JOURNAL OF VIROLOGY, Oct. 1998, p. 7934–7940 0022-538X/98/$04.0010 Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 10
An Orphan Seven-Transmembrane Domain Receptor Expressed Widely in the Brain Functions as a Coreceptor for Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus AIMEE L. EDINGER,1 TREVOR L. HOFFMAN,1 MATTHEW SHARRON,1 BENHUR LEE,1 YANJI YI,2 WONKYU CHOE,3 DENNIS L. KOLSON,3 BRANKA MITROVIC,4 YIQING ZHOU,4 DARYL FAULDS,4 RONALD G. COLLMAN,2 JOSEPH HESSELGESSER,4 RICHARD HORUK,4 AND ROBERT W. DOMS1* Department of Pathology and Laboratory Medicine,1 Department of Medicine (Pulmonary and Critical Care Division),2 and Department of Neurology,3 University of Pennsylvania, Philadelphia, Pennsylvania 19104, and Department of Immunology, Berlex Biosciences, Richmond, California 948044 Received 25 February 1998/Accepted 22 June 1998
Both CD4 and an appropriate coreceptor are necessary for infection of cells by human immunodeficiency virus type 1 (HIV-1) and most strains of HIV-2. The chemokine receptors CCR5 and CXCR4 are the major HIV-1 coreceptors, although some virus strains can also utilize alternative coreceptors such as CCR3 to infect cells. In contrast, most if not all simian immunodeficiency virus (SIV) strains use CCR5 as a coreceptor, and many SIV strains can use CCR5 independently of CD4. In addition, several orphan seven-transmembrane receptors which can serve as HIV-1 and SIV coreceptors have been identified. Here we report that APJ, an orphan seven-transmembrane domain receptor with homology to the angiotensin receptor family, functions as a coreceptor for a number of HIV-1 and SIV strains. APJ was expressed widely in the human brain and in NT2N neurons. APJ transcripts were also detected by reverse transcription-PCR in the CD4-positive T-cell line C8166, but not in peripheral blood leukocytes, microglia, phytohemagglutinin (PHA)- or PHA/interleukin-2stimulated peripheral blood mononuclear cells, monocytes, or monocyte-derived macrophages. The widespread distribution of APJ in the central nervous system coupled with its use as a coreceptor by some HIV-1 strains indicates that it may play a role in neuropathogenesis. less efficiently than either CCR5 or CXCR4. However, use of alternative coreceptors may help explain certain facets of HIV1 tropism and pathogenesis in vivo. For example, neurologic disease is a serious and relatively frequent consequence of HIV-1 infection, with microglia representing the primary targets of virus infection in the central nervous system (CNS) (4, 66, 70). Microglia express both CCR3 and CCR5, and it has been suggested that utilization of CCR3 by a virus strain may correlate with neurotropism (37), although not all virus strains that can infect microglia can use CCR3 as a coreceptor (67). Whether the use of CCR3 or other alternative coreceptors is significant in vivo is under active investigation. Here we show that an orphan seven-transmembrane receptor, APJ (50), functions as a coreceptor for a number of HIV1 and simian immunodeficiency virus (SIV) strains. APJ served as a coreceptor for some X4 and R5X4 virus strains, while two R5 isolates used APJ slightly less efficiently. Several SIV strains were also able to use APJ as a coreceptor. We confirmed that APJ is expressed widely in the human brain (45) and also found that it is expressed in NT2N neurons, a widely used model for human neurons. In addition, we detected APJ transcripts in C8166 cells but not in other T-cell lines or in peripheral blood leukocytes (PBLs), microglia, peripheral blood mononuclear cells (PBMCs), monocytes, or macrophages. The use of APJ by a number of virus strains coupled with its expression in the CNS suggests that utilization of this receptor has the potential to impact viral neuropathogenesis.
The entry of human immunodeficiency virus type 1 (HIV-1) into cells involves binding of the viral envelope (Env) protein to CD4 followed by an interaction with one of several coreceptors (reviewed in references 5, 8, 22, and 47). Binding of Env to the appropriate coreceptor is thought to trigger conformational changes in Env that mediate fusion between the viral membrane and that of the host cell. The major HIV-1 coreceptors are the chemokine receptors CCR5 and CXCR4, as all HIV-1 strains examined to date use one or both of these molecules as second receptors (21). CCR5 supports infection by R5 (macrophage-tropic [M-tropic]) virus strains, while CXCR4 supports infection by X4 (T-cell-tropic [T-tropic]) virus isolates (1, 6, 11, 19, 27, 28, 34). R5X4 (dualtropic) viral Env proteins can, in conjunction with CD4, use either CCR5 or CXCR4 for cellular entry. The differential utilization of CCR5 and CXCR4 by HIV-1 strains coupled with their expression patterns in CD4-positive cells largely explains viral tropism at the level of entry. In addition to CCR5 and CXCR4, a number of other chemokine and orphan seven-transmembrane domain receptors have been shown to function as coreceptors for one or more virus strains in vitro (11, 20, 27, 32, 38, 41, 57, 60, 62). In general, these alternative coreceptors support virus infection
* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, University of Pennsylvania, 806 Abramson, Philadelphia, PA 19104. Phone: (215) 898-0890. Fax: (215) 573-2883. E-mail:
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MATERIALS AND METHODS Cell-cell reporter gene fusion assay. The assay has been described in detail elsewhere (49, 59). Briefly, effector cells were prepared by infection of quail QT6 cells with a recombinant vaccinia virus encoding T7 polymerase (vTF1.1) and then transfection with a plasmid bearing the envelope gene of interest under the control of the T7 promoter. Env constructs SIVmac251, SIVmac239, SIVmac316, SIVmac316mut (which contains two amino acid substitutions relative to SIVmac316: 321 P3S and 325 M3I), DH12, RF, BK132, ADA, JR-FL, IIIB, and HIV-2/ST were introduced into effector cells via recombinant vaccinia virus rather than by transfection. QT6 target cells were prepared by transient transfection with plasmids encoding CD4 and the coreceptor of interest under the control of the cytomegalovirus (CMV) promoter as well as luciferase under the control of the T7 promoter. Effector and target cells were mixed the day after transfection, and cell-cell fusion was quantified by measuring luciferase activity in cell lysates 7 to 8 h following mixing. Virus infections. Two different assays were used to assess the ability of APJ to support virus infection. Luciferase reporter viruses were prepared by transfecting 293T cells with the indicated Env constructs and with the NL4-3 luciferase virus backbone (pNL-Luc-E2R2) (9, 13). Due to poor incorporation efficiency, we used 89.6-VSV in place of wild-type 89.6 Env. The 89.6-VSV Env, kindly provided by John K. Rose (Yale University), contains the vesicular stomatitis virus G-protein cytoplasmic domain in place of the normal 89.6 Env cytoplasmic domain and results in more efficient pseudotype formation. Target cells for infection were 293T, U87, quail QT6, or feline CCCS cells with CD4 and coreceptors introduced by calcium phosphate transfection. Infections were performed in media containing DEAE-dextran (8 mg/ml) or Polybrene (4 mg/ml) (for U87 cells). Cells were lysed 3 to 4 days postinfection by resuspension in 0.5% Nonidet P-40 in phosphate-buffered saline and assayed for luciferase activity. A second experimental approach used to measure virus infection involved a PCRbased entry assay. For these experiments, 50 ng of p24 of DNase-treated, cellfree virus was used to infect QT6 cells stably expressing human CD4 and transiently expressing the desired coreceptor. After 2 days, the cells were washed and lysed, and HIV-1-specific long terminal repeat (LTR) DNA sequences were detected by PCR using primers LTR-plus and LTR-minus (59-ACAAGCTAGT ACCAGTTGAGCC-39 and 59-CACACACTACTTGAAGCACTCA-39). Products were resolved by electrophoresis on 2% agarose gels, transferred to Hybond N1 (Amersham), and detected by using a 39-End Labeling Biotin kit (DuPont; probe 59-ATCTACAAGGGACTTTCCCGC-39), followed by exposure. Primary cells. Human PBMCs were isolated from blood of normal volunteers by using Ficoll-Hypaque, depleted of monocytes by serial adherence to plastic, stimulated with phytohemagglutinin (PHA-L; 5 mg/ml; Sigma) for 3 days, and then resuspended with interleukin-2 (IL-2; 20 U/ml; Boehringer Mannheim Biochemicals, Indianapolis, Ind.). RNA was extracted after 3 days of PHA stimulation and also after 1 week in IL-2. Monocytes were purified from PBMCs by selective adherence to gelatin followed by plastic and then maintained in culture to allow differentiation into monocyte-derived macrophages (MDM) as previously described (12). RNA was extracted from undifferentiated monocytes immediately after purification and from MDM after 1 week in culture. Brainderived microglia and oligodendrocytes from fresh human brain tissue, obtained from temporal lobe resections from patients with medication-resistant epilepsy, were provided by Francisco Gonzalez-Scarano and prepared as described elsewhere (68). NTera 2 human teratocarcinoma cells were grown and differentiated into postmitotic neurons (NT2N) by retinoic acid exposure as previously described (54). The differentiated cultures contain .95% postmitotic neurons, with #5% remaining undifferentiated cells. RT-PCR. To isolate total cellular RNA, 5 3 106 to 10 3 106 cells were resuspended in 1 ml of Trizol (GIBCO-BRL) and processed as recommended by the manufacturer. Total RNA was then treated with 1 ml (10 to 50 U) of DNase (RNase free; Boehringer Mannheim) per 10 mg of RNA for 30 min at 37°C in the presence of 5 mM MgCl2, with subsequent inactivation at 65°C for 10 min in the presence of 5 mM EDTA; RNA concentration was calculated based on the optical density at 260 nm. The Titan reverse transcription (RT)-PCR system (Boehringer Mannheim) was used to evaluate RNA expression patterns. The specific internal upstream 59-TACACAGACTGGAAATCCTCG-39 and downstream 59-TGCACCTTAGTGGTGTTCTCC-39 primers used resulted in an amplified product of 481 bp. To control for contamination of the RNA sample with genomic DNA despite treatment with DNase, all RNA samples were also amplified with Titan enzyme mix in which the RT but not PCR activity had been destroyed by treatment at 95°C for 10 min (this inactivation protocol was found to eliminate the ability to amplify an RNA but not a DNA template). In each RT-PCR, RNA isolated from U87-APJ stably transfected cells was included as a positive RNA control and plasmid DNA was included as a second positive control. b-Actin primers are described in reference 62. Total cellular RNA was prepared from NT2N neurons, microglia, and oligodendrocyte lysates (106 cells) by using an RNA preparation kit (RNeasy kit; Qiagen, Inc., Chatsworth, Calif.) according to the manufacturer’s instructions. The RNA preparation was suspended in Tris-EDTA (1 M Tris-HCl [pH 8.0], 1 M EDTA) and treated with RNase-free DNase I for removal of genomic DNA (40 U/10 mg of RNA; Boehringer Mannheim) in the presence of 200 U of RNasin (RNase inhibitor; Boehringer Mannheim) per ml for 30 min at room temperature. First-strand cDNA was synthesized from 0.5 mg of total RNA with
FIG. 1. HIV-1 Env-mediated cell-cell fusion. QT6 cells expressing CD4, the indicated coreceptor, and luciferase under control of the T7 promoter were mixed with cells expressing T7 polymerase and the indicated HIV-1 or HIV-2 Env protein. The degree of cell-cell fusion was determined 8 h postmixing by measuring luciferase activity. The results were normalized by setting the extent of fusion obtained when CD4 and either CCR5 (for R5 Env proteins) or CXCR4 (for X4 and R5X4 Env proteins) were coexpressed to 100%. The extent of fusion obtained with the major HIV-1 coreceptors was generally 40 to 100 times above background levels (CD4 with vector). Error bars here and in subsequent figures represent the standard error of the mean derived from multiple independent experiments.
random hexamer primers and SuperScript II RNase2 reverse transcriptase (Moloney murine leukemia virus reverse transcriptase modified; SuperScript T Preamplification System for First-Strand cDNA Synthesis; GIBCO BRL, Grand Island, N.Y.). cDNA synthesis was performed (20-ml volume) in 20 mM Tris-HCl (pH 8.4)–50 mM KCl with 5 mM MgCl2, 2.5 mM random hexamers, 1 mM each deoxynucleoside triphosphate, and 2.5 U of reverse transcriptase per ml at 42°C for 50 min, followed by heat inactivation and RNase H treatment. Primers spanning an intron in the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were used to verify that RNA was free of DNA contamination. RNA preparation and Northern blot analysis of APJ mRNA. Membranes containing poly(A)1 RNA from various human brain regions were obtained from Clontech. A Prime-It II random primer labeling kit (Stratagene, La Jolla, Calif.) was used to label the cDNA probe with [a-32P]dATP (3,000 Ci/mmol), using the Klenow enzyme. The a-32P-labeled cDNA was purified by using Quick Spin columns (Boehringer Mannheim). Then 107 cpm of a-32P-labeled cDNA was hybridized overnight in hybridization buffer containing 25 mM Na2PO4, 50 mM Tris (pH 7.4), 63 SSPE (13 SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7]), 0.1% sodium dodecyl sulfate (SDS), 100 mg of singlestranded DNA per ml, and 13 Denhardt’s solution. The membranes were washed twice in 13 SSPE–0.1% SDS at 42°C for 10 min and changed to a high-stringency wash solution of 0.23 SSPE–0.1% SDS at 42°C for 10 min. The membrane was then exposed to a Fuji Imaging plate for 4 h. Images of the plate were captured on a BAS1000Mac Bio-Imaging Analyzer (Fuji) and processed with MacBAS software. Images were printed on a Pictography 3000 (Fuji) digital printer.
RESULTS Cell-cell fusion. We used a cell-cell fusion assay to determine if APJ could function as a coreceptor for HIV-1 or SIV (49, 59). Env protein was expressed in QT6 cells either by transfection or by infection with a recombinant vaccinia virus. In addition, T7 polymerase was introduced through infection with a recombinant vaccinia virus. These effector cells were then mixed with target quail QT6 cells previously transfected with plasmids encoding CD4, the desired coreceptor (under control of the CMV promoter), and luciferase under control of the T7 promoter. In this assay, cell-cell fusion results in cytoplasmic mixing and luciferase production, which can be easily quantified. As shown in Fig. 1, coexpression of either CCR5 or CXCR4 with CD4 resulted in efficient fusion by R5 or X4 Env proteins, respectively. R5X4 Env proteins such as HIV-1 89.6 mediated fusion with cells bearing either CCR5 or CXCR4.
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TABLE 1. HIV-1 and SIV Env-mediated cell-cell fusion Virus
Env
Tropism
HIV-1
DH12 RF YU2 JR-FL SF162 91US005.11 93BR019.10 92UG031.7 93BR029.2 92UG037.8 92TH022.4 92RW020.5 SIVmac251 SIVmacBK28 SIVmac239 SIVmac316 SIVmac316mut SIV17E/Fr SIV17E/Cl SIVmac1A11 SIVagmSab1.4 SIVsmB670 clone 3 SIVsmB670 clone 12 SIVsm62A SIVsm62D SIVsm543-3 SIVsm543-B10 SIVsmPBj6
R5X4 R5X4 R5 R5 R5 R5 R5 R5 R5 R5 R5 R5
SIV
T M T M M M
T M M
Fusiona CCR5
CXCR4
APJ
111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111
111 111 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
1 2 1 2 1 1 1 2 2 1 11 1 2 1 1 111 111 11 1 1 1 1 1 1 11 1 1 1
a Cell-cell fusion was determined as described in the legend to Fig. 1 and in Materials and Methods. Data are expressed relative to the level of fusion obtained with the principal coreceptor (indicated by boldface except for DH12, which used both equally well). 111, .70%; 11, 40 to 69%; 1, 10 to 39%; 2, ,10%.
Fusion was not observed when CD4 was expressed alone (data not shown). When APJ was coexpressed with CD4 in QT6 cells, cell-cell fusion was mediated by the R5X4 Env protein 89.6 and by several X4 Env proteins at levels $70% of that observed with CXCR4 (Fig. 1). For one primary X4 Env protein (93ZR001.3 [36]), fusion with cells expressing APJ was more efficient than with CXCR4. A majority of R5 Env proteins mediated fusion with APJ-expressing cells, but at levels much lower than that observed with CCR5 (Fig. 1 and Table 1). The HIV-2/ST Env protein also mediated very inefficient fusion with cells expressing both CD4 and APJ. Whether such inefficient fusion is biologically significant is questionable. However, ADA and the primary isolate 92TH022.4 exhibited fusion mediated by APJ at roughly half the level observed when CCR5 served as the viral coreceptor. We also examined the ability of APJ to support fusion by a panel of SIV Env proteins. Unlike HIV-1, both M- and Ttropic SIV strains utilize CCR5 as a coreceptor, while CXCR4 either is not used by SIV or is used rarely (10, 30, 44). In addition, the orphan receptors STRL33, GPR15, and GPR1 can be used as coreceptors by both T- and M-tropic SIV strains (20, 32). We found that APJ supported fusion by several M- and T-tropic SIV Env proteins, but at levels lower than that observed with CCR5; exceptions were the M-tropic SIVmac316 Env and a variant thereof (316mut), which efficiently used APJ as a coreceptor in cell-cell fusion assays (Table 1). In addition, APJ typically supported fusion less efficiently than the orphan receptors GPR1, GPR15/BOB, and STRL33/Bonzo (data not shown). Finally, we have found that many SIV strains can in-
fect cells in a CD4-independent, CCR5-dependent manner (31). Therefore, we tested the ability of HIV-1, HIV-2, and SIV Env proteins to mediate fusion with QT6 cells expressing APJ alone. We found that APJ coreceptor activity was strictly CD4 dependent, as cells expressing APJ alone did not support cell-cell fusion with any of the Env proteins tested (data not shown). Infection of APJ-positive cells. We tested the ability of APJ to support virus infection with two different assay systems to more rigorously assess its ability to function as a coreceptor. In one series of experiments, we used a luciferase reporter virus assay in which various Env proteins are pseudotyped onto a common background. Human 293T cells are transfected with a plasmid that expresses Env under control of the CMV or simian virus 40 promoter and with a plasmid containing a proviral genome with an inactive Env gene and luciferase in place of Nef (9, 13). If infection of cells with these pseudotyped viruses progresses to the point of viral DNA integration, there is luciferase production which can be easily measured 3 days after infection. Human U87 cells expressing CD4 and the indicated coreceptor were infected with several HIV-1 pseudotypes (Fig. 2). Pseudotypes bearing the ADA, 89.6, and HxB Env proteins mediated infection of APJ-positive cells, though less efficiently than what was observed in the cell-cell fusion assay. We also performed infections with viral pseudotypes bearing the SIV CP-MAC and 17E/Fr Env proteins. As background levels of infection with U87 cells were relatively high, quail QT6 cells were used as targets. Both SIV Env proteins mediated infection of APJ-positive cells, though at levels below that observed when CCR5 was expressed. Unfortunately, some Env proteins could not be tested due to poor pseudotype formation or, as in the case with the HIV-1 93ZR001.3 and 92UG024.2 Env proteins, because very high background values were obtained in the absence of any exogenous coreceptor. In these cases, it is likely that the Env proteins were able to mediate infection by using endogenously expressed coreceptors. We also used a PCR-based entry assay to determine if APJ could support infection by HIV-1 IIIB and 89.6. As shown in Fig. 3, both HIV-1 IIIB and HIV-1 89.6 could enter QT6 cells expressing both CD4 and APJ, although entry was less efficient than with the major HIV-1 coreceptors. Thus, two independent techniques demonstrated that APJ served as a coreceptor for infection by both HIV-1 89.6 and HIV-1 IIIB.
FIG. 2. Virus infection assays. U87 (for HIV-1) or quail QT6 (for SIV) cells expressing CD4 and the indicated coreceptor were infected with luciferase virus pseudotypes bearing the indicated HIV-1 or SIV Env protein, and luciferase activity was determined 2 to 3 days after infection. The results are averages of triplicate infections for HIV and duplicates for SIV. Similar results were obtained in additional independent infection experiments.
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FIG. 3. Entry of virus into cells expressing CD4 and APJ. QT6 cells stably expressing CD4 and transiently expressing the desired coreceptor were infected with DNase-treated, cell-free virus. Virus-specific DNA was detected by PCR as described in Materials and Methods 2 days after infection; the products were resolved on a 2% agarose gel and detected following hybridization.
Expression of APJ. APJ was originally cloned from human genomic DNA, and analysis of rat tissues using a probe based on the rat homologue revealed that APJ is expressed widely in the brain (50). More recently, APJ was shown to be expressed in some areas of the human brain (45). Because APJ is used as a coreceptor by some virus strains, we reexamined its distribution in the human brain by Northern blot analysis. Consistent with the results of Matsumoto et al. (45), we found that high levels of APJ transcripts were present in the corpus callosum, spinal cord, and medulla (Fig. 4A). We found lower levels of APJ transcripts in other regions of the human brain as well. In peripheral tissues, the APJ transcript was readily detected in spleen but absent in PBLs (Fig. 4B). Lower levels of transcript were detected in other peripheral tissues. To investigate the distribution of APJ in cells commonly used to propagate HIV1, we performed RT-PCR analysis on a large number of cell lines. A U87 cell line that stably expressed APJ was generated and used as a positive control. We found that APJ was expressed in C8166 cells, but APJ-specific reaction products could not be detected in the other cell lines examined (Jurkat, CEMss, SupT1, Hut78, CEMx174, Molt4Cl8, PM1, U937, and THP-1 [Fig. 5 and data not shown]). In addition, we failed to obtain evidence for APJ expression in PBMCs stimulated with PHA, PHA and IL-2, or anti-CD3 and IL-2, in monocytes, or in MDM (Fig. 5). APJ-specific reaction products were also not obtained from primary human microglia. Oligodendrocytes were negative for APJ in one experiment, while a light APJspecific band was detected on two other occasions. Interestingly, differentiated NT2N neurons as well as their undifferentiated NT2 cell precursors expressed readily detectable levels of APJ message. While the APJ-specific band obtained from NT2 cells was more intense than that from the NT2N neurons, the amplified b-actin band from NT2 cells was also stronger, suggesting that this sample contained more RNA. NT2N neurons are obtained from the NT2 teratocarcinoma cell line by retinoic acid treatment. NT2N neurons are postmitotic, form fully polarized axons and dendrites, and express numerous proteins characteristic of human neurons (15, 53, 54). These findings suggest that neurons and oligodendrocytes may be the source of the APJ signal detected in the brain by Northern blotting.
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gression (14, 64). Considering the frequency with which mutations occur in the Env of HIV-1, it is not surprising that receptors related to CCR5 and CXCR4 can function as coreceptors for at least some virus strains. Thus far, nine of these alternative coreceptors have been identified: CCR2b, CCR3, CCR8, CX3CR1, GPR1, GPR15, STRL33, US28, and ChemR23 (11, 20, 27, 32, 38, 41, 55, 57, 60, 62). We now report the discovery of another alternative coreceptor, APJ, which is able to serve as a coreceptor for HIV-1 and SIV. The identification of alternative coreceptors is important for several reasons. First, identification of alternative coreceptors may help reveal conserved structural motifs that interact with either CD4 or the viral Env protein. The amino-terminal domain of CCR5 is an important determinant of coreceptor function (2, 3, 25, 29, 51, 58, 61), with a recently identified NYYT motif being particularly important (33). APJ contains an amino-terminal NYYG sequence, which may help account for its ability to function as a coreceptor for some virus strains. Second, use of alternative coreceptors may help explain certain facets of viral pathogenesis. For example, CCR3 is expressed in fetal human microglia and may support infection of these cells by neurotropic virus strains (37), although not all viruses that infect microglia use this coreceptor (67). The presence of abundant APJ transcripts throughout the human brain coupled with its ability to support virus infection in vitro indicates that this receptor has the potential to influence viral neuropathogenesis
DISCUSSION The HIV-1 Env protein is the primary determinant of viral tropism, with its role in tropism being exerted largely at the level of coreceptor use. Viruses isolated during the early stages of infection almost invariably use CCR5 as a coreceptor, while isolates obtained later may utilize CXCR4 as well as other coreceptors in place of or in addition to CCR5. Changes in viral tropism as a result of altered coreceptor use can broaden virus host range and have been linked to accelerated disease pro-
FIG. 4. Expression of APJ in human tissues. Membranes containing poly(A)1 RNA from various human brain regions (A) and peripheral tissues (B) (obtained from Clontech) were incubated with a labeled cDNA probe specific for APJ overnight and then exposed to a Fuji Imaging plate for 4 h. (A) Lanes: 1, amygdala; 2, caudate nucleus; 3, corpus callosum; 4, hippocampus; 5, whole brain; 6, substantia nigra; 7, subthalamic nucleus; 8, thalamus; 9, cerebellum; 10, cerebral cortex; 11, medulla; 12, spinal cord; 13, occipital lobe; 14, frontal lobe; 15, temporal lobe; 16, putamen. (B) Lanes: 1, spleen; 2, thymus; 3, prostate; 4, testis; 5, ovary; 6, small intestine; 7, colonic mucosa; 8, total PBLs.
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FIG. 5. Expression of APJ in primary cells and in cell lines. RNA from the indicated cells was used in one-tube RT-PCRs and 10 ml of each 25-ml reaction mixture was run on a 2% agarose gel. Alternatively, Superscript was used to generate cDNA from DNase-treated RNA obtained from microglia, oligodendrocytes (OLIGO), NT2 cells, and NT2N cells. The size of the predicted APJ band is 481 bp. Both plasmid DNA and U87-APJ RNA are included as positive controls; water was used as template for a negative control. MONO, monocytes; MAC, macrophages.
or Env-mediated cytotoxicity. Finally, CCR5 is an attractive target for antiretroviral compounds since individuals who lack CCR5 are highly resistant to virus infection (17, 39, 42, 63). Further, small-molecule inhibitors of CXCR4 have already been described (23, 24, 48, 65). Thus, alternative coreceptors could assume greater importance in vivo if means are found to inhibit HIV from using either CCR5 or CXCR4. The efficiency with which APJ can function as a viral coreceptor in cell-cell fusion assays was impressive, with several virus strains using APJ nearly as efficiently as the major HIV1 coreceptors CCR5 and CXCR4 (Fig. 1 and Table 1). In contrast, most other alternative coreceptors, including US28, CCR2b, CCR8, GPR1, CX3CR1, STRL33, and GPR15, support HIV1 Env-mediated cell-cell fusion relatively inefficiently. The efficiency with which a coreceptor supports either cell-cell fusion or virus infection can depend on surface expression levels (40, 52, 60). Thus, at high levels of expression many virus strains can use CCR3 as a coreceptor, while at lower levels relatively few virus strains can utilize CCR3 (60). Since APJ-specific antibodies and ligands are not yet available, it was not possible for us to compare the expression levels of APJ in our in vitro systems to the levels present in vivo. Nonetheless, the efficient use of APJ in cell-cell fusion by some virus strains is unusual among alternative coreceptors. In addition to cell-cell fusion, APJ also supported virus infection (Fig. 2 and 3). The efficiency with which APJ supported virus infection was generally less than what was observed in cell-cell fusion assays. The reasons for this are not clear but may be due to differences in cell type or in Env densities on the surface of cells and virus particles. We found that APJ functioned more efficiently as a coreceptor for R5X4 and X4 virus isolates than for R5 viral Envs. Like CXCR4, the first and second extracellular loops of APJ are acidic, in marked contrast to the corresponding regions in CCR5. The first and second extracellular loops of CXCR4 have been shown to be critical determinants for CXCR4 coreceptor function (7, 26, 43), and the acidic nature of these domains has been linked to the ability of some cationic compounds to block CXCR4 function (24). In addition, the V3 loop of T-tropic virus isolates is characteristically more basic than the V3 domains of M-tropic strains (18, 35) and so may interact with the acidic extracellular loops of CXCR4 and perhaps APJ as well. APJ coreceptor activity was strictly CD4 dependent for both
HIV-1 and SIV strains. While CD4-independent primary HIV1 strains have yet to be described, CD4-independent utilization of CCR5 is a relatively common property of primary SIV strains and accounts for the ability of some viruses to infect primary, CD4-negative, CCR5-positive cells such as brain capillary endothelial cells (31). Thus, expression of CCR5 in CD4negative cells may be relevant for viral pathogenesis. In the case of APJ, it appears thus far that it can function as a coreceptor only when CD4 is present. APJ was originally cloned from human genomic DNA by using primers designed to identify the vasopressin receptor and related molecules. The rat homologue was also cloned and found to have 74% amino acid identity to the human receptor (50). Northern analyses using RNA isolated from multiple rat tissues and a probe based on the rat APJ sequence detected APJ transcripts only in the brain. APJ transcripts were detected in the striatum, hippocampus, cerebellum, and cortex, and in situ hybridization experiments were in good agreement with these findings (50). A more recent study found that APJ was also expressed in multiple areas of the human brain (45). We confirmed this finding and also determined that APJ was expressed in the spleen and several other peripheral tissues, though at lower levels than found in the spleen or CNS. The detection of APJ transcripts by RT-PCR in the C8166 T-cell line and in the spleen by Northern blot analysis suggests that APJ may be expressed in some T-cell subsets. It will be important to reevaluate its expression in CD4-positive T cells, macrophages, and microglia by alternate methods when specific antisera are available. In addition, the high levels of APJspecific reaction products obtained from NT2N neurons by RT-PCR suggests that APJ may be expressed in human neurons, which would account for its widespread distribution in human brain. As for other alternative coreceptors, the significance of APJ for viral pathogenesis in vivo cannot fully be determined at this time. To do so, it will be necessary to identify the specific cell types in which APJ is expressed and determine the surface levels of APJ required to support virus infection. The identification of a natural ligand for APJ or the development of specific antibodies will make it possible to study APJ expression in primary cells and to determine if it can support virus infection. Of particular interest will be more detailed analyses of APJ expression in the CNS. Neurologic disorders are a frequent
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and significant feature of HIV-1 and SIV infection, and changes in coreceptor use may help account for the development of neurotropic strains (46, 56). Further, some Env proteins can induce signaling through CCR5 or CXCR4, potentially influencing postentry steps in virus replication or perhaps contributing to cytopathic effects in uninfected cells which might also play a role in AIDS dementia (16, 69). Thus, a larger number of neurotropic virus strains will have to be evaluated for the ability to use APJ to infect cells or to induce signaling via this receptor. In particular, primary CNS isolates should be screened for the ability to use this alternative coreceptor for infection and also for the ability to induce signaling as a result of Env-APJ interactions. ACKNOWLEDGMENTS We thank Franciso Gonzalez-Scarano, Julie Strizki, Joseph Sheih, and Andrew Albright for providing human microglia and oligodendrocytes and Jack Rose (Yale University) for supplying the 89.6-VSV Env construct. The following reagents were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: rVV/ST from Mark J. Mulligan; and 93ZR001.3, 92UG024.2, US005.11, 93BR019.10, 92UG031.7, 93BR029.2, 92UG037.8, 92TH022.4, and 92RW020.5 env clones from the WHO Network for HIV Isolation and Characterization and Beatrice Hahn. This work was supported by NIH grants AI-40880 to R.W.D. and AI-35502 to R.G.C. A.L.E. was supported by the MSTP program and by NIH grant 2T32 GM07170, T.L.H. was supported by the Franklin Scholars Program, and B.L. was supported by the Measey Foundation Fellowship for Clinicians. REFERENCES 1. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. M. Murphy, and E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1a, MIP-1b receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272: 1955–1958. 2. Alkhatib, G., M. Locati, P. E. Kennedy, P. M. Murphy, and E. A. Berger. 1997. HIV-1 coreceptor activity of CCR5 and its inhibition by chemokines: independence from G protein signaling and importance of coreceptor downmodulation. Virology 234:340–348. 3. Atchison, R. E., J. Gosling, F. S. Monteclaro, C. Franci, L. Digilio, I. F. Charo, and M. Goldsmith. 1996. Multiple extracellular elements of CCR5 and HIV-1 entry: dissociation from response to chemokines. Science 274: 1924–1926. 4. Bagasra, O., E. Lavi, L. Bobroski, J. P. Pestamer, R. Tawadros, and R. J. Pomerantz. 1996. Cellular reservoirs of HIV-1 in the central nervous system of infected individuals: identification by the continuation of in situ polymerase chain reaction and immunohistochemistry. AIDS 10:573–585. 5. Berger, E. A. 1997. HIV entry and tropism: the chemokine receptor connection. AIDS 11:S3–S16. 6. Berger, E. A., R. W. Doms, E.-M. Fenyo ¨, B. T. M. Korber, D. R. Littman, J. P. Moore, Q. J. Sattentau, H. Schuitemaker, J. Sodroski, and R. A. Weiss. 1998. HIV-1 phenotypes classified by co-receptor usage. Nature 391:240. 7. Brelot, A., N. Heveker, O. Pleskoff, N. Sol, and M. Alizon. 1997. Role of the first and third extracellular domains of CXCR-4 in human immunodeficiency virus coreceptor activity. J. Virol. 71:4744–4751. 8. Broder, C. C., and R. G. Collman. 1997. Chemokine receptors and HIV. J. Leukoc. Biol. 62:20–29. 9. Chen, B. K., K. Saksela, R. Andino, and D. Baltimore. 1994. Distinct modes of human immunodeficiency virus type 1 proviral latency revealed by superinfection of nonproductively infected cell lines with recombinant luciferaseencoding viruses. J. Virol. 68:654–660. 10. Chen, Z., P. Zhou, D. D. Ho, N. R. Landau, and P. A. Marx. 1997. Genetically divergent strains of simian immunodeficiency virus use CCR5 as a coreceptor for entry. J. Virol. 71:2705–2714. 11. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu, C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, C. Gerard, and J. Sodroski. 1996. The b-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135–1148. 12. Collman, R., N. F. Hassan, R. Walker, B. Godfrey, J. Cutilli, J. C. Hastings, H. Friedman, S. D. Douglas, and N. Nathanson. 1989. Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV1). Monocyte-tropic and lymphocyte-tropic strains of HIV-1 show distinctive patterns of replication in a panel of cell types. J. Exp. Med. 170:1149–1163. 13. Connor, R. I., B. K. Chen, S. Choe, and N. R. Landau. 1995. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 206:935–944.
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