HIV-1 Integrase Interactions with Viral RNA

HIV-1 integrase (IN) plays two essential roles in the virus life cycle: i) during early steps of infection, IN catalyzes the covalent insertion of viral complementary DNA into target cell chromatin; ii) during late steps of viral replication, IN binds to the viral RNA genome (vRNA) to yield properly mature, infectious virions. The structural and mechanistic bases for IN catalytic activities have been characterized extensively, and the catalytic activity of IN has been exploited as a therapeutic target. By contrast, the non-catalytic function of IN remains largely underexplored. Initial mutational studies demonstrated that certain IN amino acid substitutions impair proper virion maturation by mislocalazing ribonucleoprotein complexes (RNPs) outside of the protective capsid. This phenotype has been termed class II IN substitutions to delineate them from class I IN mutations that specifically impair the catalytic activity of the viral protein. More recent studies have discovered that IN binds vRNA in virions, and that these interactions are essential for proper virion maturation. IN exhibits distinct preference for select vRNA elements including the trans-activation response (TAR) element. Biochemical assays revealed that IN tetramers rather than dimers or monomers effectively bind to cognate vRNA segments. Furthermore, mutational studies have identified IN residues that selectively impaired IN-vRNA binding without substantially affecting other functions of the protein. The non-catalytic function of IN during virion maturation is an important, yet clinically unexploited, therapeutic target. A new and promising class of HIV-1 inhibitors called allosteric IN inhibitors or ALLINIs potently impair IN-vRNA interactions in WT, but not escape mutant, virus. Consequently, ALLINI treatments yield eccentric non-infectious virions with mislocalized RNPs that phenocopy the class II IN mutations. Despite these remarkable advances, the critical gaps in knowledge remain. Specifically, structural and mechanistic foundations for how IN preferentially binds select vRNA elements and encapsulates the vRNA genome within the mature capsid are unknown. To begin addressing these important questions, we propose the following two specific aims. In Aim 1, we will develop a highly innovative and powerful biochemical model system for assembly of mature virus-like particles (mVLPs), which will examine the ability of recombinant IN to effectively encapsulate vRNA inside capsid-like particles (CLPs). Furthermore, we will test whether class II IN mutations and ALLINI treatments can prevent IN-mediated encapsulation of vRNA within CLPs. In Aim 2, we will determine cryo-EM structures of IN tetramers bound to TAR RNA. Follow up biochemical and virology experiments will validate our structural findings. Collectively, the proposed research will substantially advance our understanding of the non-catalytic function of IN during virion morphogenesis and benefit the ongoing efforts to develop improved ALLINIs for their clinical applications. Project Number: 1R21AI191966-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Szu-Wei Huang (+1 co-PI) | Institution: UNIVERSITY OF COLORADO DENVER, Aurora, CO | Award Amount: $258,200 | Activity Code: R21 | Study Section: HIV Molecular Virology, Cell Biology, and Drug Development Study Section[HVCD] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19196601A1