Poster abstracts

Poster number 97 submitted by Christopher McKee

Cellular cofactor LEDGF profoundly affects HIV-1 integrase subunit-subunit interactions

Christopher J. McKee (MCDB, The Ohio State University), Jacques J. Kessl (College of Pharmacy, The Ohio State University), Mohd J. Dar (Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute), Alan Engelman (Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute), Karin Musier-Forsyth (Department of Chemistry, The Ohio State University), Mamuka Kvaratskhelia (College of Pharmacy, The Ohio State University)

Abstract:
The mandatory integration of the reverse-transcribed HIV-1 genome into the host chromosome is catalyzed by the viral protein integrase (IN). The activity of integrase is regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a key cellular cofactor critical for the integration of the viral genome. To better understand IN interactions with the cellular cofactor, we employed our mass spectrometric protein foot-printing approach. In parallel experiments, surface accessible maps for the full length IN-LEDGF complex were compared with that of free IN and IN complex with the binding defective D366N mutant LEDGF. For this, Lysine and Arginine specific small molecule modifiers were employed and affected residues were identified following tryptic digestion and MALDI-TOF analysis. The footprinting method revealed specific positions in IN located at the dimer interface that were specifically shielded from modification in the presence of wild type LEDGF and not D336N/LEDGF. To better understand interactions between individual subunits in the IN multimer and how LEDGF modulates these interactions, we have devised SDS-PAGE and fluorescence based assays. The results of these studies indicate that in the free IN multimer the individual subunits exchange rapidly, while the addition of LEDGF prevents the subunit exchange by “locking” IN in the multimeric state. Taken together, our data provide new insight into the nature of HIV-1 IN interactions with LEDGF and their potential as a therapeutic target.

Keywords: HIV-1, Integrase, Mass Spectrometry