title: A sensitive, quantitative assay for human immunodeficiency virus type 1 integration.
abstract: Quantitative methods to measure human immunodeficiency virus type 1 (HIV-1) integration promise to be important tools in dissecting the mechanisms whereby latent reservoirs of provirus are established, most notably in the resting T cells of patients receiving antiretroviral therapy. Here we describe a fluorescence-monitored, nested PCR assay that is able to quantify the relatively rare integration events that occur within these cells. Following DNA extraction, a nonkinetic preamplification step is performed with primers that bind genomic Alu elements and HIV-1 gag sequences, under conditions where primers, deoxynucleoside triphosphates, and enzyme are not limiting. This is followed by a kinetic PCR that quantitates HIV-1 long terminal repeat sequences. A T-cell-based integration standard which reflects the randomness of HIV-1 integration is also described. The assay is 10 to 100 times more sensitive than previously reported quantitative Alu PCR-based integration assays. It is specific for integration events, since no proviruses are detected in cells infected either in the presence of an integrase inhibitor or with an integrase-deficient virus. This method promises to provide important new insights into the processes underlying the accumulation and persistence of latent HIV-1 reservoirs and may eventually be useful clinically in monitoring the eradication of latent virus by novel therapies.
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D002460:Cell Line; D004279:DNA, Viral / Q000502:physiology*; D015683:Gene Products, gag / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D016014:Linear Models; D011533:Proviruses / Q000235:genetics*; D012680:Sensitivity and Specificity; D016662:Virus Integration / Q000502:physiology*
chemical_list: D004279:DNA, Viral; D015683:Gene Products, gag