After entry into the cytoplasm, HIV-1 must transit the cytoplasm, reverse transcribe, uncoat, carry out 3' processing of the viral DNA ends, traverse the nuclear pore, negotiate the nuclear milieu, integrate the processed 3' termini into a chromosome, and complete gap repair. Along the way, the virus must avoid or neutralize numerous host cell defenses, many of which are likely unknown. This broad interval in the viral life cycle remains a black box in many ways. Pre-integration trafficking into and through the nucleus is one of the most significant problems in HIV/AIDS research. Researchers studying these early events have recently implicated a number of host cell factors that are either exploited by lentiviruses (identified ones include LEDGF, Transportin-3/TNP03, CPSF6, Nup358 and several other nucleoporins, Cyclophilin A) or that must be evaded (restriction factors, other innate immunity systems). How these factors fit together into a sequential mechanistic pathway is murky at present. The cast of characters is without doubt incomplete. Intriguingly, there are suggestions that different lentiviruses negotiate nuclear import in variant and possibly flexible ways. Some of the host cell factors, most clearly LEDGF, also appear to impact integration site patterns and transcriptional activity, which has significance for the latency field. In the previou cycle of this grant we focused on the cofactor role of the lentiviral integrase interactor LEDGF as well as pre- and post-nuclear entry impacts of LEDGF integrase binding domain (IBD)-mediated dominant interference. While studying this key HIV-1 dependency factor, we pushed forward to additional host dependency and restriction factors involved in the post-entry HIV-1 replication steps that culminate in integration. We also founded a new germline transgenesis technology in an AIDS-susceptible species. The present renewal application is based on this extensive preliminary data. We propose biochemical and cultured cell experiments to understand observations we have made on the lentiviral cofactor and dominant interference functions of LEDGF. This includes most recently an interplay between LEDGF and one of the less understood HIV-1 accessory proteins, Vpr. Importantly, as our revised title for this cycle indicates, we will also include specific other host dependency factors involved in HIV- 1 pre-integration steps. We will use biochemistry, virology, integration site mapping, and site-specific gene targeting with transcription activator-like effector nucleases (TALENs) to determine mechanisms of viral pre- nuclear trafficking, nuclear import and integration. Importantly, this renewal will take our LEDGF research in vivo as well. Unlike numerous viral diseases for which mice can provide susceptible models, the in vivo pathogenesis roles of lentiviral dependency factors have never been approachable by prospective, controlled genetic manipulation (germline gene addition, knockout, knock-in) of a susceptible species. We will establish and analyze the first-ever knockout of an HIV-1 dependency factor in an AIDS-susceptible species, by targeting LEDGF, use of which is absolutely conserved by all lentiviruses.
Current approaches to HIV-1 disease are limited by drug resistance, toxicities, persisting systemic inflammation, and a lack of effective vaccines. Therapies that target the post-entry virion are presently limited to RT and IN catalysis inhibitors but these nucleic acid modifying steps are only two of many complex post-entry transformations that virion contents must undergo. New strategies that target other steps are sought, including ones that activate latent proviruses to facilitate cure. This project will contribute to strategy development by deciphering how HIV-1 depends upon host cell proteins in its complex journey from the cytoplasm to the integrated proviral state. The research is designed to reveal new basic understanding of replication and pathogenesis, at the cellular and systemic levels in vivo, to identify and define therapeutic targets, and to generate insights into how host factor interactions during post- entry steps impacts integrated provirus location and expression.
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