title: Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes.
abstract: HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3' ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.
authors.affiliation: Center for AIDS Health Disparities Research, Meharry Medical College; Department of Biochemistry and Cancer Biology, Meharry Medical College.
authors.forename: Muthukumar
authors.initials: M
authors.lastname: Balasubramaniam
authors.affiliation: Center for AIDS Health Disparities Research, Meharry Medical College; Department of Biochemistry and Cancer Biology, Meharry Medical College.
authors.forename: Benem
authors.initials: B
authors.lastname: Davids
authors.affiliation: Center for AIDS Health Disparities Research, Meharry Medical College; Department of Biochemistry and Cancer Biology, Meharry Medical College.
authors.forename: Amma B
authors.initials: AB
authors.lastname: Addai
authors.affiliation: Center for AIDS Health Disparities Research, Meharry Medical College; School of Graduate Studies and Research, Meharry Medical College; Department of Microbiology and Immunology, Meharry Medical College.
authors.forename: Jui
authors.initials: J
authors.lastname: Pandhare
authors.affiliation: Center for AIDS Health Disparities Research, Meharry Medical College; Department of Biochemistry and Cancer Biology, Meharry Medical College; Tennessee Center for AIDS Research (TN-CFAR), Meharry Medical College; cdash@mmc.edu.
authors.forename: Chandravanu
authors.initials: C
authors.lastname: Dash
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D016679:Genome, Viral; D019427:HIV Integrase / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009698:Nucleoproteins; D016662:Virus Integration / Q000502:physiology
chemical_list: D004279:DNA, Viral; D009698:Nucleoproteins; D019427:HIV Integrase