Determinants of Architecture on Retroviral Intasome Mechanics

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Retroviruses are obligate intracellular parasites that must integrate a copy of their viral genome (cDNA) into a host chromosome. Integration is accomplished by the retrovirus-encoded integrase (IN) that forms a catalytic complex with two viral cDNA long terminal repeat (LTR) ends, termed an intasome. Retroviral intasomes maintain a conserved intasome core that may be expanded into higher order IN multimer architectures. For example, the prototype foamy virus (PFV) intasome is a simple IN tetramer, while the mouse mammary tumor virus (MMTV) and Rous sarcoma virus (RSV) intasomes are IN octamers. Even higher IN multimers have been reported for the lentiviruses that include HIV-1 and Maedi-Visna virus (MVV). While numerous biochemical and cellular studies have detailed retroviral integration, the assembly mechanics and cost-benefit of different multimeric IN architecture on intasome biophysical properties is a substantial knowledge gap in retrovirology. Our previous work detailed the dynamic target search, integration kinetics, DNA lesion interactions, IN domain requirements and nucleosome targeting by PFV intasomes. Real-time single molecule studies were also performed with MMTV intasomes. Several important differences were identified between the PFV tetramer and MMTV octamer intasomes including distinct target search and strand transfer kinetics as well as the ability of MMTV to form multivalent complexes on a target DNA. These observations have prompted several key questions: What are the contributors that determine IN multimeric architecture? What are the factors of IN multimeric architecture that influence target search and strand transfer? How does intasome architecture influence chromatin DNA binding and target site selection? The PFV, MMTV, RSV and MVV intasomes are convenient biophysical models for probing intasome architecture since they naturally exist as an IN tetramer, octamer or 16-mer with published structures and assembly protocols. We have found that swapping the non-conserved peptides that link the signature conserved retroviral IN protein N-terminal domain (NTD), catalytic core domain (CCD) and C-terminal domain (CTD), converts them into active intasomes with a multimeric architecture of that often mimics the donor intasome. How and why these non- conserved linker peptides influence intasome architecture is unknown. We propose to utilize multiple highly quantitative single molecule imaging tools to understand the contributions of IN multimeric architecture on retroviral mechanics with the following Specific Aims: 1.) examine IN-multimer assembly and integrase activities that distinguish intasome architectures, 2.) determine the role of intasome architecture on the dynamic interactions with defined duplex and chromatin target DNA, and 3.) determine the role of intasome architecture on targeting host chromatin features in vivo. These studies will interrogate the contributors to IN multimer architecture and intasome dynamics with the goal of identifying new retroviral mechanics and therapeutic targets.