Alan N. Engelman
Research in the Engelman laboratory focuses on the integration step in the retroviral life cycle. In order to replicate, retroviruses must integrate the viral DNA (vDNA) product of reverse transcription into a host cell chromosome. In addition to the viral integrase (IN) protein, host cell proteins play essential roles in integration. IN functions in the context of preintegration complexes (PICs) that contain the vDNA, host cell, and other viral proteins.
PICs isolated from infected cells catalyze vDNA integration in vitro. Although work with purified IN proteins has revealed details of IN structure and function, little is known about the organization and structure of PICs. We previously defined the HIV-1 intasome as the nucleoprotein complex within PICs that mediates integration. Current projects focus on defining the stoichiometries and folding topologies of viral and cell proteins in purified PICs. In vivo roles of host cell factors in HIV-1 integration are evaluated using RNAi and gene knockout strategies. Biochemical approaches are used to reconstitute retroviral intasomes for structure determination by x-ray crystallography.
One cell protein that directly binds to HIV-1 IN, lens epithelium-derived growth factor (LEDGF), greatly stimulates recombinant IN catalytic activity in vitro. LEDGF was knocked out in mice to generate a clean ex vivo model system. HIV-1 titer was reduced approximately 80-fold on LEDGF knockout cells, revealing a crucial role for the host factor in infection. Different retroviruses differentially target genomic features like genes and promoter regions during integration - lentiviruses like HIV-1 preferentially target active genes fairly equally along their lengths. The frequency and distribution of HIV-1 integration were severely disrupted in LEDGF knockout cells: global integration was reduced approximately tenfold, with significant redistributions of residual proviruses to gene-poor regions and transcriptional start sites, mimicking the natural distributions of other retroviruses. LEDGF displays affinity for chromatin, and LEDGF fusion proteins that contain novel chromatin binding domains redirect HIV-1 to integrate at these locations. These findings highlight a critical role for LEDGF at the genomic targeting step of lentiviral integration and suggest potential translational applications for the LEDGF-IN interaction.
Department of Cancer Immunology and AIDS
450 Brookline Ave., CLS 1010
Boston, MA 02215