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Basic Science

VPAC1 is a cellular neuroendocrine receptor expressed on T cells that actively facilitates productive HIV-1 infection

Branch, Donald R.a,b,c; Valenta, Linda J. E.a,c; Yousefi, Shidaa; Sakac, Darinkac; Singla, Ruchid; Bali, Meenakshic; Sahai, Beni M.d; Ma, Xue-Zhongb,c

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From the aDepartment of Medicine, and Institute of Medical Science, University of Toronto, the bDivision of Cellular and Molecular Biology, Toronto General Research Institute of the University Health Network, and cCanadian Blood Services, Toronto Centre, Toronto, Ontario, and the dCadham Provincial Laboratory and the Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.

Requests for reprints to: D. R. Branch, Canadian Blood Services, 67 College Street, Toronto, Ontario M5G 2M1 Canada.

Received: 23 March 2001;

revised: 3 August 2001; accepted: 7 August 2001.

Sponsorship: Supported in part by Medical Research Council of Canada (MRC) grant MT-14980. S. Yousefi is a recipient of a Natural Sciences and Engineering Research Council (NSERC) of Canada Fellowship award. X.-Z. Ma is a recipient of an Ontario HIV Treatment Network Fellowship award.

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Objective: A lack of productive HIV-1 infection of Kit225 compared to Jurkat T cells, despite similar levels of CD4 and HIV-1 chemokine co-receptors, was found to correlate with the expression of vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide receptor-1 (VPAC1). We therefore examined a role for this seven-transmembrane G protein-coupled neuroendocrine receptor in modulating HIV-1 infection.

Methods: Reverse transcription–PCR was used to show the level of VPAC1 expression in different T-cell lines. A signal-blocking antibody to VPAC1 was used to examine its inhibiting effect on HIV-1 infection. Transfection of VPAC1 cDNA in both sense and anti-sense orientation was used to assess the role of VPAC1 in HIV-1 infection. HIV-1 infection was monitored by gag p24 ELISA using HIV-1IIIB or by luciferase activity using pseudo envelope-typed HXB2-NL4-3-luciferase. Analysis of HIV-1 gag DNA and 2-LTR circles was utilized to examine a possible mechanism for the effect of VPAC1.

Results: Using VPAC1 signal blocking antibody, we showed that up to 80% of productive infection with HIV-1IIIB was inhibited. We also demonstrated that HIV-1 gp120 has sequence similarity to the natural ligand for VPAC1 and postulate that it can activate this receptor directly. Transfection of VPAC1 cDNA in the anti-sense orientation resulted in a significant loss, up to 50% of productive infection. In contrast, transfection of cells with VPAC1 in the sense orientation increased the productive infection by more than 15-fold and caused a profound increase in syncytium formation. Furthermore, stimulation of VPAC1 on primary cells facilitated in vitro infection with HIV-1 HXB2-NL4-3. Analysis of HIV-1 gag DNA indicated that VPAC1 does not affect viral entry; however, cells that show negligible expression of VPAC1 may not be productively infected as indicated by a lack of 2-LTR circle formation.

Conclusion: We have discovered a cellular receptor, VPAC1, that is a novel and potent facilitator of HIV-1 infection and thus, is a potentially important new target for therapeutic intervention.

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Infection with HIV and AIDS progression continues to increase worldwide despite years of research and other efforts to control its spread [1–3]. The pathogenesis of HIV infection is linked closely to the replication of the virus in vivo [4] and depends on a variety of viral and host-associated factors [5,6]. Host factors include certain chemokine receptors, which together with the membrane-bound CD4, act as co-receptors for HIV-1 entry into cells [7–9]. Discoveries of these host factors have initiated many studies to develop therapies against HIV infection that focus on blocking these co-receptors. Indeed, the complete blockage of HIV infection and progression to AIDS with current drug treatment regimens appears to be difficult, if not impossible. The clinical inadequacy of current antiretroviral drugs is a consequence of many factors, including a rapid occurrence of viral drug resistance [10]. Therefore, studies directed at inhibiting infection using strategies that target co-receptor expression or other host factors, some of which may have yet to be discovered, seems a realistic approach to combating this disease [11]. Indeed, agents that block HIV-1 co-receptor binding and/or fusion are currently under development.

In the late 1980s, a role for the neurotransmitter, vasoactive intestinal peptide (VIP), in HIV pathogenesis was proposed based on its amino acid sequence similarity to a five amino acid portion of the viral envelope glycoprotein gp120 [12]. Although these early reports were not fully investigated, recent studies have supported this sequence similarity of VIP to gp120 [13] and one study has shown VIP to be an activator of transcription from the integrated HIV-1 long terminal repeat (LTR) [14]. However, as yet, there have been no studies of any potential role for the receptors of VIP in HIV infection.

VIP receptors are neuroendocrine receptors belonging to the secretin subfamily of the seven-transmembrane G protein-coupled receptor superfamily [15]. Thus far, three related receptors for VIP have been described, based on the binding affinity of VIP and a related peptide, pituitary adenylate cyclase-activating polypeptide (PACAP). Vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide receptors (VPAC) 1 and 2 bind VIP and PACAP with similar affinity, whereas pituitary adenylate cyclase 1 (PAC1) binds PACAP with higher affinity than VIP. VPAC1 and VPAC2 share approximately 50% homology and have been identified in humans and rodents [15,16]. Both VPAC1 and VPAC2 bind VIP with similar affinity and the activation of either receptor leads to an increase in intracellular calcium and of the second messenger cyclic AMP (cAMP) [17]. A recent report suggests that significant activation from the HIV-1 LTR is induced only by peptides specific for VPAC1 [14]. Thus, the aim of this study was to test the hypotheses that HIV-mediated signal transduction through VPAC1 facilitates HIV-1 pathogenesis and, therefore, is a new host factor involved in HIV pathogenesis and a potentially novel therapeutic target for HIV treatment strategies.

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The human T-cell lines Hut78, Jurkat, and SupT-1 were obtained from American Type Culture Collection (Manassas, Virginia, USA). These cells were chosen because they express VPAC1 at high, medium, and low levels, respectively [18]. Kit225 T cells were a gift from G. Mills (MD Anderson Cancer Center, Houston, Texas, USA). The human kidney epithelial cell line 293T was a gift from D. Littman (New York Medical University, New York, New York, USA). Normal donor whole blood was obtained from the Canadian Blood Services and primary peripheral blood mononuclear cells (PBMC) were isolated on density gradients as described previously [19].

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Antibodies, peptides and cDNA

Rabbit antibodies [20] that recognize the first extracellular loop of VPAC1 (aa 191–222) and VPAC2 (aa 174–195) and inhibit signal transduction without affecting ligand binding were a gift from E. Goetzl (University of California at San Francisco, San Francisco, California, USA). The control sera, also provided by E. Goetzl, consisted of anti-VIPR1 or anti-VPAC2 antiserum adsorbed with the immunizing peptide [20]. VIP peptides, secretin, and helodermin were from Peninsula Laboratories Inc. (Belmont, California, USA). Forskolin was from Sigma-Aldrich Chemical (Oakville, Ontario, Canada). Full-length VPAC1 complementary DNA (cDNA) were either from M. Laburthe (Pasteur Institute, Paris, France) or were generated in our laboratory by PCR cloning, based on the published sequence [17], from primary PBMC. VPAC1 cDNA was inserted into pcDNA3 (Invitrogen, Carlsbad, California, USA) in either sense or anti-sense orientation.

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T-cell tropic HIVIIIB was obtained from NIH AIDS Research and Reference Reagent Program (Rockville, Maryland, USA) and used in infection experiments at a multiplicity of infection (m.o.i.) of 0.3 infectious virions (i.v.)/cell. To generate the T-tropic pseudo envelope-typed HIV–luciferase construct, 15 μg of the plasmid NL4-3 luc (in which the firefly luciferase gene has been inserted into the viral nef gene) and 10 μg of a plasmid containing the T cell-tropic envelope gene (pHBX2 env) were precipitated as described ([21]; plasmids were gifts from D. Littman). 293T cells (2 × 106) were co-transfected with the aforementioned plasmids, incubated at 37 °C for 2 days, and the virus in supernatants was pelleted by ultracentrifugation over 20% sucrose for 1 h at 19 000 rpm (33 000 × g) and resuspended in TNE buffer (20 mM Tris, pH 7.5; 1 mM EDTA; 100 mM NaCl).

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FACS analysis

Two-color fluorescent antibody cell sorting (FACS) analysis of CD4 and CXCR4 expression was accomplished as described previously for cell surface antigen detection [22]. Briefly, 1 × 106 cells were incubated with 10 μl mouse anti-CXCR4 (12G5; NIH AIDS Research and Reference Reagent Program) followed by 2 μL fluoresceine isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin (Biosource, Camarillo, California, USA) and then with 10 μL phycoerythrin-labeled mouse anti-human CD4 (Serotec, Raleigh, North Carolina, USA). Fluorescence was measured by FACScan (Coulter Canada Inc., Mississauga, Ontario, Canada).

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Reverse transcription (RT)–PCR

Primers for amplifying human VPAC1 were designed as follows: forward primer, 5′-TGCTTGCCGT CTCCTTCTTCTCTG-3′; reverse primer, 5′-CT TTATGATCCAGAAGGTGCTTCCTGGCCTAC-3′. Messenger RNA (mRNA) was isolated using QuickPrep Micro mRNA purification kit (Amersham Pharmacia Biotech Inc., Baie d'Urfe, Quebec, Canada) from approximately 5 × 106 cells; 1 μg of the mRNA was reverse transcribed to make cDNA using First-Strand cDNA Synthesis kit (Amersham Pharmacia Biotech Inc.). A plasmid containing a full-length VPAC1 insert was used as a positive control. One microliter of sample was amplified in 25 μL reaction mixture containing 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2 , 0.25 mM each dATP, dCTP, dGTP, dTTP, 2 μM each primer, and 2.5 U Taq polymerase. After an initial incubation of 5 min at 95°C in a Perkin Elmer model 9600 thermocycler, the target sequence was amplified by 35 cycles of 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min. PCR products were separated by electrophoresis through a 2% agarose gel.

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Transfection of cells was carried out by incubating 40 μg DNA for 10 min on ice with 1 × 107 cells/ml and 400 μl of the cell suspension was electroporated in a 2 mm gap cuvette using one pulse of square waves of 90 V for 70 msec. Cells expressing VPAC1 or empty vector-containing cells were selected in the presence of geneticin (G418 1 mg/ml; Canadian Life Technologies, Oakville, Ontario, Canada).

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Immunomagnetic precipitation

Two rabbit antibodies to VIP were obtained from Sigma-Aldrich and ICN (Costa Mesa, California, USA), respectively. Mouse monoclonal antibody to HLA-A,B,C was purchased from Serotec and a mouse monoclonal anti-CD3 (UCHT-1) was produced in our laboratory from a hybridoma (a gift from G. Mills). The procedure was essentially as described previously [23]. Briefly, the antibodies (40 μg) were bound to 2.5 × 108 magnetic sheep anti-rabbit or sheep anti-mouse Ig particles (Dynal Inc., Lake Success, New York, USA). The immunomagnetic particles were washed and 5000 pg (standardized by p24 content) of HIVIIIB were added to 12.5 × 106 immunomagnetic particles. The mixture was incubated for 1 h and then washed extensively. The samples were then subjected to a standard ELISA assay for p24 (Coulter Canada Inc.).

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Western immunoblot

HIVIIIB (1800 pg; estimated by p24 content) was lysed in radioimmunoprecipitation assay lysis buffer ([24]; 1% Nonidet P-40, 0.1% SDS, 0.1% Na3 deoxycholate, 50 mM HEPES, pH 7.3, 150 mM NaCl, 2 mM sodium orthovanadate, 50 μM ZnCl2 , 2 mM EDTA, 2 mM phenylmethyl sulfonyl fluoride). The lysate was boiled in the presence of reducing sample buffer and applied to a 10–20% polyacrylamide gradient tricine gel (Invitrogen, Grand Island, New York, USA). The separated proteins were transferred to a nitrocellulose membrane and probed for VIP using anti-VIP (1/1000; ICN) followed by goat anti-rabbit horse radish peroxidase and enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech. Inc.). The membrane was stripped and re-probed with anti-gp120 (NIH AIDS Research and Reference Reagent Program).

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2-LTR circle detection and viral entry

The 2-LTR circles were assayed by nested PCR [25]. Cells (2 × 106) cells were infected with HIVIIIB (m.o.i., 0.3 i.v./cell) for 2 h, washed, and cultured for 8 and 24 h. The DNA was isolated and amplified in the presence of forward (m667) and reverse (U32) primers, and Taq polymerase, by 20 cycles of 94°C for 60 sec, 45°C for 45 sec, and 72°C for 60 sec, and a final 7 min extension at 72°C. Aliquots of PCR products were further amplified using 25 cycles of 94°C for 60 sec, 55°C for 45 sec, and 72°C for 60 sec under the same amplification conditions except that 11-digoxygenin-dUTP was added to the mixture and the two primers were replaced by U5-2LTR and U3-2LTR. A portion of this PCR product was analyzed on a 2% agarose gel. To semi-quantify the 2-LTR-derived product, an aliquot of PCR product was hybridized with a 5′-biotinylated AA55 probe, then captured in microwells and assayed by colorimetry after the reaction with enzyme-linked anti-digoxygenin antibody followed by enzyme substrate, according to the manufacture (Boehringer, Laval, Quebec, Canada). The amount of 2-LTR-derived PCR product, up to an optical density of 0.7, was linearly proportional to the input volume of a 2-LTR containing template preparation (r > 0.95).

To determine the amount of viral entry and to normalize 2-LTR circles with HIV-1 entry into cells, the amount of total HIV was determined by amplification of a region of gag using SK38/SK39 primer pairs [26] under the conditions described above for the nested PCR, except that amplification was achieved by 40 cycles of 94°C for 60 sec and 45°C for 60 sec. A portion of the PCR product was analyzed on a 2% agarose gel and gag-specific product was quantified by hybridization with 5′-biotinylated SK19 probe, as described above. A parallel assay using 8E5 cells that contain one HIV provirus per cell, showed linear proportionality between copies of HIV provirus template and the amount of gag- specific PCR product (r > 0.95).

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HIV-1 activates VPAC1 signal transduction

During infectivity studies with lab strain HIVIIIB carried out in the absence of VPAC1 agonists, we observed that productive HIV-1 infection was variable using different cells and this corresponded with the mRNA level of VPAC1 but not with CD4 or viral co-receptor expression (Fig. 1, and data not shown). An observed increase in productive infection of cells with higher levels of VPAC1 raised the intriguing possibility that HIV-1 may facilitate its own infection by activating VPAC1. To investigate this possibility, we treated cells having medium and high level expression of VPAC1 with an antibody specific to VPAC1 that blocks signal transduction [20]. When the signal transduced through VPAC1 was inhibited by the antibody, productive HIV-1 infection was substantially reduced and this effect was proportional to the level of VPAC1 in the cells (Fig. 2). Indeed, Jurkat cells that express a much lower level of VPAC1 than Hut78 cells showed a more dramatic decrease in productive infection. There was no affect on infection when anti-VPAC2 antibody was used. These results support direct activation of VPAC1 by HIV-1 when it infects cells, and the activation of VPAC1 signaling facilitates HIV-1 infection.

Fig. 1
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Fig. 2
Fig. 2
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HIV-1 gp120 is homologous to VIP

It has been reported previously that HIV-1 gp120 and VIP have some amino acid sequence similarity [12,13]. Our results using the anti-VPAC1 signal blocking antibody support this hypothesis. To further establish whether HIV-1 may directly activate VPAC1 in a similar manner to the natural ligand, VIP, we examined whether the HIV-1 envelope protein has epitope similarity to VIP. Using immunomagnetic pull-down experiments as described previously to show host protein incorporation into the HIV envelope [6,23], we show that antibodies to VIP can precipitate HIV-1 (Fig. 3a). To establish whether this was due to incorporation of VIP into the envelope of HIV-1 or was due to epitope similarity between HIV-1 gp120 and VIP, we lysed purified virions and performed Western immunoblotting with anti-VIP and anti-gp120. Fig. 3b shows that HIVIIIB virions contain no VIP; however, anti-VIP reacts strongly with gp120. These two experiments combined indicate that an amino acid motif of the gp120 protein has significant similarity to VIP confirming previous reports [12,13] and explains how HIV-1 can activate VPAC1 directly.

Fig. 3
Fig. 3
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Anti-sense cDNA to VPAC1 inhibits HIV-1 infection

To investigate whether productive HIV-1 infection would be down-regulated if the expression of VPAC1 could be reduced, we transfected Hut78 cells with a vector-driven anti-sense cDNA to VPAC1. The level of VPAC1 mRNA and cell surface protein expression in a cloned Hut78 transfectant (V9) was significantly reduced (Fig. 4a) but not completely eliminated. When V9 cells were infected with HIVIIIB, productive HIV-1 infection was reduced by half relative to parent Hut78 or Hut78 transfected with the empty pcDNA3 vector (Fig. 4b). The diminished level of productive HIV-1 infection could not be attributed to decreased levels of the viral co-receptors (Fig. 4c).

Fig. 4
Fig. 4
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Over-expression of VPAC1 enhances HIV-1 infection

To further substantiate that VPAC1 was responsible for the facilitating effects on HIV-1 infection, cells engineered to over-express VPAC1 were infected with either laboratory strain HIVIIIB or with a replication-deficient T cell-tropic pseudo-envelope typed HXB2-NL4-3 virus containing a luciferase reporter gene. All cells that we examined expressed VPAC1; therefore, we selected SupT-1 for these experiments as they are well characterized as expressing very low levels of endogenous VPAC1 [18]. VPAC1-transfected SupT-1 cells produced 14-times more virions than the control cells, estimated by production of viral gag p24 antigen (Fig. 5a). VPAC1-transfected cells also integrated virus much more efficiently (> 15-fold) than control cells as estimated by luciferase readout of the replication-deficient HIV-luciferase construct (Fig. 5a). VPAC1-transfected cells also exhibited a striking increase in syncytium formation (Fig. 5b). These findings could not be attributed to modulation of co-receptor expression on the VPAC1-transfected cells (Fig. 5c).

Fig. 5
Fig. 5
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2-LTR circles and VPAC1

It has been reported previously that the second messenger cAMP can activate transcription from the HIV-1 LTR [27]. As VIP has been reported to activate HIV-1 LTR transcription [14] and can activate adenylate cyclase and increase intracellular cAMP levels [15,16], it is possible that VPAC1-induced cAMP production is responsible for the increased HIV-1 replication. We have shown that HIV-1 and VIP can induce a similar increase in cAMP, and that this is less than the increase induced by forskolin, an agent which raises intracellular cAMP to its maximal level independently of receptor activation (Table 1). However, when forskolin was used to treat cells prior to infection it mediated only a 1.6-fold increase in productive HIV-1 infection that was comparable to a twofold increase in productive infection measured when cells were treated with VIP (data not shown). These results suggest that these agents probably mediate a similar signal (an increase in cAMP), which can slightly increase productive HIV-1 infection. However, this marginal effect with agents that increase cAMP cannot adequately explain the dramatic increases in infection when over-expression of VPAC1 is used (see Fig. 5a). Thus, our data of a 14–15-fold increase in HIV-1 infection mediated by cells over-expressing the VPAC1 cannot be accounted for by cAMP signaling alone.

Table 1
Table 1
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In order to begin to assess the role of VPAC1 signal transduction in HIV-1 pathogenesis, we performed an experiment that examines the early stages of the HIV-1 life cycle in cells expressing high and low levels of VPAC1 (Fig. 6). Using Kit225 cells that express very low levels of VPAC1 and Jurkat cells (Fig. 1) we evaluated viral entry and 2-LTR circle formation, an indicator of HIV-1 nuclear migration. The results of this experiment indicated two things: the ability of HIV-1 to enter the cell is independent of the level of VPAC1 expression, and cells with negligible levels of VPAC1, such as Kit225, do not exhibit the formation of viral 2-LTR circles. This suggests VPAC1 affects the nuclear migration of HIV-1.

Fig. 6
Fig. 6
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VPAC1 activation can support primary cell infection

Resting primary cells express CD4 and CXCR4 and, thus, allow for HIV entry. However, primary cells require activation for integration and productive infection by the virus. To assess a possible role for VPAC1 in infection of primary cells, PBMC were isolated and infected with pseudo-envelope typed HXB2-NL4-3-luciferae-encoding virions after pretreatment of the cells with phytohemagglutinin (PHA), VIP, helodermin, or secretin. PHA, a known activator of human T cells, as expected, allowed for successful integration of the recombinant virus as demonstrated by an increase in luciferase activity (Fig. 7). Remarkably, secretin, but not VIP or helodermin, was able to facilitate HIV-1 infection of primary cells independently of PHA stimulation (Fig. 7). Because, secretin has a preference for binding to VPAC1 over VPAC2 [28,29], this result suggests that specific activation of VPAC1 on primary cells can facilitate their infection with HIV-1. Additional studies are currently underway to analyze further this secretin affect on primary cell infection.

Fig. 7
Fig. 7
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The identification of the HIV-1 chemokine co-receptors has generated much interest in further characterizing these proteins and ultimately to inhibit their activation or to block their expression [11]. Although this work is important, such a one-sided approach to a disease as insidious as AIDS is dangerously limiting. Thus, we have been searching for additional host proteins involved in the pathogenesis of HIV infection [30,31]. Identification of novel endogenous proteins that modulate HIV-1 infection would have the potential to be new targets for therapeutic strategies aimed at the eventual control or eradication of HIV/AIDS worldwide. In this report, we describe one such host factor, VPAC1, a neuroendocrine cellular receptor found on CD4 and CD8 T cells, monocytes and other cells [32–36].

Our results indicate that activation of VPAC1 in T cells leads to a signaling event that can increase productive infection with HIV-1 (Fig. 2). Importantly, our results show that HIV-1 itself can activate VPAC1 to promote its own infection. This apparently occurs because of a high degree of amino acid similarity of a portion of the V2 loop of gp120 to VIP, the natural ligand for VPAC1 [12,13,37]. The results shown in Fig. 3 support this theory. Thus, HIV-1, through its envelope protein, may engage VPAC1 and activate this receptor. Whether or not all HIV-1 envelopes will activate VPAC1 remains to be determined as we have examined only IIIB and HXB2.

Previous studies with infection of primary peripheral blood cells has required activation of these cells with PHA/interleukin-2 to allow for productive HIV-1 infection [38]. However, our results suggest that specific activation of VPAC1 may also allow for productive HIV-1 infection of primary cells. VIP binds with similar affinity to VPAC1 and VPAC2 [14,32,39] whereas secretin has increased binding affinity for VPAC1 compared to VPAC2 [28] and helodermin has a higher affinity for VPAC2 [29]. Thus, our results (Fig. 7) when using these agents to treat primary peripheral blood-derived cells suggests that specific activation of VPAC1 by secretin, through its preferential interaction with VPAC1, can facilitate HIV-1 infection of primary cells in vitro. Although this is an impressive result and argues that adequate stimulation of VPAC1 alone may facilitate infection of primary cells, it is somewhat perplexing that HIV-1 itself cannot induce infection of these cells, especially since, based on our cell line results, its gp120 envelope has sequence similarity to VIP and can also activate VPAC1. However, this apparent paradox may be explained by the differential affinities of secretin, compared with those of VIP or HIV-1, for the VPAC receptors on primary cells or to the levels of VPAC receptor expression on these cells [18]. Indeed, resting human peripheral blood CD4 T cells have both VPAC1 and VPAC2 [34] thus, there may be sufficient differential expression of VPAC1 and/or cross-talk between the receptors on these cells [18] to explain these unusual results.

We do not yet know the mechanism of the facilitation of HIV-1 infection by VPAC1. However, because this effect can be diminished using a signal-blocking antibody (see Fig. 2), it is presumed that a specific intracellular signal is responsible. Although the sequence similarity between gp120 and VIP suggests the potential for direct HIV-1-mediated signaling through VPAC1, other possible interpretations of our results are possible. For example, HIV-1 binding to other surface molecules such as CD4, co-receptor, or others, might stimulate VIP and/or secretin production that then acts in an autocrine/paracrine manner to elicit the observed effects. Nevertheless, our results argue that whatever the signaling mechanism, it probably does not involve solely increases in cAMP. Indeed, our data suggest that VPAC1 may be involved in a mechanism of facilitation of HIV-1 infection that occurs prior to viral integration and, thus, would not involve cAMP affects on viral transcriptional regulation. 2-LTR circle formation occurs only after transport of the viral DNA into the nucleus and is necessary for successful integration of the viral DNA into the host genome [25]. Our results (Fig. 6), suggest that signal transduction through VPAC1 may assist viral DNA integration by modulating transport of the viral DNA into the host cell nucleus and/or the formation of 2-LTR circles. This hypothesis requires further examination and experiments are currently underway to address this.

We have identified VPAC1 to be a cellular receptor that has a novel function in its ability to facilitate HIV-1 infection. VPAC1 does not belong to the chemokine family of HIV-1 co-receptors [9]. Instead, VPAC1 is a member of the secretin family of neuroendocrine receptors [32]. VPAC1 is found on all cells that are susceptible to infection with HIV-1, including microglial cells in the brain [35,36]. Studies are currently underway to determine whether or not VPAC1 is necessary and/or sufficient for productive HIV-1 infection, although we have shown that VPAC1 is not required for viral entry (Fig. 6). We have yet to investigate a role for VPAC1 in HIV-1 infection of monocytes; however it is known that monocytes express VPAC1 [35]. Importantly, we have presented additional data (Fig. 7) indicating that VPAC1 may play an important role in primary cell infection.

The identification of VPAC1 as a potent facilitator of HIV-1 infection has the potential to open up new avenues for understanding HIV pathogenesis and possibilities for novel therapeutic strategies. It also emphasizes the importance of seeking out and evaluating other potentially significant host-related factors involved in HIV-1 infection. By exploring these factors we may elucidate a more complete understanding of the pathogenesis of HIV and this may eventually lead to successful new strategies for the control or eradication of this terrible disease.

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We are grateful to M. Laburthe (Paris), E. Goetzl (UCSF), V. KewalRamani and D. Littman (New York University) and G. B. Mills (MD Anderson Cancer Center) for gifts of cells, antibodies, and plasmids. We thank J. Piovesan for excellent technical assistance in performing the cAMP analyses and J. Zhou for her work on this project. We also thank T. Jin, E. Fish, M. Voralia, D. Phipps, and A.-K. Somani for their support and critical review of the manuscript.

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VPAC1; HIV-1; modulation of infection; therapeutic target; 2-LTR circles

© 2002 Lippincott Williams & Wilkins, Inc.


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