Retroviral DNA is integrated in the host cell genome by vial integrase protein IN and is required for replication in most cases. However, Dr. Engleman and others previously found that HIV with mutations in the active site of IN can accumulate non-integrated DNA to higher levels than wt virus, and accordingly are able to express viral proteins to a greater extent in the absence of integration than do deletion mutants of IN. Based on these results, Dr. Engleman has found that HIV with the active site mutations in IN can replicate in some, but not all cells. By putting the origin of replication of SV40 in the viral genome, and expressing the SV40 large T antigen in trans, however, nearly all cells including primary macrophages can support replication apparently in the absence of integration. The proposed work will try to determine the mechanism of replication in these viruses, the levels of expression in these systems, the parameters of why some cell types can replicate virus in the absence of integration while others can not. Further aims are to develop gene therapy vectors based on non-integrating retroviruses.
