A simple fluorescence based assay for quantification of human immunodeficiency virus particle release

BMC Biotechnology

Methodology Article

Barbara Müller¹  Johannes Hermle¹  Anke-Mareil Heuser¹  Maria Anders¹ 

[1] Department of Infectious Diseases, Virology, University Hospital Heidelberg, Im Neuenheimer Feld 324, D-69120, Heidelberg, Germany;

关键词: Human Immunodeficiency Virus;    Post Transfection;    Human Immunodeficiency Virus Replication;    Late Domain;    Tissue Culture Supernatant;   

DOI  :  10.1186/1472-6750-10-32

 received in 2009-11-10, accepted in 2010-04-20,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundThe 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.ResultsUsing a fluorescently labelled HIV-derivative, which carries an eYFP domain 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 HIV replication, as well as for random screening approaches to search for potential inhibitors of HIV-1 assembly or release.ConclusionsThe fast and simple fluorescence based quantification of HIV particle release yielded reproducible results which were comparable to the well established ELISA measurements, while in addition allowing the parallel determination of intracellular Gag expression. The protocols described here can be used for screening of siRNA libraries or chemical compounds, respectively, for inhibition of HIV in a 96-well format.

【 授权许可】

Unknown   
© Hermle et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.