Cell activation and HIV-1 replication in unstimulated CD4+T lymphocytes ingesting exosomes from cells expressing defective HIV-1
© Arenaccio et al.; licensee BioMed Central Ltd. 2014
Received: 14 April 2014
Accepted: 14 May 2014
Published: 12 June 2014
A relevant burden of defective HIV-1 genomes populates PBMCs from HIV-1 infected patients, especially during HAART treatment. These viral genomes, although unable to codify for infectious viral particles, can express viral proteins which may affect functions of host cells as well as bystander ones. Cells expressing defective HIV-1 have a lifespan longer than that of cells producing infectious particles. Hence, their interaction with other cell types, including resting lymphocytes, is expected to occur frequently in tissues where HIV actively replicates. We investigated the effects of the expression of a prototype of functionally defective HIV-1 on bystander, unstimulated CD4+ T lymphocytes.
We observed that unstimulated human primary CD4+ T lymphocytes were activated and became permissive for HIV-1 replication when co-cultivated with cells expressing a functionally defective HIV-1 (F12/Hut-78 cells). This effect depended on the presence in F12/Hut-78 supernatants of nanovesicles we identified as exosomes. By inspecting the underlying mechanism, we found that ADAM17, i.e., a disintegrin and metalloprotease converting pro-TNF-α in its mature form, associated with exosomes from F12/Hut-78 cells, and played a key role in the HIV-1 replication in unstimulated CD4+ T lymphocytes. In fact, the treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in unstimulated CD4+ T lymphocytes. TNF-α appeared to be the downstream effector of ADAM17 since the treatment of unstimulated lymphocytes with antibodies against TNF-α or its receptors blocked the HIV-1 replication. Finally, we found that the expression of NefF12 in exosome-producing cells was sufficient to induce the susceptibility to HIV-1 infection in unstimulated CD4+ T lymphocytes.
Exosomes from cells expressing a functionally defective mutant can induce cell activation and HIV-1 susceptibility in unstimulated CD4+ T lymphocytes. This evidence highlights the relevance for AIDS pathogenesis of the expression of viral products from defective HIV-1 genomes.
The high frequency of mutations occurring in HIV-1 retrotranscription can lead to the production of defective HIV-1 genomes. They can persist in peripheral blood mononuclear cells (PBMCs) of HIV-1 infected patients since host cells are expected to have a lifetime longer than that of cells infected by replication-competent quasispecies. First evidences about the significant presence in vivo of defective HIV-1 genomes came from the observation that the number of PBMCs containing HIV-1 DNA greatly exceeds that of cells expressing infectious HIV-1 [1, 2]. Later, 46% of HIV-1 genomes detected in PBMCs from 10 infected patients was found deleted, while PBMCs from 3 patients harbored only deleted or rearranged HIV-1 genomes . Sequence analysis of the HIV-1 RT gene in PBMCs and rectal tissue of highly active anti-retroviral therapy (HAART)-treated patients revealed a great number of stop codons in all samples analyzed . More recently, the analysis of 213 proviral clones from treated patients demonstrated the presence of 88.3% of genomes with identifiable defects .
Proteome of F12/HIV-1 a
Amino acid substitutions
Viral protein product
Apparently intact 
Premature stop codon at aa 76 
Apparently intact 
Exosomes are nanovesicles released by all cell types. They are lipid bilayer vesicles of 50–100 nanometers which form intracellularly upon inward invagination of endosome membranes . This event leads to the formation of intraluminal vesicles which then become part of multivesicular bodies. Subsequently, they can undergo to lysosomal degradation or, alternatively, be released into extracellular space upon fusion of multivesicular bodies with plasma membrane. Exosomes can be released also through direct extrusion of plasma membrane . Current protocols of purification and marker analysis cannot discriminate between endosome-produced nanovesicles and vesicles with similar size but originating from cell membranes. For the sake of clarity, cell-produced nanovesicles are here defined exosomes irrespectively to their origin.
It is now accepted that exosomes are part of the intercellular communication network while they were originally thought to secrete only waste cell material . Exosomes incorporate messenger RNAs, microRNAs, and proteins which are functional in target cells . Exosomes from HIV-1 infected cells incorporate Gag  and Nef HIV-1 proteins [21, 22]. HIV-1 Gag molecules associate with exosomes by virtue of their higher-order oligomerization, while Nef is incorporated in exosomes upon anchoring into lipid raft microdomains through its N-terminal myristoylation and a stretch of basic amino acid residing in its alpha-helix 1.
Here, we demonstrate that the cell activation induced by exosomes from F12/Hut-78 cells in unstimulated human primary CD4+ T lymphocytes couples with HIV-1 replication. These effects relied on the expression of NefF12.
Unstimulated CD4+T lymphocytes become susceptible to HIV-1 infection when co-cultivated with cells expressing a defective HIV-1 strain
Considering that HIV-1 replication in lymphocytes needs cell activation, we hypothesized that the expression of the defective HIV-1 generates a signal leading to lymphocyte activation. To identify the nature of such a signal, we repeated the co-culture experiments in the presence of a number of drugs or antibodies. Among the drugs we tested, HIV-1 replication in bystander cells was successfully blocked by 1 μM of both GW4869 and Spiroepoxide (Figure 1B), i.e., two structurally unrelated inhibitors of neutral sphingomyelinase blocking exosome release [23–28]. This result prompted us to focus on exosomes as possible effectors of the induction of HIV-1 susceptibility in unstimulated CD4+ T lymphocytes.
Characterization of exosomes from F12/Hut-78 cells
Nanovesicles from F12/Hut-78 cells were formally identified as exosomes by FACS analysis demonstrating the presence of both monosialotetrahexosylganglioside (GM1) [31, 32], using FITC-conjugated cholera toxin fraction B (CTX-B), and CD63 (Figure 2B). In addition, western blot analysis revealed that, consistently with what previously described for exosomes from Gag-  or Nef-expressing cells [21, 22], both HIV-1 CAp24 and Nef proteins associate with exosomes from F12/Hut-78 cells (Figure 2C).
Primary unstimulated CD4+T lymphocytes release both IL-2 and TNF-α in response to the treatment with exosomes from F12/Hut-78 cells
These data implied that the treatment of unstimulated CD4+ T lymphocytes with exosomes from F12/Hut-78 cells leads to cell activation.
The treatment with exosomes from F12/Hut-78 cells renders unstimulated CD4+T lymphocytes susceptible to HIV-1 replication
Similar to what we observed for TNF-α release, the extents of HIV-1 replication in CD4+ T lymphocytes correlated with the exosome input (Figure 4B). Notably, the treatment with 10 μM AZT led to a strong reduction of HIV-1 CAp24-expressing CD4+ T lymphocytes (Figure 4C). This result formally excluded that possible carry-over from exosome- and/or virus-associated CAp24 interfered with the results we obtained by FACS analysis, meanwhile indicating that HIV-1 was indeed expressed in unstimulated CD4+ T lymphocytes challenged with exosomes from F12/Hut-78 cells.
Next, we were interested in establishing whether HIV-1 expressing CD4+ T lymphocytes released infectious virus. To this end, CD4+ T cells were challenged with exosomes from Hut-78 or F12/Hut-78 cells and then with HIV-1 in the presence or not of AZT. Three days later, CD4+ T cell cultures were washed, and co-cultures with Rev-CEM reporter cells, i.e., a cell line expressing GFP in the presence of both HIV-1 Tat and Rev products , were set up. After additional 3 days, the percentages of GFP-expressing Rev-CEM cells were evaluated by FACS (Figure 4D). No significant increase in the percentage of GFP+ cells was observed in Rev-CEM from co-cultures with CD4+ T lymphocytes treated with control exosomes, whereas a sharp increase was detectable when the lymphocytes were treated with exosomes from F12/Hut-78 cells. Such an increase was no more detectable when challenged CD4+ T lymphocytes were cultured in the presence of AZT until the addition of the reporter cells. These results indicated that unstimulated CD4+ T lymphocytes release infectious virus when treated with exosomes from F12/Hut-78 cells. In addition, the lack of infected Rev-CEM cells in the co-cultures with CD4+ T lymphocytes challenged with control exosomes indicated that HIV-1 replicating in Rev-CEM cells did not originate from the viral input used to infect the unstimulated CD4+ T lymphocytes.
We concluded that the exosome-dependent activation of CD4+ T lymphocytes rendered them susceptible to productive HIV-1 infection.
ADAM17 is involved in both activation and HIV-1 replication in unstimulated CD4+T lymphocytes treated with exosomes from F12/Hut-78 cells
Both TNF-α neutralization and blocking TNF-α receptors-1 and -2 inhibit the HIV-1 replication in primary unstimulated CD4+T lymphocytes treated with exosomes from F12/Hut-78 cells
To further strengthen the role of TNF-α, experiments with neutralizing antibodies against the TNF-α receptors (TNFR)-1 and 2, either alone or in combination, were performed. According to what we observed with anti-TNF-α antibodies, the treatment with anti-TNFR1 and –TNFR2 antibodies alone or in combination inhibited the HIV-1 replication in unstimulated CD4+ T lymphocytes challenged with exosomes from F12/Hut-78 cells (Figure 7B). This result suggests that the activation of both TNFR1- and TNFR2-related intracellular signals is required for the HIV-1 replication in unstimulated CD4+ T lymphocytes.
Overall, these results supported the data we obtained with TAPI-2, and are consistent with the idea that TNF-α was the downstream effector of exosome-associated ADAM17.
Unstimulated CD4+ T lymphocytes replicate HIV-1 when targeted by exosomes released by cells expressing NefF12
These results indicate that the expression of NefF12 in exosome-producing cells is sufficient to render unstimulated CD4+ T lymphocytes susceptible to HIV-1 infection.
Unstimulated CD4+ T lymphocytes become susceptible to HIV-1 infection when co-cultivated with activated CD4+ T lymphocytes infected by a NefF12-expressing, non-producer HIV-1
These results add significance to what we previously observed with 293 T transfected cells in terms of the key role of NefF12 expression in the HIV-1 replication in bystander CD4+ T lymphocytes.
Formal demonstration of the presence of grossly defective HIV-1 genomes in PBMCs of AIDS patients was achieved in mid ‘90s . Physical mapping of defective genomes indicated that the frequency of deletions is proportional to their proximity to the central part of the viral genome . Subsequent studies demonstrated the presence of genetically damaged sequences in transcriptionally active proviral HIV-1 . Moreover, HAART treatment further selects for mutated/deleted viral genomes in both PBMCs and rectal tissues [4, 41]. The identification of an in vitro cell system comprehensively reproducing the events which in vivo lead to the formation of defective HIV-1 DNA is still an unmet challenge. The functionally defective F12/HIV-1 genome  could recapitulate some features of different defective HIV-1 species which in vivo remain transcriptionally active.
Our investigations started from the observation that unstimulated CD4+ T lymphocytes became susceptible to HIV-1 infection upon trans-well co-culture with F12/Hut-78 cells. This experimental setting was useful to avoid misinterpretations due to possible effects induced by cell-to-cell contact. However, it is fairly conceivable that it mirrors events occurring in free co-cultures and, more important, in tissues most relevant for HIV replication, e.g., gut mucosa and lymph nodes.
Unstimulated CD4+ T lymphocytes challenged by exosomes from F12/Hut-78 cells released both IL-2 and TNF-α. The fact that no IL-2 production was detectable until 36–48 hours after exosome challenge (data not shown) suggested that its release was consequence of the cellular activation induced by an autocrine/paracrine stimulus of TNF-α. The prompt release of TNF-α appears to be the key event leading to the activation of unstimulated CD4+ T lymphocytes challenged by exosomes from F12/Hut-78 cells.
It was reported that HIV-1 does not replicate in quiescent CD4+ T lymphocytes due to its inability to complete retrotranscription and integration steps [33, 43, 44]. On the other hand, TNF-α has manifold effects on HIV infection: for instance, it activates resting CD4+ T lymphocytes meanwhile stimulating TAK, i.e. a cell kinase required for the Tat trans-activation . It can also re-activate latent HIV-1 through the action of its downstream effectors . Our evidences that unstimulated CD4+ T lymphocytes became susceptible to HIV-1 infection when the exosome-mediated stimulus was applied before but not after HIV-1 infection are highly reminiscent of previously published data on resting CD4+ T lymphocytes stimulated with anti-CD3/CD28 antibodies before or after HIV-1 challenge . In this experimental setting, a great increase of both fully retrotranscribed and integrated products was found in pre-stimulated compared to post- and non-stimulated cells. On the basis of these literature data, we hypothesize that the TNF-α released upon exosome challenge helps the viral genome to complete early replication events. Upon provirus integration, the activation of the downstream effectors of both TNF-α and IL-2 would contribute to foster HIV-1 transcription.
We found active ADAM17 in F12/Hut-78 cells and exosomes released by them, but not in uninfected cells. Despite the cells accumulate much higher amounts of inactive ADAM17 compared to the active form, only the latter seemed to be uploaded in exosomes. The mechanism of selective upload of active ADAM17 deserves further investigations.
We cannot formally exclude that NefF12 delivered in target cells by exosomes could contribute to the cell activation/HIV-1 replication in unstimulated lymphocytes. However, the fact that NefF12 allele lacks many Nef signaling functions [13, 14] runs against this hypothesis. Most important, the domain critical for ADAM17 activation and uploading into exosomes (i.e., the N-terminal 11–40 region) [35, 38] is conversely well conserved in NefF12 .
Our results allow to propose a model where cells expressing defective HIV-1 genomes release ADAM17-loaded exosomes which, when ingested by bystander quiescent CD4+ T lymphocytes, can induce the cleavage of pre-stored pro-TNF-α. The mature cytokine may act in an autocrine/paracrine way by activating intracellular signals supporting the progression of the life cycle of infectious HIV-1. Thus, our results suggest that defective HIV-1 genomes which are transcriptionally active could play a relevant role in supporting the spread of replication-competent HIV. Furthermore, they strengthen previous evidences about the key role of TNF-α in HIV pathogenesis .
This mechanism would be of particular importance in HAART-treated patients where defective HIV-1 genomes accumulate in the presence of the block of the replication of infectious virus. In this scenario, the activation of resting CD4+ T lymphocytes induced by ADAM17-uploaded exosomes may foster the spread of replication-competent latent HIV, which was estimated to represent 11.7% of genomes , as well as the emergence of drug-resistant HIV quasispecies and, when the therapy is interrupted, infectious HIV. In addition, a sustained production of exosomes inducing the release of active TNF-α could contribute to the overall immune activation still present in successfully HAART-treated patients.
Exosomes released by cells expressing a defective HIV-1 genome activate bystander, resting CD4+ T lymphocytes and render them susceptible to HIV-1 infection. Exosome-associated ADAM17 plays a key role in both cell activation and HIV-1 replication observed in CD4+ T lymphocytes, and TNF-α is its downstream effector. Overall, our data reveal a so far neglected effect of defective but transcriptionally active HIV-1 genomes on bystander lymphocytes which could play a relevant role in the spread of HIV-1 in infected persons.
Cell cultures and isolation
Hut-78, D10/Hut-78, F12/Hut-78 , and Rev-CEM cells  were grown in RPMI medium plus 10% heat-inactivated fetal calf serum (FCS). Human embryonic kidney 293 T cells were grown in Dulbecco‘s modified Eagle’s medium plus 10% heat-inactivated fetal calf serum (FCS). CD4+ T lymphocytes were isolated from PBMCs of healthy donors by negative selection using an immunomagnetic-based kit (Miltenyi), and cultivated in RPMI medium plus 10% FCS. The cell cultures were checked for their purity through FACS analysis for CD4, CD8, and CD14 markers. Cell preparations having more than 3% of CD8+ cells and /or 1% of CD14+ cells were discarded. For activation, 2 μg/mL of phytohemagglutinin (PHA) were added to CD4+ T lymphocyte cultures. TAPI-2 was purchased from Santa Cruz Biotechnology. Recombinant human TNF-α was from R&D Systems. AZT was obtained from NIH AIDS Research and Reference Reagent Program. For anti-TNF-α neutralization experiments, 1 μg of either anti-TNF-α neutralizing antibodies (polyclonal rabbit antibodies, Fitzgerald Industries) or normal rabbit IgGs was added to CD4+ T lymphocyte immediately after exosome and HIV-1 challenges. The same amounts of antibodies were then re-added after 24 hours of culture. For TNFR-blocking experiments, anti-TNFR1 clone #16085 and anti-TNFR2 clone #22210 neutralizing monoclonal antibodies (both from R&D Systems) were used. CD4+ T lymphocytes were incubated for 1 hour at 4°C with 1 μg of the antibodies either alone or in combination or, as control, with the same amount of isotype control IgGs, and then challenged with exosomes and HIV-1. Antibodies were then re-added 24 hours later.
Molecular clones, transfections, and HIV-1 infections
Preparations of T-tropic HIV-1 were obtained from the supernatants of 293 T cells 48 hours after transfection with the pNL4-3 HIV-1 molecular clone. The VSV-G pseudotyped NL4-3/NefF12 HIV-1 strain  was obtained by co-transfecting the HIV-1 molecular clone with a pcDNA3.1 (Invitrogen)-based vector expressing the VSV-G in a 5:1 molar ratio. Both NefF12 and NefG2A alleles were cloned in the pcDNA3.1 vector after PCR amplification from the HIV-1 molecular clones expressing the respective Nef mutant [12, 48]. Transfections were performed using Lipofectamine 2000 (Invitrogen). Supernatants were clarified and concentrated by ultracentrifugation on a 20% sucrose cushion as previously described . This method ensured that exosomes from transfected cells were excluded from the vesicle pellet . Virus preparations were titrated in terms of HIV-1 CAp24 content using quantitative enzyme-linked immunosorbent assay (ELISA, Innogenetic). Infections with HIV-1 were carried out by spinoculation at 400 × g for 30 min at room temperature (r.t.) using 500 ng CAp24 equivalent of HIV-1 for 106 cells.
Trans-well co-cultures were carried out in 12-well plates using Cell Culture Insert Falcon Membrane (25 mm diameter, 0.4 μm pore size, Becton Dickinson). They were set up by putting 106 F12/Hut-78 or parental Hut-78 cells in the upper chamber, while 2 × 106 unstimulated CD4+ T lymphocytes were seeded in the bottom chamber. The co-cultures were then run overnight in the presence or not of 1 μM of the inhibitors of exosome release GW4869 (Sigma) and Spiroepoxide (Santa Cruz). Then, CD4+ T lymphocytes were recovered and infected with HIV-1, washed, and left in culture 3 days in the presence or not of 10 μM AZT. Afterwards, lymphocytes were analyzed by FACS for the HIV-1 expression through the detection of intracytoplasmic HIV-1 CAp24.
Nanovesicle purification and challenge
Cell culture supernatants containing exosome-depleted FCS were processed following already described methods for exosome purification. In detail, supernatants were centrifuged at 500 × g for 10 min and filtered with 0.22 μM pore size. Then, the supernatants underwent differential centrifugations consisting in a first ultracentrifugation at 10,000 × g for 30 min. Supernatants were then harvested and ultracentrifuged at 70,000 × g for l h. The pelleted vesicles were resuspended in 1 × PBS, and ultracentrifuged again at 70,000 × g for 1 h. Afterwards, the pellet was resuspended in 200 to 400 μL of 1 × PBS and, in some cases, subjected to discontinuous iodixanol (Axis-Shield) gradient. It was performed essentially as described . Briefly, concentrated vesicles were ultracentrifuged at 200,000 × g for 1.5 hours at 4°C in an SW41 Ti rotor (Beckman) through a 6 to 18% iodixanol density gradient formed by layering iodixanol in 1.2% increments. Then, 0.7 ml fractions were collected starting from the top. In some instances, half of each fraction was diluted with 2 volumes of 0.9% sodium chloride and ultracentrifuged for 30 min at 95,000 rpm in a TL-100 tabletop ultracentrifuge. Finally, the pellet was resuspended in 50 μl of Tris–HCl pH 7.4 10 mM, NaCl 100 mM, EDTA 1 mM, 0.1% Triton X-100.
AchE activity assay
The vesicle-associated AchE activity was evaluated through the Amplex Red kit (Molecular Probes) following the manufacturer’s recommendations. The AchE activity was measured as mU/mL, where 1 mU is defined as the amount of enzyme which hydrolyzes 1 pmole of acetylcholine to choline and acetate per minute at pH 8.0 at 37°C.
FACS analysis of cells and nanovesicles
For the detection of intracytoplasmic HIV-1 CAp24, cells were treated with trypsin for 15 min at 37°C. Then, they were labeled using the KC57-RD anti-CAp24 monoclonal antibody (Coulter) upon permeabilization with Cytofix/Cytoperm solutions (BD Pharmingen) as previously described .
Double staining of nanovesicles was performed by incubating them with 5 μl of surfactant-free white aldehyde/sulfate latex beads overnight at r.t. on a rotating plate. The binding of exosomes with the beads was necessary for both antibody labeling and FACS analysis. Afterwards, nanovesicle-bead complexes were washed and incubated at 37°C for 2 hours with FITC-conjugated CTX-B. Then, the samples were washed and incubated with PE-conjugated anti-CD63 monoclonal antibody (BD Pharmingen) 1 h at 37°C. Finally, the beads were washed, resuspended in 1 × PBS-2% v/v formaldehyde, and FACS analyzed.
Western blot assay
Western blot analysis on cell lysates was performed by washing cells twice with 1 × PBS (pH 7.4) and lysing them for 20 min on ice with lysis buffer (20 mM HEPES pH 7.9, 50 mM NaCl, 10 mM EDTA, 2 mM EGTA, 0.5% nonionic detergent IGEPAL CA-630, 0.5 mM dithiothreitol, 20 mM sodium molybdate, 10 mM sodium orthovanadate, 100 mM sodium fluoride, 10 μg/mL leupeptin, 0.5 mM phenylmethylsulfonyl fluoride). Whole cell lysates were centrifuged at 6,000 × g for 10 min at 4°C. The protein concentration of cell extracts was determined by the Lowry protein quantitation assay. Aliquots of cell extracts containing 30 to 50 μg of total proteins were resolved by 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred by electroblotting on a 0.45 μm pore size nitrocellulose membrane (Amersham) overnight using a Bio-Rad Trans-Blot. For western blot analysis of exosomes, they were lysed and analyzed as described for cell lysates. For immunoassays, membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Triton X-100 for 1 hour at room temperature, then incubated overnight at 4°C with specific antibodies diluted in PBS containing 0.1% Triton X-100. The antibodies used in immunoblots were: polyclonal rabbit anti-HIV-1 Gag CAp24 #4250 (NIH AIDS Research and Reference Program), sheep polyclonal anti-Nef ARP444 (a generous gift from Dr. Mark Harris), rabbit polyclonal anti-ADAM17 from Cell Signaling, monoclonal anti-ICAM-1 15.2 from Santa Cruz Biotech., monoclonal anti-CD63 from R&D Systems, and monoclonal anti-β-actin AC-74 (Sigma). Immune complexes were detected with horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse antibodies (GE Healthcare) and enhanced chemiluminescence reaction (Euroclone).
IL-2 and TNF-α detection
The release from CD4+ T lymphocytes of IL-2 and TNF-α was detected by ELISPOT and ELISA, respectively. For IL-2, cells were treated with exosomes and then cultivated for 48–72 hours in ELISPOT microwells (Millipore) previously coated with a monoclonal antibody against human IL-2 (Mabtech). Afterwards, the cells were removed, and a biotinylated antibody against human IL-2 was added, followed by the addition of a streptavidin-alkaline phosphatase. The plate was finally developed using BCIP/NBT substrate (Sigma). Spot-forming cells were analyzed and counted using an ELISPOT reader (Amplimedical Bioline A-EL-VIS GmbH). The measurement of TNF-α was performed through ELISA kits from Immunological Sciences following the manufacturer’s recommendations.
When appropriate, data are presented as mean + standard deviation (SD). In some instances, the paired Student’s t-Test was used and confirmed using the non-parametric Wilcoxon rank sum test. P < 0.05 was considered significant.
This work was supported by grants from AIDS National and “Ricerca Finalizzata” projects, both from the Ministry of Health, Italy. Rev-CEM cells, rabbit anti-HIV-1 CAp24 polyclonal antibodies #4250, and AZT were obtained from NIH AIDS Research and Reference Reagent Program. We thank Andreas Baur for helpful comments. We are indebted to Pietro Arciero for excellent technical support and Stefania De Menna for both secretarial and administrative supports.
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