Project Summary Anti-retroviral drugs have transformed HIV-1 infection from a death sentence to a chronic illness, but the rate with which HIV-1 evolves to acquire drug-resistance mandates constant research and development of new antiretroviral compounds. The HIV-1 integrase (IN) enzyme is a dynamic protein that plays a vital role in the HIV-1 lifecycle, and is the target of some of the most widely used, clinically approved antiretroviral drugs. The long-standing view that integration is the sole role of IN was challenged when a recent study surprisingly found that in addition to binding proviral DNA in target cells, IN also binds viral genomic RNA in virions. Intriguingly, inhibition of IN-RNA interactions through mutations within the C-terminal domain (CTD) or by allosteric integrase inhibitors (ALLINIs) results in mislocalization of the viral genomic RNA outside the capsid lattice in virions. These findings demonstrate that IN is essential during virion maturation, and this function may also be a druggable target. However, it is unknown how IN ensures proper placement of viral RNA within virions. While ALLINIs are proposed to inhibit IN-RNA interactions indirectly by inducing aberrant IN multimerization, CTD mutations are shown to directly inhibit IN binding to viral RNAs without affecting its multimeric state. As such, it is currently unclear whether IN multimerization or IN-RNA interactions drive proper particle maturation. IN-RNA interactions alone cannot adequately explain why IN is important for virion morphogenesis, as there are many Class II IN mutations scattered along the length of the IN protein which cause the same phenotype without changing the residues that directly bind RNA. I hypothesize that IN takes on a specific multimeric state or states during virion maturation which allows its binding to the viral genomic RNA. Here, I propose to characterize a panel of 25 distinct mutant IN viruses that are reported to have defects in replication and/or virion morphology or are predicted to affect IN multimerization (Aim 1). Using these mutants and employing biochemical approaches, I propose to determine whether IN multimerization or IN-RNA interactions drive virion morphogenesis (Aim 2). Finally, I plan to determine when during particle genesis IN binds to RNAs and why these eccentric particles are noninfectious (Aim 3). A better understanding of how IN contributes to proper virion maturation would greatly aid the development of new antiretroviral compounds that target a novel function of this essential enzyme.