Human gene therapy vol:19 issue:10 pages:1114-1115
ESGT Meeting location:Brugge, Belgium date:13-16 November 2008
Lentiviruses and the viral vectors derived thereof can insert their viral genome into the chromosome of a non-dividing cell. During this process the viral integrase is assisted by various cellular cofactors. Our group identifies and validates cellular cofactors of nuclear import and integration. LEDGF/p75 was originally identified in our group as a binding partner of HIV-1 integrase and as a chromosomal tether. Data suggest a role for LEDGF/p75 in targeting integration.
LEDGF/p75 was identified to interact with HIV integrase by co-IP. Validation of its role during HIV replication and lentiviral vector transduction was obtained by RNAi knock-down and mutagenesis. Direct interaction between recombinant proteins was demonstrated by pull down and Alphascreen assays. Using eGFP-integrase containing viral particles, viral PICs could be visualized in the nucleus.
LEDGF/p75 truncation mutants lacking the chromatin binding domain strongly inhibit HIV replication by competition for interaction with integrase. Stable overexpression of these truncation mutants in cells allowed the selection of HIV strains that overcome the transdominant inhibition. In these resistant strains integrase was mutated at key positions of the LEDGF/p75-integrase interface, providing critical evidence for the importance of LEDGF/p75 in HIV integration. Knock down of LEDGF/p75 severely affected integration sites of lentiviral vectors. The PWWP domain of LEDGF is responsible for the interaction with the chromosome and chimeric LEDGF proteins containing alternative DNA binding domains can substitute for LEDGF. Using Y2H we identified JPO2 and POGz as cellular binding partners of LEDGF/p75.
LEDGF/p75 is the cellular tethering factor involved in selection of integration sites. The interaction with integrase is specific for lentivirinae and lentiviral vectors. Our results facilitate the design of safer vectors for gene therapy by fusing the integrase binding domain of LEDGF with sequence specific DNA binding motifs.