The search for an HIV cure has been among the highest HIV research priorities for several decades. While much knowledge was gained regarding the persistent HIV reservoir, HIV eradication remains elusive. There is an emerging consensus that the HIV reservoir is not a fixed assortment of epigenetically silenced proviruses but rather a dynamic compendium of mostly defective proviruses. We recently showed that most infections in primary human CD4+ T memory cell subsets result in non-productive, latent infections. These findings were made possible thanks to our newly developed HIV vector system, which permits distinguishing and separating primary human cells harboring non-productive and productive proviruses. Transcriptome analysis of specific CD4+ T cell subsets identified transcript isoforms differentially expressed in the context of productive and non- productive infections. We propose an integrated experimental approach that will systematically dissect non- productive infections in primary human CD4+ T cells. First, we will analyze the proviral structure of non- productive and productive infections (Specific Aim 1), which will include both integration site analysis as well as detection of defective proviruses. We will identify and validate the transcriptional programs specific for productive and non-productive infections using advanced bulk and single cell long read transcriptome analysis approaches (Specific Aim 2). We will validate findings using genome editing tools and a panel of viral mutants lacking functional accessory genes. Lastly, we will dissect the role of innate immune responses in the context of non-productive infections (Specific Aim 3) by determining to what extent stimulation with gamma cytokines or Type-1 interferon changes the propensity of CD4+ T cell subsets to support non-productive infections. Collectively, the proposed studies will deliver insights into the interplay between non-productive HIV infections and host cell environment pointing to the molecular mechanisms that drive non-productive infections in primary human CD4+ T cells. The results obtained will also provide essential information on potential HIV latency biomarkers and directly inform of innovative HIV cure strategies.