Integration of viral DNA into host DNA is essential for retrovirus replication. Retrovirus infection of cells results in the formation of a cytoplasmic 160S nucleoprotein complex containing newly synthesized linear bluntended viral DNA. The viral integrase (IN) within this 160S nucleoprotein complex trims the DNA genome of its 3' ends by two nucleotides. This complex is subsequently transported to the nucleus and IN integrates the viral termini by a concerted mechanisms into host DNA. The long-term objectives of this application is to investigate protein/protein and protein/DNA interactions of IN within model nucleoprotein complexes capable of concerted integration. The higher- order interactions between IN, viral DNA, and target DNA are undefined. We will focus on how IN can juxtaposition both viral termini for concerted integration via subunit/subunit interactions. We will investigate the mechanism involved in assembly and maintenance of stable prointegration complexes capable of efficiently performing concerted integration. The differential role of individual subunits in these complexes will be examined. We have developed a new assay for measuring concerted integration employing avian myeloblastosis virus IN, a linear 528 bp DNA donor possessing viral termini, and circular pGEM as target. Our efficient concerted integration reactions can be easily monitored by restriction enzyme analysis and agarose gel electrophoresis. The second objective will focus on understanding the biochemical properties of IN with particular emphasis on investigating protein/protein interactions which control formation of IN dimers and tetrameres, and how these structures control the catalytic functions of IN in model nucleoprotein complexes. Solution chemistry encompassing folding of guanidine.HC1 denatured Rous sarcoma virus (RSV) IN derived from a T7 expression system may provide insight into why expressed In of various viruses are not capable of performing concerted integration efficiently in contrast to AMV IN. In collaborative studies, we will try to determine the atomic structure of RSV IN. Both objectives are focused on understanding how IN functions within model nucleoprotein completes capable of faithfully performing the concerted integration reaction. Understanding integration may be critical to providing answers on how to prevent replication of retroviruses in humans.
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