CCR6 Functions as a New Coreceptor for Limited Primary Human and Simian Immunodeficiency Viruses

More than 12 chemokine receptors (CKRs) have been identified as coreceptors for the entry of human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2), and simian immunodeficiency viruses (SIVs) into target cells. The expression of CC chemokine receptor 6 (CCR6) on Th17 cells and regulatory T cells make the host cells vulnerable to HIV/SIV infection preferentially. However, only limited information is available concerning the specific role of CCR6 in HIV/SIV infection. We examined CCR6 as a coreceptor candidate in this study using NP-2 cell line-based in-vitro studies. Normally, CD4-transduced cell line, NP-2/CD4, is strictly resistant to all HIV/SIV infection. When CCR6 was transduced there, the resultant NP-2/CD4/CCR6 cells became susceptible to HIV-1HAN2, HIV-2MIR and SIVsmE660, indicating coreceptor roles of CCR6. Viral antigens in infected cells were detected by IFA and confirmed by detection of proviral DNA. Infection-induced syncytia in NP-2/CD4/CCR6 cells were detected by Giemsa staining. Amount of virus release through CCR6 has been detected by RT assay in spent culture medium. Sequence analysis of proviral DNA showed two common amino acid substitutions in the C2 envelope region of HIV-2MIR clones propagated through NP-2/CD4/CCR6 cells. Conversely, CCR6-origin SIVsmE660 clones resulted two amino acid changes in the V1 region and one change in the C2 region. The substitutions in the C2 region for HIV-2MIR and the V1 region of SIVsmE660 may confer selection advantage for CCR6-use. Together, the results describe CCR6 as an independent coreceptor for HIV and SIV in strain-specific manner. The alteration of CCR6 uses by viruses may influence the susceptibility of CD4+ CCR6+ T-cells and dendritic cell subsets in vivo and therefore, is important for viral pathogenesis in establishing latent infections, trafficking, and transmission. However, clinical relevance of CCR6 as coreceptor in HIV/SIV infections should be investigated further.


Introduction
Human and Simian Immunodeficiency viruses (HIV and SIV) gain access to susceptible cells by binding their mature envelope (env) glycoprotein, gp120 to CD4 as receptor [1] and then to a coreceptor, preferably one of two chemokine receptors (CKRs), CCR5 and CXCR4 [2]. The binding events induce activation of another viral protein, gp41, which eventually create membrane fusion on the cell surface [3]. The major coreceptors utilization by infectious viruses determines their tropism and classified HIV/SIV primarily into three groups: R5 viruses (use CCR5 alone as coreceptor), R5X4 viruses (use both CCR5 and CXCR4) and X4 viruses (use CXCR4 alone), which are less frequent. However, viruses begin their primary infections through CCR5 receptor, and change their tropism to CXCR4, probably expand to more coreceptor-usage [4].
Typical chemokine family comprises 20 receptors, assigned to four subfamilies. The majority of the CKRs, namely CCR1, CCR2b, CCR3, CCR4, CCR5, CCR8, CCR9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6 and CX3CR1 have been identified already as coreceptors used by various HIV and SIV strains [5][6][7]. Uses of alternative coreceptors in CCR5-depleted condition were reported recently in vivo in sooty mangabeys infection [8]. Therefore, viral infection using alternative coreceptor other than CCR5 exists in vivo. However, only limited information is available concerning the usage of CC chemokine receptor 6 (CCR6), as HIV/SIV coreceptor [9]. CCR6 was identified initially as an orphan receptor and named as STRL22 [10] and subsequently renamed. The receptor is known to express on Th17 cells, regulatory T cells and memory T cells [11][12][13]. One recent study shows the coexpression of CCR6 in the presence of CCR4 and CXCR3 makes T cells more permissive to HIV infection [14]. Therefore, we sought to examine extensively whether CCR6 can support the coreceptor activity independently for HIV-1, HIV-2 or SIV strains and/or isolates. We examined a panel of 11 laboratory-adapted strains and seven primary isolates of HIV/SIV for their ability to gain entry into cells expressing CCR6 in collaboration with CD4. To our knowledge, this is the only detail report to describe CCR6 as an independent coreceptor for HIV and SIV.

Detection and Amplification of CCR6 Open Reading Frame (ORF)
The reference DNA sequence for the coding region of CCR6 was obtained from the GenBank database. Oligonucleotide primers were designed to cover its ORF and synthesized (Proligo K.K., Tokyo, Japan) to detect reverse-transcribed mRNA by PCR. Names of PCR primers, their sequences, orientations and positions in the respective open reading frames are as follows: CCR6-F, 5Á TGAGCGGGGAATCAATGAATTTCAGCGAT 3´(sense: from the 1 st to the 30 th position) and CCR6-R, 5T CAGCTTTCTATCACATAGTGAAGGACG 3´(antisense: 1110 th -1137 th ) [GenBank accession number, U 60000.1]. As controls, the mRNA expressions of CD4 (M12807.1), CCR5 (U54994.1), CXCR4 (AY242129.1) and gleceraldehyde-3phospahte dehydrogenase (GAPDH, M17851.1) were also examined. Total RNA was isolated from human cell lines and PBMC using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacture's protocol. Preparation of cDNA followed by amplification of CCR6-ORF has been done by the procedure described earlier [26]. As control, mRNA of GAPDH was amplified by RT-PCR and examined by electrophoresis through 1% (w/v) agarose gel.

Cloning of CCR6 as Coreceptor Candidate
DNA fragments for the full length ORFs of CCR6 were cloned into a TA-cloning plasmid, pGEM-T Easy (Promega, Madison, WI), and the derivative plasmids were designated as pGEM-T Easy/CCR6. DNA sequencing of TA cloned derivatives was performed by a 5500 DNA sequencer (Hitachi, Tokyo, Japan) using fluorescent primers labeled with Texas Red. Cloned CCR6 ORFs were separated from TA plasmids by restriction-digestion and recloned into an expression plasmid, pMX-puro [27]. Plasmid with correctly oriented CCR6-insert was transfected into NP-2/CD4 cells to produce and establish NP-2/CD4/CCR6 indicator cell lines as described earlier [15,26]. As HIV/SIV-susceptible positive-control cells, NP-2/CD4/CCR5 and NP-2/CD4/CXCR4 were established. NP-2/CD4 and NP-2 cell lines were used as negative-controls.

HIV/SIV Infection Assay
For viral infection assay, NP-2/CD4/CCR6, NP-2/CD4/CCR5, NP-2/CD4/CXCR4 and NP-2/CD4 cells were seeded into wells of 24-well plates at a density of 5×10 4 cells/ml. On the following day, medium was taken off and viral inoculums were added to the seeded-cells. After 6 hour incubation, the cells were washed three times with EMEM containing 10% FBS to remove free virus, filled finally with 500 µL fresh medium per well and cultured at 37°C in 5% CO 2 incubator. The cells were passaged every 3-5 days and maintained up to 8 weeks. The expression of HIV/SIV antigens in the infected cells was detected by indirect immunofluorescence assay (IFA) [28]. Viral induced cell fusions were observed under light microscopy. Syncytiaformation was detected by Giemsa staining (Muto Pure Chemicals, Tokyo, Japan). Subsequently, virus-releases through different coreceptors were quantified by measuring reverse transcriptase (RT) activities in culture supernatants of respective cell lines following the gold standard method [16].

PCR Amplification, Cloning and Sequencing of Viral DNA
We conducted PCR to detect proviral DNA for the Envcoding regions using the genomic DNA of infected cells as templates [29]. For HIV-2MIR, the primer pair used was HIV2-F (forward 5´-ATGTGATAAGCACTATTGGGATGATA-3´) and HIV2-R (reverse 5´-CATGCTTGTTTAGGTCTTGTATTGAT-3´), located between positions of 7084-7109 and 7433-7458, respectively (nucleotide positions of HIV-2CRIK, DQ307022.1). This primer pair amplified the V3 loop of env gene along with some of the C2 region. For SIVsmE660, a primer set was designed for the V1, V2, and V3 regions of env gene as described elsewhere [30]. Detected DNA bands were cloned into a TA-cloning plasmid, pGEM-T Easy, and inserts were sequenced and blasted in the NCBI nucleotide database (http:// blast.ncbi.nlm.nih.gov) to confirm the DNA integrity of the inoculated viruses.

Phylogenetic Analyses
The amino acid sequences of 20 CKRs was collected from UniProtKB/Swiss-Prot database. Alignments of their subregions, such as NTRs, extracellular loop-2 (ECL2s), and ECL-3s were performed using the BioEdit program (version 7) and their phylogenetic trees were constructed with the MEGA (Version 5.1, Molecular Evolutionary Genetics Analysis) for windows.

Ethics Statement
This study was approved by the ethics committee of the Gunma University Faculty of Medicine and written consent was obtained from enrolled healthy blood donor.

Detection of CCR5, CXCR4 and CCR6 Expressions
We detected mRNA expression of CCR6 in various cells including T cell line, hepatic cells and PBMC by RT-PCR using the primers specific for the ORF ( Table 1). The outcome shows a good harmony to previous findings that reported of CCR6 expression selectively on Th17 cells and regulatory T cells, memory T cells, B cells and dendritic cells [11,31]. Expression of CCR5 was found in ATL-1K, Jurkat, Daudi and in PBMC. CXCR4 expression was similar to that of CCR5 as well as in C8166. CD4 expression was detected in all T cell lines used here and in PBMC, but not in Daudi, as expected (Table 1). NP-2 cells expressed neither CD4 nor coreceptor candidates, therefore, works as an ideal host for coreceptor-study.

CCR6 is a coreceptor for HIV/SIV
A coreceptor activity of CCR6 for HIV-1HAN2, HIV-2MIR and SIVsmE660 primary isolates was noticed when viral antigenpositive NP-2/CD4/CCR6 cells were detected by IFA ( Figure  1). We detected proviral DNA as alternative evidence in establishing viral infection using CCR6-coreceptor. A few percentages of NP-2/CD4/CCR6 cells became HIV-1HAN2 antigen-positive in 4-7 days after its inoculation ( Figure 1): viral antigen-positive cells, however, did not increase during cultivation continued for 7 weeks (Figure 2A). In contrast, HIV-2MIR showed accelerated replication using CCR6 and more than 60% cells became antigen positive within 3 weeks after inoculation ( Figure 2B). Similarly, SIVsmE660 started very slow and steady infection through CCR6-coreceptor and about 2% of the cells became IFA-positive after cultivation for up to four weeks. Then the infection progressed rapidly and more than 80% of the cells became SIV antigen-positive seven weeks after infection ( Figure 2C). In contrast, all HIV/SIV strains, other than X4 HIV strains IIIB and SBL6669, replicated well in NP-2/CD4/CCR5 cells: most cells became HIV/SIV antigen-positive 3-7 days after inoculation. NP-2/CD4/CXCR4 cells showed high susceptibilities to the viral strains except for HIV-1BaL, SIVmac251, SIVsmE660 and primary HIV-1 isolate 92US723 (Table 2). NP-2/CD4 cells were completely resistant to infection by all HIV and SIV strains tested. Retroviral cytopathic effects (CPE) in cell-line-system produce large amount of the viruses. The amount of released virus was assessed by reverse transcriptase (RT) activities in spent culture media [33] of viral inoculated cells. For RT assay, culture fluid of infected NP-2/CD4/CCR5 cells at day 7 was collected when 70-90% cells were viral antigen-positive by IFA. However, culture media of NP-2/CD4/CCR6 was examined at day-22 for HIV-2MIR and day-52 for SIVsmE660 when 60-80% respective cells were infected along with CPE. The RT activities of HIV-1HAN2 using CCR5 and CXCR4 as coreceptors were 6.0×10 5 cpm/mL and 5.8×10 5 when 80-90% cells become antigen positive by IFA. However, it produced an RT activity of 4.8×10 4 cpm/mL in replication mediated by CCR6 when only 2.5% of cells were antigen-positive ( Figure 2D.). This virus could not increase either antigen-positive cells or RT activity with longer incubation period. On the other hand, HIV-2MIR had produced RT activities of 3.0×10 5 cpm/mL 4.1×10 5 cpm/mL at the point of 85-90% infection through CCR5 and CXCR4 respectively. At 70% of infections via CCR6, RT level was 2.5×10 5 cpm/mL there ( Figure 2E). The RT release of SIVsmE660 using CCR5 and CCR6 as coreceptors were 2.3×10 6 and 1.5×10 6 cpm/mL, respectively when 80-85% cells become antigen positive ( Figure 2F). The virus did not  With the increase of viral-infected NP-2/CD4/CCR6 cells, ballooning of fused cells were detected under a light microscope as the sign of cytopathic effect [34]. The HIV-2MIR produced more prominent cell-fusions than that of SIVsmE660 through CCR6. HIV-1HAN2 did not produce any ballooning until the end of the assay we conducted ( Figure 3A). Majority of HIV and SIV produced CPE as well as syncytia through CCR5/ CXCR4-transduced NP-2/CD4 cells. Clusters of multinucleated giant cells (MGC) in syncytia were quantified after fixation and staining with Giemsa. Both HIV-2MIR and SIVsmE660 induced syncytia formation in NP-2/CD4/CCR6 cells, however, HIV-1HAN2 could not form syncytium there ( Figure 3B). Clusters were variable in sizes and consisting of 10-20 nucleuses per syncytium. Coreceptor activities of CCR5/ CXCR4 frequently generated viral-induced MGC, while coreceptor-negative NP-2/CD4 cells did not form any cluster of nuclei.

Sequence analyses of the proviral DNAs
Due to the high mutation rate of HIV/SIV, the viral populations of CCR5-user may not be similar to those of  CCR6-user. We analyzed the genetic divergence of proviral DNAs of HIV-2MIR and SIVsmE660 propagated in NP-2/CD4/ CCR5 and NP-2/CD4/CCR6 cells. For HIV-2MIR, we amplified the C2-V3 region of env genes and sequenced two clones from each of MIR-R5 and MIR-R6 strains. An overall good integrity was observed in the majority of the nucleotide sequences between R5 and R6 MIR strains. There were four common nucleotide substitutions in the C2 region of MIR-R6 compared to MIR-R5, however, no such changes were found inV3 region ( Figure S1). The nucleotide substitutions resulted two amino acid substitutions, namely, tryptophan (W) to cysteine (C) and lysine (K) to arginine (R) in the C2 region common in both clones ( Figure 4A). Another change of amino acid from lysine (K) to asparagine (N) was found in one clone of R6-MIR but not in other clone. For SIVsmE660, the env gene for the V1-V3 region of R6-variant exhibited six nucleotide substitutions common in two clones relative to that of the parental smE660-R5 ones ( Figure S2). Some of the nucleotide substitutions were synonymous that did not result amino acid changes. However, non-synonymous substitutions of nucleotides resulted three amino acid changes such as, arginine (R) to glycine (G) and vice-versa in V1 region, another change from glycine (G) to glutamic acid (E) in C2 region ( Figure 4B). Combining, no amino acid substitution were found in the V3 region of either HIV-2MIR or SIVsmE660 while growing through NP-2/CD4/ CCR6 cells. In contrast, both of the viruses showed common amino acid substitutions in their C2 regions in using CCR6 as coreceptors.

Discussion
All HIV/SIV strains or isolates examined in this study were known to use CCR5 and/or CXCR4 as major coreceptors. Many HIV isolates have been found to use FPRL1 [35], CCR2b [36], CCR3 [37], CCR8 [28], CXCR1 [15], CXCR2 [15], CXCR5 [38], CXCR6 [39], CXCR7/RDC1 [40], CX3CR1 [41], D6 [42], APJ [43] and GPR1 [44] as entry co-factors. Most of the reported coreceptors were either CKRs or orphan GPCRs in nature. We did not find detail report of CCR6 to carry out coreceptor function independently so far. Therefore, to examine its novelty among coreceptor family for HIV/SIV is quite inspiring. The expression history of CCR6 in various memory T cells, dendritic cells and Th17 [13] in presence or absence of CD4 and CCR5/CXCR4 has made us more interested to examine its role in HIV infection through this study. Accordingly, the abundant expression of CCR6 in PBMC and the T-cell line C8166, ATL1K was detected, but not in Jurkat cell line (Table 1). We reported the coreceptor role CCR6 for some limited HIV and SIV strains/isolates by various in vitro examinations. Another variant of CCR6, known as CKR-L3 [45], was shown to exhibit similar coreceptor function (S. Islam, et al., unpublished data). Active involvement of CCR6 in HIV infection was mentioned in earlier studies where CCR6expressing Th1 and Th17 cells became more permissive to HIV/SIV infection than that of its CCR6-negative subsets [14,46]. Role of CCR6 was further clarified when ligands to CCR6 was reported to inhibit the viral infection [47].  The function of CCR6 as coreceptor has been revealed by multiple distinct and independent examinations through NP-2/CD4/CCR6 cell-line system. However, NP-2/CD4/CCR6 cells took much longer time to become infected by viruses compared to NP-2/CD4/CCR5 or NP-2/CD4/CXCR4 cells, indicating that CCR6 is much weaker coreceptor than CCR5 or CXCR4. Moreover, CCR6 was found to support infection for three isolates out of 18 HIV/ SIV used in the study and hence justified as a low-efficient entry co-factor than major ones. Further study with high number of various HIV/SIV strains and isolates could clarify our present findings more distinctly. However, CCR6 might be important window for viral infection when alternative coreceptors are blocked or down regulated, as described for alternative coreceptors in CCR5-null situation [8,48].
To find whether a common property is present among the HIV/SIV coreceptors, we examined and analysed the amino acid sequence of NH 2 -terminal region (NTR) of CCR6, CCR5, CXCR4 and other CKRs. Almost all reported HIV/SIV coreceptors possess tyrosine (Y) residues accompanied by aspartic acid (D), asparagine (N) or gluatamic acid (E) residues in their NTRs and tyrosine are reported to play important roles in their coreceptor activities [6,49]. The latest reported coreceptor, N-formylpeptides, FPRL1 that contains two tyrosine residues plus glutamic acids in its NTR, was shown to support the entry of various HIV/SIV strains and isolates [35]. Likely, CCR6, the latest reported coreceptor, also contains three Ys, four Ds and four Es in its NTR ( Figure 5A). To discover the relatedness of CCR6-NTR among CKRs, we constructed a phylogenetic tree taking all 20 CKR-NTRs using BioEdit program (Version 7). In the tree, the NTRs were clustered into several distinct branches according to the subfamilies of CKRs. We found CCR6 to form a closely related cluster with CXCR6 ( Figure 5B), which was reported HIV-and SIV-coreceptor [39,48], therefore justified its candidacy. Beside NTR, the second or third extracellular loop of coreceptor also play important role in HIV entry [50,51]. Therefore, we prepared two other phylogenetic trees using second and third ECLs of CCR6 to the respective positions of other CKRs. The second ECL of CCR6 placed it close to CX3CR1 ( Figure 5C), which is another reported coreceptor [41]. The phylogenetic tree drawn from third ECLs clustered CCR6 again to CXCR6 ( Figure 5D), hence reassured its position close to a HIV-coreceptor. Therefore, the hypothesis based on amino acid analyses of NTR and ECLs of CCR6 presented itself a HIV/SIV-coreceptor, as supported by our laboratory investigations.
The third variable region (V3) of HIV-1 gp120 envelope glycoprotein carries essential features for coreceptor binding [52] and determines the types of coreceptor utilization for entry [53]. We deduced the amino acid sequences from the viral DNA sequence of V3 and adjacent env regions of HIV-2MIR and SIVsmE660 isolates propagated in NP-2/CD4/CCR6 cells. Upon examination of the V3 and C2 regions of the HIV-2MIR-CCR6 variants in comparison to MIR-CCR5, the V3 region of the CCR6-variants did not show any divergence compared to the parental CCR5-isolate. However, two non-synonymous amino acid substitutions in the C2 region of CCR6-using variant were found. We assume the C2 region could be responsible for the selective use of CCR6 as coreceptor for HIV-2MIR. Similarly, CCR6-using SIVsmE660 exhibited no amino acid substitution in the V3 envelope region. The findings showed good agreement with previous studies describing the region corresponding to the V3 of HIV-1 is conserved in SIV [54] and the genetic variation of SIV is different from HIV-1 [55]. However, this study detected the V1 and V2 as the possible regions in the envelope glycoprotein to exhibiting the genetic variation of SIVsmE660 for selecting CCR6 as new coreceptor and our observation showed harmony to previous reports [56,57].

Conclusions
Identification of CCR6 as an independent HIV/SIV coreceptor contributed our understanding of virus-host interactions. The expression of CCR6 influences migration of memory CD4+ Tcell subsets into the intestine, brain, and other tissues [58], and may contribute disseminating viral infection. The newly identified coreceptor may promote HIV transmission and disease progression in part, particularly when other major coreceptors are affected. The assumption has been reflected in a recently conducted study in sooty mangabeys that identified the use of alternative coreceptors when CCR5 is mutated [8]. Taking all findings in account, one can presume that the usage spectrum of CKRs or GPCRs as coreceptors by HIV/SIV may be much broader than those of identified to date. However, it remains to be investigated why CCR6 confined its coreceptoractivity limitedly for some HIV/SIV isolates but not for others. was made to present amino terminal region (NTR) and extracellular loops (ECLs) of CCR6 structure. Subsequently three phylogenetic trees were prepared based on NTR, ECL2 and ECL3 of 20 chemokine receptors (CKRs). Branch of CCR6 on each tree was indicated by arrow. CKRs that are reported to function as HIV/SIV coreceptors are marked by "▲". The phylogenetic tree for NTRs of CKRs (B) showed CCR6 homologous to CXCR6. On the other hand, the tree made from ECL2s of all CKRs (C) placed CCR6 close to CX3CR1. However, the tree prepared from ECL3s of all CKRs (D) positioned CCR6 similar to CXCR6 again.