- Research article
- Open Access
Generation and characterization of a stable cell population releasing fluorescent HIV-1-based Virus Like Particles in an inducible way
© Muratori et al; licensee BioMed Central Ltd. 2006
- Received: 14 September 2006
- Accepted: 27 December 2006
- Published: 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.
- Sodium Butyrate
- Reference Reagent Program
- Virion Incorporation
- Peripheral Blood Mononucleated Cell
- Moloney Leukemia Virus
Fluorescent viral particles represent an extremely useful tool for the study of virus-cell interaction. For this reason many efforts have been devoted to the recovery of fluorescent viruses of different species, including herpesviruses [1, 2], adenoviruses , rhabdoviruses , and the African swine fever virus . Concerning HIV, on the one hand, virions which lead to only the infected cell becoming fluorescent were generated by replacing the nef gene with the enhanced green fluorescent protein (gfp) gene  or by inserting a gfp-ires cassette upstream to nef . On the other hand, HIV particles, which themselves were fluorescent due to the incorporation of a fluorescent fusion protein, were generated by inserting the gfp gene between the matrix and capsid Gag sequences . Of note, however, such engineered HIV-1 derivatives, although replication-competent, suffer of limitations in their replication efficiency. As an alternative to insertion of the GFP gene into the viral genome, fluorescent HIV-1 virions were generated by co-expressing a wild-type HIV-1 genome with the GFP-Vpr fusion product [9, 10]. In this case, the fluorescent signal is detectable only in the first replication cycle, and persist until it is intracellularly degraded. Up until now, fluorescent viral particles have been generated by transient transfection procedures and cell lines stably releasing such particles have not yet been available.
Similarly to Vpr, the HIV Nef protein is incorporated in HIV-1 particles, even if at lower levels, i.e. approximately 10 molecules per virion [11–13]. The Nef virion incorporation depends on its localization at the cell membrane. This, in turn, depends on its N-terminus myristoylation marked by the presence of a glycine residue at the position 2, as well as on the presence of two clusters of basic amino acids at the N-terminal region, i.e. two lysines at the positions 4 and 7 and four arginines within the region of amino acid 17–22 .
HIV is thought to bud at level of the so called "detergent-resistant microdomains" (rafts) , that are regions within the cell membranes enriched in cholesterol and sphingolipids. Several authors demonstrated the presence of Nef in rafts [16–19], even if the relative biologic significance has to be clarified yet. In any case, Nef has been found associating poorly (from 2 to 5%) to lipid rafts , and this may account for the low amounts of Nef incorporated into the virions. Hence, a more efficient Nef localization at the lipid raft is expected to increase its viral incorporation. This was indeed the case of the HIV-1 Nef double mutant V153L, E177G we recently described, whose strong raft localization leads to a virion incorporation about 100-fold more efficient than its wt counterpart .
Palmitoylation is a reversible post-translational protein fatty acylation generally occurring on cysteine residues located close to domains interacting with cell membranes. Such a modification regulates the intracellular protein trafficking and the disposition at the cell membrane rafts. In this regard, it was recently demonstrated that the Nef palmitoylation produced by mutating the Gly 3 to Cys increases the raft localization up to 12-fold compared with wt Nef [20, 22]. By consequence, it could be anticipated that palmitoylated Nef undergoes virion incorporation with an efficiency significantly higher than the wild-type counterpart.
Here, we report that a fusion protein consisting of palmitoylated Nef mutant and the green-fluorescent protein (NefG3C-GFP) was efficiently incorporated into HIV-1 particles. In order to generate a stable source of fluorescent HIV-1 virus-like particles (VLPs), we have then stably introduced an expression vector for NefG3C-GFP into a previously described inducibly regulated 293-based HIV-1 packaging cell line. Upon induction of these cells (18-4s), high amounts of fluorescent HIV-1-based VLPs were produced which may be employed for both basic research and antiviral screening.
NefG3C-GFP is incorporated in HIV-1 virions at high levels
Since NefG3C strongly associates with cell membranes [20, 22], it is conceivable that at least part of the NefG3C-GFP we detected was associated with microvesicles and/or exosomes rather than to viral particles. To test this possibility, we transfected 293T cells with the NefG3C-GFP expressing vector alone or together with the pCMVΔR8.74 HIV-1 packaging construct. Forty-eight hours later, the supernatants were harvested, concentrated and purified as above described, and equal volumes of each preparation were analyzed by anti-Nef Western blot. As shown in Figure 1C, no detectable amounts of NefG3C-GFP were found in supernatants from 293T cells transfected in the absence of the HIV-1 packaging construct. This strongly suggests that the most part of NefG3C-GFP molecules we detected in supernatants from the cells co-transfected with the HIV-1 packaging construct were indeed associated with viral particles.
These data strongly support the hypothesis that Nef palmitoylation results in a significant increase in virion incorporation levels which was the basis to attempt the generation of cells stably releasing VLPs with incorporated NefG3C-GFP.
Isolation and induction of the 18-4s cell line stably expressing the NefG3C-GFP fusion product
We have used the 293 Rev-Gag-Pol  (henceforth referred to as 293/RGP) as a platform for the generation of stable cells releasing fluorescent HIV-1 based VLPs. 293/RGP cells are packaging cells for HIV-1 derived vectors inducibly expressing and releasing HIV-1 VLPs. In the 293/RGP cells, the HIV-gag-pol gene and the HIV-rev are separately expressed under the control an ecdysone-inducible promoter (for a review, see ) so that particle production requires addition of the ecdysone analogue ponasterone A (PonA) to the medium.
We then sought to establish the best experimental conditions for the induction of the expression of HIV-1 products in 18-4s cells. Following the methods previously described for the 293/RGP cells , we treated 18-4s cells with 5 mM sodium butyrate and/or PonA 2 μM, and monitored the expression of HIV-1 CAp24 and NefG3C-GFP by FACs two and three days later. Specifically, 293/RGP and 18-4s cells were treated with: i) PonA alone; ii) both PonA and sodium butyrate for one day and, thereafter, with PonA alone, or iii) with PonA and sodium butyrate. As shown in Figure 2B, the treatment with PonA alone resulted in inefficient expression of Gag products in 18-4s cells, while more than 50% of the parental 293/RGP cells was induced to express Gag products. The addition of sodium butyrate was mandatory for significant Gag induction in 18-4s cells. In fact, a pulse of sodium butyrate for 24 h resulted in 60% of the cells expressing Gag whereas its continuous presence for the 48 h induction period further increased the percentage of positive cells to 77%. Additionally, the expression of NefG3C-GFP was increased 3–4 fold. Sodium butyrate treatment increased also the percentage of Gag positive 293/RGP cells from 57% to more than 80%, and improved the relative mean fluorescence intensity by 2–2.5 fold. Of note, in 18-4s cells the treatment with sodium butyrate alone led to an increase of the NefG3C-GFP expression similar to that induced also in the presence of PonA (not shown). No remarkable differences in terms of expression of both CAp24 and NefG3C-GFP were observed between the cells harvested at the day 2 and at the day 3 (not shown). At this time, however, the cell cultures suffered of some cell mortality (not shown).
Finally, the intracellular localization of NefG3C-GFP we analyzed by fluorescence microscope seemed qualitatively similar to that already described for wt Nef  in the presence, however, of a stronger localization at the cell membrane (not shown).
Dose-response of the PonA induced production of VLPs from 18-4s cells
Characterization of NefG3C-GFP VLPs
Furthermore, we were interested in estimating the number of molecules incorporated in the VLPs released from induced 18-4s cells. To this end, we compared the intensity of the signals from decreasing amounts of recombinant (r) Nef with those from decreasing amounts of VLPs whose contents in CAp24 were preventively and rigorously established by quantitative ELISA (Figure 4D). On the basis of the densitometric analysis carried out on the Western blot films (not shown), we estimated that 20 ng of 18-4s VLPs contain approximately 1.25 ng of NefG3C-GFP molecules. Considering the purity of rNef preparation (i.e., 95%), the differences in the molecular weight between CAp24 and NefG3C-GFP, and that it was reported that HIV-1 particles incorporate approximately 5,000 molecules of CAp24 per virion , we calculated the presence of about 150 NefG3C-GFP molecules per VLP. Such an estimation cannot take account of possible differences in the antibody recognition between NefG3C-GFP and wt rNef. These, however, are not expected to be of a major relevance since the anti-Nef polyclonal Abs preparation we employed was recovered using a full-length wt rNef derivative as immunogen.
In conclusion, our data indicate that, upon induction, 18-4s cells produce HIV-1 VLPs whose morphology was not influenced by the high levels of incorporated NefG3C-GFP molecules.
Cytofluorimetric detection of fluorescent NefG3C-GFP VLPs
Next, to assess whether the NefG3C-GFP system could be applied also to other retroviruses, we evaluated the levels of incorporation of NefG3C-GFP molecules in Moloney Leukemia Virus (MLV) particles, that were shown to efficiently incorporate HIV-1 Nef . To this aim, we compared the fluorescence associated to VLPs produced in the presence of wt Nef-GFP with that of NefG3C-GFP. 293T cells were co-transfected with the vector expressing the respective GFP-related product together with a Gag-Pol MLV packaging construct, and equivalent amounts of purified VLPs, as measured by the Mn++ dependent reverse transcriptase assay, were incubated with aldehyde latex beads. The FACs analysis (Figure 7B) clearly showed low levels of fluorescence associated with wt Nef-GFP MLV VLPs, while strong GFP-related signals arose from the beads binding the NefG3C-GFP MLV VLPs. This result represents the proof-of-principle that the NefG3C-GFP approach could be of utility also for non HIV-1 retroviral systems.
Pseudotyped 18-4s VLPs efficiently deliver NefG3C-GFP molecules in target cells
Conversely, and as expected, the viral entry driven by HIV-1 Env appeared significantly less efficient, considering that 1 μg/105 cells of HIV-1 Env pseudotyped VLPs rendered fluorescent about 40% of target cells that also internalized lower amounts of NefG3C-GFP, as indicated by the lower mean fluorescence intensity (Figure 8C). The idea that the fluorescence detected in target cells indeed relied on authentic viral fusion events was strengthen by the inhibition we observed upon the pre-incubation of HIV-1 Env VLPs with either the 17b anti-Env gp 120 (Figure 8C), or the 2F5 anti-Env gp41 neutralizing mAbs (not shown).
Taken together, our data support the idea that the 18-4s cells could be a useful reagent for the study of early events correlating with the viral entry mediated also by heterologous virus receptors.
To the best of our knowledge, this is the first report describing the isolation of a stable cell population releasing fluorescent HIV-1 based VLPs. The biological features of 18-4s cells do not diverge significantly from those of the 293/RGP parental cells , except for a more stringent requirement of sodium butyrate for the VLP production. Sodium butyrate is known acting on methylated promoter sequences and/or on surrounding heterochromatinic DNA, ultimately leading to the re-activation of silenced genes. This is thought occurring through the hyperacetylation of histone and non-histone nuclear proteins [30, 31]. We observed that, in the one hand, the parental 293/RPG cells significantly decreased the levels of VLP release with passages. This was possibly due to a progressive transcriptional silencing of HIV-1 regulatory and/or structural genes, and was efficiently reverted by sodium butyrate. This treatment, on the other hand, was absolutely required for the release of VLPs from 18-4s cells, likely due to the silencing of HIV-1 genes already operative at the time of the cell isolation.
The FACs analysis revealed that 18-4s cells are composed by two clearly distinguishable sub-populations in terms of the expression of both NefG3C-GFP and ΔNGFr. In particular, on the basis of the mean fluorescent intensities we measured by FACs, the bright sub-population seemed to accumulate both products about 5-fold more efficiently than the dull one. Concerning the most relevant expression of NefG3C-GFP, we noticed that with passages, the bright sub-population tended to reduce, meanwhile appearing a detectable fraction of GFP negative cells. Again, this is likely the consequence of the silencing of the NefG3C-GFP sequences rather than to their loss since also in this case the treatment with sodium butyrate resulted in the restoration of the original GFP profile (not shown). We have no obvious explanations for the mechanisms underlying the production of the two sharply distinct 18-4s cell sub-populations, even if it may be conceivable that this reflects the differences in the number and/or sites of the integration of the plasmid vectors. In addition, the consequent protein overproduction might be associated with an impaired protein degradation efficiency, thus resulting in steady-state protein levels overriding the real differences in the numbers of integrated vectors.
The 2–3 fold decreased levels of VLP release from the 18-4s cell line compared with the parental cells appeared consistent with the reduced viral production we observed in transient transfection experiment (not shown), and was possibly due to some steric hindrance occurring during the assembling/release process as the consequence of the high levels of NefG3C-GFP incorporation. Of note, the data from EM analysis showed that this did not lead to significant morphologic alterations of VLPs that in both cases appeared as both mature and immature particles. In this regard, we cannot formally exclude that the significant presence of immature viral particles depended at least in part on a slow kinetic of maturation rendering more difficult the detection of mature particles through the electron microscope observation of ultra-thin cell sections we performed.
The analyses we carried out by FACs and by fluorescence and confocal microscopes consistently indicated that the cells targeted by pseudotyped 18-4s VLPs become fluorescent as early as 2 hours after the challenge. Generally speaking, the great part of HIV-1 particles undergo cell internalization through a receptor independent endocytosis leading to lysosome degradation [10, 32], whose inhibition, in fact, increases HIV infection [33, 34]. In the analysis of the VLP entry we performed, it is worthy of note that the 18-4s VLPs pseudotyped with the fusion defective VSV-G or with "null" VLPs did not induce a significant increase of the cell fluorescence compared with the mock challenged cells. These results, together with those obtained with chemical inhibitors or neutralizing antibodies, strongly suggests that the cell fluorescence we detected basically relied on the completion of the virus fusion events with the release into the cytoplasm of the fluorescent molecules, a conclusion also supported by our results from the confocal microscope analysis.
We feel the relevance of the 18-4s cells stands in two main fields of application. The first could be the study of the mechanisms and dynamic of viral entry, and the second regards the screening of antiviral compounds targeting the late HIV replication events. In particular, the possibility to pseudotype HIV-1 VLPs with a wide array of viral receptors could allow gaining more insights on the not yet fully elucidated mechanism of entry of viruses of different species. This is for instance the case of Human hepatitis C virus, whose cell receptor/co-receptors have still to be unambiguously identified (for a review, see ). In this regard, the cell fluorescence generated by the VLP entry would be a more stringent marker of viral entry compared with the in use systems relying on the lentivirus-mediated transduction of gene markers whose expression can be restricted by host cell factors. On the other hand, the rather simple protocol for the detection of fluorescent VLPs by FACs we developed renders 18-4s cells a both safe and powerful tool for large-scale screenings of new anti-HIV compounds electively targeted to the assembly/release processes. Thus, 18-4s cells are anticipated to be a useful reagent for multiple purposes that will be freely available upon request.
Furthermore, from the point of view of the basic virology, the possibility to analyze viral particles by FACs as here described opens the way towards new rapid and accurate approaches in the investigation on the molecular composition of enveloped viruses in terms of both viral and virus-associated cell products.
Finally, on the basis of the results we obtained with the MLV-based VLPs, we expect that the NefG3C-GFP approach could be applied also to different retroviral systems close to HIV-1(e.g., HIV-2, SIV) and MLV.
Fluorescent VLPs produced by the 18-4s cell line can be considered a novel reagent useful for studies of virus entry also in fields other than HIV. In addition, by exploiting a FACs-based assay for the detection of the VLP fluorescence we here originally describe, 18-4s cells would be of a great utility for screening new generations of antiviral compounds targeting late events of HIV-1 replication.
Molecular constructs, cell lines and cultures
The vector expressing the NefG3C-GFP mutant was generated from the pcNefsg25GFP vector using the quickchange kit (Stratagene, La Jolla, CA), and the mutation was checked by sequencing. 18-4s cells were recovered by the liposome (Lipofectamine-2000, Invitrogen, Carlsbad, CA)-mediated transfection of the HIV-1 packaging 293/Rev-Gag-Pol cells  with vectors expressing NefG3C-GFP and the human Nerve Growth factor receptor truncated in its intracytoplasmic domain . 293T, 293/Rev-Gag-Pol, and 18-4s cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% decomplemented Fetal Calf Serum (dFCS). CEMss and U937 cells were cultivated in RPMI supplemented with 10% dFCS. Human primary lymphocytes were isolated from peripheral blood mononucleated cells (PBMC) isolated from 20–40 year-old healthy male blood donors. Lymphocytes were selected from PBMC using an immunomagnetic-based selection kit from Miltenyi Bioytec (Auburn, CA). PBLs were activated with 2 μg/ml phytohemagglutinin (Sigma-Aldrich, Milan, Italy), and cultivated in RPMI containing 20% of dFCS in the presence of 100 U/ml of recombinant human IL-2 (Roche, Nutley, NJ). Monocytes were isolated by 1 h adherence of PBMC, followed by immunomagnetic depletion of non-monocytic cells carried out through a Miltenyi selection kit. Monocytes were cultivated in 48-well plates in RPMI supplemented with 20% dFCS.
VLP and HIV-1 production, purification, titration, and challenge
Preparations of VLPs were obtained from either 293T, 293/RGP, or 18-4s cells. In the first instance, 293T cells were co-transfected by liposomes (Lipofectamine-2000, Invitrogen) with the pCMVΔR8.74 HIV-1 packaging vector  and the immediate-early CMV promoted vectors expressing either NefG3C-GFP, wt Nef-GFP , GFP-Vpr , or the NL4-3 wt Nef. From 48 to 72 hours later, the supernatants were harvested, clarified, and concentrated by ultracentrifugation on 20% sucrose cushion 100,000 g, 2 h at 4°C. Alternatively, VLP preparations were obtained from the supernatants of either 293/RGP or 18-4s cells two or three days after the induction with 5 mM sodium butyrate (Sigma-Aldrich, St. Louis, MO) and/or 2 μM of Ponasterone A (PonA) (Sigma-Aldrich). Pseudotyped VLPs from 18-4s cells were obtained by transfecting 5 to 10 μg of the vectors expressing the respective viral receptor (i.e., VSV-G or X4 Env HIV-1 from the HXB-2 isolate, both obtained from the NIH AIDS Research and Reference Reagent Program) and, 4 to 6 hours later, by inducing the cells with sodium butyrate and PonA. Twenty-four hours later, the supernatants were replaced with fresh medium containing the inducers, and VLP containing supernatants were harvested 48 and 72 hours after the induction, clarified, and purified as here above described. The purification of 18-4s VLPs was performed by a iodixanol gradient. Briefly, VLPs purified by a 20% sucrose cushion were loaded on a 6 to 35 % discontinuous gradient of Optiprep (Axis-Shield, Oslo, Norway). Samples were ultracentrifuged at 37,000 rpm at 4°C for 90 min on a SW41 rotor, and 700 μl fractions were collected from the top. These where then 1:5 diluted with PBS, pelletted, re-suspended in 100 μl, and finally 30 μl analyzed by both anti-Nef and anti-Gag Western blot.
VLP preparations were titrated by measuring the HIV-1 CAp24 contents by quantitative ELISA (Abbott, Abbott Park, Illinois), and by the reverse transcriptase assay . For the cell challenges, VLPs were adsorbed by spinoculation at 150 g for 30 min at room temperature (r.t.). Afterwards, the cell cultures were re-fed by adding fresh medium, and incubated at 37°C. Finally, the cells were treated for 15 min with trypsin at 37°C, fixed, and analyzed by FACs. Inhibition experiments were carried out by pre-treating cells at 37°C for 2 h with either bafilomycin A1 (Sigma-Aldrich), or chloroquine (Sigma-Aldrich) for the VSV-G VLPs, or by pre-incubating HIV-1 Env VLPs for 1 hour at 4°C with the 17b or 2F5 neutralizing mAbs (200 ng for 1 μg of VLPs), both obtained from the NIH AIDS Research and Reference Reagent Program
HIV-1 NL4-3 virus preparations were recovered by transiently transfecting 293T cells with the pUc/NL4-3 infectious molecular clone . HIV-1 particles recovered from the supernatants were purified and titrated as here above described.
Western blot analysis
Both cells and purified VLP preparations were lysed in PBS, 1% Triton-X100 in the presence of anti-proteolytic agents. For the preparation of cytoplasmic extracts, whole cell lysates were centrifuged at 6,000 g for 10 min at 4°C, and the supernatants frozen at -80°C. Aliquots of 30 μg of total cell proteins were separated in 10% SDS-PAGE, thereby undergoing the immunoblot analysis (Western blot). rNef preparations were obtained and quantified as previously described . The following mono- or polyclonal Abs served for the revelation of both VLP- and cell-associated HIV-1 products: pooled strongly reactive antisera from HIV-1 positive AIDS patients; ARP 444 sheep anti-Nef antiserum from Mark Harris, University of Leeds, Leeds, UK; polyclonal anti-VSV-G protein from Immunology Consultant Laboratories (Newberg, OR); anti-HIV-1 Env gp120 4G10 mAb from the NIH AIDS Research and Reference Reagent Program; and anti-GFP mAb from Clontech (Clontech Laboratories, Palo Alto, CA). In some experiments, VLPs were treated with subtilisin A to digest contaminating cell debris and proteins possibly non specifically adsorbed to the VLP envelope. Briefly, purified virus suspensions were supplemented with an equal volume of 2 mg of subtilisin A (Sigma-Aldrich)/ml in 40 mM Tris-HCl (pH 8.0), 2 mM CaCl2, and incubated 2 hours at 37°C. The digestion was stopped by addition of phenylmethylsulfonyl fluoride to 5 μg/ml.
Fluorescence, confocal, and electron microscope analyses
For the analysis at the fluorescence microscope, 18-4s cells were placed on poly-L-Lysine (Sigma-Aldrich) coated cover glass. Cells were then fixed in 4% paraformaldehyde (Sigma-Aldrich) in PBS and quenched for 10 min with 0.1 M glycine in PBS. Cover slips were then mounted to slides using anti-fade mounting medium, after, in the cases of the 4'-6-Diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA.) labeling, cell permeabilization with 0.1% Triton-X100 (Sigma-Aldrich) in PBS. Finally, the cells were observed using a Zeiss Axioskop 2 plus fluorescence microscope. For the confocal microscope analysis, cells were fixed with 2% v/v of formaldehyde in PBS, and both phase contrast and fluorescence images were taken by an Olympus IX-81 device. For transmission electron microscopy (TEM) examination, cells were fixed in 2.5% cacodylate-buffered (0.2 M, pH 7.2) glutaraldehyde for 20 min at r.t. and post-fixed in 1% OsO4 in cacodylate buffer for 1 h at r.t. Specimens were then dehydrated through graded series of ethanol solutions and embedded in Agar 100 resin (Agar Aids, Cambridge, U.K.). After staining with uranyl acetate and lead citrate, ultra-thin sections were observed with a Philips 208 electron microscope at 80 kV.
FACs analysis of cells and VLPs
For the detection of the human Nerve Growth factor receptor, 3 × 105cells were incubated with a 1:100 dilution of the 20.4 mAb (ATCC, Rockville, MD, USA) or, as control, with a isotype-matched, specie-specific irrelevant mAb for 1 h at 4°C. Thereafter, cells were washed and labeled for 1 h at r.t. with a l:100 dilution of phycoerythrin (PE)-conjugated goat anti-mouse IgG. Cell populations were finally fixed with 2% formaldehyde in PBS and analyzed by FACs. For the detection of HIV-1 Gag-related products, the cells were treated with Permeafix (Ortho Diagnostic, Raritan, NJ) for 30 min at r.t., and then labeled for 1 h at r.t. with a l:100 dilution of PE-conjugated anti-CAp24 KC-57 mAb (Coulter Corp. Hialeah, FL). For the VLP detection by FACs, 5 μl of surfactant-free white aldehyde/sulfate latex beads (Invitrogen Molecular Probes, Eugene, ON) were added to 100 μl of clarified supernatants. The mixture was incubated at r.t. for 1–2 h on a rotating plate, and therefore the samples were washed once with PBS buffer. For the direct FACs analysis, the washed beads were finally resuspended in PBS-2% formaldehyde. Alternatively, the VLP-coupled beads were treated with Permeafix for 30 min at r.t., and then incubated either with a l:100 dilution of PE-conjugated of the anti-CAp24 KC-57 mAb, or with a 1:30 dilution of the anti-Nef mAb # 6.2 from the NIH AIDS Research and Reference Reagent Program.
This work was supported by grants from the AIDS project of the Ministry of Health, Rome, Italy, and by EU, 6th Framework Programme, "New HIV Targets", contract n. LSHP-CT-2004-012169. The ARP444 sheep anti-Nef antiserum was a kind gift of Mark Harris (University of Leeds, UK). The vectors expressing the VSV-G, the HIV-1 X4 Env receptor, and the MLV Gag-Pol packaging construct, as well as the 6.2 anti-Nef, the 17b anti-Env gp120, and the 2F5 anti-Env gp41 mAbs were obtained from NIH AIDS Research and Reference Reagent Program. The GFP-Vpr expressing vector was a kind gift of Tom Hope, Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL. We are indebted to Massimo Sanchez, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy for the technical assistance in cell sorting procedures, to Elena Toschi, National AIDS Center, Istituto Superiore di Sanità, Rome, Italy, for helping in both fluorescence and confocal microscope analyses, and to Federica M. Regini for the excellent editorial assistance.
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