17th International HIV Drug Resistance Workshop 10-14 June 2008, Sitges, Spain

CELLULAR COFACTORS OF HIV INTEGRASE AS NOVEL ANTIVIRAL TARGETS

Antivir Ther. 2008; 13(Suppl. 3):P4 (abstract no. P2)

Z Debyser
KU Leuven Flanders, Belgium

BACKGROUND: Lentiviruses 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 has identified and validated cellular cofactors of nuclear import and integration to develop novel targets for anti-HIV therapy. LEDGF/p75 was originally identified in our group as a binding partner of HIV-1 integrase and as a chromosomal tether. Its role during HIV replication was independently confirmed by RNA interference (RNAi) knockdown, mutagenesis and in mouse knockout cells. Truncation mutants of LEDGF/p75 lacking the chromosome attachment site strongly inhibit HIV replication by competition for interaction with integrase. Data suggest a role for LEDGF/p75 in targeting integration. As for nuclear import of HIV, no cellular cofactor has been identified unambiguously.

METHODS: Using cells overexpressing truncation mutants, we selected HIV strains that overcome the 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, fluorescence cross-correlation spectroscopy and AlphaScreen. The mutations were compared with the reported crystal structure of the integrase core in complex with the integrase-binding domain of LEDGF/p75. Yeast two-hybrid analysis was used to identify cellular partners of LEDGF/p75. AlphaScreen is used for drug discovery.

RESULTS: Detection of integrase mutations in the resistant strains at key positions in the LEDGF/p75–integrase interface provides crucial 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. Resistance selection occurs, but at the cost of a reduced affinity of integrase for LEDGF/p75 and impaired replication kinetics in human peripheral blood lymphocytes. Resistance mutations do not overlap with those observed in clinical trials with strand-transfer inhibitors. We identified JPO2 and POGz as cellular binding partners of LEDGF/p75. Counterscreens are established to identify small molecules inhibiting integrase interaction without cellular toxicity. Using yeast two-hybrid analysis we identified a novel cofactor of integrase mediating nuclear import of HIV.

CONCLUSION: Our results provide a striking example of the power of viral molecular evolution and biological relevance to the crystal structure of the LEDGF/p75 integrase interface. Demonstration of the exclusive role of LEDGF/p75 in HIV integration justifies our ongoing effort in developing small-molecule inhibitors targeting the interaction between integrase and LEDGF/p75. Cofactor-based anti-HIV therapy may become a new paradigm in antiviral research.

2008-06-10
P2

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