Figure 5
From: LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions
Systematic evaluation of the multiple effects of LEDGINs on HIV-1 replication. (A-E) (RT)-qPCR analyses for gRNA and viral DNA species evaluating early replication events (entry to integration) are shown. (A) HIVCX05045 enters cells as efficient as the control (HIVDMSO) virus as measured by RT-qPCR analysis of gRNA isolated 2 h after synchronized infection of MT-4 cells. As expected, addition of DS10000, but not efavirenz, inhibits entry of both HIVDMSO and HIVCX05045. Heat-inactivated virus preparation was used as negative control. Mean values ± standard deviation from three independent experiments are shown. (B, C) qPCR analysis for early and late RT product kinetics in MT-4 cells infected with HIVDMSO or HIVCX05045 in the presence or absence of efavirenz (EFAV) or raltegravir (RAL) added during infection as RT and integration inhibitor controls. There is a five-fold less RT products in cells infected with HIVCX05045 compared to HIVDMSO and efavirenz blocked the RT activity of both viruses. (D, E) 2-LTR circle and integrated copies kinetics are shown. HIVDMSO has normal nuclear import as well as integration which are evidenced by the accumulation of 2-LTR circles in cells treated with raltegravir. In stark contrast to HIVDMSO, the PICs of HIVCX05045 did not translocate into the nucleus as shown by background integrants. This result was further supported by the lack of the accumulation of 2-LTR circles when cells were treated with raltegravir (HIVCX05045+RAL). Mean values ± standard deviations for triplicate measurements. (F) Analysis of HIV preintegration complex (PIC) nuclear import. Percentage of nuclear versus total PICs in cells infected with HIVCX05045 (gray bars, n = 72) or HIVDMSO (black bars, n = 71) is shown. More than 50% of cells infected with HIVCX05045 did not contain any fluorescently labeled PIC in the nucleus. In contrast, 2-7% of fluorescently labeled PICs were detected in the nucleus of more than 50% of cells infected with HIVDMSO. In the inset, the cumulative probability of nuclear PICs is plotted for HIVCX05045 (gray) and HIVDMSO (black). The empirical distribution function was computed using the Kolmogorov-Smirnov test, p < 0.0001).