison test for each group compared to WT were used to calculate significance. *, p < 0.05; **, p < 0.01, and ****, p < 0.0001. (c) Co-cultures were maintained until 16 dpi. The kinetics of supernatant p24 levels are shown by the red line (left y-axis), and viable cells as counted with trypan blue exclusion are shown by the blue line (right y-axis). The average values (±SE) of three assays are shown.