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: 2.59). 02/2006; 6(1):52. DOI: 10.1186/1472-6750-6-52
Source: PubMed

ABSTRACT 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.

Download full-text


Available from: Maurizio Federico, Jul 28, 2015
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Among strategies aimed at developing new nanoparticle-based vaccines, exosomes hold much promise. They are nanovesicles released by basically all eukaryotic cell types originating from intraluminal vesicles which accumulate in multivesicular bodies. Exosomes have immunogenic properties whose strength correlates with the amounts of associated antigens. Engineering antigens to target them in exosomes represents the last frontier in terms of nanoparticle-based vaccines. Here we report a new method to incorporate protein antigens in exosomes relying on the unique properties of a mutant of the HIV-1 Nef protein, Nef(mut). This is a biologically inactive mutant we found incorporating into exosomes at high levels also when fused at its C-terminus with foreign proteins. We compared both biochemical and antigenic properties of Nef(mut) exosomes with those of previously characterized Nef(mut) -based lentiviral virus-like particles (VLPs). We found that exosomes incorporate Nef(mut) and fusion protein derivatives with similar efficiency of VLPs. When an envelope fusion protein was associated with both exosomes and VLPs to favor cross-presentation of associated antigens, Nef(mut) and its derivatives incorporated in exosomes were cross-presented at levels at least similar to what observed when the antigens were delivered by engineered VLPs. This occurred despite exosomes entered target cells with an apparent lower efficiency than VLPs. The unique properties of HIV-1 Nef(mut) in terms of exosome incorporation efficiency, carrier of foreign antigens, and lack of anti-cellular effects open the way toward the development of a flexible, safe, cost-effective exosome-based CD8(+) T cell vaccine platform.
    Vaccine 10/2012; 30(50). DOI:10.1016/j.vaccine.2012.10.010 · 3.49 Impact Factor
  • Source
    • "To dissect among these different possibilities, the intracellular fate of challenging virus was followed using GFP-labeled HIV-1-based VLPs (Muratori et al., 2006) pseudotyped with either wt or fusiondefective VSV-G (Fig. 6A). The fluorescence of these VLPs relies on the high incorporation levels of the product of fusion between GFP and a HIV-1 Nef mutant acquiring a palmitoylation site at its N-terminus as the consequence of the G to C substitution at the amino acid 3. First, the possible effects of PIs on the virus attachment on target cells were investigated. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The inhibitors of HIV-1 protease (PIs) have been designed to block the activity of the viral aspartyl-protease. However, it is now accepted that this family of inhibitors can also affect the activity of cell proteases. Since the replication of many virus species requires the activity of host cell proteases, investigating the effects of PIs on the life cycle of viruses other than HIV would be of interest. Here, the potent inhibition induced by saquinavir and nelfinavir on the replication of both vesicular stomatitis and influenza viruses is described. These are unrelated enveloped RNA viruses infecting target cells upon endocytosis and intracellular fusion. The PI-induced inhibition was apparently a consequence of a block at the level of the fusion between viral envelope and endosomal membranes. These findings would open the way towards the therapeutic use of PIs against enveloped RNA viruses other than HIV.
    Virology 05/2011; 417(1):37-49. DOI:10.1016/j.virol.2011.05.002 · 3.28 Impact Factor
  • Source
    • "C Nef mutants incorporate at high levels in HIV-1- derived VLPs and act efficiently as carrier molecules (Peretti et al., 2005; Muratori et al., 2006). In Fig. 1, the design of the Nef mut -based VLP system is summarized. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Here we report a novel strategy for the induction of CD8(+) T cell adaptive immune response against viral and tumor antigens. This approach relies on high levels of incorporation in HIV-1 VLPs of a mutant of HIV-1 Nef (Nef(mut)) which can act as anchoring element for foreign proteins. By in vitro assay, we found that VLP-associated Nef(mut) is efficiently cross-presented by antigen presenting cells. Inoculation in mice of VLPs incorporating the HPV-16 E7 protein fused to Nef(mut) led to an anti-E7 CD8(+) T cell response much stronger than that elicited by E7 recombinant protein inoculated with incomplete Freund's adjuvant and correlating with well-detectable anti-E7 CTL activity. Most relevantly, mice immunized with Nef(mut)-E7 VLPs developed a protective immune response against tumors induced by E7 expressing tumor cells. These results make Nef(mut) VLPs a promising candidate for new vaccine strategies focused on the induction of CD8(+) T cell immunity.
    Virology 10/2009; 395(1):45-55. DOI:10.1016/j.virol.2009.09.012 · 3.28 Impact Factor
Show more