This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The experimental focus of this proposal is to investigate the composition, assembly and transport of the human immunodeficiency virus (HIV) preintegration complex (PIC). The PIC is a large nucleoprotein complex that facilitates transport of the viral DNA into the nucleus of cells upon completion of reverse transcription. The viral components of the PIC have been characterized, but the size of the PIC suggests it contains a large amount of cellular factors, perhaps both protein and nucleic acid. Structural studies of PICs are hampered by several factors, including: 1) Low quantity in the cell; 2) a relatively short life-span; and 3) difficulty of purification. We propose to develop methods to produce and purify a sufficient quantity of PICs to proteomically analyze PIC composition and characterize nuclear transport. Composition will be analyzed by gel electrophoresis and tandem mass spectrometry. Nuclear transport will be assessed by an in vitro nuclear import and integration assay. This data will give insight into the architecture and cellular transport of the PIC, the first step in developing a new class of anti-retroviral therapeutics.
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