Experimental assessment of K locus effects on the gray wolf response to virus

In North American gray wolves, black coat color is dominantly inherited via a three base pair coding deletion in the canine beta defensin 3 (CBD103) gene. This three base pair deletion, called the KB allele, was introduced through hybridization with dogs and subsequently underwent a selective sweep that increased its frequency in wild wolves. Despite apparent positive selection, KBB wolves have significantly lower fitness than wolves with the KyB genotype, even though the two genotypes show no observable differences in black coat color. Thus, the KB allele is thought to have pleiotropic effects on as-yet unknown phenotypes. Given the role of skin-expressed CBD103 in innate immunity, we hypothesized that the KB allele influences the gene regulatory response to viral infection. To test this hypothesis, we developed a panel of primary epidermal keratinocyte cell cultures from 24 wild North American gray wolves (both Kyy and KyB genotypes) and generated immortalized Kyy and CBD103 knockout lines. We assessed the transcriptome-wide responses of wolf keratinocytes to polyinosinic:polycytidylic acid (polyI:C), which mimics infection by a double-stranded RNA virus, and to live canine distemper virus. Keratinocytes with the KyB genotype and with the Kyy genotype had similar gene regulatory responses to viral infection, suggesting that this response does not explain pleiotropic effects of the KB allele on fitness. This study supports the feasibility of using cell culture methods to investigate the phenotypic effects of naturally occurring genetic variation in wild mammals.