Characterization of the Human Chemerin Receptor − Chemr23

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Virology 355 (2006) 6–17 www.elsevier.com/locate/yviro

Characterization of the human chemerin receptor − ChemR23/CMKLR1 − as co-receptor for human and simian immunodeficiency virus infection, and identification of virus-binding receptor domains ⁎ Ulrika E.A. Mårtensson a, , Eva Maria Fenyö b, Björn Olde a, Christer Owman a

a Division of Molecular Neurobiology, Wallenberg Neuroscience Center, Lund University, SE-223 62, Sweden b Division of Medical Microbiology, Lund University, SE-223 62, Sweden Received 13 April 2006; returned to author for revision 18 May 2006; accepted 10 July 2006 Available online 10 August 2006

Abstract

Studies were conducted to elucidate co-receptor spectrum and function of the inflammatory receptor, CMKLR1/ChemR23, which was recently identified as the receptor for the cystatin-like chemoattractant, TIG2, also named chemerin. An infection model was applied based on stably transfected NP-2.CD4 host cells expressing various co-receptor constructs and exposed to panels of HIV-1, HIV-2 and SIV primary isolates. In a panel of 27 HIV-1 isolates tested, 12 isolates could use CMKLR1/ChemR23. As expected from a relatively high sequence homology with the extracellular domains of CCR3, HIV-1 isolates showing R3 tropism were particularly efficient in using CMKLR1/ChemR23. In addition, 5 out of 7 HIV-2 isolates and 13 out of 15 SIV (SMM-3 origin) used CMKLR1/ChemR23, in accordance with the previously documented ability of these isolates to use several co-receptors. In order to define important extracellular epitopes for the viral interaction, a hybrid receptor model was applied. This was based on the fact that the rat receptor, although structurally very similar to the human orthologue, was inefficient as viral co- receptor. When the rat receptor was “humanized” to include regions unique to the human receptor (N-terminus or second extracellular loop), exposure to HIV-1, HIV-2 and SIV isolates resulted in infection. The relative importance of the two critical receptor regions differed between HIV- 1/HIV-2 on the one hand and SIV on the other. The results strongly support that the chemerin receptor, in the presence of CD4, functions as a “minor co-receptor” promoting infection by these classes of viruses. © 2006 Elsevier Inc. All rights reserved.

Keywords: G-protein coupled receptor; Chemerin receptor; HIV-1; HIV-2; SIV; Hybrid receptor model

Introduction co-receptor for SIV (Marcon et al., 1997). Besides the major co- receptors, a large number of additional, so-called minor, co- Human immunodeficiency virus 1 and 2 (HIV-1 and -2) and receptors have been described that mediate viral entry “in vitro” simian immunodeficiency virus (SIV) require the CD4 receptor (Edinger et al., 1997). ChemR23, first mentioned under the (Dalgleish et al., 1984; Klatzmann et al., 1984) together with name of chemoattractant-like receptor 1 (CMKLR1) (Gantz et particular seven-transmembrane G-protein coupled receptors al., 1996), is primarily expressed in dendritic cells and (GPCRs) for entry into, and infection of, human immune cells. macrophages, and has been proposed to function as a minor The chemokine receptors, CXCR4 and CCR5, are the major co- co-receptor promoting infection by select HIV and SIV isolates receptors for HIV and SIV. HIV uses both (Choe et al., 1996; (Samson et al., 1998). Upon the recent identification of the Deng et al., 1996; Doranz et al., 1996; Dragic et al., 1996; Feng endogenous receptor ligand as chemerin, the receptor is now et al., 1996) as major co-receptors whereas CCR5 is the main also described as “the chemerin receptor” (Wittamer et al., 2003). Expression of the receptor mRNA has also been detected in trophoblast cells from early placentas (Mognetti et al., 2000) ⁎ Corresponding author. Fax: +46 46 2220568. as well as in human fetal and simian adult astrocytes (Croitoru- E-mail address: [email protected] (U.E.A. Mårtensson). Lamoury et al., 2003).

In this study, we have performed a detailed characterization rCMKLR1.hECL2-EGFP constructs to the cell surface was of the importance of the chemerin receptor as a co-receptor for confirmed by confocal microscopy (Fig. 3). Since the HIV-1, HIV-2 and SIV using an infection model with primary hCMKLR1 mAb recognized rCMKLR1.hNterm-EGFP, it isolates. Earlier work from our laboratory, forming part of this indicates that it is specific for the N-terminus. Native NP-2 study, was performed before CMKLR1 was identified as the cells were used as negative controls in the flow cytometric chemerin receptor. For reason of consistency, the designation analysis (data not shown). CMKLR1 will therefore be used throughout the present study as a synonym for the chemerin receptor. The human glioma cell Characterization of hCMKLR1 as a co-receptor line, NP-2, was chosen as host cell line for recombinant receptor expression since it lacks endogenous co-receptors, except in the As a general characterization of hCMKLR1 as co-receptor case of Bonzo/CXCR6/STRL33 for which low mRNA expres- for HIV-1, 15 isolates of different genetic subtypes (A, B, C, D, sion has been detected (Soda et al., 1999). The domains E, F) were tested. Only 3 isolates, of subtype D, were shown to important for the virus interaction with the chemerin receptor infect the test cells (Table 2). Since all HIV-1 isolates use either, were mapped by the use of recombinant hybrid receptor or both, of the major co-receptors, CCR5 and CXCR4, and as a constructs. further control for the infectivity of the various isolates tested, infection experiments were in the entire study carried out with Results cells expressing CCR5 and CXCR4, respectively. Table 2 also shows that three positive isolates, in addition, used the hybrid Stable expression of recombinant receptors in NP-2.CD4 cells receptor, FC-4b, for infection. Cells expressing CD4 alone were always negative. Flow cytometric studies were performed on NP-2 cells stably The sequence similarity between hCMKLR1 and some expressing the CD4 receptor, alone or in combination with the structurally related, more well-established, co-receptors was following receptors: CCR5, CXCR4, CCR3, FC-4b (a chimera elucidated by amino acid alignments of the extracellular where the N-terminal region originates from CXCR4 and the C- receptor domains (Fig. 4). The highest sequence similarity terminal region originates from CCR5) (Antonsson et al., 2003), was found for CCR3, particularly within the first cellular loop hCMKLR1, rCMKLR1, hCMKLR1-EGFP, rCMKLR1-EGFP, (ECL1 in Fig. 3). Against this background, 12 isolates of the R3 rCMKLR1.hECL2-EGFP or rCMKLR1.hNterm-EGFP. The phenotype (clade B) were tested for their ability to infect cells receptor mAbs and isotype control antibodies that were used expressing hCMKLR1. The isolates have been further char- appear in Table 1. An equally high expression level of CD4 acterized in previous studies (Karlsson et al., 2003, 2004). As (data not shown) was confirmed for all cell lines. Other many as nine of these isolates used hCMKLR1 for infection transfected receptors were also expressed at high level (Figs. (Table 3). All isolates also used the hybrid FC-4b receptor 1A–D), except CCR3 that showed low expression (Fig. 1E). efficiently (not included in Table 3). As before, cells expressing However, expression was enough for its use as positive control CD4 alone were all negative. in the ELISA assays following the infection experiments with hCMKLR1 was also tested for its ability to support infection CCR3-using (i.e., R3) isolates. Although hCMKLR1 and of CD4+ cells with HIV-2. Five isolates out of 7 tested utilized rCMKLR1 show 86% sequence identity (Fig. 2), the this receptor (Table 4). In all cases tested, FC-4b also supported hCMKLR1 antibody was not specific for rCMKLR1, HIV-2 infection. rCMKLR1-EGFP or rCMKLR1.hECL2-EGFP and, hence, SIVsm (strain SMM-3), originally isolated from a naturally expression of these receptors could not be confirmed by flow infected sooty mangabey monkey, and 14 sequential SIV cytometry. Instead, localization at the rCMKLR1-EGFP and isolates from seven cynomolgus macaques inoculated with this isolate, were used to test the SIV usage of hCMKLR1. The SIVsm/SMM-3 strain, as well as 14 of the sequential isolates, Table 1 used hCMKLR1 (Table 5). As expected from previous studies Antibodies used for the cytometric analysis (Bron et al., 1997; Chen et al., 1997; Edinger et al., 1997; Hill et Receptor Primary Secondary Isotype primary Isotype secondary al., 1997; Kirchhoff et al., 1997; Marcon et al., 1997), all mAb mAb mAb mAb isolates tested were positive for CCR5. In contrast to the case of CD4 CD4-RPE IgG1-RPE HIV-2, SIV showed little or no usage of the CCR5/CXCR4 CCR5 CCR5- IgG2Ak-RPE RPE receptor chimera, FC-4b. All SIV isolates where amplified in CXCR4 5/5B5 F(ab′)2-RPE 7B1-IgG2A F(ab′)2-RPE human PBMCs. In a separate study, some isolates were FC-4b 5/5B5 F(ab′)2-RPE 7B1-IgG2A F(ab′)2-RPE amplified also in monkey PBMCs, which gave the same results. CCR3 CCR3- IgG2A-RPE RPE Mapping the virus-binding domain of CMKLR1 hCMKLR1 hCMKLR1 F(ab′)2-RPE IgG3 F(ab′)2-RPE The 5/5B5 and 7B1 antibodies were generated in house (Antonsson et al., 2003; Although the human and rat orthologues, rCMKLR1 and Pettersson et al., 2000). The hCMKLR1, IgG3, CCR3 and IgG2A antibodies were purchased from R&D Systems, whereas CD4, IgG1, CCR5 and IgG2Aκ, hCMKLR1, show 86% overall identity at amino acid level (Fig. were bought from PharMingen-BD Biosciences. Goat anti-mouse immunoglo- 2), only the human variant acted as co-receptor for most isolates bulin F(ab′)2-RPE was purchased from Daco Cytomation. tested (Table 6). The largest sequence differences were detected 8 U.E.A. Mårtensson et al. / Virology 355 (2006) 6–17

Fig. 1. (A–E) Flow cytometric analysis of stable expression of receptors in NP-2 cells. Each cell line was stained for the CD4 receptor (data not shown) and CCR5, CXCR4, CCR3, FC4-b, hCMKLR1, rCMKLR1, hCMKLR1-EGFP, rCMKLR1.hNterm-EGFP, rCMKLR1-EGFP and rCMKLR1.hECL2-RGFP, respectively. Dotted-line histograms represent cells stained with isotype control antibodies whereas continuous-line histograms indicate staining with specific receptor antibodies. Non-transfected NP-2 cells were used as negative control for all antibodies tested. U.E.A. Mårtensson et al. / Virology 355 (2006) 6–17 9

Fig. 2. Amino acid alignment of rCMKLR1 (accession no. AJ002745) and hCMKLR1 (accession no. U79526 for hCMKLR1a, having two additional amino acids as indicated within parenthesis, and U79527 for hCMKLR1b). The alignment was performed using the Clustal X program. Shaded boxes represent regions of identity between species. The human N-terminus and extracellular loops were assessed using the program TMHMM 2.0 and indicated by horizontal lines. in the N-terminus and in the second extracellular loop (Fig. 2). GPCRs, we have devoted our interest to various species In order to find out whether a particular region of hCMKLR1 variants of a receptor which has provisionally been was important for virus entry, we exchanged either the second designated CMKLR1—the first name for the human receptor extracellular loop or the N-terminus of rCMKLR1 for the (Gantz et al., 1996). It has later been described as CMKRL3 corresponding human sequences. In the absence of a mAb in rat (Owman et al., 1996), DEZ in mouse (Methner et al., recognizing rCMKLR1, we tagged the constructs with EGFP in 1997), ChemR23 again in human (Samson et al., 1998) and order to confirm surface expression (Fig. 3). also listed as Gpcr27 in GPCR databases. Although being All isolates that were positive with hCMKLR1 and at the an orphan, the receptor arose particular interest when it was same time negative with wild-type rCMKLR1 (Table 6) were indicated that the human variant might function as another also tested on the chimeras. The results from quantitative data so-called minor co-receptor (along with the major co- with select isolates in which the hCMKLR1 usage was receptors, CCR5 and/or CXCR4), promoting entry of select particularly efficient are shown in Fig. 5. HIV-1 and SIV isolates into immune cells (Samson et al., Taken together, rCMKLR1 did only support infection by 1998). In the course of our ongoing project, we have some of the HIV-1 isolates. “Humanizing” the rat receptor by characterized the promoter function of the mouse receptor exchanging either the N-terminus or the second extracellular orthologue (mCMKLR1) and have provided evidence for the loop increased the infectibility with HIV-1, to a varying degree existence of a second, previously unknown, splice variant of for the two extracellular regions. Both regions seem to be the receptor gene (Mårtensson et al., 2004, 2005). By the important also for promoting infection with HIV-2 isolates. On recent identification of the natural ligand as a proinflamma- the other hand, infection experiments with the SIV isolates tory mediator called chemerin (Wittamer et al., 2003), indicated that the second extracellular loop was the primary previously known as TIG2 (Nagpal et al., 1997), it will now domain recognized for co-receptor function. be possible to explore also the physiological relevance of this receptor. Discussion In the light of the accumulated interest in CMKLR1, we have in the present study further elucidated its viral co-receptor As part of a longstanding interest in certain inflamma- spectrum and function. Among 10 HIV-1 isolates of various tory-type, functionally unknown (so-called “orphan”) subtypes previously tested by Samson et al. (1998), only one 10 U.E.A. Mårtensson et al. / Virology 355 (2006) 6–17

Fig. 3. Cell membrane localization of receptors using confocal microscopy. (A) NP-2.CD4 cell lines expressing receptors fused to EGFP in the C terminal were fixed on a multi-well chamber microscopy slide. (B) The cell lines were also stained for CD4 receptor expression. (C) Overlay of green and red fluorescence confirms membrane localization. proved to be positive. In a panel of 27 HIV-1 isolates tested in Although SIV is of non-human origin, the structure is very the present study, 12 isolates were positive. The observation that similar to HIV-1 and HIV-2 in both genetic sequence and they all belonged to either clade B or D may simply be due to biological aspects (Desrosiers and Ringler, 1989). SIV has the fact that the panel included fewer isolates of other genetic therefore become a useful model for studies of co-receptor subtypes (A, C, E and F). interactions, particularly since the virus is less restrictive in its When comparing the amino acid sequence of the extra- co-receptor spectrum. It has, therefore, conveniently been used cellular domains (i.e., the putative virus-binding regions) of to screen for co-receptor function in more general terms. In both well-known minor co-receptors, it was found that CMKLR1 the present study and in that by Samson et al. (1998), a large mostly resembled CCR3. For this reason, we chose to include number of SIV isolates of varying origin efficiently utilized 12 well-characterized R3 isolates (Karlsson et al., 2003, 2004) CMKLR1/ChemR23 for cellular entry. This further supports among the abovementioned panel of 27 HIV-1 isolates. As that CMKLR1 (i.e., the chemerin receptor) is a potent mediator could be expected, the majority of the R3 isolates turned out to of immunodeficiency virus entry. In accordance with this, be positive also for CMKLR1. CMKLR1 is expressed in dendritic cells and macrophages, HIV-2 seems to use CMKLR1 much more efficiently than which are well-known targets for HIV/SIV. However, the HIV-1 for cellular entry. Out of seven tested isolates, five were receptor also has a more widespread expression, including positive. In contrast, three HIV-2 isolates previously tested astrocytes, chondrocytes, placenta, microglia and maybe also using a cell−cell fusion assay (Samson et al., 1998), rather (depending on species) liver, lung, parathyroid gland and than the present infection model, were all negative. The thymus (Croitoru-Lamoury et al., 2003; Methner et al., 1997; discrepancy in results may be explained by the way isolates Mognetti et al., 2000; Mårtensson et al., 2004; Owman et al., were selected for testing, and/or differences in assay 1996). The relevance of these multiple expression sites when it techniques. Our findings are in line with the general notion comes to viral infection and inflammation has to await future that HIV-2 is more promiscuous in utilizing a broader experiments. repertoire of various cellular co-receptors than HIV-1 (Bron The FC-4b receptor chimera, which is a hybrid between et al., 1997; McKnight et al., 1998). The ability of HIV-2 to CCR5 and CXCR4, has proved useful in elucidating certain enter CD4− cells (Reeves et al., 1999), suggests a more evolutionary aspects of HIV-1 co-receptor use. It was therefore flexible structure of the envelope glycoprotein (Reeves and included in the present co-receptor characterization, but it did Schulz, 1997), which may confirm this statement. not reveal any further distinct feature of CMKLR1/ChemR23 U.E.A. Mårtensson et al. / Virology 355 (2006) 6–17 11

Table 2 2003; Zhou et al., 2001). Although there is a high sequence Co-receptor function of hCMKLR1 expressed in NP-2. CD4 cells as analyzed similarity between the human and rat orthologues, the rat by infection with HIV-1 isolates of different subtypes receptor was presently shown to be inefficient as a viral co- Virus Genetic Infectious dose HIV-1 p24 antigen production in receptor. Since the highest sequence differences between the rat isolate subtype (ng/ml) supernatants of cells expressing: and human receptors are found in the N-terminus and the CCR5 CXCR4 FC-4b hCMKLR1 second extracellular loop, these regions were interchanged, 93UG-086 D 2 +++ +++ +++ ++ resulting in a “humanized” rat receptor equipped with the 5 +++ +++ +++ ++ human N-terminus or second extracellular loop. The hybrids 10 +++ +++ +++ ++ were used to elucidate the isolate preference for either of these 92UG-053 D 2 − +++ +++ + 5 − +++ +++ + receptor epitopes. 10 − +++ +++ ++ While both HIV-1 and HIV-2 could use either the N-terminus 92UG-021 D 2 − + − + or the second extracellular loop of CMKLR1, SIV seemed to 10 − +++ ++ ++ prefer the second extracellular loop of the receptor for infection. − 20 +++ +++ +++ The ability of HIV-2 to use alternative epitopes for viral entry 94UG-118 D 2 +++ −−− 5 +++ −−− agrees well with the more promiscuous nature of this virus. The 10 +++ −−− sequential SIV isolates tested with the hybrid receptors all 92UG-005 D 2 +++ ++ − + displayed a high dependence of the second extracellular loop. 5 +++ + −− In conclusion, the results support the evidence that the −− 10 +++ ++ chemerin receptor can function as a minor co-receptor for select 92UG-001 D 2 − +++ −− 5 − +++ −− HIV-1 isolates, as well as more generally for HIV-2 and SIV 10 − +++ −− isolates. Among certain better-characterized co-receptors, the 94UG-117 D 2 − +++ −− HIV-1 co-receptor function of the chemerin receptor resembles 5 − +++ −− that of CCR3. Using a “humanized” hybrid rat chemerin − −− 10 +++ receptor as model, it was shown that the major determinants for 92UG-024 D 2 − + −− 10 − +++ + − HIV-1 and HIV-2 interaction with the receptor reside to a 20 − +++ + − varying degree in the N-terminus and second extracellular loop, 93UG-070 D 2 − +++ +++ − whereas the viral interaction in the case of SIV primarily 10 − +++ +++ − involves the second extracellular loop. 20 − +++ +++ − 92UG-037 A 2 +++ −−− 10 +++ − + − Materials and methods 20 +++ − + − 92BR-020 B 2 +++ −−− Virus isolates 5 +++ − + − − 10 +++ + + The HIV-1 isolates 92UG021, 92UG024, 92UG037, 92BR-025 C 2 +++ −−− 3 +++ −−− 93UG070, 92BR020, 92BR025, 93BR019, 93BR029 and 93BR-019 B 2 +++ +++ −− 92TH019 were obtained from the WHO Network for HIV 5 +++ ++ +++ − Isolation and Characterization (Rubsamen-Waigmann et al., 10 +++ +++ +++ − 1994; WHO, 1994). Other HIV-1 isolates as well as HIV-2 −−− 93BR-029 F 2 +++ isolates were previously characterized in separate studies 5 +++ − + − 10 +++ − + − (Bjorndal et al., 1997; Karlsson et al., 2003, 2004; Morner 92TH-019 E 2 + −−− et al., 1999; WHO, 1994). The primarily SIV isolates were NP-2.CD4-CCR5, -CXCR4, -FC-4b and -hCMKLR1 cells were infected with isolated from cynomolgus macaques, inoculated with SIVsm above shown concentrations of virus. The ability to use the receptors is (strain SMM-3 from P. Fultz and H. Mclure, Yerkes presented as the average of absorbance from triplicate wells obtained in enzyme- Regional Primate Research Center, Atlanta, GA, USA). linked immunosorbent assay (ELISA) (absorbance of uninfected cells is Strain SMM-3 was originally isolated from a naturally − subtracted). , p24 antigen negative (absorbance < 0.3); +, p24 antigen positive infected sooty mangabey. 56:3, 59:3, C2, C44, C68, C73 (absorbance 0.3–0.9); ++, p24 antigen positive (absorbance 1.0–1.9); +++, p24 antigen positive (absorbance > 2.0). Parental NP-2.CD4 cells were included in and B174 represent the name of the inoculated cynomolgus each experiment and were negative for HIV-1 p24 antigen. macaques whereas the figures following the colon (56:3:56) describe the day post-infection that blood samples were collected for virus isolation. co-receptor function. The reason for the broad viral usage of Virus stocks were prepared by infection of phytohemagglu- FC-4b may simply reflect the fact that the hybrid receptor is tinin (PHA)-stimulated peripheral blood mononuclear cells composed of important epitopes from both CCR5 and CXCR4. (PBMCs) mixed from two blood donors. The donor PBMCs The general picture of the structural requirements for co- were isolated by separation of buffy coats on Ficoll gradient receptor function indicates that several extracellular epitopes are (Pharmacia) in RPMI-1640+Glutamax I culture medium, involved in virus entry (Alkhatib et al., 1997; Atchison et al., supplemented with 10% fetal calf serum (FCS) and 0.5% 1996; Brelot et al., 1997; Doranz et al., 1997; Wittamer et al., penicillin/streptomycin. Pooled PBMCs (7×106) were infected 12 U.E.A. Mårtensson et al. / Virology 355 (2006) 6–17

Fig. 4. Alignments of amino acid sequences from the extracellular regions of various co-receptors. The alignments were performed using the Clustal X program. Shaded boxes represent regions of identity between co-receptors. The extracellular regions were assessed using the program TMHMM 2.0. with virus in 7 ml RPMI medium containing 10% FCS, 5 U/ml 12, assayed for virus antigen content and stored at −80 °C until of interleukin 2 (IL-2, Amersham Biosciences), 2 μg/ml used. HIV-1 was detected by measuring virus p24 antigen in a polybrene (Sigma), 50 U/ml penicillin and 50 μg/ml strepto- p24 ELISA (Vironostika HIV-1 Antigen, Biomérieux). HIV-2 mycin. Cell-free supernatants were harvested at day 8, 10 and and SIV were detected by measuring virus p26 antigen in an U.E.A. Mårtensson et al. / Virology 355 (2006) 6–17 13

Table 3 Table 4 Co-receptor function of hCMKLR1 expressed in NP-2.CD4 cells as analyzed by Co-receptor function of hCMKLR1 expressed in NP-2. CD4 cells and analyzed infection with CCR3-using HIV-1 isolates by HIV-2 isolates Virus Infectious dose HIV-1 p24 antigen Virus Infectious dose HIV-2 p26 antigen isolate (ng/ml) production in supernatants of isolate (ng/ml) production in supernatants cells expressing: of cells expressing: CCR3 hCMKLR1 FC-4b hCMKLR1 3609:5186 2 +++ +++ 1812 1.5 Pos Pos 5 +++ +++ 1010 1.1 Pos Pos 10 +++ +++ 1816 1.5 Pos Pos 778:975 2 +++ + 1808 1.5 Pos Pos 5 +++ + 1654 1.5 NT a Pos 10 +++ ++ 6669 1.5 Pos Neg 2289:5103 2 +++ − 1653 1.5 NT Neg 5 +++ + NP-2.CD4-FC4-b and -hCMKLR1 cells were infected with different HIV-2 10 +++ ++ isolates. To confirm the infectivity of the isolates used, they were tested on NP-2. 553:1004 2 +++ − CD4-CCR5 and -CXCR4 cells. All isolates used these co-receptors to a variable 5 +++ + extent (cf. Morner et al., 1999). The ability to use the receptors is presented as 1679:2129 2 +++ − absorbance obtained in enzyme-linked immunosorbent assay (ELISA). Defini- 5 +++ + tion of Pos and Neg, see Materials and methods ( Virus isolates). 10 +++ + a NT, not tested. 1679:5594 2 +++ − 5 +++ + 10 +++ + cells without co-receptor, expressing CD4 only, receiving virus. 1991:6750 2 +++ − Results shown as Neg reflect absorbance level <0.3. 5 +++ + 10 +++ + Constructions of human/rat receptor hybrids 230:7279 2 +++ − 5 +++ − 7 +++ + The coding regions of human and rat CMKLR1 (hCMKLR1 958:8567 2 +++ − and rCMKLR1) were amplified from genomic DNA by PCR 5 +++ − using Pwo DNA polymerase (Roche Diagnostics) and cloned into 10 +++ + BamHI or EcoRV/NotI sites of the bicistronic vector, pIRESpuro 446:744 2 +++ − 5 +++ − (Clontech). Constructs were sequenced using the BigDye 10 +++ − Terminator Cycle Sequencing kit (PE Applied Biosystems). 1865:3017 2 +++ − 5 +++ − 10 +++ − Table 5 1865:3305 2 +++ − Co-receptor function of hCMKLR1 expressed in NP-2. CD4 cells as analyzed 5 +++ − by infection with SIV isolates 10 +++ − Virus Infectious dose HIV-1 p26 antigen production in NP-2.CD4-CCR3 and -hCMKLR1 cells were infected with above shown isolate (ng/ml) supernatants of cells expressing: concentrations of virus. To confirm the infectivity of the isolates used, they were tested on NP-2.CD4-CCR5 and -CXCR4 cells. All isolates used these co- CCR5 FC-4b hCMKLR1 receptors to a variable extent (cf. Karlsson et al., 2003, 2004). The ability to use SIVsm 10 Pos Pos Pos the receptors is presented as the average of absorbance from triplicate wells 56:3:56 2 Pos Neg Pos obtained in enzyme-linked immunosorbent assay (ELISA) (absorbance of 56:3:91 2 Pos Neg Pos uninfected cells is subtracted). −, p24 antigen negative (absorbance < 0.3); +, 59:3:56 6 Pos Pos Pos p24 antigen positive (absorbance 0.3–0.9); ++, p24 antigen positive (absorbance 59:3:91 6 Pos Neg Pos 1.0–1.9); +++, p24 antigen positive (absorbance > 2.0). Parental NP-2.CD4 59:3:730 6 Pos Pos Pos cells were included in each experiment and were negative for HIV-1 p24 C2:264 3 Pos NTa Neg antigen. C2:498 4 Pos NT Neg C44:90 10 Pos Pos Pos C44:365 10 Pos Neg Pos C44:810 10 Pos Neg Pos “in-house” p26 ELISA (Thorstensson et al., 1991)orby C68:90 10 Pos Neg Pos analyzing virus reverse transcriptase activity using Cavidi HS- C68:1590 10 Pos Neg Pos kit Lenti RT (Cavidi Tech AB). The p26 ELISA does not allow C73:540 10 Pos Neg Pos for quantitative analysis. Instead, results in the Tables are B174:14 10 Pos Pos Pos arbitrarily expressed as positive (Pos) or negative (Neg). Pos is NP-2.CD4-CCR5, -FC-4b, and -hCMKLR1 cells were infected with above defined as the absorbance level exceeding three times above shown concentrations of virus. The ability to use the receptors is presented as the average of absorbance from triplicate wells obtained in enzyme-linked background (absorbance value >0.3). Presently, background is immunosorbent assay (ELISA). Definition of Pos and Neg, see Materials and defined as the absorbance measured in non-infected cells (i.e., methods ( Virus isolates). cells expressing co-receptor but receiving only vehicle) or in a NT, not tested. 14 U.E.A. Mårtensson et al. / Virology 355 (2006) 6–17

Table 6 Hoshino (Gunma University School of Medicine, Japan). The Co-receptor function of rat CMKLR1 expressed in NP-2.CD4 cells as analyzed cells were grown in Eagle's minimum essential medium by infection with HIV-1, HIV-2 and SIV isolates (EMEM) (Sigma) supplemented with 10% fetal bovine serum Virus type Virus isolate Infectious dose p24/p26 antigen (FBS), 1% glutamax (Invitrogen), 50 U/ml penicillin, 50 μg/ml (ng/ml) production in supernatants streptomycin and 0.5 mg/ml geneticin (Invitrogen) at 37 °C in of cells expressing: 7% CO2. NP-2.CD4 cells were stably transfected with either of hCMKLR1 rCMKLR1 the following receptor cDNAs: CCR5, CXCR4, FC-4b (a HIV-1 3609:5186 a 10 +++ − chimera between CXCR4 and CCR5), CCR3, hCMKLR1, − UG92021 10 ++ rCMKLR1, hCMKLR1-EGFP, rCMKLR1-EGFP, rCMKLR1. UG92053 10 ++ − 2289:5103 10 ++ − hECL2-EGFP and rCMKLR1.hNterm-EGFP. The respective 778:975 10 ++ + coding sequences (all verified by sequencing) were cloned into UG93086 10 ++ +++ the pIRESpuro vector (Clontech). The day before transfection, 1679:5594 10 + +++ 8×105 NP-2.CD4 cells were seeded in 10-cm dishes, and 4 μg 1991:6750 10 + ++ receptor plasmid was transfected using Lipofectamine (Invi- 553:1004 5 + ++ HIV-2 1812 a 1.5 Pos Neg trogen). Two days after transfection, cells were transferred to 1010 a 1.1 Pos Neg 15-cm dishes and selected for 3 weeks in media supplemented 1654 1.5 Pos Neg with 0.5 μg/ml puromycin (Sigma) for stable monoclonal 1808 1.5 Pos Neg receptor expression. 1816 1.5 Pos Neg SIV 59:3:56 a 6 Pos Neg 59:3:91 a 6 Pos Neg Virus infection of receptor-expressing NP-2.CD4 cells 59:3:730 a 6 Pos Neg 56:3:56 2 Pos Neg Two days before infection, 4×103 cells/well were seeded 56:3:91 2 Pos Neg into 48-well plates using medium without antibiotics. At the C44:90 10 Pos Neg time of infection, medium was removed and virus added to C44:365 10 Pos Neg μ C44:810 10 Pos Neg triplicate wells in 200 l medium containing polybrene C68:90 10 Pos Neg (2 μg/ml). Two hours after infection, medium with polybrene C68:1590 10 Pos Neg was added to a total volume of 500 μl/well. After overnight C73:540 10 Pos Neg incubation, cells were washed 3 times in 1 ml PBS (PBS B174:14 10 Pos Neg Dulbecco's) (Life technologies), and 1 ml fresh medium SIVsm 10 Pos Neg without polybrene was added to each well. Twelve days after NP-2.CD4-hCMKLR1 and -rCMKLR1 cells were infected with the above infection, medium was sampled from each well for detection shown concentrations of HIV-1, HIV-2 or SIV isolates. NP-2.CD4-CCR5 and -CXCR4 were used as positive controls. The ability to use the receptors is of viral antigen. HIV-1 and HIV-2/SIV infection was presented as the average of absorbance obtained from triplicate wells in enzyme- monitored with ELISA as described above. Cells were also linked immunosorbent assay (ELISA) (absorbance of uninfected cells is evaluated microscopically for syncytia formation. subtracted). −, p24 antigen negative (absorbance<0.3); +, p24 antigen positive – – (absorbance 0.3 0.9); ++, p24 antigen positive (1.0 1.9); +++, p24 antigen Flow cytometric analysis positive (absorbance > 2.0). Definition of Pos and Neg, see Materials and methods ( Virus isolates). 6 a Isolates tested on human/rat CMKLR1 hybrids as shown in Fig. 5. 1×10 cells per sample were washed in washing buffer (PBS without Ca2+,Mg2+ and sodium bicarbonate+0.5% milk powder+0.003% sodium azid) (Kebo) and suspended in Enhanced green fluorescent protein (EGFP) was fused in- 100 μl washing buffer containing 0.08–2 μg/μl anti-receptor frame to the C-terminus of hCMKLR1 (hCMKLR1-EGFP) monoclonal antibody (mAb) and corresponding isotype control and rCMKLR1 (rCMKLR1-EGFP), respectively, using a (Table 1). R-phycoerythrin conjugated to the antibody was used stepwise PCR procedure according to a modified protocol for detection. The cells were incubated for 30 min at 4 °C, (Olde et al., 1998) of the single-overlap extension method washed twice and re-suspended in 500 μl washing buffer for (Lefebvre et al., 1995). The same method was used to analysis. In case a secondary antibody was needed for detection, replace the N-terminus and the second extracellular loop of cells were incubated for another 30 min at 4 °C in 100 μl the rCMKLR1-EGFP with the corresponding human washing buffer containing 25 ng/μl R-phycoerythrin conjugated sequences, resulting in the chimera rCMKLR1.hNterm- goat anti-mouse immunoglobulin F(ab′)2, whereafter cells were EGFP and rCMKLR1.hECL2-EGFP. Final constructs were washed and re-suspended as above, followed by analysis in a subcloned into the EcoRV/NotI sites of pIRESpuro and their Becton-Dickinson FACS Calibur flow cytometer using the identity confirmed by sequencing. CellQuest software.

Stable expression of recombinant receptors Confocal microscopy

The human glioma cell line, NP-2, with and without stably NP-2.CD4 cells stably expressing hCMKLR1-EGFP, expressed CD4 (NP-2.CD4), was kindly provided by Dr. Hiroo rCMKLR1-EGFP, rCMKLR1.hECL2-EGFP or rCMKLR1.hN- U.E.A. Mårtensson et al. / Virology 355 (2006) 6–17 15

Fig. 5. HIV-1, HIV-2 and SIV infection of NP-2 cells expressing CD4 alone or in combination with either hCMKLR1, hCMKLR1-EGFP, rCMKLR1-EGFP, rCMKLR1.hNterm-EGFP and rCMKLR1.hECL2-EGFP. (A) Infection with a HIV-1 isolate, strongly positive for hCMKLR1 (3609:5186 [10 ng/ml] in Table 3). The level of infection was estimated by measuring the HIV-1 p24 content in the cell medium 12 day post-infection. (B) Infection with two positive HIV-2 isolates (1812 [2 ng/ml] and 10101 [2 ng/ml], Table 4). The degree of infection was detected in cell culture supernatants 12 day post-infection with p26 ELISA. (C) Infection with three positive sequential SIV isolates (59:3:56 [10 ng/ml], 59:3:91 [10 ng/ml], 59:3:730 [10 ng/ml], Table 5). Degree of infection was assayed as in panel B. Background signal from non-infected cells has been subtracted. Bars represent means+SD from triplicates. The various recombinant test receptors and receptor hybrids are depicted schematically. term-EGFP were grown on multi-well chamber slides. At 40% were mounted in 2.5% PVA-DABCO (Sigma) and examined by confluency, cells were washed 3 times in PBS (without Mg2+ and confocal microscopy (Zeiss LSM510, Göttingen, Germany). Ca2+)+0.5% BSA and fixed in 4% formaldehyde (Sigma) for 15 min at room temperature. Cells were blocked in PBS+2% Data analysis FBS for 20 min at room temperature and stained with R- phycoerythrin conjugated anti-CD4 mAb or isotype control for Statistical errors are shown as standard deviations (SD). 60 min at 4 °C. After 3 times' washing in PBS+0.5% BSA, cells Sequence alignments were carried out using the Clustal X 16 U.E.A. Mårtensson et al. / Virology 355 (2006) 6–17 program and receptor transmembrane predictions to determine Desrosiers, R.C., Ringler, D.J., 1989. 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