Structural and Biochemical Studies of HIV and MMTV Retroviral Integration onto Nucleosome-bound Target DNA - Summary/Abstract: cells The integration of viral DNA (vDNA) into host chromatin establishes an infection in target and is a hallmark of the replication cycle of all retroviruses, including the Human Immunodeficiency Virus (HIV). Integration is catalyzed by the intasome, a complex consisting of multimers of viral integrase proteins and short vDNA oligonucleotides. Most prior work used free target DNA to probe retroviral integration, largely due to experimental simplicity. However, eukaryotic host target DNA is chromatinized, with nucleosomes forming the core structural building blocks of the genome. Accordingly, our understanding of intasome-mediated integration into chromatin remains incomplete. This proposal will employ novel nucleosome reconstitution and sequencing strategies, biochemical binding and activity assays, and high-resolution cryogenic electron microscopy (cryo-EM) to mechanistically interrogate how and where retroviruses integrate vDNA into chromatin. Three Scientific Aims will be developed using HIV and, as a simpler model system, the Mouse Mammary Tumor Virus (MMTV). In Aim 1, a novel assay will be utilized to interrogate intasome binding preferences on nucleosome assemblies. In Aim 2, the preferred patterns of intasome-mediated integration into and around nucleosomes will be defined. In Aim 3, structures of intasomes bound to nucleosome assemblies will be solved using cryo-EM to dissect the mechanistic basis of integration into chromatin. Preliminary results recently obtained revealed that intasomes selectively integrate into nucleosomal super helical locations that were not previously reported, highlighting both the novelty and significance of the proposed work. Collectively, these studies will advance mechanistic studies underlying the key integration step catalyzed by retroviruses, and the results will have important implications for understanding how these viruses cause diseases that affect millions of people around the world. During the training period, the PI will benefit from the collaborative and scientifically diverse environment present within the Biological Sciences PhD program at UCSD/Salk Institute. With the aid of his two sponsors, the PI will become proficient in numerous technical skills, including but not limited to protein expression and purifications, biochemical activity assays, next generation sequencing, and cryo-EM data collection, processing, and atomic model refinement. Moreover, the PI will enhance his scientific soft skills by presenting his research at conferences, attending grant and manuscript writing workshops, mentoring undergraduate students, and teaching courses. Collectively, the NRSA Predoctoral Fellowship will prepare the PI to become an independent researcher for his next stage of academic training as a postdoctoral associate.
