HIV-1 Intasome Assembly and Function

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HIV-1 INTASOME ASSEMBLY AND FUNCTIONS PROJECT SUMMARY / ABSTRACT New HIV-1 retrovirus infections continue to drive a worldwide pandemic. Combined drug therapies have helped to blunt the clinical outcomes that afflict HIV-1 infected individuals. However, drug-resistance virus mutations that challenge these treatment regimens persist, making identification of new drug targets of crucial importance. HIV-1 integration into the human genome is essential for a productive infection. Integration is catalyzed by the retrovirus encoded integrase (IN), which forms a multimeric complex with the long terminal repeat (LTR) ends of the reverse-transcribed viral cDNA (termed an intasome and/or pre-integration complex). Structural comparisons show that all seven retrovirus genera maintain a conserved intasome core (CIC) configuration, which precisely positions the LTR-ends for catalytic strand-transfer into a genomic target site during integration. Different retrovirus family members expand the size of the CIC by appending additional IN subunits. For example, the prototype foamy virus (PFV) intasome assembles as a simple tetramer while the mouse mammary tumor virus (MMTV) forms an 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. The contributions of IN-multimer architecture to HIV-1 biology and biophysical chemistry is unknown. The assembly processes that ultimately result in an HIV-1 intasome remain enigmatic. Accumulating evidence suggests that the viral capsid containing the HIV-1 genome is imported into the nucleus where assembly of the intasome occurs in concert with capsid disassembly. Integration into the genomic chromatin then occurs 1-2 µm from the capsid disassembly site. HIV-1 integration is facilitated by host factors that include LEDGF/p75. We and others have found that LEDGF/p75 is required for efficient HIV-1 intasome assembly in vitro. These observations underpin several key unanswered questions: What are the progressions that result in an assembled HIV-1 intasome? What is the role of LEDGF/p75 in intasome assembly? What is the impact of IN- multimer architecture on intasome stability and genomic target site selection in vitro and in vivo? 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-subunit assembly progressions that control multimeric HIV-1 intasome architecture, 2.) determine the role of HIV-1 intasome architecture on the dynamic interactions with defined chromatin target DNA 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 may be exploited as therapeutic targets.