Structure based drug design targeting the well defined

Structure-based drug design targeting the well-defined interface (Cherepanov et al., 2005) between the IBD and the HIV-1 IN catalytic core resulted in the development of 2-(quinolin-3-yl)acetic Forskolin derivatives that inhibit HIV-1 replication (Christ et al., 2010). This novel class of antivirals is referred to as LEDGINs (Christ and Debyser, 2013; Christ et al., 2010, 2012; Debyser et al., 2015; Demeulemeester et al., 2014a). Novel congeners with nanomolar activity act as allosteric inhibitors, preventing the binding of both LEDGF/p75 and HRP-2 and interfering with the catalytic activity of IN (i.e. the so-called ‘early effect’) (Christ et al., 2012; Kessl et al., 2012; Tsiang et al., 2012). Recently, LEDGINs were found to inhibit late stage HIV replication as well (i.e. the ‘late effect’) (Balakrishnan et al., 2013; Desimmie et al., 2013; Jurado et al., 2013; Le Rouzic et al., 2013). The phenotype requires the binding of LEDGINs to the LEDGF/p75 binding pocket in IN (Desimmie et al., 2013; Le Rouzic et al., 2013) and is mediated by enhanced multimerisation of IN in the viral particles (Balakrishnan et al., 2013; Borrenberghs et al., 2014; Desimmie et al., 2013; Jurado et al., 2013). Contradictory results have been obtained as to whether LEDGINs affect the integration site distribution (Feng et al., 2016; Gupta et al., 2014; Sharma et al., 2014). In any case LEDGIN treatment results in a steep dose-dependent inhibition of viral replication in cell culture, supporting their clinical development (Fader et al., 2014; Fenwick et al., 2014). Here we investigated the early effect of LEDGINs and evaluated their effect on HIV integration site distribution. In addition, we monitored the effect of LEDGINs on the establishment of the latent reservoir and investigated whether retargeting of integration could lead to a silent HIV reservoir resistant to reactivation. In a stepwise approach we first demonstrate that LEDGF/p75 depletion results in reduced integration and a quiescent state of residual integrants. Next, we demonstrate that upon treatment with LEDGINs, blocking the LEDGF/p75–IN interaction, the residual proviral integration shifts away from transcription units. LEDGIN treatment also shifts the 3D localization of the integrated provirus towards the inner nucleus. LEDGIN-induced retargeting results in a silent HIV reservoir in cell lines and primary CD4+ cells. This silent reservoir is refractory to reactivation by latency reversing agents (LRAs). Pushing sufficient proviruses into latency is theoretically predicted to drive the basic reproduction number of HIV below 1, resulting in unsustainable infection (Rouzine et al., 2015). Hence, addition of LEDGINs to cART regimens during acute HIV infection may represent a new strategy to achieve a remission of HIV infection in patients.
Materials and Methods
Results
Discussion The moral duty to respond to the call for an HIV cure calls for exploration of experimental and innovative HIV cure strategies. As a complement to current purge-and-kill approaches aimed at forcing HIV out of its hiding places to obtain a sterilizing cure, we here provide experimental evidence for a strategy to push the virus towards transcriptional quiescence by interference with LEDGF/p75, the main determinant of integration site selection. General belief states that latency is an accident rather than a default pathway of an actively replicating cytopathic virus. The fact that LEDGF/p75, the tethering determinant of HIV integration, directs integration preferentially towards actively transcribed regions, ensuring productive infection, is consistent with this notion. Recently, it was proposed that HIV latency is a hardwired, evolutionarily conserved switch increasing the likelihood of successful mucosal transmission during primary infection (Razooky et al., 2015; Rouzine et al., 2015). Whereas HIV Tat is known to be the main viral determinant in controlling the HIV transcriptional state, the contribution of other viral and host determinants to the transcriptional state of the HIV provirus awaits further clarification. Here we investigated the role of LEDGF/p75 in the establishment of HIV latency. We demonstrate that LEDGF/p75 depletion, known to result in retargeting of integration away from the body of actively transcribed genes (Fadel et al., 2014; Schrijvers et al., 2012a; Shun et al., 2007), increases the fraction of quiescently infected cells and simultaneously decreases the reactivation potential of the proviruses. In light of the recently proposed role of LEDGF/p75 (and the Iws1/Spt6 complex) in post-integration HIV transcriptional repression (Gérard et al., 2015), this observed decrease may even be an underestimation of the contribution of the integration environment to the latent phenotype. Taken together, these observations suggest that disruption of the LEDGF/p75–IN interaction not only inhibits integration but could as well affect the establishment of the latent pool and the reactivation from latency.