INHIBITION OF RECOMBINANT HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REPLICATION BY A SITE-SPECIFIC RECOMBINASE

Current molecular genetic strategies to inhibit productive human immun odeficiency virus type 1 (HIV-1) replication have involved the generat ion of gene products which provide intracellular inhibition of essenti al virally encoded proteins or RNA structures. A molecular strategy to excise proviral DNA from HIV-1-infected cells and render these cells virus free would provide an attractive direct antiviral strategy, prov iding a mechanism to remove viral genes from infected cells. The poten tial of such a molecular genetic intervention was examined by using th e Cre-loxP recombination system. A recombinant HIV-1 clone, designated HIVlox, that contains lox-P within a nonessential U3 region of the lo ng terminal repeats was synthesized. The loxP motif was maintained dur ing replication of HIClox in CEM cells, as demonstrated by reverse tra nscriptase PCR analyses of genomic RNA isolated from virions. Two diff erent types of HIV-1-permissive cells, CEM cells and 293 tells express ing the CD4 glycoprotein, were transformed with a Cre expression vecto r which was shown to encode Cre DNA binding and recombinase activities . HIVlox infection of CEM or CD4(+) 293 cells expressing Cre resulted in a substantial reduction in virus replication compared to control ce lls, and evidence for the presence of the expected excision product wa s found. Site specific excision of HIV-1 can therefore be achieved by using this model system with acute infection. These studies represent one step toward the development of a novel antiviral strategy for the treatment of AIDS.