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Session 16 Oral Abstracts
Viral Genes and Cellular Co-Factors
Tuesday, 10 am - 12:30 pm
Presentation Time: 11:30 am
Room 2008


68
Characterization of the Role of LEDGF during HIV Replication
Z Debyser*1, S Emiliani2, B Van Maele3, J Vercammen4, M Maroun2, K Busschots3, P Cherepanov3, E De Clercq3, J C Rain5, and R Benarous6
1The European TRIoH Consortium, Rega Inst. for Med. Res., Katholieke Univ. Leuven, Belgium; 2Inst. Cochin, Paris, France; 3Rega Inst. for Med. Res., Katholieke Univ. Leuven, Belgium; 4Lab. for Molecular Dynamics, Katholieke Univ. Leuven, Belgium; 5The European TRIoH Consortium, Hybrigenics S.A., Paris, France; and 6The European TRIoH Consortium, Inst. Cochin, Paris, France

Background:  LEDGF (lens epithelium-derived growth factor, p75) interacts tightly with HIV-1 integrase and is essential for nuclear targeting of this protein in human cells. A large-scale yeast 2-hybrid screen independently revealed LEDGF as a binding partner of HIV-1 integrase.

Methods:  We have now mapped the molecular interaction between LEDGF and HIV-1 integrase using 2-hybrid screening. Several HIV-1 integrase point mutants deficient for the interaction with LEDGF/p75 were selected by 2-hybrid screening of a library of HIV-1 integrase random mutants. The replication kinetics of HIV-1 containing those integrase mutations in cell culture was followed by p24 measurements and real-time Q-PCR analysis. To pinpoint the replication step affected by LEDGF/p75 silencing, we analyzed the kinetics of total HIV-1 DNA, 2-LTR circles, and integrated proviral DNA using real-time PCR. The specificity of LEDGF-IN interaction for various retroviral integrases was verified in a His-tag pull-down assay. Fluorescence correlation spectroscopy was used to study the interaction between IN, LEDGF, and DNA substrates.

Results:  A more precise mapping located the LEDGF-interacting domain within the catalytic core of integrase between amino acids 56 and 182. After expresssion in eukaryotic cells, the selected integrase mutants localized in the cytoplasm. The replication of the mutant viruses in cell culture was abolished and was markedly affected at the integration step. Transient knock-down of LEDGF/p75 during HIV-1 replication in HeLaP4 cells likewise resulted in 2- to 3-fold inhibition of integration. Using a His-tag pull-down assay, we observed an interaction between LEDGF/p75 and integrase of HIV-1 and HIV-2, but not of HTLV-2 integrase.

Conclusions:  The interaction between HIV-1 integrase and LEDGF/p75 mapped to integrase amino acids 56 to 182, and residues critical for this interaction were identified. LEDGF/p75 is required for nuclear localization of integrase and integration of HIV. Silencing of LEDGF/p75 interferes with HIV-1 replication. LEDGF/p75 seems to be a specific co-factor of lentiviral integrases.

Keywords: integration; co-factors; LEDGF