Generation and characterization of a stable cell population releasing fluorescent HIV-1-based Virus Like Particles in an inducible way.

Claudia Muratori (1) , Paola d'Aloja (1) , Fabiana Superti (2) , Antonella Tinari (2) , Nathalie Sol-Foulon (3) , Sandra Sparacio (4) , Valerie Bosch (5) , Olivier Schwartz (3) , Maurizio Federico (1)

1 National Aids Center
2 Department of Technology and Health
3 Virus et Immunité
4 Department of Molecular Virology
5 Forschungsschwerpunkt Infektion und Krebs, F020

BACKGROUND: 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. RESULTS: 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 mug/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. CONCLUSION: 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.

Domaines: Génétique; Biologie cellulaire; Maladies infectieuses; Microbiologie et Parasitologie

Claudia Muratori, Paola d'Aloja, Fabiana Superti, Antonella Tinari, Nathalie Sol-Foulon, et al.. Generation and characterization of a stable cell population releasing fluorescent HIV-1-based Virus Like Particles in an inducible way.. BMC Biotechnology, 2006, 6, pp.52. ⟨10.1186/1472-6750-6-52⟩. ⟨pasteur-00134527⟩