HIV-1 Integration

点击翻译按钮获取中文摘要

Significant progress towards understanding the mechanisms involved in retrovirus integration is evident, but many facets of retrovirus integration remain unclear. The multimeric state of integrase (IN) subunits, their role in assembly and their molecular interactions with viral DNA in preintegration complexes (PIC) found in virus-infected cells are still undefined. The spatial arrangements of IN subunits near the viral DNA termini necessary to mediate assembly and concerted integration of the viral DNA termini are unknown. The regions of IN responsible for contact between the two viral termini bound by IN mediating concerted integration are unknown. Our initial efforts with non-ionic detergent disrupted HIV-1 virions to characterize concerted integration in vitro; has led to our recent success with newly-folded recombinant HIV-1 IN. Co-expression of HIV-1 IN with a plasmid expressing protein chaperones (GroEL-GroES) in bacteria allows purified recombinant HIV-1 IN to efficiently mediate concerted (full-site) integration without auxiliary viral or cellular proteins. We will use the reconstitution assay to determine what role newly-folded HIV-1 IN plays in specific assembly steps, in IN-LTR DNA interactions at the molecular level, in full-site integration properties of nucleoprotein complexes having characteristics of purified HIV-1 and murine leukemia virus PIC (or intasomes). The interactions of HIV-1 IN with wild type and mutant viral termini for full-site integration will be investigated. We will attempt to establish the multimeric state of HIV-1 IN subunits necessary and sufficient in nucleoprotein complexes mediating full-site integration. We will determine what amino acid residues of HIV-1 IN contribute to the protein-protein and protein-DNA interactions responsible for assembly and full-site integration. We will use the reconstituted HIV-1 IN-LTR donor complexes to investigate the effects of active site inhibitors to HIV-1 IN integration in vivo and in vitro. We will continue to investigate the role HIV-1 IN has in the retrovirus life cycle with emphasis on understanding full-site integration.