Allelic Variation in <i>CXCL16</i> Determines CD3⁺ T Lymphocyte Susceptibility to Equine Arteritis Virus Infection and Establishment of Long-Term Carrier State in the Stallion

<div><p>Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of horses and other equid species. Following natural infection, 10–70% of the infected stallions can become persistently infected and continue to shed EAV in their semen for periods ranging from several months to life. Recently, we reported that some stallions possess a subpopulation(s) of CD3⁺ T lymphocytes that are susceptible to <i>in vitro</i> EAV infection and that this phenotypic trait is associated with long-term carrier status following exposure to the virus. In contrast, stallions not possessing the CD3⁺ T lymphocyte susceptible phenotype are at less risk of becoming long-term virus carriers. A genome wide association study (GWAS) using the Illumina Equine SNP50 chip revealed that the ability of EAV to infect CD3⁺ T lymphocytes and establish long-term carrier status in stallions correlated with a region within equine chromosome 11. Here we identified the gene and mutations responsible for these phenotypes. Specifically, the work implicated three allelic variants of the equine orthologue of <i>CXCL16</i> (<i>EqCXCL16</i>) that differ by four non-synonymous nucleotide substitutions (XM_00154756; c.715 A → T, c.801 G → C, c.804 T → A/G, c.810 G → A) within exon 1. This resulted in four amino acid changes with EqCXCL16S (XP_001504806.1) having Phe, His, Ile and Lys as compared to EqCXL16R having Tyr, Asp, Phe, and Glu at 40, 49, 50, and 52, respectively. Two alleles (<i>EqCXCL16Sa</i>, <i>EqCXCL16Sb</i>) encoded identical protein products that correlated strongly with long-term EAV persistence in stallions (P<0.000001) and are required for <i>in vitro</i> CD3⁺ T lymphocyte susceptibility to EAV infection. The third (<i>EqCXCL16R</i>) was associated with <i>in vitro</i> CD3⁺ T lymphocyte resistance to EAV infection and a significantly lower probability for establishment of the long-term carrier state (viral persistence) in the male reproductive tract. <i>EqCXCL16Sa</i> and <i>EqCXCL16Sb</i> exert a dominant mode of inheritance. Most importantly, the protein isoform EqCXCL16S but not EqCXCL16R can function as an EAV cellular receptor. Although both molecules have equal chemoattractant potential, EqCXCL16S has significantly higher scavenger receptor and adhesion properties compared to EqCXCL16R.</p></div

The ability of EqCXCL16 to function as an entry receptor for EAV is determined by amino acid substitutions in the N-terminal ectodomain of the protein encoded by exon 1.

<p>A) Stable HEK-293T cell lines were generated by transfection with the pJ609-EqCXCL16S or pJ609-EqCXCL16R plasmid DNA followed by selection with antibiotic puromycin. These cells (panel a: EqCXCL16S, panel b: EqCXCL16R) were surface stained with Gp α-EqCXCL16 Ab or guinea pig pre-bleed (Gp Pb) sera (panels d and e) as primary antibodies, followed by goat anti-Gp IgG(H+L) conjugated to Alexa Fluor 488 and analyzed by confocal microscopy to confirm the expression of EqCXCL16 protein. Naïve HEK-293T cells were also stained with Gp α-EqCXCL16 Ab (panel c). B) Naïve HEK-293T and stable HEK-EqCXCL16S and HEK-EqCXCL16R cells were lysed in RIPA cell-lysis buffer and an equal amount of lysates were analyzed by WB using Gp Pb sera or Gp α-EqCXCL16 Ab. Arrow indicates the absence or presence of EqCXCL16 protein in the WB membrane (molecular weight approximately 30 kDa). C) Naïve HEK-293T (a), HEK-EqCXCL16S (b), and HEK-EqCXCL16R (c) cells were infected with EAV sVBSmCherry (synthetic virus expressing mCherry) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006467#pgen.1006467.ref024" target="_blank">24</a>] at a multiplicity of infection of 1 (MOI = 1). At 12 hours post infection (hpi), cells were washed with PBS, fixed in 4% paraformaldehyde (PFA) and analyzed with an inverted immunofluorescence microscope for the expression of mCherry (red) as an indicator of EAV infection. Markedly increased numbers of stable HEK-EqCXCL16S cells were shown to express mCherry compared to naïve HEK-293T and stable HEK-EqCXCL16R cells. D) The role of EqCXCL16 variants on EAV infection and gene expression was analyzed in the EAV sVBSmCherry infected cells. a) HEK-EqCXCL16S, b) HEK-EqCXCL16R, and c) naïve HEK-293T cells were infected with EAV sVBSmCherry at an MOI = 1. After fixation in 4% PFA at 12 hpi, cells were analyzed with an inverted immunofluorescence microscope for the expression of EAV nsp-1 gene using a monoclonal antibody, α-nsp-1 MAb (12A4). Images are representative of three independent experiments.</p

Binding of EAV to EqCXCL16 is determined by amino acid substitutions within the N-terminal ectodomain of the protein encoded by exon 1.

<p>A). Effect of amino acid differences between EqCXCL16S and EqCXCL16R on EAV binding as determined by a combination of VOPBA and Far-WB analysis. Naïve HEK-293T, stable HEK-EqCXCL16S and HEK-EqCXCL16R cells were lysed in RIPA buffer, the lysate proteins separated using 12% SDS-PAGE and transferred onto a PVDF membrane. Membrane bound proteins were denatured and renatured using sequentially decreasing concentrations of Gn-HCl following which the membranes were blocked and incubated either with purified EAV VBS (15 μg/ml) in protein binding buffer (a) or with protein binding buffer without purified EAV VBS (b). After washing, membranes were incubated with α-GP5 MAb 6D10 and developed using the enhanced chemiluminescence (ECL) method. Binding of EAV VBS to EqCXCL16 protein is shown in (a [arrow]). The same membrane (a) was stripped and re-probed with Gp anti-EqCXCL16 as shown in panel c. As indicated by the arrow in panel c, EqCXCL16S and EqCXCL16R were detected at the same position on the membrane where EAV GP5 was detected in panel a. B). Amino acid substitutions between the “S” and “R” EqCXCL16 isoforms and attachment of EAV to host cells. Equal numbers (2 x 10⁶) of a) naïve HEK-293T cells and stable b) HEK-EqCXCL16S and c) HEK-EqCXCL16R cells were washed, resuspended in cold PBS (pH 7.4) with 2% FBS (PBS-F) and then incubated with biotinylated EAV VBS on ice for 2 h in the dark. After adsorption, excess EAV was removed by washing in cold PBS-F, and cells were then stained with Streptavidin-FITC and DAPI solution. Significantly higher number of HEK-EqCXCL16S cells were observed to bind biotinylated EAV (panel a) compared to the HEK-EqCXCL16R (panel b) or naïve control HEK-293T cells (panel c). Statistically different results are represented with different letters, a and b. Panel d shows the graphical representation of the images from panels a, b, and c. All the images depicted were representative of three independent experiments. Data were analyzed by ANOVA; P<0.001 was considered as significant.</p

Alignment of predicted amino acid sequences within exon 1 of CXCL16 proteins from seven different mammalian species.

<p>The <i>Equus caballus</i> CXCL16R protein (amino acid residues 24–78) is depicted at the top, followed by <i>Equus caballus</i> CXCL16S protein and CXCL16 of other mammalian species.</p

Breeds of stallions and status as carriers and non-carriers for shedding of EAV in semen more than one year post EAV infection.

<p>Breeds of stallions and status as carriers and non-carriers for shedding of EAV in semen more than one year post EAV infection.</p

Gene frequencies among Thoroughbred (n = 67), Quarter Horse (n = 53), Saddlebred (n = 60), and Standardbred (n = 60) horses for alleles <i>CXCL16R</i>, <i>CXCL16Sa</i>, and <i>CXCL16Sb</i>.

<p>Gene frequencies among Thoroughbred (n = 67), Quarter Horse (n = 53), Saddlebred (n = 60), and Standardbred (n = 60) horses for alleles <i>CXCL16R</i>, <i>CXCL16Sa</i>, and <i>CXCL16Sb</i>.</p

Effect of amino acid substitutions between “S” and “R” isoforms of EqCXCL16 on binding to the EqCXCR6 receptor protein <i>in vitro</i>.

<p>Interactions between purified recombinant EqCXCL16S/R-EqCXCR6 were examined using Far-WB. Equal amounts (20 μg) of His-tagged EqCXCR6 protein or BSA as a control were separated in different lanes on 10% SDS-PAGE and transferred onto a PVDF membrane. Proteins were then sequentially denatured and renatured by using different concentrations of Gn-HCl. After blocking, the membranes were incubated with soluble EqCXCL16S (panel a) or EqCXCL16R (panel b) protein (5 μg/ml) followed by Rb α-EqCXCL16 Ab. After washing, membranes were developed using the ECL method. Binding of EqCXCL16S and EqCXCL16R to EqCXCR6 is indicated by arrows (panels a and b). These interactions occurred at the same location as that occupied by EqCXCR6; this was confirmed by stripping the membrane shown in panel b and re-probing it with anti-His antibody as shown in panel c.</p

Differences between the membrane-bound forms of each EqCXCL16 isoform in OxLDL scavenger receptor activity.

<p>Approximately 5 x 10⁵ naïve HEK-293T and stable HEK-EqCXCL16S and HEK-EqCXCL16R cells/well were plated on a 24-well plate, permitted to adhere for 24 h prior to washing and incubation with Dil-OxLDL for 3 h at 37°C. Following this, cells were washed again with PBS pre-warmed to 37°C, fixed with 4% PFA, and examined with an inverted fluorescence microscope. A marked increase in Dil-OxLDL binding and internalization was shown in HEK-EqCXCL16S (b) as compared to naïve HEK-293T (a), or HEK-EqCXCL16R (c), or HEK-EqCXCL16S cells treated with Gp α-EqCXCL16 pAb (d).</p

Two models showing the possible mechanisms of EAV infection of equine CD3⁺ T lymphocytes.

<p>Model 1: A subpopulation of equine CD3⁺ T lymphocytes expresses transmembrane (TM) form of EqCXCL16S (i.e. horses with the CD3⁺ T lymphocyte subpopulation susceptible to <i>in vitro</i> EAV infection; A) or TM-EqCXCL16R (i.e. horses that lack the CD3⁺ T lymphocyte subpopulation susceptible to <i>in vitro</i> EAV infection; B) on their cell surface and EAV could directly infect these CD3⁺ T lymphocytes expressing TM-CXCL16S but not those expressing TM-CXCL16R. Model 2: CD3⁺ lymphocytes do not express either isoform of TM-CXCL16 but EAV infects these cells via ligand-receptor (CXCL16-CXCR6) complexes occurring on the CD3⁺ T lymphocytes surface (indirect infection). In this model the CD14⁺ monocytes and macrophages from susceptible and resistant horses express TM-CXCL16S (C) or TM-CXCL16R (D). EAV binds with TM-CXCL16S but not with TM-CXCL16R and is then cleaved by metalloproteinases and the soluble CXCL16S-EAV complex (sCXCL16S-EAV) interacts with CXCR6 present on the CD3⁺ T lymphocytes and thus, infects the CD3⁺ T lymphocytes from susceptible horses (C). It is also possible that TM-CXCL16S after interaction with EAV is not cleaved but comes in contact with CXCR6 present on the CD3⁺ T lymphocytes cells and thus infects the CD3⁺ T lymphocytes. TM-CXCL16R will not bind with EAV and hence CD3⁺ T lymphocytes of resistant horses will not become infected (D).</p

Association of <i>CXCL16</i> alleles with carrier and non-carrier status of stallions for EAV shedding in semen (n = 77).

<p>Association of <i>CXCL16</i> alleles with carrier and non-carrier status of stallions for EAV shedding in semen (n = 77).</p