PROJECT SUMMARY/ABSTRACT Cell-intrinsic antiviral proteins elicited by the interferon (IFN) response comprise one of the first lines of defense against viral infection. However, the mediators of IFN inhibition and their potency vary by virus and cell type, underscoring the need to study IFN restriction during natural infection. Like other viruses, HIV-1 triggers the IFN response and demonstrates IFN sensitivity in cell culture and in chronically infected patients. Moreover, it is well characterized that HIV transmission is inefficient, typically requiring several exposures that result in a bottleneck of viral variants. Together, these findings suggest that antiviral IFN-stimulated genes (ISGs) may pose a major barrier to HIV during transmission and early infection. Nevertheless, it is still unknown which ISGs are actually induced by HIV, primarily due to the difficulty of obtaining samples during the brief window of acute infection. Data on ISG restriction of HIV in natural settings are also limited, as previous work has largely used immortalized cell lines and lab-adapted viruses. A more complete understanding of the ISGs present during acute HIV infection and their antiviral potency in the cells that fuel HIV dissemination are needed to determine how the IFN response shapes early HIV infection. To address this gap, this proposal characterizes cellular ISG expression in precious peripheral blood mononuclear cells (PBMCs) collected during early infection in vivo and defines ISG restriction in vitro against an early HIV isolate in HIV target cells, CD4+ T cells. This study will explore the hypothesis that a set of HIV-induced, functionally antiviral ISGs are responsible for IFN-mediated inhibition of HIV in CD4+ T cells. First, the in vivo arm will determine the profile of ISG transcripts upregulated in each PBMC cell type during early HIV infection through a single-cell approach. This arm utilizes rare acute HIV and post-antiretroviral therapy (ART) PBMCs from the Mombasa Cohort, a long-term open cohort of women at high risk for HIV. By comparing expression levels between PBMCs from acute infection and after ART-mediated viral suppression within donors (n=10 donors), this approach will inform which ISGs are induced by HIV infection in each cell type that contributes to HIV spread. Second, the in vitro arm of this project will quantify the antiviral function of ISGs in CD4+ T cells from multiple donors. Chosen ISGs will be inactivated via CRISPR to measure their contribution to IFN-mediated restriction of an IFN-sensitive HIV isolate. This approach will assess ISGs induced by HIV in CD4+ T cells, as defined by the in vivo arm, and will also validate ISGs described as antiviral by cell line studies. By interrogating ISG expression and HIV restriction directly in primary human samples, this project will inform the ISG milieu that HIV faces during the earliest stages of infection and which of these factors meaningfully impact HIV replication. Carryin...