HIV-1 matrix protein p17 increases the production of proinflammatory cytokines and counteracts IL-4 activity by binding to a cellular receptor

Abstract

Purified recombinant HIV-1 p17 matrix protein significantly increased HIV-1 replication in preactivated peripheral blood mononuclear cell cultures obtained from healthy donors. Because HIV-1 infection and replication is related to cell activation and differentiation status, in the present study, we investigated the role played by p17 during the process of T cell stimulation. Using freshly isolated peripheral blood mononuclear cells, we demonstrate that p17 was able to enhance levels of tumor necrosis factor α and IFN-γ released from cells stimulated by IL-2. IL-4 was found to down-regulate IFN-γ and tumor necrosis factor α, and p17 restored the ability of cells to produce both cytokines. The property of p17 to increase production of proinflammatory cytokines could be a mechanism exploited by the virus to create a more suitable environment for HIV-1 infection and replication. Our data show that p17 exerts its biological activity after binding to a specific cellular receptor expressed on activated T lymphocytes. The functional p17 epitope involved in receptor binding was found to be located at the NH₂-terminal region of viral protein. Immunization of BALB/c mice with a 14-aa synthetic peptide representative of the HIV-1 p17 functional region (SGGELDRWEKIRLR) resulted in the development of p17 neutralizing antibodies capable of blocking the interaction between p17 and its cellular receptor. Our results define a role for p17 in HIV-1 pathogenesis and contribute to our understanding of the molecular mechanism of HIV-1 infection and the development of additional antiviral therapeutic strategies.

Productive HIV-1 replication is regulated by the status of host cells, through incompletely defined mechanisms and by the virus itself. HIV-1 infection and replication are linked to cell proliferation, activation, and differentiation, which in turn, are regulated by a variety of extracellular (e.g., cytokines, chemokines, hormones) and intracellular (e.g., transcription factors and DNA replication factors) host factors, and also by regulatory viral proteins. These viral proteins enhance HIV-1 replication in infected cells, facilitate infection of other cells, or interfere with given immune functions. For instance, Tat promotes the infection of certain cells and HIV-1 expression in latently infected cells (1); Vpr regulates cell permissiveness to HIV-1 replication (2); and by binding to certain cellular proteins, Nef contributes to T cell activation, thus influencing both HIV-1 replication and T cell survival (3, 4).

The HIV-1 matrix protein p17 is a structural protein that is important in the life cycle of the retrovirus. It has a role in the early stages of virus replication and participates in the preintegration of the DNA complex into the nucleus of host cells (5, 6). Moreover, p17 is involved in viral RNA binding (7) and transport to the plasma membrane (8–10), in incorporation of the HIV-1 envelope into virions (7), as well as in particle assembly (8). It has been reported that p17 can be the target of neutralizing antibodies against HIV-1 (11–13) and that high levels of p17 antibodies are correlated with slower progression to AIDS (14, 15). The findings of a protective role of immunological response against p17 and the presence of neutralizing epitopes on p17 are surprising, because immunoelectron microscopy and computer modeling studies of HIV-1 suggest that p17 is located to the interior surface of the viral membrane (16), and these findings allow speculation on a possible activity of p17 externally from the virus particle in supporting virus replication.

We previously demonstrated that the HIV-1 matrix protein p17 was able to interact with human IFN-γ without affecting its biological properties (17, 18). Apparently this interaction is caused by structural similarities between p17 and IFN-γ (19). More recently, we have shown that p17 increases proliferation of IL-2 or phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) and enhances HIV-1 replication (20).

In an attempt to elucidate the mechanism(s) underlying p17-enhanced HIV-1 infection, we tested the possibility that p17 facilitates virus replication into target cells by enhancing the production of cytokines known to positively affect viral entry and reverse transcription as well as proviral reactivation. Therefore, we examined the effect of p17 on the production of HIV-1 inductive proinflammatory cytokines, IFN-γ and tumor necrosis factor α (TNF-α) (21, 22). Our results show that p17 up-regulates the secretion of both IFN-γ and TNF-α by PBMCs stimulated with IL-2. Moreover, we show that p17 can completely reverse the capability of IL-4 to dramatically reduce the secretion of these proinflammatory cytokines. Finally, we demonstrate that p17 biological activity is generated by the interaction between viral protein and a specific receptor expressed on activated T cells, via a structure located at the p17 NH₂-terminal region.

Materials and Methods

Recombinant HIV-1 p17 Protein.

The coding sequence of HIV-1 isolate BH-10 p17 (amino acids 1–132) (23) was amplified by PCR with specific primers that allowed us to clone the p17 sequence into the BamHI site of the prokaryotic expression vector pGEX-2T (Amersham Pharmacia). The glutathione S-transferase (GST) fusion proteins were expressed in Escherichia coli and purified by using glutatione Sepharose 4B beads (Amersham Pharmacia). The viral protein was cleaved from GST while still bound to gluthatione-agarose beads by thrombin, as described (24). The p17 protein was further purified (>98%) by reverse-phase FPLC, reaching a purity more than 98%. The absence of endotoxin contamination in the recombinant HIV-1 p17 preparation (<0.1 endotoxin units/ml) was assessed by Limulus amoebocyte assay (BioWhittaker). Purified HIV-1 p17 was also biotinilated by using AH-NHS-Biotin (SPA, Milan) according to the manufacturer's instructions.

Cell Culture.

PBMCs were obtained from healthy individuals, who gave their informed consent to this research according to the Helsinki declaration. Cells were seeded in 96-well U-bottom culture plates (Nunc) at a density of 10⁶ cells/ml and were cultured for the indicated period at 37°C in RPMI 1640 medium (Sigma) supplemented with 10% heat-inactivated human AB serum (Sigma), 100 units/ml penicillin, and 100 μg/ml streptomycin (complete medium).

Cytokine Assays.

PBMCs were cultured in triplicate in 96-well U-bottom culture plates in complete medium with IL-2 (Genzyme) at a concentration of 20 units/ml in the presence or absence of different concentrations of recombinant purified p17 (ranging from 2.5 to 100 ng/ml). Inhibition of TNF-α and IFN-y secretion was performed by adding IL-4 (Peprotech, Rocky Hill, NJ) (20 ng/ml) to the IL-2-, IL-12- (Genzyme) (10 ng/ml), or IL-15- (Genzyme) (10 ng/ml) stimulated PBMCs cultured in the presence or absence of p17. Cell-free culture supernatants were collected 3 days after cell activation and assayed for the presence of TNF-α and IFN-γ by ELISAs (BioSource International, Camarillo, CA) according to the manufacturer's instructions.

Flow Cytometric Analysis of HIV-1 p17 Binding to Cell Surface.

Freshly collected PBMCs or PBMCs stimulated for different periods of time with IL-2 (100 units/ml) were incubated for 30 min on ice with different amounts of biotinilated p17, ranging from 10 ng/ml to 1.6 μg/ml. PBMCs were then incubated for 30 min on ice with phycoerithrin (PE)-conjugated streptavidin (Becton Dickinson). In some experiments, cells were also stained with FITC-conjugated anti-CD4, anti-CD8, and anti-CD19 mAbs (Becton Dickinson). Data were analyzed with cellquest software (Becton Dickinson). Data obtained were used to construct a Scatchard plot as described (25).

Purification of CD4⁺ and CD8⁺ T Cell Subsets.

CD4⁺ and CD8⁺ cells were purified from PBMCs of healthy individuals by positive selection using anti-CD4 and anti-CD8 magnetic beads (MiniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany). Purity was always higher than 96% for each T cell subset. Resting cells or cells stimulated for different periods of time with IL-2 (100 units/ml) were stained and analyzed for expression of p17 receptors on their surface.

mAbs to HIV-1 p17.

Female BALB/c mice were primed with 100 μg of p17 emulsified in complete Freund's adjuvant and boosted at 15-day intervals with 100 μg of protein in incomplete adjuvant. At 3 days after boost four mice were killed and serum was collected and stored at −70°C. At the same time, the spleens were obtained for cell fusion. The fusion protocol, using NS-0 mouse myeloma cells in the presence of 50% polyethylene glycol, was as described (26). Hybridoma supernatants were tested by ELISA with 96-well polystyrene microtiter plates coated with recombinant p17 (0.25 μg per well). The reactivity of different mAbs to native p17 was assessed by Western blot analysis, using commercial HIV-1 Western blot kits (Sanofi, Paris).

HIV-1 p17 Blocking Assays.

PBMCs were cultured in triplicate in 96-well U-bottom culture plates in complete medium with IL-2 (at a concentration of 20 units/ml) in the presence or absence of different concentrations of recombinant purified p17 (ranging from 2.5 to 100 ng/ml). Inhibition of TNF-α and IFN-y secretion was performed by adding IL-4 (20 ng/ml) to the IL-2-stimulated cells. mAbs to p17, at concentrations ranging from 0.5 to 10 μg/ml, were added to cells at the beginning of culture. Cell-free culture supernatants were collected 3 days after cell activation and assayed for the presence of TNF-α and IFN-γ. mabs to p17 were also used at the above concentrations to block p17 binding to cell receptors, as assessed by flow cytometry.

Epitope Mapping.

On the basis of the sequence of p17 molecules we synthesized 62 peptides 10 aa long. Such peptides were representative of the entire length of the p17 molecule and overlapped with a shift of 2 aa. The IFN-γ peptide EAENLKKYFN, recognized by the mAb IGMB-15 (27), was synthesized as a positive control of peptide synthesis and mAb reactivity. The peptides were synthesized by using the SPOTs Synthesis kit (Genosys Biotechnologies, Pampisford, U.K.) on a 8 × 12-cm cellulose membrane, in blue spots areas derivatized with a dimer of β-alanine-NH₂ groups. mAbs were tested for their reactivity against peptides according to the manufacturer's instruction.

Synthesis of Peptide and Mice Immunization.

The p17 peptide SGGELDRWEKIRLR and its ovalbumin-coupled counterpart were purchased from Primm (Milan). Female BALB/c mice were primed i.p. with 50 μg of ovalbumin-conjugated p17 peptide or 50 μg of ovalbumin in complete Freund's adjuvant and boosted at 15-day intervals with 50 μg of the respective immunogen in incomplete Freund's adjuvant. Mice were also immunized with recombinant p17 as described above. At 3 days after boost four mice were killed and serum was collected and stored at −70°C. Reactivity of sera was tested against recombinant p17 by solid-phase ELISA. Block of p17/receptor interaction was assessed by flow cytometry. PBMCs were incubated with biotilinated p17 in the absence (K) or the presence (Ab) of polyclonal antibodies. Percentage of inhibition was calculated as: % of p17 receptor-positive cells stained in K − % of p17 receptor-positive cells stained in Ab/% of p17 receptor-positive cells stained in K.

Results

Effect of p17 on TNF-α and IFN-γ Secretion by PBMCs Stimulated with IL-2.

To determine whether p17 might influence the production of the proinflammatory IFN-γ and TNF-α, which are known to create a more suitable environment for HIV-1 replication, we performed a series of experiments to measure the secretion of these cytokines after stimulation of PBMCs with IL-2 in the absence or presence of the viral protein. Different doses of IL-2, ranging from 2.5 to 100 units/ml, were tested. In all of the subjects analyzed, 20 units/ml led to a consistent induction of both TNF-α and IFN-γ secretion in the supernatant of PBMC cultures. Addition of p17 at different doses increased the production of these cytokines by IL-2-treated PBMCs. The maximum increases were reached when p17 was used at 50 ng/ml, even if p17 showed to be biologically active at a concentration as little as 5 ng/ml. At a p17 concentration of 50 ng/ml, increase was more pronounced for TNF-α ranging from 36% to >100% than for IFN-γ, whose enhancement ranged from 29% to 65%. Use of p17 alone in the absence of mitogen activation had no capacity to induce IFN-γ or TNF-α production (Fig. 1).

Figure 1.

Effect of p17 on TNF-α and IFN-γ production by PBMCs cells upon stimulation with IL-2. Cells were incubated with IL-2 for 72 h in the presence or absence of p17. Unstimulated (medium) and p17-treated cells were used as control of the basal production of the cytokines during the cultures. Supernatants were collected, and TNF-α and IFN-γ production was measured by ELISA. Data are representative of three independent experiments with similar results.

Reversal of IL-4 Induced Inhibition on TNF-α and IFN-γ Production by p17.

Addition of IL-4 to cultures of PBMCs stimulated with IL-2 decreased both TNF-α and IFN-γ production. The decrease in the IFN-γ secretion ranged from 62% to 83%, whereas that of TNF-α ranged from 68% to 84%. To analyze whether p17 was able to counteract the inhibitory effects of IL-4, the viral protein was added to PBMCs at the beginning of culture concomitantly to IL-2 and IL-4. After 72 h of culture, the supernatants were collected and analyzed for both IFN-γ and TNF-α secretion. In all experiments performed, the results obtained showed that p17 was able to restore the capability of PBMCs to produce TNF-α and IFN-γ, with a recovery ranging from 88% to 100% and from 77% to 89%, respectively (Fig. 2).

Figure 2.

Effect of p17 on IL-4 activity directed toward TNF-α and IFN-γ secretion. PBMCs were stimulated with IL-2, IL-12, or IL-15 in the presence or absence of IL-4 alone or in combination with p17. Supernatants were collected after 72 h from mitogen stimulation and assayed for the presence of TNF-α and IFN-γ by ELISA. Data are representative of three independent experiments with similar results.

To establish whether the main target of p17 activity was the IL-2-dependent activation pathway, we analyzed its effect on two IL-2-related cytokines such as IL-12 and IL-15. In fact, IL-12 synergizes with IL-2 in different activities and in particular in driving proliferation and differentiation toward a Th1 effector type (28). On the contrary, IL-15 has overlapping biological effects with IL-2 that are partly caused by the shared use of the IL-2 receptor subunit gamma chain (29).

IL-12 and IL-15 induced both TNF-α and IFN-γ secretion and the addition of p17 did not increase the production of either cytokine (data not shown). As shown in Fig. 2, similar to what was observed in IL-2-stimulated cultures, the addition of IL-4 to PBMCs stimulated with IL-12 and IL-15 decreased both TNF-α and IFN-γ secretion. The decrease in the TNF-α production by IL-12-stimulated cultures ranged from 64% to 81% and from 59% to 72% by IL-15-stimulated PBMCs. The IL-4-induced inhibition in IFN-γ secretion by IL-12-stimulated cultures ranged from 61% to 85% and from 52% to 74% by IL-15-stimulated PBMCs. The addition of p17 to IL-4-treated cultures did not restore the release of the two cytokines observed in cultures stimulated with IL-12 or IL-15 alone, suggesting that the activity of p17 mainly influences the IL-2-mediated activation pathway.

Binding Assay.

To determine whether the activity of p17 was caused by interaction with a receptor expressed on the target cells, biotin-conjugated p17 was allowed to react with viable PBMCs obtained from healthy donors. PBMCs were used either soon after blood donation or after 72 h of IL-2 stimulation. Flow cytometric analysis showed binding of p17 on a small percentage of freshly collected PBMCs from healthy individuals. As shown in Fig. 3, staining of cells with mAb to CD4, CD8, and CD19 revealed that almost the majority of B cells constitutively expressed p17 receptors on their surface, whereas both CD4⁺ and CD8⁺ T cells tested negative. The stability of the p17 receptor-positive phenotype by B cells and the acquisition of p17 receptor expression by T cells was evaluated in PBMC cultures stimulated with IL-2. Almost 100% of B cells were p17 receptor-positive after 72 h of IL-2 stimulation. At the same time we observed a consistent percentage of p17 receptor-positive CD4⁺ and CD8⁺ T cells. Time-course experiments were then performed to study the kinetics of the de novo p17 receptor expression on purified CD4⁺ and CD8⁺ T cell subsets after IL-2 stimulation. As representatively shown in Fig. 4, the appearance of p17 receptors was observed as soon as after 1 h of stimulation on a small percentage of both CD4⁺ and CD8⁺ T cells. The percentage of cells positive for p17 receptor expression increased over time, reaching a peak at 48 h of culture.

Figure 3.

Binding of p17 to a cellular receptor. Biotin-conjugated p17 was allowed to react with freshly collected PBMCs (A–C) or with PBMCs stimulated for 72 h with IL-2 (D–F). Detection of p17 on the cell surface was performed by using PE-conjugated streptavidin as specific reagent. Cells were then stained with a mAb mixture containing FITC-conjugated anti-CD19 (A and D), anti-CD4 (B and E), and anti-CD8 (C and F). Data are displayed as bivariate dot plots. The percentage of cells in each quadrant is given in the upper right corner of each panel.

Figure 4.

Kinetics of p17 receptor expression on T cell subsets. Purified CD4⁺ (■) and CD8⁺ (●) T cells were cultured in the presence of IL-2. Cells were recovered at the indicated time points and then allowed to react with biotin-conjugated p17. Detection of p17 on the cell surface was performed by using PE-conjugated streptavidin as specific reagent.

Biotin-conjugated p17 bound to its receptor with an apparent K_d of 2.5 × 10⁻⁸ M as determined by Scatchard analysis (Fig. 5).

Figure 5.

Binding of p17 to its cellular receptor is dose dependent. Stimulated PBMCs were incubated with different doses of biotin-conjugated p17 and counterstained by using PE-conjugated streptavidin. Cells expressing p17 receptors were analyzed by flow cytometry. Hundred % of positive cells refers to the concentration of p17 that was able to detect the maximum expression level of its receptor. (Inset) Scatchard plot of binding data generated by staining cells (at numbers ranging from 1.25 × 10⁵ to 1 × 10⁶) with different p17 concentrations (ranging from 10 ng/ml to 1.6 μg/ml).

HIV-1 p17 Blocking Assays.

We investigated whether anti-p17 mAbs could affect p17 biological activity and the interaction of viral protein with its cell receptor. Among different mAbs to p17, one named MBS-3, was capable of blocking the effects of p17 on the secretion of proinflammatory cytokines. In fact, IL-2- and p17-stimulated PBMCs reduced the production of IFN-γ and TNF-α to levels comparable to those obtained in cultures stimulated with IL-2 alone when mAb MBS-3 was added to cultures at the beginning of mitogen stimulation (data not shown). As shown in Fig. 6, the addition of mAb MBS-3 to cultures stimulated with IL-2 plus IL-4 and p17 completely blocked the capability of viral protein to reverse the inhibitory activity of IL-4. Other anti-p17 mAbs, and among them one named MK-1, did not show any neutralizing activity. Moreover, the addition of mAb MBS-3 at the beginning of mitogen stimulation did not interfere, in the absence of p17, with proinflammatory cytokine synthesis and IL-4 inhibitory activity. Finally, mAb MBS-3, but not mAb MK-1, completely blocked p17 binding to its receptor (Fig. 7).

Figure 6.

Neutralization of p17 biological activity by the anti-p17 mAb MBS-3. PBMCs obtained from three healthy individuals were stimulated with IL-2 in the presence or absence of IL-4 alone or in combination with p17. Anti-p17 mAbs MBS-3 and MK-1 were added to PBMC cultures stimulated with IL-2 plus IL-4 in the presence of p17. Supernatants were collected after 72 h from mitogen stimulation and assayed for the presence of TNF-α and IFN-γ by ELISA.

Figure 7.

Inhibition of the interaction between p17 and its cellular receptor by the anti-p17 mAb MBS-3. Biotin-conjugated p17 was allowed to react with freshly collected (A–C) and IL-2-stimulated PBMCs (D–F) in the absence (A and D) or the presence of anti-p17 mAbs MBS-3 (B and E) or MK-1 (C and F). Binding of p17 to cells was detected by using PE-conjugated streptavidin as specific reagent. Data were analyzed by using cellquest software and displayed as histograms. The percentage of cells expressing the p17 receptor is given in the upper right corner of each panel.

Epitope Mapping.

To define the p17 functional region, epitope mapping was performed with a series of decapeptides that completely spanned the HIV-1 p17 protein. The solid-phase peptides were individually screened for reactivity to mAb MBS-3. Data obtained showed that the binding region was included between amino acids 9 and 22 (SGGELDRWEKIRLR). We evaluated the capability of the peptide, as a soluble molecule, to interfere with mAb MBS-3 binding to solid-phase p17. The peptide was found to block in a dose-dependent manner the mAb MBS-3/p17 interaction (data not shown).

Development of p17 Neutralizing Antibody in Mice Immunized with a p17 Peptide Analogue.

BALB/c mice were then immunized with the p17 peptide SGGELDRWEKIRLR coupled to ovalbumin to evaluate the capability of the p17 functional region to elicit an anti-p17 neutralizing antibody response. All animals immunized with the ovalbumin-coupled p17 peptide produced antibodies capable of recognizing recombinant HIV-1 p17, as assessed by solid-phase ELISA. The seropositive mice were also Western blot-positive for HIV-1 p17. The titer of p17 antibodies in mice immunized with the peptide ranged from 1:400 to >1:64,000. On the other hand, all animals immunized with recombinant p17 produced antibodies to viral protein at titers ranging from 1:8,000 to >1:64,000. Sera obtained from immunized mice were then tested, at a final dilution of 1:50, for p17 neutralizing activity. As shown in Table 1, the neutralizing activity correlated with the ELISA titers to recombinant p17.

Table 1.

Capability of p17 polyclonal antibodies to interfere with the p17/receptor interaction

Immunogen	Sera titer*	% inhibition†
p17	<1:64,000 (n = 4)	20–70
	>1:64,000 (n = 6)	100
Ovalbumin-p17 peptide	1:400 (n = 2)	0
	1:400–1:64,000 (n = 4)	20–60
	>1:64,000 (n = 4)	100
Ovalbumin	0 (n = 5)	0

^*Serial dilution of sera was used to perform titration in a solid-phase ELISA assay. 

^† Calculated as described in Materials and Methods. 

Discussion

Productive or nonproductive HIV-1 replication is regulated in part by the status of host cells by means of mechanisms not yet completely known. Several regulatory viral proteins enhance HIV-1 replication in infected cells or act extracellularly to facilitate infection of other cells or to interfere with immune functions (1–4). In a previous report we showed that HIV-1 matrix protein p17 was able to increase both cell proliferation and virus replication (20), and we demonstrate here that p17 exerts other biological activities favoring conditions for HIV-1 replication.

HIV-1 p17 was found to enhance the production of proinflammatory cytokines, namely IFN-γ and TNF-α from IL-2-stimulated PBMCs, and both of these cytokines promote HIV-1 production. In particular, TNF-α has been implicated in the pathogenesis of HIV-1 as a direct modulator of virus expression (21, 22). IFN-γ belongs to a group of cytokines that exert pleiotropic effects. However, a potential role for this cytokine in the in vivo regulation of HIV-1 infection is suggested by the following observations: elevated levels of plasma IFN-γ detected in patients with HIV-1 in the absence of concurrent opportunistic infections (30); increased amounts of IFN-γ secreted by CD4⁺ and CD8⁺ T cells from HIV-1-infected patients (31); and high number of IFN-γ-producing cells detected in lymph node biopsies of HIV-1-infected patients with persistent generalized lymphoadenopathy (32). Moreover, IFN-γ is known to positively influence TNF-α production by monocyte/macrophages (33).

The activity of p17 was tested on PBMCs stimulated with other cytokines that have overlapping biological effects with IL-2, including IL-12 and IL-15. Our data showed that p17 enhances IFN-γ and TNF-α secretion only when PBMCs were stimulated by IL-2, but not by the other two cytokines. The finding with IL-15 is in agreement with Li et al. (34), who demonstrated that this cytokine is a critical growth factor in initiating T cell division, whereas IL-2 controls the magnitude of T cell growth, thus providing data that IL-2 and IL-15 regulate distinct aspects of primary T cell expansion in vivo. Moreover, the absence of p17 activity on IL-12-stimulated PBMCs may be explained by the fact that IL-12 and IL-2 activate distinct components of the JAK/STAT signaling pathway during T cell activation (35, 36). These results suggest that the main target of p17 is the IL-2 exclusive pathway and does not involve the other activatory signaling shared with IL-12 and IL-15.

It is well known that IL-4 is an anti-inflammatory cytokine that can suppress IFN-γ and TNF-α production in PBMCs stimulated by IL-2 and/or IL-12 (37–39). Our data show that IL-4 inhibits the IL-2, IL-12, and IL-15 effects on IFN-γ and TNF-α production. However, the addition of p17 to PBMC cultures concomitantly to IL-4 restores completely the ability of PBMCs to secrete both IFN-γ and TNF-α only when they were stimulated by IL-2. This finding underlines again the capability of p17 to exert its biological activity mainly on the IL-2-dependent activation/proliferation pathway and suggests a role of the viral protein in creating a suitable environment for HIV-1 replication even in the presence of inhibitory factors.

In a previous report we found, by using radiolabeled p17, that activity of the viral protein was apparently not mediated by its interaction with a cellular receptor (20). We therefore hypothesized that p17 activity depended on its interaction with a protein released by activated cells. However, data reported here show that p17 counteracts the inhibitory activity of IL-4 only after IL-2 but not IL-12 and IL-15 stimulation. Because IL-4 inhibits proinflammatory cytokine release in IL-2 as well as IL-12- and IL-15-stimulated PBMCs, it was difficult to explain the biological activity of p17 as exerted by an extracellular released factor. Therefore, we investigated a possible p17 interaction with a cellular receptor by a procedure already used for studying the interaction between cytokines and their cellular receptors, which consists in the conjugation of the receptor ligand with biotin. Detection of biotinylated p17 on the surface of cells is therefore achieved by immunofluorescence assay, using PE-conjugated streptavidin as a specific reagent. Indeed, flow cytometric analysis showed that p17 was binding to a cell surface receptor on freshly collected peripheral B cells but not on peripheral CD4⁺ and CD8⁺ T cells. The p17 receptor was regulated in an activation-dependent fashion on T cells. In fact, p17 stained mitogen-stimulated T cells. This finding is in line with our previous results (20) showing that p17 was not inducing proliferation of—and here proinflammatory cytokine production by—resting T cells but acted synergistically with a proliferative signal provided by IL-2. Discrepancies in the results obtained by using biotin- or ¹²⁵I-labeled p17 were probably caused by conformational changes of the viral protein occurred during the radiolabeling procedure we used.

As shown, high affinity binding takes place on nonactivated peripheral blood-derived B cells. Interestingly, in HIV-1-infected individuals with lymphoadenopathy, p17 can be readily detected in germinal centres where, in addition to resting B cells, mainly activated B and some T cells are present (40). Future experiments should clarify the role of p17 in polyclonal activation of B cells in HIV-1 infection.

De novo expression of p17 receptors on the surface of IL-2-stimulated T cells was found to occur at the very early stages of T cell activation. Binding of p17 to mitogen-activated T cells was more pronounced on a proportion of cells or absent on cells within the CD4⁺ and CD8⁺ subset, suggesting the presence of cells more or less responsive or even not responsive to the p17 biological activity. Specificity of p17 binding to its cellular receptor was assessed by completely blocking it by anti- p17 mAb MBS-3 but not by other anti-p17 mAbs generated in our laboratory. The finding that mAb MBS-3, but not other anti-p17 mAbs, interferes with the p17-dependent increase of IFN-γ and TNF-α release, as well as with the capability of p17 to counteract the IL-4 inhibitory activity, suggests that the biological activity of the viral protein occurs after binding to its receptor. Moreover, it is likely that the effects of p17 on cells and therefore, on HIV-1 replication, are caused by the activation of specific components of the signaling pathways during T cell activation.

The finding that p17 becomes active on PBMCs at picomolar concentrations and that it is exported from infected cells in this concentration range (C.P., C.S., and M.A.D.F., unpublished work) makes the mechanism we observed in vitro also possible in vivo. HIV-1 p17 could reach optimal concentrations into body fluids also through a mechanism of virus disintegration or by immune lysis of infected cells where autocrine or paracrine regulation of HIV-1 replication would ensue. In this context, anti-p17 antibodies may act by blocking p17 activity, therefore decreasing HIV-1 replication. This hypothesis would explain the finding that the presence of anti-p17 antibodies represent a serological marker of disease progression during HIV-1 infection in both adults and children (14, 15).

The functional epitope of p17 is located at the NH₂-terminal region of the protein and comprised between amino acids 9 and 22, as assessed by epitope mapping analysis and confirmed by the finding that mice immunized with a p17 peptide analogue of the p17 functional region generate p17 neutralizing antibodies. The functional epitope we identified is already known to be a potent B cell epitope of HIV-1 (41). However, its importance is highlighted by cross-sectional and longitudinal studies showing that asymptomatic HIV-1-infected patients have antibodies against a peptide that includes this functional epitope, whereas they declined completely to nondetectable levels in AIDS patients (42), suggesting that antibodies against the p17 functional region may be protective. However, further studies will allow us to more precisely establish whether neutralizing human anti-p17 antibodies directed against the biologically active structure we identified are present in a proportion of HIV-1-infected patients at different stages of viral infection, and whether they may be correlated with a better clinical stage.

In conclusion, the HIV-1 matrix protein p17 has all of the characteristics of a viral cytokine capable of influencing activation and proliferation of HIV-1 target cells, after binding to a cellular receptor. This property makes p17 an additional member of the group of regulatory proteins that use host mechanisms to the advantage of HIV-1 infection and replication. The future characterization of the receptor and knowledge on its role in promoting p17 activity will improve our understanding of the mechanisms used by HIV-1 to better replicate into different target cells and suggest ways to approach anti-HIV-1 therapeutic strategies.

Abbreviations

PBMC

peripheral blood mononuclear cell

TNF-α

tumor necrosis factor α

PE

phycoerithrin