Background:  Retroviruses per definition insert their viral genome into the host cell chromosome. Although the key player of retroviral integration is viral integrase, a role for cellular co-factors has been proposed. However, the interplay between host proteins and HIV is poorly understood. LEDGF/p75 was originally identified in our group as a binding partner of HIV-1 integrase in human cells. Its role during HIV replication remained under debate until recently, when independently confirmed results from RNAi knock-down and mutagenesis experiments demonstrated a role for LEDGF/p75 in HIV replication. Preliminary results suggest a chromosomal tethering role for LEDGF/p75. In support of this supposition, truncation mutants of LEDGF/p75 lacking the chromosome attachment site strongly inhibit HIV replication by competition for interaction with integrase.

Methods:  Using cells over-expressing these mutants, we have now selected different HIV strains that have overcome this inhibition. Resistant strains were sequenced and the mutations studied in the context of viral clones and recombinant integrase. Virus phenotype and replication were studied in detail. Interaction between mutant integrase and LEDGF/p75 was determined by confocal microscopy, in vitro pull-down, FCCS and AlphaScreen. The mutations were compared with the reported crystal structure of the integrase core complexed with the integrase binding domain of LEDGF/p75.

Results:  Detection of integrase mutations in the resistant strains at key positions in the LEDGF/p75-integrase interface provides critical evidence for the importance of LEDGF/p75 in HIV integration. The resistance mutations obtained corroborated in vitro results from alanine scanning of the integrase-LEDGF/p75 interface. These results provide a striking example of the power of viral molecular evolution. Resistance selection occurs, but at the cost of a reduced affinity of integrase for LEDGF/p75 and impaired replication kinetics in human PBL. Using FCCS we demonstrate that the affinity of mutant integrase for the LEDGF/p75 truncation mutant is impaired more severely than for full length protein, providing a molecular explanation for the observed resistance phenotype. Moreover, the additive inhibition by LEDGF/p75 knockdown and mutagenesis of the integrase-LEDGF/p75 interface points to the incapability of HIV to circumvent LEDGF/p75 during proviral integration.

Conclusions:  Our data provide biological relevance to the previously resolved structure of the LEDGF/p75 integrase interface. Demonstration of the exclusive role of LEDGF/p75 in HIV integration justifies efforts in developing small molecule inhibitors targeting the interaction between integrase and LEDGF/p75.