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

National AIDS Center, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy. <>
BMC Biotechnology (Impact Factor: 2.03). 02/2006; 6(1):52. DOI: 10.1186/1472-6750-6-52
Source: PubMed


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 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.
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.

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Available from: Maurizio Federico, Oct 10, 2015
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    • "Fluorescent VLPs were obtained essentially as described [53] by transient transfection of 293T cells by the Lipofectamine-2000 (Invitrogen) method using the pCMVdelta R8.74 HIV-1 packaging vector and the immediate-early CMV promoted vectors expressing NefG3C-GFP and the R5 (HIV-1 BaL) or X4 (HIV-1 HXBc2) envelope proteins, quoted as VLP-R5Env or VLP-X4Env, respectively. As control, VLPs without envelope protein were used (null-VLPs). "
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    • "For the semi-quantitative western blot analysis, serial dilutions of recombinant (rec)Nef preparations produced, purified, and quantified in our laboratory as previously described [28], were used. The subtilisin A assay was carried out as described [29]. Briefly, exosome samples were supplemented with an equal volume of 1 mg of subtilisin A (Sigma)/ml in 40 mM Tris–HCl (pH 8.0), 2 mM CaCl 2 , and incubated 1 h at 37 • C. The digestion was stopped by adding 1:1 (v/v) FCS and phenylmethylsulfonyl fluoride to 5 ␮g/ml. "
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    • "The frequency of targeting events was comparable to the one obtained using conventional means of introduction of the nuclease and repair matrix by transfection (26) or lentiviral transduction (Figure 1). Lentiviral virions can be used to ferry heterologous proteins as fusions with viral proteins such as Vpr, Nef, IN or Gag (36–39). The delivery of active proteins at the time of virion entry and trafficking into the cell has been reported (40). "
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