Abstract New therapeutic options are needed to treat people infected with multi-drug resistant HIV-1 phenotypes. Here we propose to both investigate and exploit the non-catalytic, essential function of HIV-1 integrase (IN) during virion morphogenesis as a novel therapeutic target. Our highly collaborative research team has already made important contributions toward these goals. Specifically, our studies have revealed that IN binds the viral RNA genome (vRNA) in virions and that these interactions enable formation of properly matured, infectious HIV-1 particles. Moreover, we found that IN tetramers bind with high affinity to select vRNA elements. These findings have begun to uncover mechanistic details behind the non-catalytic function of IN. We have been also investigating the mode of action of a promising class of novel antiretroviral agents termed allosteric HIV-1 inhibitors or ALLINIs. Our studies have demonstrated that ALLINIs inhibit IN-vRNA interactions in virions by inducing aberrant IN multimerization. Consequently, ALLINI treatments yield inactive virus particles with ribonucleoprotein complexes mislocalized outside of the protective capsid. Our more recent studies have focused on characterizing the antiviral mechanism of action of a lead clinical candidate ALLINI pirmitegravir (PIR, STP0404) discovered by ST Pharm, Republic of Korea. This highly safe and potent HIV-1 inhibitor has recently advanced into Phase 2 clinical trials in the USA. Here we propose to build on our highly successful research and have set out the following two multidisciplinary and complementary specific aims. In aim1, we will continue our investigation into the non-catalytic role of IN during virion morphogenesis. For this, we will use biochemical assays to delineate sequence and structural features of vRNA preferentially recognized by HIV-1 IN. Furthermore, we will determine cryo-EM structures of IN tetramers bound to cognate vRNA segments. In addition, we’ll develop a novel and powerful in vitro model system for assembly of mature virus like particles by using IN mediated encapsulation of vRNA inside capsid like particles. Taken together, these studies will markedly advance our understanding of the non-catalytic function of IN during virion morphogenesis and the underlying mechanisms for assembly of mature virions. In Aim 2, we will continue our investigation into a highly promising investigational drug PIR. Specifically, we will delineate the mechanism for how PIR impairs IN-vRNA interactions. Furthermore, our structural, biochemistry and virology experiments will uncover the underlying mechanisms for drug-resistance to PIR. In turn, the findings from these studies will inform our medicinal chemistry efforts to rationally develop improved chemotypes with an enhanced barrier to resistance. Collectively, the two specific aims will elucidate key aspects of the HIV-1 IN biology and will exploit these insights to advance novel therapies to treat people living with H...