a simple fluorescence based assay for quantification of human immunodeficiency virus particle release一个简单的基于荧光分析的量化人类免疫缺陷病毒粒子释放.pdfVIP

Hermle et al. BMC Biotechnology 2010, 10:32 /1472-6750/10/32 M E T H O D O L O G Y A R T I C L E Open Access Methodology article A simple fluorescence based assay for quantification of human immunodeficiency virus particle release Johannes Hermle, Maria Anders, Anke-Mareil Heuser and Barbara Müller* Abstract Background: The assembly and release of human immunodeficiency virus (HIV) particles from infected cells represent attractive, but not yet exploited targets for antiretroviral therapy. The availability of simple methods to measure the efficiency of these replication steps in tissue culture would facilitate the identification of host factors essential for these processes as well as the screening for lead compounds acting as specific inhibitors of particle formation. We describe here the development of a rapid cell based assay for quantification of human immunodeficiency virus type 1 (HIV-1) particle assembly and/or release. Results: Using a fluorescently labelled HIV-derivative, which carries an eYFP do main within the main viral structural protein Gag in the complete viral protein context, the release of virus like particles could be monitored by directly measuring the fluorescence intensity of the tissue culture supernatant. Intracellular Gag was quantitated in parallel by direct fluorescence analysis of cell lysates, allowing us to normalize for Gag expression efficiency. The assay was validated by comparison with p24 capsid ELISA measurements, a standard method for quantifying HIV-1 particles. Optimization of conditions allowed the robust detection of particle amounts corresponding to 50 ng p24/ml in medium by fluorescence spectroscopy. Further adaptation to a multi-well format rendered the assay suitable for medium or high throughput screening of siRNA libraries to identify host cell factors involved in late stages of H