Summary The majority of the human transcriptome consists of long non-coding RNAs (lncRNAs), which regulate the expression and function of protein-coding genes, immune cell development, differentiation, and response to pathogens. Infections induce global changes in lncRNA expression. The expression of lncRNAs in HIV-infected cells and how host lncRNAs impact the replication and persistence of HIV infection are unknown. A spontaneous functional cure of HIV-1 occurs in 0.3-0.5% of all HIV patients. These patients, termed elite controllers (EC), maintain undetectable levels of HIV in the absence of treatment, maintain stable CD4+ T cell counts, and are less likely to transmit HIV. The ECs exemplify spontaneous control of HIV, and identifying defense mechanisms in these patients holds promise for finding a functional cure for HIV infection. Previous studies focused on protein- coding genes. However, effective HIV control is likely to involve complex gene networks, including lncRNAs. We found several lncRNAs significantly suppressed in EC vs. HAART-treated chronic HIV-patients and healthy uninfected controls. The functional impact of lncRNAs suppressed in EC (SIEC) on HIV outcomes is unknown. We have observed significant global changes in cellular lncRNA expression in HIV-infected CD4+T cells compared to uninfected cells in our preliminary studies. Cellular functions of most of the HIV deregulated (HIDE) lncRNAs are yet to be determined. We employed a CRISPR/RfxCas13d (CasRx)-silencing screen to determine the functional impact of SIEC and HIDE lncRNAs on HIV replication. Our preliminary data showed that silencing of several SIEC and HIDE lncRNAs significantly regulated HIV replication. Based on these preliminary data, we hypothesize that host lncRNA expression in HIV-infected cells orchestrates natural resistance and disease outcome in HIV infection. We will determine how the suppression of specific lncRNAs in ECs is protective and elucidate molecular mechanisms of their function (Aim 1). We will also identify HIV deregulated (HIDE) lncRNAs modulating viral replication and investigate the mechanism(s) underlying lncRNA-mediated regulation (Aim 2). We will pursue these aims using innovative combinations of molecular and biochemical techniques, such as CRISPR/CasRx-silencing, transcriptomics, RNA antisense purification, and Mass-Spectrometry in cell line models as well as primary CD4+ T cells. The proposed research is significant because it will identify cellular lncRNAs that influence spontaneous control of HIV and disease outcomes, deliver unprecedented insight into lncRNA-mediated regulation of HIV replication, lay the groundwork for the development of new approaches to intervention.