Generation and characterization of a stable cell population releasing fluorescent HIV-1-based Virus Like Particles in an inducible way
1 National AIDS Center, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy
2 Department of Technology and Health, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy
3 Virus and Immunity Group, Department of Virology, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France
4 Department for Molecular Virology, University of Heidelberg, Otto-Meyerhof-Zentrum, Im Neuenheimer Feld 350, 69120 Heidelberg, Germany
5 Forschungsschwerpunkt Infektion und Krebs, F020, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
BMC Biotechnology 2006, 6:52 doi:10.1186/1472-6750-6-52Published: 27 December 2006
The availability of cell lines releasing fluorescent viral particles can significantly support a variety of investigations, including the study of virus-cell interaction and the screening of antiviral compounds. Regarding HIV-1, the recovery of such biologic reagents represents a very hard challenge due to the intrinsic cytotoxicity of many HIV-1 products. We sought to overcome such a limitation by using a cell line releasing HIV-1 particles in an inducible way, and by exploiting the ability of a HIV-1 Nef mutant to be incorporated in virions at quite high levels.
Here, we report the isolation and characterization of a HIV-1 packaging cell line, termed 18-4s, able to release valuable amounts of fluorescent HIV-1 based Virus-Like Particles (VLPs) in an inducible way. 18-4s cells were recovered by constitutively expressing the HIV-1 NefG3C mutant fused with the enhanced-green fluorescent protein (NefG3C-GFP) in a previously isolated inducible HIV-1 packaging cell line. The G3C mutation creates a palmitoylation site which results in NefG3C-GFP incorporation into virions greatly exceeding that of the wild type counterpart. Upon induction of 18-4s cells with ponasterone A and sodium butyrate, up to 4 μg/ml of VLPs, which had incorporated about 150 molecules of NefG3C-GFP per viral particle, were released into the culture supernatant. Due to their intrinsic strong fluorescence, the 18-4s VLPs were easily detectable by a novel cytofluorometric-based assay developed here. The treatment of target cells with fluorescent 18-4 VLPs pseudotyped with different glycoprotein receptors resulted in these becoming fluorescent as early as two hours post-challenge.
We created a stable cell line releasing fluorescent HIV-1 based VLPs upon induction useful for several applications including the study of virus-cell interactions and the screening of antiviral compounds.