HIV integrase/DNA complexes and concerted integration

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DESCRIPTION (provided by applicant): In HIV-1 infected cells, the viral RNA is reverse transcribed into a 10 kbp linear blunt-ended DNA followed by the formation of the cytoplasmic preintegration complex (PIC). The viral integrase (IN) within the PIC cleaves a dinucleotide from the 3' OH ends of the viral DNA prior to nuclear transport. IN inserts the two recessed ends in a concerted fashion into the host chromosome, producing the provirus. The cellular co- factor, LEDGF/p75, influences the ability of the PIC to select sites on the host chromosomes for integration. The IN-IN and IN-DNA interactions within the PIC critical for concerted integration and the effect of cellular co-factors on this process are not well defined at the biochemical and biophysical levels. We recently identified a nucleoprotein complex on native agarose gels where IN non-covalently juxtaposes two viral DNA ends that produces the synaptic complex (SC) that possesses properties associated with the PIC. We will study the assembly properties of the SC, investigate the ability of IN to protect the terminal U5 and U3 DNA sequences from DNaseI digestion and determine the molar ratios of chemically cross-linked dimers, tetramers and a larger-size multimer of IN located in the SC. A unique ~32 bp DNaseI protective footprint by IN in the SC suggests a structural relationship to the larger-size multimer. We will investigate the relationships between these cross-linked species to identify the unique inter-subunit residues responsible for formation of the tetramer, as determined by mass spectrometry studies of cross-linked tryptic dipeptides. In- gel fluorescence resonance energy transfer-derived distance measurements and atomic force microscopy will determine the topology of the two viral DNA ends within the SC. We expect to generate a structural paradigm describing the SC and its relationship to the PIC. We will define the cellular co-factor LEDGF/p75 physical interactions with the SC and determine if it affects the SC by measuring the distance between fluorophore- labeled DNA substrates in the SC. We will develop a model system to determine if LEDGF/p75 influences host-site selection on chromatinized supercoiled target DNA for concerted integration. We will study known Class II IN mutants in vitro that are catalytically active and possess post-nuclear entry defects. We will also determine the functionality of the unique residues identified in the cross-linked IN tetramer observed in the SC. Site-directed mutagenesis of these IN residues will be performed for probing of the PIC in vivo and the SC in vitro. The knowledge gain from the R21 studies will be used to further investigate the PIC and the involvement of cellular co-factors in integration. Completion of the R21/R33 studies will foster a necessary and achievable understanding of concerted integration both in vitro and in vivo. PUBLIC HEALTH RELEVANCE: We are building a platform of structural information to understand the HIV-1 synaptic complex (SC) capable of concerted integration. We will determine the IN subunit interactions within SC to define what residues are responsible for formation of the IN tetramer. The functionality of these residues will be investigated by site-directed mutagenesis to probe the PIC in vivo and the SC in vitro. We will determine if LEDGF/p75 physically interacts with SC and test whether it influences host-site selection on chromatinized supercoiled target DNA that contains HIV-1 host-site consensus sequences.