Final Report Summary - TRXN-PURGE (Mechanisms of transcription in HIV latency; novel strategies to activate)
Combination anti-retroviral therapy (cART) is extremely effective in suppressing HIV replication to undetectable levels, halting disease progression and preventing the onset of AIDS. However, long-lived latent HIV-1-infected cells persist in a latent state despite the presence of cART and present the major roadblock for a cure as HIV rebounds once cART is interrupted (de Crignis et al., 2017; Ne et al., 2018). Latency is the result of a block in HIV gene expression, causing major challenges to immune elimination of infected cells. One strategy for HIV Cure is to shock or induce latent HIV infected cells to express virus via treatment with latency reversal agents (LRAs) such that they can be seen and eliminated by the immune system (Ne et al., 2018; de Crignis et al., 2017). However, current LRAs have proven to be ineffective clinically, highlighting the need for novel, more potent, more specific and less cytotoxic LRAs. In Trxn-PURGE we have developed methodology for and performed two independent unbiased screens to identify functionally relevant molecules physically associated with the latent HIV LTR and essential for maintenance of HIV latency. In Aim1, we used a tagged Cas9 gRNA guided tether to the latent HIV LTR downstream of the transcription start site in order to isolate the latent HIV promoter bound to its interactors (Ne et al., in prep). To accomplish this, we developed a two-tiered methodology which as a first step purified the tagged bait and its crosslinked associated complex, followed by removal of the non-chromatin bound proteins as a critical subsequent step. This method has resulted in the Mass Spec identification of a list of candidates distinctly associated with the latent and active HIV promoters and contains factors previously known to be associated with the HIV LTR as validation of its quality as well as novel interactors which can be targeted for inhibition to achieve latency reversal (Ne et al., submitted). We have focused on characterizing a number of novel transcription and chromatin associated factors as well as a group of associated molecules that play a role in HIV post-transcriptional RNA processing, stability and surveillance, highlighting the importance of post-transcriptional regulation in controlling HIV gene expression. In Aim 2 we used a gene trap mutagenesis approach in Haploid latently infected cells and identified a list of nonessential candidates that are involved in HIV transcription silencing, which can be targeted for latency reversal (Roling et al., in prep). Interestingly, several factors overlap between candidates identified in Aims 1 and 2, underlining the relevance and veracity of the screens. In Trxn-PURGE, we also searched for novel putative LRAs from a biochemically diverse natural source of small molecules, that of fungal supernatants (Aim 3). We found three fungal supernatants that strongly reversed latency and developed an orthagonal liquid chromatography coupled to Mass Spec/NMR approach together with mycology and latency reversal bioassays to identify the active component in the supernatant. After multiple rounds of fractionation, Mass Spec and NMR was used to identify GTX as a LRA purified from the fungus Aspergillus fumigatus, that targets 7skNSRNP for degradation, freeing up PTEFb and targeting the transcription elongation step to activate HIV (Stoszko et al., submitted). We also identified BAF inhibitors as a novel class of LRAs which target the repressive HIV LTR BAF complex causing de-repression of HIV (Stoszko et al., 2016) and characterized its effects on immune cells toxicity and function (Zhao et al., 2019). From a screen of approximately 350,000 molecules, we identified, in collaboration, a novel macrolactam scaffold BAF inhibitor class of molecules, which target the ARID1A subunit specifically, and which strongly reactivated latent HIV (Marian et al., 2018). We filed a patent to protect this finding. While solving technical hurdles we encountered in Aims 1 and 2, we examined the role of noncoding variants found to be associated with HIV susceptibility and found allele-specific long distance regulation of the IL-32 gene confers susceptibility to HIV infection, with IL-32 gamma producing a pro-inflammatory environment conducive to infection (Palstra et al., 2018). Aside from the mechanistic and molecular characterization (Aim4) of candidate therapeutic molecular targets and compounds (Stoszko et al., 2016; Abner et al., 2018; Marian et al., 2018; Zhao et al, 2019; Ne et al., submitted; Roling et al., in prep) a major objective of Trxn-PURGE was the establishment of a translational infrastructure at Erasmus MC where basic insights and findings in the HIV transcription and latency field would be translated and clinically tested. This involved the establishment of ex vivo primary CD4+T cell models of latency as well as examination of effect on the reservoir after ex vivo treatment of cells obtained from HIV infected volunteers on c-ART. This been a very active and fruitful area where the basic findings in the project have been examined in a translational context and in one case is the process of clinical testing; we obtained medical ethical approval to examine the effect of the BAF inhibitor pyrimethamine, an FDA/EMA approved drug used in the treatment of toxoplasmosis, on depletion of the latent reservoir in HIV infected patient volunteers in a proof of concept clinical study, which is currently ongoing.