Quantitation of Cell-Free HIV by Reverse Transcriptase Activity

This assay measures the activity of reverse transcriptase (RNA-dependent-DNA polymerase), an enzyme that synthesizes DNA using RNA as the template. The detection of reverse transcriptase activity (RT) indicates the presence of HIV-1 in in vitro cultures of human peripheral blood mononuclear cells (PBMCs). Because active HIV-1 infection results in lysis of lymphocytes and release of free virions, the associated enzyme activity can be found in cell culture supernatants. Virus is concentrated from the supernatant and separated from soluble cellular contaminants by pelleting in polyethylene glycol and resuspended in a minimal volume of suspension buffer prior to assay.