As it is becoming increasingly clear that currently pursued HIV-1 reactivating strategies may not result in therapeutic HIV-1 eradication, alternative eradication strategies need to be explored. We here propose to develop terminal transcriptional silencing (TTS) strategies specific for latent HIV-1 infection events. TTS strategies would have their biological equivalent in retro/lentiviral silencing. However, TTS would not target de novo infection events, but have to transcriptionally silence pre-existing, latent provirus in the absence of viral proteins, mostly RNA or non-integrated DNA that usually trigger the antiviral cellular response. Evidence for the existence of cellular innate immune mechanisms that specifically target lentiviral infection is provided by the fact that lentiviral silencing is a major roadblock for lentiviral-based gene therapy. The existence of innate cellular defense mechanisms that can control pre-existing virus-style genome expression is suggested by the existence of defense mechanisms against a related, evolutionary ancient class of ?genomic intruders? called retrotransposons. These defense mechanisms individually evolved in response to each different retrotransposon class. Unfortunately, because of this specificity, mechanisms that continuously actively control retrotransposons to suppress their genomic spread, actually are inactive against HIV-1. Specificity of TTS strategies against HIV-1 infection is essential as, beyond the general consideration of toxicity issues of any drug intervention, TTS strategies cannot cause (i) any interference with innate control mechanisms that suppress retrotransposon activity or cause (ii) any interference with mechanisms that control epigenetic silencing of genes in human cells. For the R61 phase, we propose an iterative approach that combines high content analysis methods (ATAC-seq, RNA-seq, kinome array analysis) with pharmacological perturbation screens to identify drug targets that specifically control the cellular innate antiviral response to HIV-1 infection. The proposed research will take advantage of (i) a preexisting, large selection of HIV-1 reporter cells, (ii) the finding that certain clinical HIV-1 and in particular HIV-2 strains cause a much more potent innate TTS response than the commonly used laboratory adapted HIV-1 clones, and (iii) the finding that macrophages are much more efficient in executing TTS than T cells. The deliverable of the R61 phase will be (1) the identification of druggable targets that if pharmacologically addressed, enable TTS in latently HIV-1 infected T cells and (2) HTS-compatible drug screening assays for the identification of TTS-inducing drugs against these targets. In the R33 phase, we will perform the actual drug screens to identify TTS-inducing compounds. Identified candidate compounds would be tested in primary cell models of latent infection and cell material derived from HIV-1 patients. By the end of the R33 phase we expect to have identified at least one clinically relevant TTS strategy comprising of one or several drugs.