Expression and Coreceptor Function of APJ for ... - ScienceDirect

Virology 276, 435–444 (2000) doi:10.1006/viro.2000.0557, available online at http://www.idealibrary.com on

Expression and Coreceptor Function of APJ for Primate Immunodeficiency Viruses Bridget A. Puffer,* Matthew Sharron,* Christine M. Coughlan,* Fre´de´ric Baribaud,* Carrie M. McManus,* Benhur Lee,* Jim David,† Ken Price,† Richard Horuk,‡ Monica Tsang,† and Robert W. Doms* ,1 *Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; †R&D Systems, Minneapolis, Minnesota 55413; and ‡Berlex Biosciences, Richmond, California 94804 Received July 3, 2000; returned to author for revision July 14, 2000; accepted July 26, 2000 APJ is a seven transmembrane domain G-protein-coupled receptor that functions as a coreceptor for some primate immunodeficiency virus strains. The in vivo significance of APJ coreceptor function remains to be elucidated, however, due to the lack of an antibody that can be used to assess APJ expression, and because of the absence of an antibody or ligand that can block APJ coreceptor activity. Therefore, we produced a specific monoclonal antibody (MAb 856) to APJ and found that it detected this receptor in FACS, immunofluorescence, and immunohistochemistry studies. MAb 856 also recognized APJ by Western blot, enabling us to determine that APJ is N-glycosylated. Using this antibody, we correlated APJ expression with coreceptor activity and found that APJ had coreceptor function even at low levels of expression. However, we found that APJ could not be detected by FACS analysis on cell lines commonly used to propagate primate lentiviruses, nor was it expressed on human PBMC cultured under a variety of conditions. We also found that some viral envelope proteins could mediate fusion with APJ-positive, CD4-negative cells, provided that CD4 was added in trans. These findings indicate that in some situations APJ use could render primary cell types susceptible to virus infection, although we have not found any evidence that this occurs. Finally, the peptide ligand for APJ, apelin-13, efficiently blocked APJ coreceptor activity. © 2000 Academic Press

1999). Thus, differential use of CCR5 and CXCR4 cannot by itself account for SIV cellular tropism. Second, SIV Env proteins can bind directly to CCR5 in the absence of CD4 (Edinger et al., 1999; Martin et al., 1997). Third, a number of primary SIV strains, particularly macrophage-tropic and neurotropic isolates, exhibit a considerable degree of CD4-independence, as they are able to infect CCR5positive cells that lack CD4 (Edinger et al., 1997a, 1999). This has led us to the suggestion that CCR5 may have been the primordial receptor for the primate lentiviruses (Edinger et al., 1997a; Martin et al., 1997). In addition to the major coreceptors, a number of related chemokine and other seven transmembrane domain receptors can function as viral coreceptors (reviewed in Berger et al., 1999). These alternative coreceptors are only rarely used as efficiently as the major coreceptors, they are used by smaller numbers of virus strains, their use is not obviously related to virus tropism, and their ability to support virus infection sometimes requires levels of expression that are not attained in vivo. However, use of alternative coreceptors could influence viral tropism, particularly in specialized compartments like the central nervous system and thymus. In addition, use of alternative coreceptors could assume greater importance in the face of effective small molecule inhibitors of CCR5 and CXCR4. It is therefore worthwhile to study the alternative coreceptors, to identify which of the approximately 15 identified to date might be most important in vivo.

INTRODUCTION Entry of primate immunodeficiency viruses into target cells requires the interaction of the viral envelope protein (Env) with cellular receptors (Berger et al., 1999; Doms and Peiper, 1997; Moore et al., 1997). For primary HIV-1 strains, CD4 binding triggers conformational changes in the gp120 subunit of Env that enables it to interact with an appropriate coreceptor (Lapham et al., 1996; Trkola et al., 1996; Wu et al., 1996). All HIV-1 strains use the chemokine receptors CCR5 (R5 virus strains), CXCR4 (X4 virus strains), or both (R5X4 virus strains) as coreceptors (reviewed in Doms and Moore, 1998). Coreceptor binding is the probable triggering event that leads to membrane fusion-inducing structural alterations in Env and subsequent virus entry. Thus, the differential use of CCR5 and CXCR4 by virus strains coupled with the expression pattern of these receptors largely governs HIV-1 tropism at the level of entry. Like HIV-1, SIV strains also use CD4 and coreceptors to infect cells, though there are some important differences. First, nearly all SIV strains studied to date use CCR5, whereas relatively few use CXCR4 (Edinger et al.,

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To whom correspondence and reprint requests should be addressed at University of Pennsylvania, Department of Pathology and Laboratory Medicine, 806 Abramson Bldg., 34th and Civic Center Blvd., Philadelphia, PA 19104. Fax: (215) 573-2883. E-mail: doms@mail. med.upenn.edu. 435

0042-6822/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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To assess the potential relevance of an alternative coreceptor, it is important to generate specific antibodies so that the cell types in which the receptor is expressed can be identified, and to determine whether expression levels are sufficiently high to support virus infection. It is also useful to obtain antibodies or ligands that can inhibit coreceptor activity to determine whether the receptor actually mediates infection in primary cell types. In this report, we describe the development of the first monoclonal antibody directed to APJ, a seven transmembrane domain receptor shown to function as a coreceptor for some HIV-1 and SIV strains (Choe et al., 1998; Edinger et al., 1998; Zhang et al., 1998). The expression of APJ and the relationship between expression levels and coreceptor function are described. In addition, we found that the natural peptide ligand for APJ, apelin-13 (Tatemoto et al., 1998), efficiently blocked APJ coreceptor activity. These reagents can be used to study APJ expression in vivo and to determine whether APJ supports virus infection in a given cellular context. RESULTS Generation and characterization of a monoclonal antibody to APJ We immunized mice with cells stably expressing APJ to generate monoclonal antibodies (MAbs) to this HIV-1 coreceptor. Our previous attempts to generate antibodies to chemokine and related seven transmembrane domain receptors using synthetic peptides have been largely unsuccessful, whereas immunization of mice with cells stably expressing the desired receptor preserves the molecule’s native conformation and typically results in the production of conformation-dependent MAbs (Lee et al., 1999a; Sharron et al., 2000). Hybridomas were screened against the stable APJ transfectants with the parental NSO cell line serving as a negative control. A single hybridoma was identified that was specific for the APJ-positive cell type. After cloning, the IgG3 clone 72133 (catalog no. MAb 856; R&D Systems, Minneapolis, MN) was tested for the ability to recognize APJ on transiently transfected 293T cells by FACS. We found that MAb 856 recognized 293T cells expressing APJ, but not cells transfected with the pCDNA3 vector alone or with plasmids expressing either CD4, CCR1, CCR2b, CCR3, CCR4, CCR5, CXCR1, CXCR2, CXCR4, GPR1, GPR15, ChemR1, ChemR23, STRL33, or CX 3CR1/V28, confirming the specificity of this antibody (Fig. 1). Coexpression of CD4 with APJ did not affect antibody reactivity (Fig. 1). Maximum reactivity of MAb 856 for 293T cells transiently expressing APJ was obtained at a concentration of 20 ␮g/ml, and maximum binding was obtained by 15 min. We next investigated whether MAb 856 could be used in other experimental formats. We found that MAb 856 recognized APJ by Western blot, indicating that, unlike most MAbs we have generated against seven transmem-

FIG. 1. Characterization of MAb 856. 293T cells were transfected with CD4 and/or the designated chemokine/orphan receptor expression plasmid. Cells were lifted off the plate with PBS, stained with MA856 as described under Materials and Methods, and analyzed by FACS.

brane domain receptors, MAb 856 most likely recognizes a conformation-independent, linear determinant (Fig. 2). APJ could be resolved by SDS–PAGE only in the absence of boiling. If samples were boiled, the bulk of the APJ aggregated and failed to enter the separating gel. Western blot analysis of the resolved APJ protein detected three discrete bands, suggesting that the protein was posttranslationally modified. Because of the spacing of the bands, the fact that APJ contains two potential Nlinked glycosylation sites, and that other HIV coreceptors contain N-linked carbohydrates (Berson et al., 1996), we felt that this banding pattern was most consistent with carbohydrate modifications. Indeed, when cells expressing APJ were treated with tunicamycin to inhibit N-linked glycosylation, only a single band was detected which comigrated with the most quickly migrating band observed in the absence of tunicamycin (Fig. 2). Therefore, we conclude that APJ is N-glycosylated. The presence of three bands could be consistent with incomplete glycosylation (perhaps as a consequence of overexpression) in which zero, one, or two N-linked sites are used. Alternatively, carbohydrate processing in the Golgi could account for some of the mobility differences. MAb 856 recognized APJ transfectants by immunoflu-

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FIG. 2. APJ is N-glycosylated. 293T cells transfected with GFP(Mock), CCR5, or APJ constructs were cultured overnight in the presence (lanes 3 and 5) or absence (lanes 1, 2, 4, and 6–9) of tunicamycin. Lysates were prepared as described under Materials and Methods; approximately 30,000 cell equivalents of each sample were mixed with sample buffer and heated to either 37°C (lanes 1–5), 55°C (lanes 6 and 7), or boiled (lanes 8 and 9). Proteins were resolved by SDS–PAGE, transferred to PVDF membrane, and detected by MAb 856.

orescence following paraformaldehyde treatment, giving a typical cell surface staining pattern (Fig. 3B). Staining of 293T cells transfected with pCDNA3 vector demonstrated background levels (Fig. 3A). To determine whether MAb 856 could be used for immunohistochemical analysis of paraffin-embedded sections, we stained sections of human cerebellum (Fig. 3D) since APJ message has been reported to be abundant in the CNS (O’Dowd et al., 1993). We found that MAb 856 recognized a subset of cells in these sections, including pyramidal neurons in the hippocampus (data not shown). Thus, MAb 856 can be used to characterize APJ expression in tissue sections. Confirmation of the staining pattern obtained with MAb 856 by an additional technique such as in situ hybridization for APJ message should be considered until additional antibodies to APJ are obtained. APJ expression on cell lines and primary human PBMCs We used MAb 856 to stain a number of cell lines commonly used for HIV and SIV infectivity studies because of its potential to function as a viral coreceptor. We did not detect APJ expression on CEMX174, CEMss, clone 8, U937, HUT-78, U87, THP-1, PM-1, Jurkat, or C8166

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cells by FACS analysis. We previously failed to identify APJ mRNA in these cells as judged by RT-PCR analysis, with the exception of C8166 cells in which an APJ-specific reaction product was detected (Edinger et al., 1998). Therefore, we conclude that these commonly used cell lines either do not express APJ or express less than 1000 copies of this receptor per cell, which is the approximate limit of APJ detection using MAb 856 (not shown) and a quantitative FACS protocol (Lee et al., 1999b). Alternative HIV-1 coreceptors that are expressed on CD4-positive cell types in vivo are more likely to be relevant for HIV tropism and pathogenesis than receptors that are not. Previously, we failed to detect APJspecific transcripts in adult human microglia, monocytederived macrophages, and PBMCs cultured under a variety of conditions (Edinger et al., 1998). However, an independent report demonstrated the presence of APJ cDNA in human PBMC activated with PHA plus IL-2 (Choe et al., 1998). We used MAb 856 to stain freshly isolated human PBMC as well as PBMC activated with IL-2 or a combination of PHA and IL-2. While CXCR4 expression was readily detected on both CD4-positive and -negative PBMC, APJ was not detected on cells grown under any condition (Fig. 4). These results, coupled with our previous RT-PCR analyses, argue that APJ is not expressed on CD4-positive T-cells, at least not at levels that can be detected by MAb 856. Antiviral activity of the APJ ligand apelin An endogenous 36-amino-acid-long peptide ligand for APJ, termed apelin, has been identified and isolated from bovine stomach extracts. When apelin was added to CHO cells stably expressing APJ, it resulted in an increase in extracellular acidification (Tatemoto et al., 1998). In addition, a 13-amino-acid-long C-terminal fragment of apelin, apelin-13, exhibited increased activity (Tatemoto et al., 1998). To determine whether apelin-13 could inhibit APJ coreceptor function, we synthesized this peptide. Addition of apelin-13 to human 293T cells expressing APJ that had been loaded with the calcium-sensitive dye Fura-2 resulted in an increased fluorescent signal, consistent with the increases in intracellular calcium observed after ligand binding to many other seven transmembrane domain receptors (Fig. 5). This confirmed that our synthetic apelin-13 was biologically active and demonstrated that apelin induces calcium signaling in APJ-expressing cells. Apelin-13-induced APJ receptor signaling could not be inhibited by preincubating APJ-expressing cells with MAb 856, indicating that this antibody does not inhibit ligand binding. We also assessed the ability of apelin-13 to induce a calcium flux in human PBMC, but failed to observe a response. In contrast, the addition of RANTES led to a readily observed signal (data not shown). This finding provides addi-

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FIG. 3. MAb 856 detects APJ by immunofluorescence and immunoperoxidase staining. Transfected 293T cells were stained with mouse IgG (A) or MAb 856 (B) and processed for immunofluorescence as described under Materials and Methods. Human cerebellum sections were incubated with mouse IgG (C) or MAb 856 (D) and processed for immunoperoxidase staining.

tional evidence that APJ is not expressed in human PBMC. Having confirmed that the synthetic apelin-13 was biologically active, we asked whether it could inhibit APJ coreceptor activity. Because relatively few virus strains can use APJ to infect cells efficiently, we used a cell–cell fusion assay so that APJ coreceptor activity for several Env proteins could be easily monitored (Edinger et al., 1998). As shown in Fig. 6, quail QT6 cells expressing CD4 and APJ supported fusion by cells expressing either the SIVmac316, HIV-1 89.6, or HIV-1 HXB Env proteins. Apelin-13 blocked APJ-dependent cell–cell fusion in a concentration-dependent manner (Fig. 6). By contrast, apelin-13 did not inhibit SIVmac316 or HIV-1 89.6 use of CCR5, nor did it inhibit HIV-1 89.6 or HXB use of CXCR4 (Fig. 6), confirming its specificity for APJ. The apelin-36 full-length peptide also blocked Env fusion via the APJ receptor (data not shown). Unlike apelin-13, MAb 856 did not block APJ coreceptor activity under any condition tested (data not shown). These results are in agreement with a recent study by Zou et al. (2000), who showed that apelin peptides of various lengths could block infection of APJ-positive cells by either HIV-1 or HIV-2.

APJ coreceptor activity as a function of receptor expression levels Generally, for a coreceptor to be relevant in vivo, it must be expressed on CD4-positive cells and it must be expressed at levels sufficiently high to support virus infection. Some alternative coreceptors, such as STRL33, support virus infection at nonphysiological expression levels, at least for some virus strains (Sharron et al., 2000). To determine the levels of APJ surface expression required for coreceptor activity, we transiently transfected human 293T cells with a constant amount of a CD4 expression plasmid and serial dilutions of plasmids expressing APJ, CCR5, or CXCR4. The next day, the ability of these cells to support cell–cell fusion by the HIV-1 89.6, HXB, and SIVmac316 Env proteins was tested (Figs. 7B, 7C, and 7D, respectively). We confirmed by FACS analysis that receptor expression was indeed related to the amount of plasmid used (Fig. 7A). Consistent with previous work, we found that, whereas coreceptor activity diminished as receptor levels decreased, both CCR5 and CXCR4 supported detectable levels of cell–cell fusion, even at very low levels of receptor expression (Fig. 7B–D). Cells expressing APJ exhibited a nearly identical

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accomplished either by the addition of soluble CD4 or by CD4-positive cell types. To determine whether APJ-positive, CD4-negative cells could exhibit coreceptor activity when CD4 is provided in trans, we used the cell–cell fusion assay. Cells expressing the HIV-1 89.6, HIV-1 HXB, or SIVmac316 Env proteins did not fuse with cells expressing APJ alone (Fig. 8). Addition of sCD4, however, enabled cells expressing the HIV-1 89.6 and HXB Env proteins, but not cells expressing SIVmac316, to fuse with APJ-positive cells. Thus, as has been observed for CXCR4 and CCR5, APJ coreceptor activity can be supported in some circumstances by CD4 added in trans. DISCUSSION The discovery of the major primate immunodeficiency virus coreceptors has greatly expanded our understanding of viral tropism and pathogenesis and for defining new drug targets. While the importance of CCR5 and CXCR4 for HIV-1 and CCR5 for SIV is clear, the in vivo relevance of the approximately 15 alternative coreceptors identified to date is not. Alternative coreceptors have been discovered using a variety of experimental approaches, but in all cases they have been defined as being competent to mediate infection of one or more

FIG. 4. APJ is not expressed on human PBMC. Human PBMC were cultured with IL-2 or IL-2 with PHA for 4 days prior to staining with MAbs specific for the APJ, CXCR4, or CD4, and FACS analysis. APJ or CXCR4 expression is shown on CD4⫹ or CD8⫹ T cells as indicated. The dotted line represents the staining obtained with the isotypematched control antibody; the solid line indicates the specific antibody.

pattern: there was not a threshold level below which coreceptor activity was lost (Figs. 7B, 7C, and 7D). Therefore, we conclude that APJ can function as an HIV-1 coreceptor, even when expressed at low levels. Rescue of APJ coreceptor activity by soluble CD4 Although APJ could function as a coreceptor at expression levels likely to be found in vivo, we have not detected APJ expression on CD4-positive cell types. However, APJ could still prove to be a relevant coreceptor in vivo under some circumstances. Specifically, a number of primary SIV and HIV-2 strains have been identified that can infect cells in a CD4-independent fashion (Edinger et al., 1997a; Reeves et al., 1999). In addition, Goldsmith and colleagues showed that primary fetal human astrocytes, which are CD4-negative but do express CXCR4, could be infected by X4 virus strains, provided that CD4 was supplied in trans (Speck et al., 1999). This could be

FIG. 5. Apelin-13 induces an increase in intracellular calcium. 293T cells were transfected with APJ, loaded with FURA-2 calcium indicator dye, and resuspended in D-PBS with Ca 2⫹ and Mg 2⫹. The cells were then placed in a cuvette at 37°C, and fluorescence detected in response to apelin, and thrombin receptor agonist (TAP) peptides. TAP was used as a positive control to confirm that FURA-2-loaded cells were both viable and responsive. Emissions were monitored at 340 and 380 nm with excitation set at 510 nm.

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FIG. 6. Inhibition of APJ coreceptor activity by apelin-13. QT6 cells expressing CD4 and the indicated coreceptor were incubated with apelin-13 for 15 min prior to addition of effector cells expressing the indicated Env protein. Targets and effectors were mixed and incubated for 6–7 h at 37°C, then cells were lysed with 0.1% Triton/PBS and lysates were assayed for luciferase activity. Fusion for each envelope in the absence of apelin was normalized to100% to make it possible to compare envelopes with widely different fusion activities for this alternative coreceptor. Data from one of four representative experiments are shown. The results from the other three experiments were similar.

virus strains when expressed in a cell line along with CD4. In at least some cases, alternative coreceptors share considerable sequence homology with CCR5, or contain structural motifs that have been shown to be important for coreceptor function (Choe et al., 1998; Dragic et al., 1998; Farzan et al., 1998). APJ falls into the latter category, sharing an acidic, tyrosine-rich motif found in the N-terminal domains of many coreceptors, including CCR5 and CXCR4, even though the receptor shares greater overall similarity with the angiotensin receptor family (Choe et al., 1998; O’Dowd et al., 1993). Given these structural similarities and the sequence variability characteristic of the V1/2 and V3 regions of Env that are largely responsible for coreceptor choice (reviewed in Hoffman and Doms, 1999), it is perhaps not surprising that molecules other than CCR5 and CXCR4 can mediate entry for some virus strains, particularly when the receptors are overexpressed. The real question is whether any of these receptors are currently relevant in vivo, or could become relevant if small molecule receptor antagonists provide selective pressure for viral evolution. For an alternative receptor to be generally relevant in vivo, it needs to be expressed in CD4-positive cells at levels sufficient to mediate virus infection. Receptor ex-

FIG. 7. APJ expression levels and coreceptor activity. Duplicate wells of QT6 target cells were transfected with T7-luciferase, CD4, and coreceptor at the designated concentrations. At 24 h posttransfection cells were either examined by FACS for coreceptor expression levels (A) or mixed with effector cells expressing the 89.6, HXB, or SIVmac316 Envs (B, C, and D, respectively), and fusion activity was assayed by luciferase activity. Data from one of three representative experiments are shown. The results from the other two experiments were essentially identical, although maximal MCF values varied between experiments because transient transfections were employed.

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FIG. 8. Effects of CD4 provided in trans on APJ coreceptor activity. A fusion assay was performed using QT6 target cells transfected with APJ and T7 luciferase in the presence or absence of CD4 expression vector. At 24 h posttransfection, effector cells expressing the designated Env and T7 polymerase were added to the targets and the media was supplemented with soluble CD4 (sCD4). Approximately 6–7 h after mixing, cells were lysed and assayed for luciferase activity. Data from one experiment of three replicates are shown. All values were normalized to levels of fusion detected when effectors were mixed with target cells transfected with APJ and CD4. mCD4 indicates cellularly expressed, membrane-bound CD4.

pression levels clearly influence coreceptor activity. In general, coreceptor activity falls as receptor expression levels decrease, though the major coreceptors (CCR5 and CXCR4) can support some degree of infection even at low levels of expression (Edinger et al., 1999; Kozak et al., 1997; Kuhmann et al., 2000; Platt et al., 1998; Sharron et al., 2000). This may not be true for some alternative coreceptors, which may function only at very high levels of receptor expression. In the case of STRL33, some virus strains can use this receptor for infection only at levels of expression that far exceed levels that are found in vivo (Sharron et al., 2000). Whether this is true for additional coreceptors has not yet been determined, often because specific antibodies are not available. By developing a MAb specific for APJ, we were able to determine that APJ, like the major coreceptors, functions even at very low levels of expression. Thus, levels of APJ found in vivo are likely to be sufficient for coreceptor activity, provided that CD4 is present, since we have not observed CD4-independent use of APJ by any HIV-1 or SIV strain examined to date. APJ expression has been studied through analysis of APJ message, often by RT-PCR, with conflicting results with regards to expression of APJ in CD4-positive primary cell types (Choe et al., 1998; Edinger et al., 1998). Differences observed could be the result of either donor variability or the highly sensitive nature of the RT-PCR technique. Using MAb 856, we found that APJ was not expressed at detectable levels in numerous cell lines or in CD4-positive T-cells from four donors, cultured under a variety of conditions. In addition, apelin-13 did not induce a detectable calcium response in human PBMC,

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providing additional evidence that APJ is either not expressed in human PBMC or is present at very low levels. These findings, coupled with our inability to detect APJ message in monocyte-derived macrophages or microglia (Edinger et al., 1998), lead us to conclude that APJ is not likely to be of major importance as a coreceptor in vivo. This is in contrast to another alternative coreceptor that we have studied in detail, STRL33. We found that STRL33 was expressed in some CD4-positive T-cells, that it was expressed at levels sufficient to support infection for some viruses, and we identified one primary HIV-1 strain that could infect some CD4-positive T-cells independently of CCR5 and CXCR4 by using STRL33 (Sharron et al., 2000). Although it is possible that MAb 856 may not detect all conformations of APJ, the negative FACS results coupled with our inability to detect APJspecific message argues strongly that this alternative coreceptor is not expressed in the most important cell types for HIV-1 infection in vivo. MAb 856 and apelin-13, however, will be important in elucidating the normal function of APJ. Though APJ is not likely to be a relevant HIV-1 coreceptor in vivo, it could potentially support virus infection in specialized contexts. APJ message is relatively abundant in the CNS, an important site of HIV-1 infection (O’Dowd et al., 1993). Indeed, we found APJ expression in a subset of pyramidal neurons using MAb 856 and immunoperoxidase staining. The ability of MAb 856 to detect APJ in this format will make it possible to carefully study APJ expression in solid tissues. There is reasonable evidence that CD4-negative cell types in the CNS can be infected by HIV-1, though this is not a universal finding (reviewed in Kolson et al., 1998). When infection of CD4-negative cell types is observed, the efficiency of this process is not known nor are the mechanisms of virus entry. Goldsmith and coworkers (Speck et al., 1999) showed that some HIV-1 strains could infect CD4-negative human astrocytes in vitro, provided CD4-positive cells were also present. This, and experiments in which soluble CD4 could render some CD4-negative cells susceptible to infection, provides at least one mechanism for CD4-independent infection (Schenten et al., 1999; Speck et al., 1999). It will be difficult to determine whether this occurs in vivo, though if neurotropic virus strains in general are better able to infect cells via this pathway, then the case for this infectious entry pathway being relevant in vivo becomes stronger. In the case of SIV, many virus strains exhibit at least some degree of CD4independence, but this is almost invariably mediated by CCR5 (reviewed in Edinger et al., 1999). Thus, we consider CD4-independent, APJ-dependent SIV infection to be an unlikely event. Finally, engagement of coreceptors by the viral Env protein could potentially trigger signaling events that could impact cell health and viability. This, again, is a somewhat controversial area and one that is experimentally difficult to address. Nonetheless, it will be

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worthwhile to determine whether APJ signaling induced by apelin or Env has any physiological consequences in primary cells derived from the CNS. The generation of MAb 856 and the ability of the APJ ligand apelin-13 to induce an increase in intracellular calcium and block coreceptor activity will make it possible to study these questions, and point out the importance of developing antibodies and identifying natural ligands for other viral coreceptors, whether real or an in vitro artifact. MATERIALS AND METHODS Antibodies The antibodies used in this study included FITC-conjugated anti-CD4, tricolor-conjugated anti-CD8, and goat anti-mouse F(ab⬘) 2 IgG (H ⫹ L) R-phycoerythrin conjugated (all from Caltag Laboratories, Burlingame, CA). Antibodies from R&D Systems included anti-human CXCR4 MAb173 and anti-APJ MAb 856. Other antibodies included anti-CXCR4 MAb 12G5 (Endres et al., 1996) and anti-CCR5 MAb 2D7 (Pharmingen, San Diego, CA). MAb 856 (mouse anti APJ clone 72133; R&D Systems) was made by immunizing Balb/c mice using syngenic mouse myeloma (NSO) cells stably expressing the full-length human APJ sequence (O’Dowd et al., 1993). Western blot analysis 293T cells transfected with a pCDNA3 APJ expression plasmid were cultured overnight in the presence or absence of 5 ␮g/ml tunicamycin. At 24 h posttransfection, cells were lysed in RIPA buffer (1% deoxycholate, 1% Triton X-100, 150 mM NaCl, 250 mM Tris, pH 8.0) and the nuclear fraction was removed by low-speed centrifugation. SDS–PAGE (6⫻) sample buffer containing reducing agent was added and lysates were heated to 37, 55, or 95°C immediately prior to resolution of proteins on 10% acrylamide gels. Proteins were transferred to PVDF membranes (Millipore, Bedford, MA) and nonspecific protein binding was blocked with blotto for 1 h. MAb 856 was added at a 1:1000 dilution for 1 h, then the blot was washed several times with 0.2% Triton X-100 in PBS. Anti-mouse HRP conjugate (Promega, Madison, WI) was added at a 1:10,000 dilution for 1 h, washing was repeated, and proteins detected with Supersignal (Pierce, Rockford, IL).

and T7-luciferase. At 24 h postinfection or transfection effector cells were added to the target cells and fusion quantitated 7 h postmixing by lysing cells with 0.05% Triton X-100 in PBS and measuring luciferase activity. Ligand blocking or soluble CD4 studies were performed by adding ligand or CD4 to target cells approximately 15 min prior to addition of effector cells. Calcium mobilization assay Calcium signaling response was measured in 293T cells transfected with plasmids expressing CCR5 or APJ, or empty vector via calcium phosphate. At 24 h posttransfection approximately 1 ⫻ 10 6 cells were loaded with FURA2-AM (Molecular Probes, Eugene, OR) for 1 h, then washed with PBS without Mg 2⫹ or Ca 2⫹ and gently lifted from the plate by pipetting. Cells were pelleted and resuspended in Dulbecco’s PBS with Mg 2⫹ and Ca 2⫹ (BioWhittaker, Walkersville, MD) at 2 ⫻ 10 6 cells per ml, and fluorescence was measured with an Aminco-Bowman luminescence spectrometer in a stirring cuvette. Excitation and emission were monitored at 340 and 380 nm and extracellular calcium concentration was calculated as described (Grynkiewicz et al., 1985). Apelin-13 peptide was added at 3 ␮g/ml and thrombin receptor agonist peptide (TAP) was added to a concentration of 27 ␮M following addition of ligand as a control, to confirm that cells were permissive for induction of calcium signaling. Immunohistochemistry Paraffin-embedded tissue was dehydrated in graded alcohol baths and then deparaffinized in xylene. After rehydration the sections were quenched for 20 min in 0.8% hydrogen peroxide in methanol. Sections were then incubated in 2% normal horse serum (Vector Laboratories, Burlingame, CA) followed by an overnight incubation at 4°C in primary antibody. The sections were washed and incubated with a biotinylated secondary antibody (1:750) (Vector Laboratories) followed by incubation with avidin-biotin-peroxidase complex (Vector Laboratories). After washing, the slides were developed with 3⬘,3⬘-diaminobenzidene to give a brown reaction product (Sigma, St. Louis, MO) and then dehydrated and mounted with Cytoseal (VWR Scientific, Boston, MA). Immunofluorescence staining

Cell–cell fusion assay This assay has been described previously (Rucker et al., 1997). Briefly, effector 293T or QT6 cells were infected with vaccinia virus expressing T7 polymerase (vTF1.1) (Alexander et al., 1992) and with a vaccinia expressing the HIV-1 89.6 (vBD3) (Doranz et al., 1996), SIVmac316 (vCB75) (Edinger et al., 1997b), or HIV-1 HXB (vPE16) (Earl et al., 1991) Env proteins. Target QT6 quail cells were transfected with plasmids expressing CD4, coreceptor,

All cells were fixed in 4% paraformaldehyde/PBS/4% sucrose for 20 min at 4°C. Permeabilization was carried out by treating the fixed samples with methanol (⫺20°C) for 10 min at 4°C and then 0.2% Triton X-100 for 10 min at 4°C. Blocking was carried out in 10% goat serum/PBS for 30 min at room temperature (RT). The antibody was reconstituted to a final concentration of 9 ␮g/ml in 10% goat serum/PBS and incubated with the samples for 1 h at RT. Mouse IgG was used as a control at the same

IN VIVO SIGNIFICANCE OF APJ CORECEPTOR FUNCTION

concentration as the APJ antibody. The coverslips were washed with PBS three times and then incubated for 1 h at RT with goat anti-mouse Alexa FITC at a final concentration of 4 ␮g/ml. The coverslips were washed with PBS three times and then mounted in Fluoromount (Southern Biotechnology Associates, Birmingham, AL), sealed, and photographed. Images were captured using a Nikon Eclipse E600 microscope attached to a Nikon camera and the software package Phase 3 Imaging Systems, using a 40 ⫻ 10 non-oil-immersion lens. FACS analysis Stimulated PBLs cultured for 4 days with either IL-2 or IL-2/PHA were incubated for 30 min on ice in FACSstaining buffer (PBS, 3% FBS, 0.05% sodium azide) with 10 ␮g/ml of the designated antibody. Cells were washed in 1 mL FACS-staining buffer, then incubated with the recommended dilution of anti-mouse PE antibody. Washing was repeated and cells were incubated with recommended concentrations of the CD4 and CD8 antibodies. Cells were washed again and fixed in 1% paraformaldehyde. Fluorescence staining was analyzed on a FACScan (Becton Dickinson, San Jose, CA) with the Cellquest software package. ACKNOWLEDGMENTS R.W.D. was supported by NIH R01 40880, by a Burroughs Wellcome Fund Award for Translational Research, and by an Elizabeth Glaser Scientist Award from the Pediatric AIDS Foundation. C.M. was supported by NIH NRSA T32NS07180, F.B. was supported by a fellowship from the Swiss National Science Foundation, and B.L. was supported by K08 HL03923-01.

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