HIV-1 Intasome Assembly and Function

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PROJECT SUMMARY / ABSTRACT New HIV-1 infections continue to drive a worldwide pandemic. Combinatorial anti-retroviral therapies (cART) have helped to blunt the clinical outcomes of HIV-1 infected individuals. However, drug-resistance mutations continue to challenge cART regimens underscoring the importance of identifying new viral drug targets. HIV-1 integration into the human genome is essential for a productive infection. Integration is catalyzed by the retrovirus encoded integrase (IN) that forms a complex with the long terminal repeat (LTR) ends of the viral cDNA, produced by reverse transcription of the HIV-1 genomic RNA. The resulting intasome precisely positions the LTR-ends for catalytic strand-transfer into a genomic target site. Structural comparisons show that all seven retrovirus genera maintain a central Conserved Intasome Core (CIC) containing a tetramer of IN. Some retrovirus family members expand the structure surrounding the CIC by appending additional IN subunits. For example, the prototype foamy virus (PFV) intasome assembles into a simple IN-tetramer while the mouse mammary tumor virus (MMTV) forms an IN-octamer by attaching IN-dimers to either side of the CIC. IN octamer, decamer, dodecamer (12-mer) and hexadecamer (16-mer) intasomes have been reported for HIV-1. Remarkably, the contributions of IN-multimer architecture to HIV-1 biology is largely unknown. The IN-assembly progressions that result in a fully formed HIV-1 intasome are similarly unknown. During infection, reverse transcription and intasome assembly occurs at or near the nuclear membrane. The host cofactor LEDGF/p75 appears to facilitate chromatin localization of the HIV-1 intasomes, and deletion of LEDGF/p75 reduces HIV-1 integration at least 10-fold. We have found the non-conserved peptides linking well- known conserved IN domains control HIV-1 IN-multimer architecture, and that LEDGF/p75 is necessary for efficient HIV-1 intasome assembly in vitro. These observations underpin several key unanswered questions: What are the factors that guide IN multimer progressions resulting in a fully assembled HIV-1 intasome? What is the function of LEDGF/p75 in HIV-1 intasome assembly and/or chromatin interactions? How does HIV-1 IN- multimer architecture impact genomic target site selection in cellulo? We propose to utilize innovative real-time single molecule imaging and analysis to understand the contributions of IN-multimer architecture on HIV-1 mechanics with the following Specific Aims: 1.) determine the IN assembly progressions that control HIV-1 intasome architecture, 2.) determine the role of HIV-1 intasome architecture on the dynamic interactions with defined target DNA and chromatin in vitro, and 3.) determine the role of HIV-1 intasome architecture on targeting host chromatin features in cellulo. These studies are designed to interrogate the animated processes that support HIV-1 intasome architecture with the goal of identifying additional retroviral progressions that might be exploited as therapeutic targets.