The HIV-1 Pre-Integration Complex: Structural and Functional Role of INI1 and LEDGF, Cellular Cofactors of Viral Integrase

Abstract

Virally encoded integrase (IN) proteins perform several important steps in the life cycle of retroviruses. During the early events of viral replication the RNA genome is converted into its cDNA copy which upon interaction with cellular and viral proteins generates the pre-integration complex (PIC). IN is a permanent component of the PIC. The Integrase Interactor Protein 1 (INI1) a homolog of yeast SNF5 and the lens epithelial derived growth factor (LEDGF) have been shown to interact with HIV-1 IN. In order to understand the mechanisms of the INI1 - mediated inhibition and/or activation functions in the early stage of HIV-1 infection, we analyzed the structure-function relationships of a quaternary complex comprising the full length wild type HIV-1 IN, the full length wild-type LEDGF, the INI1 IN binding domain (173-290) and viral U5 DNA. The stoichiometry of the components is 4/2/2/2, as shown by mass spectrometry and FCS. We determined for the first time the binding constants of U5 vDNA for IN by fluorescence anisotropy and found that the dissociation constants of IN/LEDGF and IN/LEDGF/INI1 for U5 vDNA remained in the same order of magnitude, while INI1 when bound to the IN/LEDGF complex inhibited the 3’processing reaction. CryoEM and in vitro functional analysis show that INI1, located within the cellular DNA binding site, inhibits the 3’ processing but not specific viral DNA binding. INI1 stabilizes the highly flexible integrase in a nonproductive conformation. Taken together, our data suggest that the role of INI1 could be to stabilize the highly flexible IN protein in a conformation that prevent non-specific interaction and auto integration during nucleosome targeting. Our results provide the basis for a novel type of integrase inhibitors (conformational inhibitors).