PROJECT SUMMARY Although untreated HIV/AIDS infections are fatal, humans nevertheless have an array of powerful innate antiviral responses that help suppress replication and exert strong selective pressures on the virus during transmission and during viral rebound following ART interruption. These observations suggest that innate immune responses could be of therapeutic value if we can understand them better and discover ways to strengthen them. To infect a cell, HIV-1 must run a gauntlet of such innate immune sensors and restrictions. Studies in Project 2, Cellular Defenses Against HIV, will reconstitute and characterize the mechanisms of cellular innate immune sensing and restriction of HIV-1 that occur during the first half of the viral life cycle. Studies in Aim 1 (SERINC Structure, Mechanism, and Antiviral Activity) will build on our recent cryoEM structure of hSERINC3, and our discoveries that restricting SERINCs are nonspecific phospholipid scramblases and that loss of phosphatidyl serine (PS) asymmetry elicits antiviral effects. We hypothesize that SERINCs inhibit HIV-1 entry by altering the natural asymmetry of the virion lipid bilayer. We propose to determine how SERINCs flip lipids, and to test whether SERINCs exert their antiviral activities by altering PS distribution and disrupting Env conformations. Studies in Aim 2 (HIV-1 Recognition and Innate Signaling) will leverage our ability to reconstitute cGAS innate immune sensing of replicating HIV cores in vitro and our discovery that HIV-1 capsid inhibitors can promote innate signaling in infected myeloid cells. We now propose to determine: 1) how viral core structure and stability affect cGAS-mediated detection of reverse transcription, 2) how capsid-binding factors such as PQBP1 contribute to cGAS sensing in myeloid cells, 3) whether and how CA inhibitors can promote cGAS detection of HIV-1, 4) how cell-specific factors regulate cGAS activity, and 5) how the resulting downstream responses restrict HIV-1 infection. Studies in Aim 3 (TRIM Restrictions) will follow from our observation that TRIM5 proteins form hexagonal cages around incoming HIV-1 capsids. Experiments in this Aim are designed to fill fundamental gaps in our understanding of the viral inhibition and signaling processes that follow this initial TRIM5 recognition step, and to discover how host cofactors facilitate or modulate TRIM5 restriction. Studies in Aim 4 (Reconstitution of Other Restrictions) will build on our ability to reconstitute SAMHD1 and APOBEC3G restriction of replicating HIV-1 cores in vitro, and we now propose to reconstitute MxB restriction. These reconstituted reactions will be used to fill gaps in our understanding of restriction mechanisms, cofactors, and regulation, and to examine how capsid inhibitors can enhance MxB activity.