Abstract While a sterilizing cure of HIV-1 infection has been reported in two individuals after a stem cell transplant with CCR5∆32 homozygous cells, a spontaneous functional cure of HIV-1 occurs in 0.3-0.5% of all infected individuals. These individuals, termed elite controllers (EC), maintain undetectable levels of HIV-1 replication in the absence of treatment, despite the repeated isolation of replication-competent virus from viral reservoir cells. These individuals provide living evidence that spontaneous control of HIV-1 infection is possible, and the identification of virological and immunological mechanisms underlying such a remarkable disease outcome holds promise for inducing a functional cure of HIV-1 infection in broader HIV-1 patient populations. Here, we propose the novel hypothesis that undetectable viral loads in EC are related to specific genetic and epigenetic features of proviral HIV-1 DNA in reservoir cells. Based on strong preliminary data, we posit that intact HIV-1 proviruses from EC are preferentially located in non-genic chromosomal regions that do not permit effective viral gene transcription, resulting in a proviral landscape in deep latency and with very limited responsiveness to reactivation stimuli in vivo. In addition, we propose that intact proviruses from EC are also frequently integrated into chromosomal regions that show epigenetic characteristics of repressive chromatin, evidenced by enhanced distance to accessible chromatin and enrichment with inhibitory histone marks and hypermethylated DNA. To investigate this, we plan to longitudinally analyze the frequency and chromosomal location of intact proviruses in blood and lymphoid tissues of EC, using novel experimental and biocomputational analysis approaches (Specific Aim 1). In addition, we will conduct a detailed analysis of epigenetic chromatin features at the integration sites of intact proviruses, using next-generation sequencing assays for genome-wide characterization of chromatin accessibility, RNA transcription, activating and inhibitory histone marks, and DNA methylation (Specific Aim 2). Finally, we will perform functional experiments to evaluate responsiveness to viral reactivation stimuli, using novel single-cell assays to simultaneously characterize proviral sequence, chromosomal integration sites and HIV-1 transcriptional activity of single viral reservoir cells (Specific Aim 3); these highly innovate single-reservoir-cell assays will allow to functionally test the hypothesis that chromosomal locations of intact proviruses in EC maintain a state of deep latency and confer resistance to viral reactivation stimuli. If successful, this project will significantly expand our current understanding of how natural, drug-free control is possible, and be highly informative for inducing natural control in broader patient populations.