74lb Identification of LEDGF/p75 as an Essential Factor for HIV-1 Replication that Binds Integrase: a Novel Target for Anti-HIV-1 Drug Discovery.?§E ? S. Emiliani1, J.C. Rain2, M. Maroun1, F. Moisant2, E. Segeral1, L. Selig2, P. Legrain2, R. Benarous*1 1Institut Cochin, INSERM, CNRS Paris, France and 2Hybrigenics S A, Paris, France
Background : Although many interactions between HIV-1 and cellular proteins have been reported, very few of them have been shown to impact significantly viral replication. Methods : To identify key cellular factors essential for HIV-1 replication, we perf ormed i) comprehensive large-scale two-hybrid screens with proteins of the R5 HIV-1 isolate YU2 using highly complex cDNA libraries from CEM cells, ii) RNAi-mediated silencing of genes coding for selected cellular partners of viral proteins, iii) specific two-hybrid screens of HIV-1 random fragments library with cellular proteins impacting HIV-1 replication. Results : Among the new cellular partners of HIV-1 proteins, we identified a novel nuclear partner of Integrase, LEDGF/p75 (Lens Epithelium-Derived Growth Factor), involved in transcriptional regulation and cellular protection against stress-induced apoptosis. Interaction was confirmed by co-immunoprecipitation using anti-LEDGF antibodies. Silencing of LE DGF/p75 re sulted in severe impairment of HIV-1 replication to an extent more than 80%. By contrast, silencing of Ini1, another Integrase partner, had no inhibitory effect on virus production. Two-hy brid screens of the HIV-1 random fragments library using LEDGF/p75 as bait allowed the precise mapping of the 56-182 aa region from the Integrase catalytic core domain involved in the interaction. Interestingly, Bushman and coll. reported recently that the LEDGF gene was overexpressed upon HIV-1 infection, and Debyser and coll. found that recombinant LEDGF/p75 enhanced strand transfer activity of HIV-1 IN in vitro.Conclusions : LEDGF/p75 was identified as an Integrase partner. LEDGF/p75 is a chromatin associated protein that acts as a general transcription co-regulator involved in cell survival and stress response. The identification of novel key cellular factors implicated in HIV replication is probably due to the complexity of the cellular and viral libraries screened, to the comprehensiveness of the screens and to the comparison of the results obtained with the random- and oligo dT-primed cDNA libraries. Functional implication of the cellular interacting proteins was established via the systematic use of gene silencing experiments in viral replication assays. Thus, LEDGF/p75, as a key cellular factor for HIV-1 replication, represents a potential target for the development of novel anti HIV-1 drugs.o ^
