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  • Recently developed LEDGINs small molecule inhibitors of

    2018-11-14

    Recently developed LEDGINs, small molecule inhibitors of LEDGF/p75–IN interaction, allowed us to test this hypothesis. Indeed here we demonstrate: (i) a LEDGIN-mediated shift in lentiviral integration site distribution resembling LEDGF/p75 depletion (out of the body of actively transcribed genes, with increased integration in the vicinity of CpG islands) ii) strongly reduced but authentic residual integration, (iii) a LEDGIN-mediated shift in 3D nuclear location of HIV provirus away from the nuclear rim; (iv) a relative increase in the fraction of quiescent proviruses and (v) a dose-dependent block in HIV reactivation from latency both in cell lines and primary CD4+ T-cells. It was recently proposed that pushing enough proviruses into quiescence could drive the basic reproduction number of HIV below 1, resulting in unsustainable infection (Rouzine et al., 2015). LEDGIN treatment apparently succeeds in rendering (almost) 100% of the virus into a quiescent state refractory to reactivation (Figs. 5c & f and 6). Although final proof will only be obtained in clinical trials, our cell culture data in relevant cell lines and primary cells provide evidence for the feasibility of this strategy. The importance of the site of integration in the human genome for basal transcriptional activity of HIV was evidenced more than a decade ago (Jordan et al., 2001). Now it is known that genomic target site selection during lentiviral integration is a multi-step process where biases are introduced at different levels, which each in part affect the stochastic proviral gene expression levels. At least three levels can be recognized. First, nuclear topology and proximity to the nucleopore affect integration site selection (Di Primio et al., 2013; Marini et al., 2015). Next, chromatin readers such as LEDGF/p75 or HRP-2 that recognize epigenetic marks associated with transcriptional activity tether the preintegration complex to active gene regions. No data exist suggesting a protein gradient. Therefore, as proposed by Marini et al. (2015), preferential lentiviral integration within the proximity of the nuclear periphery probably reflects the encounter by the HIV PIC of the first LEDGF/p75 bound chromatin close to nucleopores. Finally, bias for target DNA Tubastatin A HCl recognition by integrases also influences local and global integration patterns (Demeulemeester et al., 2014b, 2015; Serrao et al., 2015). In theory, interference with any of those mechanisms could shift the resulting proviral reservoir from transcriptionally active to quiescent. Both LEDGF/p75 depletion and LEDGIN treatment affect integration, integration site selection (Fig. 3, Supplementary Fig. 3a–c) and nuclear location (Fig. 4). Probably the reduced reactivation due to altered chromatin context is not entirely reflected by the features measured in the integration site analysis. As shown, aberrant integrations or LTR deletions are most likely not contributing, or only to a minor extent (Supplementary Fig. 5 and Table 2). In the absence of LEDGF/p75, HRP2 determines HIV integration site selection. The fact that LEDGINs also inhibit the interaction between HRP-2 and HIV-1 IN (Schrijvers et al., 2012a), can explain the more pronounced effect of LEDGINs on reactivation from quiescence compared to LEDGF/p75-depletion. Identification of the exact nature of the altered chromatin context responsible for the observed phenotype awaits further experimentation. HIV latency is of multifactorial nature and the transcriptional state of integrated provirus is not only influenced by molecular determinants but also depends on the infected host cell and its activation state (Dahabieh et al., 2015). Therefore the respective quiescent fraction and responsiveness to different LRAs might alter depending on the cell model used since promoter activity may differ between cell types. In order to study the effect of integration site distribution on HIV latency we used NL4.3-based (HIV-tCD34) and LAI-based (OGH) single round reporter viruses in a concise reactivation setup in T-cell lines and activated CD4+ T-cells. LEDGINs block HIV replication during integration (referred to as ‘the early effect’) as well as during assembly (i.e. ‘the late effect’), with inhibition during the late step having a 10- to 100 fold higher potency than the early step (Debyser et al., 2015). In the experiments with single round virus, we used micromolar concentrations of LEDGIN, required to inhibit the early step. Interestingly, recent experimental results suggest that addition of submicromolar concentrations of LEDGINs during virus production results in viruses that after integration are again refractory to LTR-driven gene expression (Supplementary Fig. 7), suggesting that during multiple round replication also low concentrations of LEDGINs induce quiescent proviral pools. Our data were corroborated with wt NL4.3 virus in IL-2/PHA activated primary CD4+ T-cells (Fig. 7). These multiple round experiments allowed the use of submicromolar LEDGIN (CX014442) concentrations. It will be of interest to extend these observations in the future to other latency models. Preliminary data in a more sophisticated model for HIV latency based on infection of CCL19-activated resting CD4+ cells (Spina et al., 2013) corroborates a LEDGIN-mediated shift of the HIV reservoir into a quiescent state refractory to reactivation (unpublished data). Evidence is growing that initiation of cART early after Tubastatin A HCl infection is effective in reducing the size of the viral reservoir (Ananworanich et al., 2012, 2015; Hocqueloux et al., 2013; Hoen et al., 2007; Malatinkova et al., 2015). Early treatment initiation with ART will likely become standard clinical practice in HIV care. This is supported by the outcome of the first large-scale international “Strategic Timing of AntiRetroviral Treatment” (START) study, showing a considerably lower risk of developing AIDS and other serious conditions when compared to later treatment initiation (INSIGHT START Study Group, 2015).