DETECTION OF INTRACELLULAR HIV-1 REV PROTEIN BY FLOW-CYTOMETRY

The Rev trans-activator protein plays a pivotal role in human immunode ficiency virus type 1 (HIV-1) replication by allowing expression of th e viral structural proteins. We have developed a protocol to quantitat ively assay intracellular steady state levels of Rev Ag (Rev wild type and RevM10 proteins) by flow cytometry. Three fixation and permeabili zation techniques were compared. These protocols varied in the magnitu de of the signal which could be detected, and in the ability to distin guish between Rev Ag positive and negative populations. This technolog y is applicable to a variety transduced or transfected cell types (spe cies, lineage), and for cell lines and primary cells acutely infected with HIV-1. The assay is therefore a valuable tool both to analyze Rev protein expression levels in HIV-infected cells and to optimize deliv ery of the dominant-negative RevM10 gene for clinical gene therapy app lications. In addition, a second, independent intracellular protein (H IV-Tat) has been detected using the same approach.