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


CONCLUSIONS
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.
Muratori
Claudia
C
National AIDS Center, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy. muratori@iss.it <muratori@iss.it>
D'Aloja
Paola
P
Superti
Fabiana
F
Tinari
Antonella
A
Sol-Foulon
Nathalie
N
Sparacio
Sandra
S
Bosch
Valerie
V
Schwartz
Olivier
O
Federico
Maurizio
M
D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D014771:Virion / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins