A formylpeptide receptor, FPRL1, acts as an efficient coreceptor for primary isolates of human immunodeficiency virus

Background More than 10 members of seven-transmembrane G protein-coupled receptors (GPCRs) have been shown to work as coreceptors for human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), and simian immunodeficiency viruses (SIVs). As a common feature of HIV/SIV coreceptors, tyrosine residues are present with asparagines, aspartic acids or glutamic acids in the amino-terminal extracellular regions (NTRs). We noticed that a receptor for N-formylpeptides, FPRL1, also contains two tyrosine residues accompanied by glutamic acids in its NTR. It was reported that monocytes expressing CCR5 and FPRL1 in addition to CD4 are activated by treatment with ligands or agonists of FPRL1. Activated monocytes down-modulate CCR5 and become resistant to infection by HIV-1 strains. Thus, FPRL1 plays important roles in protection of monocyptes against HIV-1 infection. However, its own coreceptor activity has not been elucidated yet. In this study, we examined coreceptor activities of FPRL1 for HIV/SIV strains including primary HIV-1 isolates. Results A CD4-transduced human glioma cell line, NP-2/CD4, is strictly resistant to HIV/SIV infection. We have reported that when NP-2/CD4 cells are transduced with a GPCR having coreceptor activity, the cells become susceptible to HIV/SIV strains. When NP-2/CD4 cells were transduced with FPRL1, the resultant NP-2/CD4/FPRL1 cells became markedly susceptible to some laboratory-adapted HIV/SIV strains. We found that FPRL1 is also efficiently used as a coreceptor by primary HIV-1 isolates as well as CCR5 or CXCR4. Amino acid sequences linked to the FPRL1 use could not be detected in the V3 loop of the HIV-1 Env protein. Coreceptor activities of FPRL1 were partially blocked by the forymyl-Met-Leu-Phe (fMLF) peptide. Conclusion We conclude that FPRL1 is a novel and efficient coreceptor for HIV/SIV strains. FPRL1 works as a bifunctional factor in HIV-1 infection. Namely, the role of FPRL1 in HIV-1 infection is protective and/or promotive in different conditions. FPRL1 has been reported to be abundantly expressed in the lung, spleen, testis, and neutrophils. We detected mRNA expression of FPRL1 in 293T (embryonal kidney cell line), C8166 (T cell line), HOS (osteosarcoma cell line), Molt4#8 (T cell line), U251MG (astrocytoma cell line), U87/CD4 (CD4-transduced glioma cell line), and peripheral blood lymphocytes. Roles of FPRL1 in HIV-1 infection in vivo should be further investigated.


Conclusion:
We conclude that FPRL1 is a novel and efficient coreceptor for HIV/SIV strains. FPRL1 works as a bifunctional factor in HIV-1 infection. Namely, the role of FPRL1 in HIV-1 infection is protective and/or promotive in different conditions. FPRL1 has been reported to be abundantly expressed in the lung, spleen, testis, and neutrophils. We detected mRNA expression of FPRL1 in 293T (embryonal kidney cell line), C8166 (T cell line), HOS (osteosarcoma cell line), Molt4#8 (T cell line), U251MG (astrocytoma cell line), U87/CD4 (CD4-transduced glioma cell line), and peripheral blood lymphocytes. Roles of FPRL1 in HIV-1 infection in vivo should be further investigated.

Background
More than 10 members of seven-transmembrane G protein-coupled receptors (GPCRs) support the entry of human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), and simian immunodeficiency viruses (SIVs) into target cells as coreceptors in collaboration with the primary receptor CD4 [1,2].
We noticed that as a common feature of most CKRs, as well as HIV/SIV coreceptors, tyrosine residues with asparagines, aspartic acids or glutamic acids are present in the amino-terminal extracellular regions (NTRs) [28,29]. A receptor for N-formylpeptides, FPRL1, also contains two tyrosine residues accompanied by glutamic acids in its NTR [30]. FPRL1 has been reported to be expressed in the lung, spleen, and testis, and in neutrophils, and to play an important role in the activation of neutrophils [31]. Monocytes expressing FPRL1 in addition to CD4 are activated by treatment with ligands or agonists of FPRL1. Activated monocytes down-modulate CCR5 and thus become resistant to infection by R5 HIV-1 strains. [32]. In this report, we demonstrate that FPRL1 itself has the capacity to support the entry of various HIV/SIV strains, including primary HIV-1 isolates, into target cells as a novel coreceptor.

FPRL1 as a novel candidate HIV/SIV coreceptor
Major HIV/SIV coreceptors, CCR5 and CXCR4, contain tyrosines and these tyrosines in NTRs of CCR5 and CXCR4 have been demonstrated to be necessary for their coreceptor activities [28,33]. All CKRs reported to have HIV/SIV coreceptor activities harbor tyrosines in their NTRs. Most non-CKR GPCRs that were reported to function as HIV/ SIV coreceptors also harbor tyrosines accompanied by aspartic acids, glutamic acids or asparagines in their NTRs (Additional file 1). Therefore, to discover a novel candidate coreceptors of HIV/SIV, we constructed a phylogenetic tree of peptide receptors for 36 GPCRs containing reported HIV/SIV coreceptors (20 CKRs, and 16 non-CKR GPCRs) using the ClustalW program [72] (Fig. 1). The peptide receptors were clustered into several distinct branches corresponding to the subfamilies of GPCRs. In this phylogenetic tree, we found that CKRs were closely related to each other and that all of the formylpeptide receptors reported so far, FPRL1, FPRL2 and FPR1, constituted a distinct subgroup, closely located at positions near CKRs and anaphylatoxin receptors, some of which have been demonstrated to act as coreceptors for HIV/SIV [22]. FPRL1, unlike FPRL2 or FPR1, has tyrosine residues accompanied by asparagines, aspartic acids, and glutamic acids in its NTR (see Additional file 1). Therefore, we focused on a formylpeptide receptor, FPRL1, as a novel candidate coreceptor for HIV/ SIV.
Phylogenetic tree of peptide receptors belonging to the GPCR family Figure 1 Phylogenetic tree of peptide receptors belonging to the GPCR family. The phylogenetic tree for 20 CKRs and 16 GPCRs related to CKRs was constructed by the Clus-talW program [72] according to the methods described in the DDBJ website (National Institute of Genetics, Center for Information Biology and DNA Databank of Japan, http:// www.ddbj.nig.ac.jp). FPRL1 is indicated by the arrow. GPCRs reported to function as HIV/SIV coreceptors are indicated by "*".

Amino acid sequences of the V3 domain of the HIV/SIV Env protein
Cell line-adapted HIV-1 strains, GUN-4V (GPR1-X4) and GUN-7WT (R5-X4), could not use FPRL1 as a coreceptor, whereas their related strains, GUN-4WT and GUN-7V, with one or two amino acid substitutions in the V3 region, could use FPRL1 ( Fig. 3 and see Additional file 2). This finding raised the possibility that a determinant of the FPRL1 use of HIV-1 as a coreceptor lies in the V3 region. Therefore, we determined the amino acid sequences of the V3 regions of primary HIV-1 strains propagated in NP-2/ CD4 cells expressing one of the coreceptors. DNA regions coding for the V3 domain of gp120 were amplified by PCR using cellular DNA of NP-2/CD4/FPRL1 cells infected with AG204, AG206, HCM305, HCM309, HCM342, or mSTD104 strains and NP-2/CD4/CXCR4 cells infected with the HCM303 strain as templates. These amplified DNAs were cloned into the TA-cloning vector pTarget and their nucleotide sequences were determined. Cell count When their nucleotide sequences were compared with those of HIV-1 isolates submitted to the Genbank and reported previously [34], several nucleotide substitutions were observed in the V3 sequences of the primary isolates infecting NP-2/CD4/CCR5, NP-2/CD4/CXCR4, or NP-2/ CD4/FPRL1 cells (data not shown).

Expression of GPCR mRNA in various types of human cells detected by RT-PCR
No amino acid substitution, however, was detected in the V3 domains, because all the nucleotide substitutions detected in the V3 domains were synonymous (see Additional file 3). Single-amino acid substitutions were detected in the C3 domain of AG206, HCM303, and HCM342 strains propagated in FPRL1-expressing cells. These findings indicate that subtype C or AE HIV-1 strains propagated in PBLs and those propagated in NP-2/CD4 cells expressing CCR5, CXCR4 or FPRL1 have the identical amino acid sequences in the V3 domains. Nevertheless, five HIV-1 strains using FPRL1, GUN-1WT, GUN-7WT, HCM305, HCM309, and HCM342, had threonine at the 13th amino acid position of the V3 region, while two HIV-1 strains which did not use FPRL1 as a coreceptor, GUN-4WT and HCM303, had serine at this position. The amino acids at this position may be responsible for determining FPRL1 use by these HIV-1 strains.
HIV-1 samples produced by NP-2/CD4/FPRL1 cells that had been infected with the AG204, AG206, HCM308, and HCM342 strains could use CCR5 as a coreceptor (data not shown). These results suggest that the primary HIV-1 samples are not a mixture of FPRL1-tropic virus and R5-tropic virus, and that HIV-1 isolates using FPRL1 can also use CCR5 as a coreceptor.
Use of FPRL1, CCR5, CXCR4 or GPR1 as a coreceptor by various cell line-adapted HIV-1 strains

Partial inhibition of the coreceptor activity of FPRL1 by the fMLF peptide
It has been reported that the coreceptor functions of CCR5 and CXCR4 for HIV-1 infection can be inhibited by their ligands, RANTES and SDF-1β, respectively [37,38]. Some ligands have been used as starting materials to find and develop anti-HIV reagents. We examined the inhibitory effects of an FPRL1 ligand, fMLF peptide, on infection with HIV/SIV. NP-2/CD4/FPRL1 cells were pretreated with the fMLF peptide (100 μg/ml). As a control, NP-2/CD4/CCR5 cell were also pretreated with a chemokine, RANTES, a ligand for CCR5. As shown in Fig [37].

Expression of FPRL1 mRNA in a wide variety of cells
We investigated the expression of FPRL1, CCR5, CXCR4, and GPR1 mRNA in cells originating from various types of human tissues by RT-PCR. CD4 and GAPDH mRNA were detected as controls. estimated to be comparable to those of CXCR4 in these cells. FPRL1 mRNA was, however, not detected in HepG2 (hepatoblastoma), Huh7 (hepatoma), or NP-2/CD4 (CD4-transduced glioma) cells. CCR5 mRNA was detected in Molt4#8, U251MG, and 293T cells, even though the levels were much lower than those of FPRL1 mRNA. CXCR4 mRNA was detected clearly in C8166 and Molt4#8 cells, and weakly in 293T, HepG2, HOS, Huh7, U251MG cells, and PBLs. CD4 mRNA was detected in BT-20N, C8166, Molt#3, NP-2/CD4, U87/CD4 cells, and PBLs. Similar amounts of GAPDH mRNA were detected in all cells tested here. No signal was seen in RNA samples without reverse transcriptase treatment, indicating that the chromosomal DNA did not contaminate the cDNA preparations. FPRL1 are expressed abundantly in various types of cells derived from not only lymphoid tissues, but also the brain.

Discussion
The genomic diversity of HIV-1 is thought to be generated by the low fidelity of its reverse transcription and frequent recombination of the genome [39]. Mutation of amino acid sequences in the V3 domain of the Env can give HIV-1 the ability to use various GPCRs as coreceptors. Coreceptors other than CCR5 and CXCR4, that are related to the clinical involvement of the HIV-1 infection have not yet been clarified. We planned to identify novel corecep-tors that can be frequently used not only by cell lineadapted HIV-1 strains, but also by primary HIV-1 isolates.
In this study, we focused on a formylpeptide receptor, FPRL1, which is located genetically close to CKRs in the phylogenetic tree we made, containing three tyrosines in its NTR ( Fig. 1 and see Additional file 1) as a candidate for a novel coreceptor.  (Fig. 3). Thus, FPRL1 use by cell line-adapted HIV-1 strains does not correlate with their use of CCR5, CXCR4 or GPR1. We showed that the R5-X4 phenotype of the GUN-4WT and GUN-7WT strains can be changed to the X4-GPR1 phenotype of GUN-4V and GUN-7V variants by one or two amino acid substitutions at the V3 loop [59]. Therefore, the V3 loop is thought to be a determinant of FPRL1 use, as well as GPR1 use, by HIV-1. Like other coreceptors for HIV-2 and SIV strains, FPRL1 was also efficiently used by R5-X4-dualtropic HIV-2 and SIV strains (Fig. 6).
The ratio of FPRL1 use was as high as 80% for the primary HIV-1 isolates belonging to subtypes AE and C. This ratio for subtype B primary HIV-1 isolates was, however, much lower according to our preliminary examination (data not shown), suggesting that FPRL1 use may be linked to infection with subtypes AE and C HIV-1.
It is intriguing that only a few cell line-adapted HIV-1 strains could use FPRL1 as a coreceptor, whereas many primary HIV-1 isolates propagated in PBLs could do so. It is probable that a population of HIV-1 that uses FPRL1 as a coreceptor in addition to CCR5 or CXCR4 may have The susceptibilities of NP-2/CD4/GPCR cells to HIV-1 strains Figure 5 The susceptibilities of NP-2/CD4/GPCR cells to HIV-1 strains. Cells were infected with two primary HIV-1 isolates, AG204 and mSTD104, and a cell line-adapted strain, Ba-L. Six days after infection, cells positive for HIV-1 antigens were detected by IFA using a fluorescence microscope. Percentage of cells judged to be positive for IFA are shown.

NP-2/CD4/ FPRL1
NP-2/CD4/ CCR5 been lost in the course of propagation of HIV-1 strains in vitro using cell lines because it was markedly smaller than the HIV-1 population which did not use FPRL1, but did use CCR5 or CXCR4.
It has been shown that determinants for CCR5, CXCR4, and GPR1 uses by HIV-1 strains lie in the V3 domain of the Env protein [53,41]. Amino acid substitutions that are apparently linked to FPRL1 use could be identified in the V3 domain of GUN-1WT, GUN-4V, and GUN-7WT strains. That is, the amino acid substitutions at the tip of the V3 domain from proline to serine or from proline to threonine can affect FPRL1 use by HIV-1. Therefore, we examined the possibility that any specific amino acid substitution of the Env protein may give HIV-1 the ability to use FPRL1 as a coreceptor in addition to CCR5 or CXCR4 use. Primary isolates, AG204, AG206, AG208, HCM305, HCM308, HCM342, and mSTD104, were inoculated to, and propagated in, NP-2/CD4/FPRL1 cells in addition to NP-2/CD4/CCR5 or NP-2/CD4/CXCR4 cells. Then, HIV-1 DNA in these cells was subjected to DNA sequencing.
A few nucleotide substitutions were detected in the the V3 domain of the env gene between HIV-1 propagated in NP-2/CD4/FPRL1 cells and NP-2/CD4/CCR5 or NP-2/CD4/ CXCR4 cells (see Additional file 3). The deduced amino acid sequences of the V3 domain of HIV-1 isolates propagating in NP-2/CD4/FPRL1 cells were identical to those propagated in NP-2/CD4/CCR5 or NP-2/CD4/CXCR4 cells. Furthermore, HIV-1 produced by NP-2/CD4/FPRL1 cells that had been infected with the HIV-1 isolates could infect NP-2/CD4/CCR5 or NP-2/CD4/CXCR4 cells (data not shown). Therefore, these primary isolates could use FPRL1 as a coreceptor in addition to CCR5 and/or CXCR4. It is possible that the determinants of FPRL1 use can not be separated from CCR5, CXCR4 or GPR1 use in HIV-1 strains. There is still another possibility that amino acid mutations in regions other than the V3 domain give HIV-1 strains the ability to use FPRL1 as a coreceptor.
fMLF peptides are bacterial products that have potent chemotactic activities for phagocytes. It was reported that FPRL1 activated by the fMLF peptide or peptides derived from the Env glycoprotein gp120 of HIV-1 interferes with the coreceptor function of CCR5 and CXCR4 by downregulating them, and as a result, these peptides prevent HIV-1 infection [32,42,43]. Desensitization or down-regulation of CCR5 by the fMLF peptide has also been observed in human immature dendritic cells, on which both FPRL1 and CCR5 are expressed [42].
In this study, a partially inhibitory effect of the fMLF peptide on the FPRL1-mediated infection with HIV/SIV strains was observed, while anti-CD4 MoAb NuTH/I could almost completely block it (Figs. 7A and 7B). The Use of FPRL1, CCR5, CXCR4 or GPR1 as a coreceptor by HIV-2 and SIV strains  regions of the coreceptors interacting with HIV or SIV are reported to be distributed in extracellular domains such as NTR and ECLs of GPCRs [44]. In contrast, the highly hydrophobic fMLF peptide has been reported to bind to the membrane-spanning region of FPRL1 [45]. Therefore, we assume that the difference in the binding site between the fMLF peptide and HIV-1 is one of the reasons why the fMLF peptide could not efficiently interfere with the interaction of FPRL1 with HIV-1.
The fMLF peptide also partially inhibited infection of NP-2/CD4/CCR5 cells with several HIV-2 or SIV strains (Fig.  7B). The amino acid sequences of the membrane-spanning regions are more conserved among GPCRs than those of NTRs and ECLs. Between FPRL1 and CCR5, the amino acid sequence homology of their membrane-spanning regions is about 30%, although that for their NTRs and ECLs is about 8.5% (data not shown). Therefore, the fMLF peptide might have a partially inhibitory effect on infection of NP-2/CD4/CCR5 cells with the CBL23 and mndGB-1 strains. The low, but apparent inhibitory effect of the fMLF peptide on HIV-2 infection mediated by CCR5 suggests that it can be a starting material to develop a novel anti-HIV drug.
Expression of the major coreceptor CCR5 or CXCR4 has been detected in various cells ( Fig. 2A) [46]. Expression of FPRL1 mRNA was also detected in human cells derived from a wide variety of origins, such as PBLs, C8166 (T cells), Molt4#8 (T cells), 293T (embryonic kidney cells), HOS (osteosarcoma cells), U251MG (astrocytoma cell), and U87/CD4 (glioma cell) ( Fig. 2A). In addition to these cells, expression of the FPRL1 gene has been reported to be detected in various types of cells such as neutrophils or phagocytes, and in many organs [31,46], even though their roles in human cells other than neutrophils remain to be elucidated. Roles of coreceptors other than CCR5 and CXCR4 in HIV-1 infection and the pathogenesis of AIDS should be investigated further.

Conclusion
FPRL1 is a novel and efficient HIV/SIV coreceptor. In particular, it should be noted that FPRL1 is efficiently used by primary HIV-1 isolates. FPRL1 works as a bifunctional factor in HIV-1 infection. Namely, FPRL1 works not only as an inhibitory factor but also as an enhancing factor for HIV-1 to enter target cells. Expression of FPRL1 gene was detected in various types of tissues and cells. HIV1-infection mediated through FPRL1 may, therefore, play any important roles in progression of complicated symptoms of AIDS. The clinical significance of FPRL1 in HIV-1 infection in vivo should be further studied.

Detection of GPCR mRNA
Total RNA was isolated from human cells using an RNA extraction kit, SepaGene (Sanko-Junyaku Inc., Tokyo, Japan), in accordance with the manufacturer's protocol. cDNA for the total cellular RNA was constructed as described elsewhere [49]. mRNA expression for CCR5, CD4, CXCR4, GPR1, FPRL1, and GAPDH was detected by PCR of cDNA preparations using the sense and antisense primer pairs described above [49]. Amplified cDNA was detected by 1% (w/v) agarose gel electrophoresis.

Cloning of the FPRL1 gene
A DNA fragment encoding the entire open reading frame (ORF) of the FPRL1 gene was amplified by RT-PCR using the FPRL1-specific primers, FPRL1CN and FPRL1CR, and cDNA constructed from the total RNA isolated from C8166 cells. The ORF DNA of the FPRL1 gene was cloned into the TA-cloning plasmid pDrive (QIAGEN K. K., Tokyo, Japan) and the plasmid obtained was designated pDrive/FPRL1. The DNA fragment containing FPRL1 ORF was isolated from the pDrive/FPRL1 plasmid by EcoRI digestion and subcloned into the expression plasmid pCX-bsr [69]. The FPRL1 plasmid obtained was designated pCX-puro/FPRL1. The cloned FPRL1 gene was sequenced and found to be 100% homologous in terms of amino acid sequences to the reported gene [M76672] [30].

Determination of the effects of an anti-CD4 monoclonal antibody, GPCR ligands, and tyrosine sulfation on HIV-1 infection
To determine CD4 dependency of HIV infection, NP-2/ CD4/CCR5 and NP-2/CD4/FPRL1 cells were cultured in E-MEM containing a serially diluted anti-CD4 monoclonal antibody (MoAb), Nu-TH/I (Nichirei Inc., Tokyo, Japan), at 37°C for two hours. The cells were incubated in E-MEM with or without Nu-TH/I MoAb at 37°C for two hours and then exposed to HIV-1 in an amount corresponding to 1 × 10 4 cpm of RT activity. After removing the inocula, the cells were incubated at 37°C in E-MEM containing 10% (v/v) FCS for four days.
CCR5 and FPRL1 contain several tyrosine residues in their NTRs and ECLs, but only NTRs harbor the signal sequence for tyrosine sulfation [73]. To clarify the effect of tyrosine sulfation in NTR of CCR5 and FPRL1, NP-2/CD4/CCR5 and NP-2/CD4/FPRL1 cells were incubated in E-MEM containing an inhibitor of tyrosine sulfation, NaClO3 (10 mM), for 48 hours and then inoculated with viruses.

Detection of HIV-/SIV-infected cells
The susceptibilities of the cells described above to HIV/ SIV were determined by indirect immunofluorescence assay (IFA), which detects HIV-1-, HIV-2-, or SIV-antigens expressed in infected cells, as previously reported [71]. A pool of sera derived from HIV-1-infected humans or SIVmac-infected macaques was used as the first antibody [16,49]. Infection was checked on days 2, 4, and 6 after inoculation.

Phylogenetic analysis
The multiple alignment of the amino acid sequences of 20 CKRs and 16 GPCRs closely related to CKRs and their phylogenetic tree was constructed using the ClustalW program [72]. GPCR