The Major for Mouse Adenovirus Susceptibility Maps to of the Hematopoietic Cell Surface-Expressed LY6 Family This information is current as of September 29, 2021. Katherine R. Spindler, Amanda R. Welton, Efrem S. Lim, Suman Duvvuru, Irene W. Althaus, Jennifer E. Imperiale, Adam I. Daoud and Elissa J. Chesler J Immunol 2010; 184:3055-3062; Prepublished online 17 February 2010; Downloaded from doi: 10.4049/jimmunol.0903363 http://www.jimmunol.org/content/184/6/3055 http://www.jimmunol.org/ References This article cites 69 articles, 39 of which you can access for free at: http://www.jimmunol.org/content/184/6/3055.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Major Locus for Mouse Adenovirus Susceptibility Maps to Genes of the Hematopoietic Cell Surface-Expressed LY6 Family

Katherine R. Spindler,* Amanda R. Welton,*,1 Efrem S. Lim,*,2 Suman Duvvuru,† Irene W. Althaus,* Jennifer E. Imperiale,* Adam I. Daoud,*,3 and Elissa J. Chesler‡,x

Susceptibility to mouse adenovirus type 1 is associated with the major quantitative trait locus Msq1. Msq1 was originally mapped to a 13-Mb region of mouse (Chr) 15 in crosses between SJL/J and BALB/cJ inbred mice. We have now narrowed Msq1 to a 0.75-Mb interval from 74.68 to 75.43 Mb, defined by two anonymous markers, rs8259436 and D15Spn14, using data from 1396 backcross mice. The critical interval includes 14 Ly6 or Ly6-related genes, including Ly6a (encoding Sca-1/TAP), Ly6e (Sca-2/

Tsa1), Ly6g (Gr-1), and gpihbp1 (GPI-anchored high-density lipoprotein–binding 1), as well as the encoding an Downloaded from aldosterone synthase (Cyp11b2). The Ly6 family members are attractive candidates for virus susceptibility genes because their products are GPI-anchored membrane expressed on lymphoid and myeloid cells, with proposed functions in cell adhesion and cell signaling. To determine interstrain variation in susceptibility and produce additional resources for cloning Msq1,we assayed the susceptibility phenotype of four previously untested inbred mouse strains. Susceptibility of strain 129S6/SvEvTac was subsequently localized to the Ly6 complex region, using polymorphic genetic markers on Chr 15 in a population of 271 (129S6/ 129S6 SvEvTac 3 BALB/cJ)F1 3 BALB/cJ backcross mice. We identified a major 129S6/SvEvTac susceptibility allele, Msq1 ,on http://www.jimmunol.org/ Chr 15 in the same region as Msq1SJL. The results indicate that a major host factor in mouse adenovirus type 1 susceptibility is likely to be a member of the Ly6 gene family. The Journal of Immunology, 2010, 184: 3055–3062.

he genetic background of an individual can significantly and human adenoviruses cause acute and persistent with influence the outcome of viral . Identification of high morbidity and mortality in immunocompromised hosts. Re- T genes involved in susceptibility or resistance to viruses has spiratory infection by both mouse and human adenoviruses results increased our understanding of many viral disease processes (1). in chemokine upregulation (reviewed in Ref. 10). MAV-1 primarily Unbiased genetic approaches have revealed specific host proteins infects endothelial cells, which is only rarely seen for human ad- involved in susceptibility to both RNA and DNA viruses, such as enoviruses. The endotheliotropism is particularly marked in the by guest on September 29, 2021 West Nile virus and mouse CMV (2–5). brain, spinal cord, and spleen (7, 11, 12), and MAV-1 causes hem- Inbred and outbred strains of mice vary significantly in their orrhagic encephalomyelitis accompanied by breakdown of the susceptibility to mouse adenovirus type 1 (MAV-1) (6–8). MAV-1 blood–brain barrier and altered expression of tight junction proteins has similarities to human adenoviruses in structure, or- (6, 7, 13). In addition, MAV-1 targets monocytes and , ganization, and some aspects of pathogenesis (9). Both MAV-1 which are also effectors of the host response to MAV-1 infection (14). We are investigating the mechanisms involved in MAV-1 pathogenesis, using a variety of methods, and we have shown that *Department of Microbiology and Immunology, University of Michigan, Ann Arbor, both adaptive and innate immune responses play a role in the viral † ‡ MI 48109; Genome Science and Technology Program and Systems Genetics disease process (10, 14–16). We are using a positional cloning ap- Group, Biosciences Division, University of Tennessee-Oak Ridge National Labora- tory, Oak Ridge, TN 37831; and xThe Jackson Laboratory, Bar Harbor, ME 04609 proach to identify the important host genes involved in susceptibility 1Current address: Center for Molecular Imaging, Department of Radiology, Univer- of mice to MAV-1. sity of Michigan, Ann Arbor, MI. Susceptibility to MAV-1 is a dominant quantitative trait, with 2Current address: Department of Microbiology, University of Washington, Seattle, a quantitative trait locus (QTL), Msq1, mapping to chromosome WA. (Chr) 15 (logarithm of odds [LOD] score = 21) (17). Msq1SJL 3Current address: Department of Human Evolutionary Biology, Harvard University, accounts for 40% of the trait variance between the susceptible Cambridge, MA. SJL/J strain and resistant BALB/cJ strain. Initially, the 2 LOD Received for publication October 15, 2009. Accepted for publication January 5, confidence interval for Msq1SJL was 18 Mb and included ∼300 2010. predicted genes, including Hsf1 (the gene encoding heat shock This work was supported by National Institutes of Health Grant AI068645 (to K.R.S.). factor 1 protein), Sla1 (Src-like adapter protein 1), and the Ly6 Address correspondence and reprint requests to Dr. Katherine R. Spindler, Depart- ment of Microbiology and Immunology, University of Michigan Medical School, gene-family complex (lymphocyte Ag 6). The Ly6 genes are 1150 W. Medical Center Drive, SPC 5620, 6723 Medical Science Building II, Ann particularly attractive as candidate genes for susceptibility because Arbor, MI 48109. E-mail address: [email protected] they encode GPI-anchored cell-surface proteins (18). There are Abbreviations used in this paper: Chr, chromosome; GR-1, granulocyte differentia- .20 predicted Ly6 or Ly6-related genes in this interval of Chr 15, tion Ag; HDL, high-density lipoprotein; LOD, logarithm of odds; MAV-1, mouse adenovirus type 1; NCBI, National Center for Biotechnology Information; QTL, and protein functions or expression patterns are known for some. quantitative trait locus; Sca, stem cell Ag; SNP, single nucleotide polymorphism; LY6 proteins are variably expressed on different cell types, in- SSLP, simple sequence length polymorphism; TAP, T cell-activating protein; TSA-1, cluding lymphoid and nonlymphoid cells, and during hemato- thymic shared Ag-1; uPAR, urokinase-type plasminogen activator receptor. poietic cell differentiation. Some LY6 proteins are involved in Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 lymphocyte adhesion, T cell responses, and the IFN response. The www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903363 3056 MAJOR LOCUS FOR MAV-1 SUSCEPTIBILITY MAPS TO LY6 FAMILY chicken Ly6E gene has been identified as a candidate gene for nome interval of interest, using the Tandem Repeats Database (http:// resistance to the avian herpesvirus Marek’s disease virus (19). tandem.bu.edu/cgi-bin/trdb/trdb.exe) (29). Primers were designed to am- ∼ Recently, two different approaches have identified Ly6 or Ly6- plify regions of 200 nt from template DNAs for the three mouse strains. Primer pairs that amplified newly identified polymorphic PCR products related gene involvement in HIV-1 susceptibility. A whole-genome used in this work are listed in Table I. linkage analysis using cellular susceptibility to HIV in vitro Single nucleotide polymorphisms (SNPs) potentially polymorphic for identified the Ly6 locus on human Chr 8q24 (20). A small in- the mouse strains and interval of interest were initially identified using SNP terfering RNA screen identified 250 HIV-dependency factors, 3 of databases extant in 2005–2006, GNF3 and Celera2 (30, 31). The SNPs are now compiled in the Mouse Phenome Database (www.jax.org/phenome/ which were members of the LY6 family: Gml, Ly6D, and Lypd4 SNP) and are shown in Fig. 1 by numbers with an “rs” prefix. Further SNP (21). In a similar approach, LY6E was identified as 1 of 305 hu- haplotype analysis of additional resistant and susceptible strains was done man proteins that affect West Nile virus infection (22). using the Center for Genome Dynamics Mouse SNP Database v. 1.3 To determine which of the candidate gene(s) in the Msq1SJL locus (http://cgd.jax.org/cgdsnpdb/). correlates with susceptibility to MAV-1, we used fine mapping and DNAs were isolated from tail snips and amplified by PCR in 20-ml reactions as described (32). Samples were electrophoresed on 7% poly- haplotype analysis to reduce the interval from 18 Mb to 0.75 Mb, acrylamide gels. Alternatively, DNA samples were analyzed by the Uni- a region that encompasses 14 Ly6 or Ly6-related genes. The Ly6 versity of Michigan Sequencing Core (Ann Arbor, MI), using fluorescently gene complex in mice has two major haplotypes, Ly6a and Ly6b, labeled primers. For SNP analysis, only an aliquot of the PCR reaction was that code for two lymphocyte specificities with distinct inbred electrophoresed on a gel. The remainder of each sample was purified using QIAquick PCR Purification Kit columns (Qiagen, Valencia, CA), and the mouse strain distribution patterns (23–25). These strain distribution DNA was then sequenced by the University of Michigan Sequencing Core. patterns correlate with known susceptibility or resistance to MAV-1 among inbred strains (8). We therefore identified additional strains Statistical analysis and QTL mapping Downloaded from of mice that are susceptible or resistant to MAV-1 to extend this QTL analysis was performed using R/qtl software. SSLP markers used in correlation and develop additional mapping tools. We analyzed the QTL analysis were D15Mit13, D15Mit6, D15Spn101, D15Mit70, and D15Mit107. Individual markers were quality tested for difference from progeny of a backcross between BALB/cJ mice and one additional 2 MAV-1–susceptible mouse strain, 129S6/SvEvTac. We identified the expected 1:1 ratio, using the x test for Mendelian segregation. 129S6 The D15Mit6 marker showed significant deviation from the 1:1 ratio a QTL allele, Msq1 , that maps to the same region of Chr 15 as (p = 7.87 3 10219) with a surplus of BALB/cJ homozygote allele calls SJL Msq1 (LOD = 46). Taken together, the results provide strong relative to heterozygotes. None of the other markers had significant de- http://www.jimmunol.org/ evidence that susceptibility to MAV-1 is encoded by one or more viation from expectation (p . 0.05). Mouse brain virus loads were entered members of the Ly6 gene complex. as the background-subtracted average ELISA value of triplicate measure- ments from each mouse. QTL mapping was performed using the R/qtl scanone function with single-marker regression and the imputation Materials and Methods method, using 271 backcross mice genotyped at five markers. General Viruses and mice linear modeling (ANOVA) of the allelic effect was performed using the R/qtl fitqtl function at the marker nearest to the QTL peak. Wild-type MAV-1 (26) was propagated and titrated as described (27). All virus stocks used for phenotyping were aliquots from a single preparation that were thawed only one time, for preparation of inocula. All mice were Results obtained from The Jackson Laboratory (Bar Harbor, ME) except for 129S6/ Fine mapping of the Msq1 interval by guest on September 29, 2021 SvEvTac (129S6), which were obtained from Taconic Farms (Germantown, The 2-LOD confidence interval previously identified for Msq1SJL on NY). All animal work complied with relevant federal and institutional pol- ∼ icies. Mice were maintained in microisolator housing with food and water Chr15wasestimatedtobefrom39to54cM,correspondingto 65– provided ad libitum. A selective phenotyping scheme was used on 1140 78 Mb on the physical map (17). We determined the MAV-1 sus- backcross mice from an expanded backcross, as was used to initially identify ceptibility phenotypes for 256 (BALB/cJ 3 SJL/J)F1 3 BALB/cJ SJL 3 3 the Msq1 locus (17), (BALB/cJ SJL/J)F1 BALB/cJ. In a different backcross progeny and then genotyped for markers on all 20 mouse backcross, mice were obtained by mating both (129S6 3 BALB/cJ)F and 1 . Forty backcross mice were recombinant across the (BALB/cJ 3 129S6)F1 mice as the female parent with BALB/cJ mice as the male parent. Breeding pairs were maintained on breeder chow (Purina 5008; Chr 15 2-LOD confidence interval defined by two markers used St. Louis, MO). in the original analysis, D15Mit270 (63.3 Mb) and D15Mit70 (81.0 Mb). An additional microsatellite marker that had been pre- Phenotyping viously described (D15Mit211, 66.6 Mb) was supplemented with two 2 Mice 3–5 wk of age were injected i.p. with 10 PFU of MAV-1 diluted in others that we designed, D15Spn101 and D15Spn54 (Table I), to 0.1 ml PBS. Mice were euthanized at 8 d postinfection, and organs and further genotype the recombinant mice. Five of the 256 backcross tissues were collected and stored at 270˚C. Brain viral loads were de- termined by capture ELISA, using brain homogenates as described (28); mice were recombinant across the interval. To fine map the genetic SJL this method gives results that correlate with virus quantitation by plaque location of Msq1 , we expanded the backcross, collecting 1140 assay (17). An average ELISA value from homogenates of mock-infected additional mice. A selective phenotyping scheme was used: geno- mice was background subtracted from the infected samples. To standardize types were determined for all progeny mice at D15Spn101 and assays performed at different times, a MAV-1 virus stock was included as a positive control in each assay performed; resulting experimental sample D15Spn54; subsequently, only mice recombinant across this interval values were normalized to the MAV-1 control. PBS was used as a negative were infected with MAV-1 for susceptibility phenotype measurement. control in each assay. Susceptible mice were originally defined as those We identified 19 additional backcross mice that were recombinant , 3 . 4.4 with an LD50 10 PFU, and resistant mice all had LD50 values 10 across the interval (Fig. 1); 16 of these had phenotypes concordant ∼ (8). These values correspond to OD450 0.04 and 0.31, respectively, in the with their genotypes. The three mice with discordant genotypes and capture ELISA (17). phenotypes are discussed below. Taken together, the original and Genotyping the expanded backcrosses yielded 21/1396 recombinants (1.50%) SJL All marker positions in this work correspond to National Center for Bio- across the Msq1 interval from 72.56 to 75.85 Mb of Chr 15. technology Information (NCBI) Build 37 of the Mus musculus genome To further refine the candidate interval, we developed additional sequence. Simple sequence length polymorphism (SSLP) markers that microsatellite markers and identified SNP markers that are poly- differentiate between the strains used were identified from the Chr 15 morphic for BALB/cJ and SJL/J. The 21 recombinant mice were region of interest (www.informatics.jax.org/). We confirmed that the SSLPs were polymorphic between BALB/cJ and SJL/J or 129S6 mice by each genotyped using 2 or more of these 9 markers (Fig. 1); from performing PCR on genomic DNAs of each strain. To produce additional these results we deduced a maximal 0.75-Mb interval for the SSLP markers, we identified regions of tandem repeats in the mouse ge- Msq1SJL QTL. One individual had a crossover between rs8259436 The Journal of Immunology 3057

Table I. New microsatellite loci used in this study

Polymorphic Between

BALB/cJ BALB/cJ Locus Map Positiona Forward Primer Reverse Primer and SJL/J and 129S6 D15Spn101 72,556,431 59-TCAGGCAAGATCTGCTGAAG-39 59-ACAGTTGTCCCACGTTCTGG-39 ++ D15Spn106 72,849,859 59-ATCCCACACGGAGTGAAAAC-39 59-TTTATGCACCAGTGGACAGC-39 ++ D15Spn14 75,432,404 59-GGCTGGCCTTAAACTCTCAG-39 59-AAGGCACAGCTGGACATGG-39 ++ D15Spn54 75,850,197 59-CCTGCTGGGAGAGAGACTTCA-39 59-GGAATGAAAGGGGGATCATAC-39 + 2 aMap position indicated is the first nt at the centromeric side of the repeat (based on NCBI Build 37, July 2007).

and rs3662946, defining the left end of the interval at 74.68 Mb, by LD50 assay as susceptible to MAV-1 (SJL/J and SWR/J) and and four individuals had crossovers between rs31717260 and four as resistant (129P3/J, BALB/cJ, C3H/HeJ, and C57BL/6J) D15Spn14, defining the right end of the interval at 75.43 Mb. (8). We also identified three additional susceptible strains, PL/J, Fifteen candidate genes in the 0.75-Mb interval were identified 129S6, and FVB/NJ; and one additional resistant strain, NOD/ using Ensembl and are depicted in Fig. 1 and listed in Table II (33– ShiLtJ. For two of those strains, the Ly6 lymphocyte specificity 43). Eight are genes encoding Ly6 family proteins (Ly6e, Ly6i, Ly6a, has been reported, and it correlates with that reported for other

Ly6c1, Ly6c2, Ly6g, Ly6f, and Ly6h). Six genes (gpihbp1 and five susceptible strains: PL/J and129Sv are both Ly6.2 (23, 25). Downloaded from single transcripts of unknown association, 2010109I03Rik-201, We examined the SNP haplotypes for susceptible and resistant I830127L07Rik, BC025446, AC116498.15, and 9030619P08Rik) strains across the interval from 72.9 to 75.3 Mb at 15 SNPs spaced encode predicted proteins that have LY6/urokinase-type plasmin- 0.2 to 0.3 Mb apart (30). In the region from 73.4 to 74.5 Mb, there was ogen activator receptor (uPAR) domains, and are thus termed Ly6- haplotype identity among strains; that is, the susceptible strains SJL/J, related. LY6/uPAR sequences are found in a large protein family SWR/J, FVB/NJ, and PL/J had haplotypes in the region identical including the LY6 proteins, uPAR, and other proteins (44, 45). LY6/ to those found in the resistant strains BALB/cJ, A/J, and C3H/HeJ http://www.jimmunol.org/ uPAR domains have 10 cysteine residues in a consensus pattern with (data not shown). This observation is consistent with our inability to the C-terminal cysteine followed by an asparagine. find known SNPs or design microsatellite markers that detect poly- morphisms between BALB/cJ and SJL/J mice for the interval from Susceptibility of additional inbred mouse strains to MAV-1 72.85 to 74.68 Mb. In contrast, in the region from 74.6 to 75.3 Mb, the The Ly6 complex has long been known to have two haplotypes, four susceptible strains had identical haplotypes to each other that a b Ly6 and Ly6 , that code for two lymphocyte specificities, Ly6.1 were different from the shared haplotypes of the three resistant strains and Ly6.2, respectively, determined by alloantisera to cell-mem- (data not shown). With a denser SNP database (Center for Genome brane Ags (23–25). The distribution pattern of these Ly6.1 and Dynamics Mouse SNP Database v. 1.3), the haplotype identity per-

Ly6.2 specificities among inbred mouse strains correlates with sisted from 73.4 to 74.5 Mb (data not shown). The haplotype analysis by guest on September 29, 2021 susceptibility that we had previously identified for a number of did not reduce the number of candidate genes in the interval. inbred strains. For example, Ly6.1 strains include BALB/c, A/J, A. SW, and C3H/HeJ (23), which are resistant to MAV-1 (8, 17). Susceptibility of 129S6 mice maps to Chr 15 Ly6.2 strains include SJL and SWR, which are susceptible to To determine whether susceptibility of 129S6 mice maps to the MAV-1. To test this correlation further and develop additional same region of Chr 15 as susceptibility of SJL/J mice, we crossed resources for mapping Msq1, we screened additional inbred mouse 129S6 and BALB/cJ mice. Progeny F1 animals were infected with strains for susceptibility to MAV-1. We infected mice and assayed MAV-1 and had susceptibility phenotypes (brain virus loads) in- for viral load in the brain, using capture ELISA (17) (Fig. 2). The termediate between the parental phenotypes (data not shown). results supported findings in two strains previously characterized This result is similar to the case for (BALB/cJ 3 SJL/J)F1 mice

FIGURE 1. Genetic mapping of Msq1. The top line shows SSLP and SNP markers used in fine mapping (BALB/cJ 3 SJL/J)F1 3 BALB/cJ backcross progeny in the Chr 15 interval between 72.56 and 75.85 Mb; physical positions of each marker are indicated in parentheses. The X symbols indicate the number of mice with genotypic recombination within each subinterval. The lower line is an expansion of the deduced critical interval with predicted candidate genes. Ly6 genes are indicated by black triangles, Ly6-related genes by gray triangles, and the other gene by a white triangle. Genbank transcript designations for the Ly6-related genes (Ly6R1–5) are given in Table II. Genes transcribed from the top (+) strand point to the right, and genes transcribed from the bottom (2) strand point to the left. Numbers below the line indicate the physical map position in megabases. 3058 MAJOR LOCUS FOR MAV-1 SUSCEPTIBILITY MAPS TO LY6 FAMILY

Table II. Predicted genes in the Msq1 interval

Position (Mb), Class Symbol Stranda Alternate Name(s), Function, Expression Ly6 Ly6e 74.79, + TSA-1, Sca-2, expressed on most intrathymic precursor cells of the lymphoid lineage (18) Ly6i 74.81, 2 Expressed on bone marrow cells, monocytes, macrophages, granulocytes, and myeloid precursors; and in spleen, thymus, kidney, and lung (33, 34) Ly6a 74.83, 2 Sca-1, TAP, expressed on bone marrow stem cells that are precursors to all thymic subsets (18, 35) Ly6c1 74.88, 2 Expressed on inflammatory monocytes, some NK cells, and plasmacytoid dendritic cells (36) Ly6c2 74.94, 2 Expressed on leukemia cells and on macrophages infiltrating rejected allografts (37, 38) Ly6g 74.99, + Gr-1, expressed on mature granulocytes (18) Ly6f 75.10, + mRNA expressed in nonlymphoid tissues (39) Ly6h 75.40, 2 mRNA expressed in brain (40) Ly6-related Ly6Rbb 74.71, 2 2010109I03Rik, intectin (41) Ly6R2b 74.97, 2 I830127L07Rik Ly6R3b 75.05, + BC025446 Ly6R4b 75.07, 2 AC116498.15-201 Ly6R5b 75.26, 2 9030619P08Rik Gpihbp1 75.43, + GPI-anchored HDL-binding protein 1, expressed in heart, lung, liver (42)

Other Downloaded from Cyp11b2 74.69, 2 Cytochrome P450 11b2, aldosterone synthasec aNCBI Build 37. Position listed is the start of transcription. bLy6-related 1–5; designation given in this paper for purpose of discussion. cMouse Genome Database (www.informatics.jax.org, August 2009) (43). Gr-1, granulocyte differentiation Ag; HDL, high-density lipoprotein; Sca, stem cell Ag; TAP, T cell-activating protein; TSA-1, thymic shared Ag-1.

(17) and indicates that susceptibility in 129S6 mice is a semi- a major effect QTL on Chr 15. The peak marker was D15Spn101 http://www.jimmunol.org/ dominant quantitative trait. from single-marker analysis, and imputation mapping showed the We obtained 271 backcross mice, consisting of both (129S6 3 effect maxima on Chr 15 at ∼69 Mb with a peak LOD of 46.2. The BALB/cJ)F1 3 BALB/cJ and (BALB/cJ 3 129S6)F1 3 BALB/cJ results from fitting the candidate QTL using ANOVA showed the progeny, and determined their susceptibility phenotypes. Fig. 3 percentage of phenotypic variation explained by the QTL at 69 shows the distribution of phenotypes of the entire backcross pop- Mb on Chr 15 analyzed alone was 54% (F[1, 269] = 331; p = 9.01 3 ulation. The distribution was similar when phenotypes of backcross 10249; LOD = 47.3). This interval on Chr 15 for Msq1129S6 en- SJL mice from two types of F1 parents were plotted separately (data not compasses the interval we have previously identified for Msq1 . shown). The phenotype distribution for the backcross mice is con- by guest on September 29, 2021 sistent with susceptibility of 129S6 mice being a quantitative trait Discussion determined by a single major locus. To understand the contribution of genetic variation in host defense We determined genotypes of the 271 backcross mice for five pathways to adenoviral pathogenesis and to identify which host markers on Chr 15 that differentiate between 129S6 and BALB/cJ genes play a role in MAV-1 susceptibility, we have used a positional mice. We performed QTL analysis on the 271 backcross mice, cloning approach. An 18-Mb region of mouse Chr 15 was initially using a QTL mapping package for the statistical software R, R/qtl, identified as the region with the major QTL for MAV-1 suscep- ∼ as previously described (17). We observed an association of sus- tibility; this region encoded 300 predicted genes (17). We used ceptibility with markers on Chr 15. A one-way scan for QTLs backcross mice, additional polymorphic markers, and SNP hap- using single-marker regression and the imputation method (256 lotype identity to reduce the size of the critical interval to 0.75 imputations using 5000 permutations and a 2-cM step interval) of Mb. This region encodes 14 Ly6 or Ly6-related genes and an al- the mapping analysis showed that viral load is associated with dosterone synthase gene. We cannot eliminate aldosterone syn- thase as a candidate gene, but we believe that Ly6 family members are more likely for reasons based on expression and function,

0.8 discussed below. The fine mapping eliminated the candidate genes Hsf1 and Sla1, which were previously considered candidates in the 0.6 18-Mb interval based on their biological function, as well as several Chr 15 Ly6 and Ly6-related genes, such as Ly6d, Ly6k, 0.4 Lynx1, Slurp1, and Psca. In the (BALB/cJ 3 SJL/J)F1 3 BALB/cJ backcross analysis, 3 0.2

Virus load (OD450) of 19 mice that were recombinant across the interval delineated by

0.0 D15Spn101 and D15Spn54 had phenotypes for susceptibility to SJL SWR 129 PL/J FVB NOD B6 C3H 129 BALB MAV-1 that were discordant with their genotypes for markers S6 P3 within the interval. The mice were resistant to MAV-1, as mea- Strain sured by brain viral load, and yet their genotype was heterozygous FIGURE 2. MAV-1 susceptibility of inbred strains of mice. Brain viral (BALB/cJ/SJL/J) at the four markers in the interval from 74.80 to loads were determined by capture ELISA. Each brain homogenate was 75.28 Mb. Because susceptibility to MAV-1 is dominant (17), assayed in triplicate, and each bar represents the average of the back- ground-subtracted triplicate measurements per homogenate with SD. mice with the heterozygous genotype should be susceptible. In Samples were standardized to a virus stock positive control included in each case, the phenotype–genotype discordance in these three each assay. OD450, OD at 450 nm. Strain abbreviations: SJL, SJL/J; SWR, individuals is in the direction of unexpected resistance. The virus SWR/J; 129S6, 129S6/SvEvTac; FVB, FVB/NJ; NOD, NOD/ShiLtJ; B6, did not replicate to high levels in their brains, despite being in- C57BL/6J; C3H, C3H/HeJ; 129P3, 129P3/J; BALB, BALB/cJ. jected with virus. We have previously seen such false-negative The Journal of Immunology 3059 30 Frequency 01020 0.0 0.2 0.4 0.6 0.8 Viral Load

FIGURE 3. MAV-1 susceptibility phenotypes in backcross mice. Viral loads in 271 (129S6 3 BALB/cJ)F1 3 BALB/cJ and (BALB/cJ 3 129S6)F1 3 BALB/cJ backcross mice were determined by ELISA assay as in Fig. 2 and plotted in rank order. Each bar represents the mean value for an individual mouse brain homogenate assayed in triplicate; for clarity, standard deviations are not shown but are comparable to those in Fig. 2. Genotypes at D15Spn101 are indicated by the bar colors: black, homozygous BALB/cJ; light gray, heterozygous, BALB/cJ / 129S6; and four medium-gray bars marked with dots, undetermined. Inset, the frequency of phenotypic classes of the same population. Downloaded from occurrences of low viral load in known genetically susceptible clonal Abs raised against lymphocytes. This resulted in a complex mice, at a frequency of 2–4% (K.R. Spindler, unpublished data). nomenclature for the LY6 Ags. This situation was further com- An example of a single animal of this type can be seen for the plicated by the fact that it was not known at first that LY6 Ags susceptible SJL/J mice assayed in Fig. 2. These events may be due were encoded by a multigene family, and the relationship between to experimental failures in the infection procedure. A second genes and alleles was not clear. For example, Ly6A.2 and Ly6E.1

possibility is that one or more of these three mice do not represent were initially thought to be different genes, but they are actually http://www.jimmunol.org/ a false-negative phenotyping error but were truly MAV-1 resistant, alleles of Ly6A (24). For the Ly6c1 and Ly6c2 genes, the highly despite being genetically heterozygous for the interval. That is, similar LY6C1 and LY6C2 proteins were originally thought to be cumulative resistant alleles at genes elsewhere in the genome may two allelic forms encoded by one gene (18, 49–51). The most be sufficient to produce the resistant phenotype. Susceptibility is recent comprehensive Ly6 gene-family review was published in a quantitative trait, with multiple candidate loci identified in the August 2004 and did not make extensive use of the emerging initial QTL analysis (17). For example, a locus on Chr 5 has an annotated mouse genome sequence (18). Furthermore, even in the additive effect on the phenotype. We favor false-negative infection current literature, references are made to populations of cells as the most likely explanation because we did not obtain any mice based on old designations that are ambiguous, such as LY6A/E with discordance as unexpected susceptibility. However, the nu- and LY6G/C. The Gr-1 antigenic specificity of mAb RB6-8C5 was by guest on September 29, 2021 mber of discordant individuals was small, and further analysis first reported to be shared by the Ly6c and Ly6g gene products, would be needed to resolve the possible explanations. with LY6C and LY6G being expressed on distinct cell types (52). We analyzed additional strains for their MAV-1 susceptibility However, a more recent study indicates that for this LY6G Ab phenotype by measuring brain virus loads by capture ELISA. For there is no cross-reactivity with LY6C on multiple hematopoietic strains that had been previously assayed for susceptibility by LD50 cell types (53). To assist in clarifying the Ly6 gene family no- rather than ELISA, the ELISA and LD50 results were in agreement menclature, in this discussion we use the nomenclature of genes in (SWR/J, C3H/HeJ, and 129P3/J) (8). We also identified additional the Ly6 locus established by the International Committee on susceptible and resistant strains, using ELISA. We found that the Standardized Genetic Nomenclature for Mice, as found in the strain distribution pattern of Ly6.1 and Ly6.2 specificities among Mouse Genome Database (www.informatics.jax.org, August inbred strains correlated with susceptibility to MAV-1. That is, 2009) (43). In addition, five Ly6-related transcripts have been Ly6.1 strains include BALB/c, A/J, A.SW, and C3H/HeJ (23); all of identified solely by genomic sequence annotation within the these are resistant to MAV-1 (8). Ly6.2 strains include SJL, SWR, critical interval for MAV-1 susceptibility (Table II). These Ly6- PL/J, and 129Sv (23, 25), and these are susceptible to MAV-1. related transcripts are discussed below, using abbreviated names, Analysis of a backcross of a second susceptible strain (129S6) Ly6R1–Ly6R5, because they have predicted proteins that all have with the resistant BALB/cJ strain showed that susceptibility to LY6/uPAR domains (data not shown). MAV-1 mapped to the same interval on Chr 15 as did SJL/J sus- The mRNA sequences of Ly6R1–5 are distinct from one another ceptibility. This Msq1129S6 QTL alone accounts for 54% of the and from the characterized Ly6 genes. Predicted proteins of the variation in susceptibility phenotype. The result that susceptibility Ly6R1, Ly6R2, and Ly6R3 transcripts are noted in the Mouse Genome in an additional strain maps to the same locus as Msq1SJL enabled us Database (www.informatics.jax.org/; August 2009) (43) as having to use SNP haplotype analysis in conjunction with recombinant LY6/uPAR domains, that is, sequences with 10 cysteine residues in mouse analysis to confirm the critical interval. The information also a consensus pattern with the C-terminal cysteine followed by an as- has enabled us to produce bacterial artificial chromosome-based paragine (44, 45). Ly6R2 lacks a predicted initiator methionine, but transgenic mice from a 129S6 library (T.-H. Hsu and K.R. Spindler, we have preliminary evidence of a complete Ly6R2 cDNA with an unpublished observations). Analysis of these mice for dominant initiator methionine from SJL/J mice (M.T. Stier and K.R. Spindler, MAV-1 susceptibility may reduce the candidate interval and confirm unpublished observations). We analyzed the predicted protein se- the identity of the susceptibility gene(s) within the 0.75-Mb interval. quences of the other two transcripts, Ly6R4 and Ly6R5, and noted that The Ly6 complex on Chr 15 in mice is a multigene family with the entire LY6/uPAR motif is found in the open reading frame for at least 20 related genes that likely arose by intrachromosomal Ly6R4. The protein predicted from Ly6R5 terminates soon after the gene duplication (18, 46–48). LY6 Ags were first identified by fifth cysteine residue; however, downstream in an alternate reading polyvalent antisera and subsequently through the use of mono- frame we found a sequence corresponding to the remainder of the 3060 MAJOR LOCUS FOR MAV-1 SUSCEPTIBILITY MAPS TO LY6 FAMILY

LY6 homology (data not shown). Thus, it is possible that Ly6R5 is in animal, upon antigenic stimulation (reviewed in Ref. 18). During the process of becoming a pseudogene. Few expression or functional infection of mice by lymphocytic choriomeningitis virus, LY6A data are available for the five Ly6-related transcripts in the Chr 15 expression increases on CD4+ and CD8+ T cells and persists at interval. Ly6R1 encodes a protein that has been identified as intectin, a high level on memory T cells (61). Inflammatory monocytes an intestine-specific protein involved in apoptosis of intestinal epi- express high levels of LY6C and are recruited to sites of inflamed thelial cells (41). tissue during infection by viruses, bacteria, and parasites, in Ly6 and Ly6-related genes are found in other vertebrates, in- contrast to resident monocytes, which are LY6Clo and rarely mi- cluding chickens, rats, and humans. In humans, 11 Ly6-related grate to inflamed sites (36, 62–65). Both in vivo and in vitro genes are found on Chr 8q24, syntenic to Chr 15 (54); in both stimulation of immune cells to IFNs increases expression of Ly6 mouse and humans, Ly6-related genes are also found on other genes. For example, both type I and type II IFNs stimulate LY6 chromosomes. The mouse Chr 15 interval that we have identified expression on T and B lymphocytes (66–68). IFNs also stimulate for susceptibility to MAV-1 has more genes and is longer than the nonimmune cells: IFN-g stimulates LY6 expression in kidney (69) corresponding human interval on Chr 8q24. That is, in the 0.61- and on inflamed intestinal epithelial cells in murine models of Mb between mouse Ly6e and Ly6h there are 10 Ly6 and Ly6- colitis (70). Because IFN production is a major innate immune related genes (Fig. 1), but there are no predicted genes with response to viral infection, it is reasonable to hypothesize that sequence similarity to Ly6 genes in the corresponding 0.14 Mb of viral infection may result in altered expression of LY6 proteins. human Chr 8 between Ly6e and Ly6h (55). Notably, LY6A and We suggest that this may underlie susceptibility to viral infections LY6C proteins do not have human orthologs. caused by Ly6 or Ly6-related genes.

LY6 proteins encoded in the critical interval for MAV-1 sus- In addition to our findings that Ly6 genes underlie the QTL for Downloaded from ceptibility are expressed on cells that either are targets of MAV-1 susceptibility to MAV-1, studies have implicated Ly6 genes for (monocytes/macrophages and endothelial cells) (14, 56) or are susceptibility to three other viruses. The chicken Ly6E locus is lymphoid or myeloid cells (or their precursors) that are effectors in a major candidate for susceptibility to Marek’s disease virus, innate and adaptive immunity (Table II), making them strong a herpesvirus, based on several experimental approaches (19, 71). candidates for involvement in susceptibility. Differences in se- The human Ly6 locus was identified in a whole-genome analysis

quence or expression of these proteins between susceptible and of lymphoblastoid cell susceptibility to HIV-1 (20), and three Ly6 http://www.jimmunol.org/ resistant strains may underlie the susceptibility differences among or Ly6-related genes were identified in a small interfering RNA mouse strains. LY6I, LY6C1, and LY6C2 are expressed on mon- screen for host factors required by HIV-1 (21). Ly6E was identi- ocytes, macrophages, and dendritic cells (reviewed in Ref. 18); fied as a host susceptibility factor for West Nile virus in a similar strain differences in expression patterns on Ly6.1 and Ly6.2 strains small interfering RNA screen (22). Nothing has been reported have been reported for LY6C (although it is not known whether about the mechanism by which Ly6 genes contribute to infection these differences were for LY6C1 or LY6C2 expression) (51). As by these or other viruses. In fact, infection of Ly6a knockout mice measured by immunohistochemistry, the LY6A protein is promi- by lymphocytic choriomeningitis virus demonstrated that LY6A is nently expressed in brain vascular elements in Ly6.2 strains dispensable for normal T cell function in acute and memory re- (C57BL/6 and AKR/J) (57, 58). These authors did not definitively sponses (61). We found no difference in susceptibility to MAV-1 by guest on September 29, 2021 show that the Ab staining was specific to endothelial cells, but it is infection between Ly6a knockout mice and BALB/c controls at consistent with endothelial expression of LY6A. In contrast, no either high or low virus doses (105 or 102 PFU, respectively) (I.W. expression is seen in vascular elements of Ly6.1 strains (BALB/c Althaus and K.R. Spindler, unpublished observations). Given the and C3H/HeJ) using the same Ab (58). It will be informative to number of Ly6-related proteins and the variety of cell and organ determine whether similar LY6A expression differences are seen types in which these genes are expressed, the mechanisms may be for additional Ly6.1 (MAV-1–resistant) and Ly6.2 (MAV-1– quite different for various viruses. However, the findings that susceptible) strains, as this may be a cause or effect of differences susceptibility to viruses from three different families maps to Ly6 in MAV-1 susceptibility. genes strongly suggest that the Ly6 gene products are key players Alterations in effector functionality of lymphoid or myeloid in virus–host interactions. cells might be caused by Ly6 gene differences and lead to sus- ceptibility to MAV-1. LY6E is expressed on intrathymic cells that Acknowledgments give rise to all the thymic subsets; it is differentially expressed We thank Steffi Ameres, Shanna Ashley, Marina Gerbin, and the University during T cell development and is found on immature T cells and of Michigan Genotyping Core for technical assistance in genotyping. We peripheral B cells (59, 60). LY6A is expressed on embryonic and are grateful to Dave Burke for numerous discussions on mapping strategy fetal aorta and liver, and on adult hematopoietic stem cells (re- and comments on the manuscript. We thank Tien-Huei Hsu and Matt Stier 2 2 viewed in Ref. 55). LY6A is differentially expressed on hemato- for discussion about Ly6 / genes and the manuscript. We thank William 2/2 poietic stem cells in Ly6.1 and Ly6.2 strains, with ∼100% of cells Stanford for the gift of Ly6a mice and BALB controls. We thank Tim and 25% of cells being positive, respectively. This is reminiscent Wiltshire for generously providing SNP database files prior to publication of the different levels of expression of LY6A in brain vascular and Janan Eppig for nomenclature advice. We are grateful to Phil King and Mike Imperiale for comments on the manuscript. elements, described above. Both T and B lymphocytes are im- portant for control of MAV-1 infection (15, 16). It is possible that functional differences in T cell- and/or B cell-mediated host re- Disclosures sponse to MAV-1 infection result from differences in LY6E, The authors have no financial conflicts of interest. LY6A, or less well characterized LY6 proteins in susceptible and resistant strains. Little is known about the physiological function References of the LY6 proteins, but they are thought to be involved in cell– 1. Brinton, M. A. 1997. Host susceptibility to viral disease. In Viral Pathogenesis. cell adhesion and signaling (reviewed in Refs. 18, 55). Functional N. Nathanson, R. Ahmed, R. Gonzalez-Scarano, D. E. Griffin, K. V. Holmes, roles for LY6 proteins in disease pathogenesis that may relate to F. A. Murphy, and H. L. Robinson, eds. Lippincott-Raven, Philadelphia, p. 303–328. 2. Perelygin, A. A., S. V. Scherbik, I. B. Zhulin, B. M. Stockman, Y. Li, and viral disease susceptibility are suggested by several findings. Ex- M. A. Brinton. 2002. Positional cloning of the murine flavivirus resistance gene. pression of LY6 proteins is induced on many mouse tissues in the Proc. Natl. Acad. Sci. USA 99: 9322–9327. The Journal of Immunology 3061

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