DETECTION OF HIV-1 INFECTION WITH A GREEN FLUORESCENT PROTEIN REPORTER SYSTEM

Several systems for the detection of HIV-1 have been described in whic h HIV-1-susceptible cells contain a reporter gene (chloramphenicol ace tyltransferase, beta-galactosidase, or alkaline phosphatase) under the control of the HIV-1 long terminal repeat (LTR). Upon infection by HI V-1. the expression of the viral rat product increases transcription f rom the HIV-1 LTR promoter, leading to high-level expression of the re porter gene product. Previously described reporter systems require pro cessing of the cells by lysis, fixation, or other steps following infe ction to detect the reporter gene product, In the present study, the A equorea green fluorescent protein S65T variant (GFP-S65T) was used in a reporter system for detecting HIV-1. HeLa-CD4 cells transfected with the plasmid pRH1, which encodes GFP-S65T under the control of the HIV -1 LTR promoter, and either co-transfected with a plasmid encoding the HIV-1 tat product or superinfected with HIV-1, expressed high levels of GFP-S65T, which was readily detected by fluorescence microscopy and fluorescence-activated cell-sorting analysis. The advantages of this system include its simplicity, sensitivity, and ability to detect and sort live HIV-1-infected cells using readily available instruments. Th e construction of cell lines stably transfected with pRH1 will provide a tool for titering HIV-1 and sorting HIV-1-infected cells.