Ancient and Recent Selective Pressures Shaped Genetic Diversity at AIM2-Like Nucleic Acid Sensors

Rachele Cagliani 2 Diego Forni 2 Mara Biasin 1 Manuel Comabella 0 Franca R. Guerini 5 Stefania Riva 2 Uberto Pozzoli 2 Cristina Agliardi 5 Domenico Caputo 5 Sunny Malhotra 0 Xavier Montalban 0 Nereo Bresolin 2 4 Mario Clerici 3 5 Manuela Sironi 2 0 Department of Neurology-Neuroimmunology, Centre d'Esclerosi M ultiple de Catalunya , Cemcat, Hospital Universitari Vall d'Hebron (HUVH) , Barcelona, Spain 1 Department of Biomedical and Clinical Sciences, University of Milan , Italy 2 Bioinformatics Laboratory, Scientific Institute IRCCS E. Medea , Bosisio Parini (LC), Italy 3 Chair of Immunology, Department of Physiopathology and Transplantation, University of Milan , Italy 4 Dino Ferrari Centre, Department of Physiopathology and Transplantation, University of Milan, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico , Milan, Italy 5 Laboratory of Molecular Medicine, Don C. Gnocchi Foundation ONLUS , IRCCS, Milan, Italy AIM2-like receptors (ALRs) are a family of nucleic acid sensors essential for innate immune responses against viruses and bacteria. We performed an evolutionary analysis of ALR genes (MNDA, PYHIN1, IFI16, and AIM2) by analyzing inter- and intraspecies diversity. Maximum-likelihood analyses indicated that IFI16 and AIM2 evolved adaptively in primates, with branch-specific selection at the catarrhini lineage for IFI16. Application of a population genetics-phylogenetics approach also allowed identification of positive selection events in the human lineage. Positive selection in primates targeted sites located at the DNA-binding interface in both IFI16 and AIM2. In IFI16, several sites positively selected in primates and in the human lineage were located in the PYD domain, which is involved in protein-protein interaction and is bound by a human cytomegalovirus immune evasion protein. Finally, positive selection was found to target nuclear localization signals in IFI16 and the spacer region separating the two HIN domains. Population genetic analysis in humans revealed that an IFI16 genic region has been a target of long-standing balancing selection, possibly acting on two nonsynonymous polymorphisms located in the spacer region. Data herein indicate that ALRs have been repeatedly targeted by natural selection. The balancing selection region in IFI16 carries a variant with opposite risk effect for distinct autoimmune diseases, suggesting antagonistic pleiotropy. We propose that the underlying scenario is the result of an ancestral and still ongoing hostpathogen arms race and that the maintenance of susceptibility alleles for autoimmune diseases at IFI16 represents an evolutionary trade-off. - Data deposition: The IFI16 coding sequences for Macaca fascicularis and Chlorocebus aethiops have been deposited at GenBank under the accessions KF154419 and KF154420. Introduction Mammalian nucleic acid-sensing receptors play essential roles in the recognition of infectious agents and in triggering innate and adaptive immune responses. Different classes of nucleic acid-sensing molecules have been identified; these molecules are classified on the basis of cellular localization, target specificity, and downstream signaling pathway. Among them, toll-like receptors (TLRs) are the best characterized class, and at least four members (TLR3, TLR7, TLR8, and TLR9), located at the endosomal membrane, are specialized in viral sensing (Desmet and Ishii 2012). TLRs signal through MyD88 or TRIF to induce the release either of inflammatory The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. cytokines or of type I interferons (IFNs). The cytoplasmic detection of viral RNA is also mediated by cytosolic RIG-I-like receptors (RIG-I, IFIH1, and DHX58), which elicit type I IFN responses through the mitochondrial antiviral signaling (MAVS) adaptor (Desmet and Ishii 2012). NOD2, a member of the NOD-like receptor (NLR) family, also impinges on MAVS upon sensing single-stranded RNA (ssRNA), whereas NLRP3 is activated by ssRNA or dsRNA resulting in the formation of the inflammasome complex, which is mediated by PYCARD (also known as ASC) (Desmet and Ishii 2012). Finally, the IFNinducible HIN-200 gene family, also called PYHIN gene family, comprises a class of homologous viral sensor proteins characterized by the presence of an N-terminal pyrin-domain and a 200-amino acid signature motif (HIN-200 domain) (Veeranki and Choubey 2012). In humans, four members of this family have been identified and are encoded by a cluster of genes (MNDA, PYHIN1, IFI16, and AIM2) located on chromosome 1. These proteins share a pyrin motif involved in proteinprotein interactions, as well as one (AIM2, MNDA, and PYHIN1) or two (IFI16) HIN-200 domains that mediate binding to double-stranded DNA (dsDNA) (fig. 1) (Schattgen and Fitzgerald 2011). The best studied PYHIN family members are AIM2 and IFI16. The former is a sensor of cytosolic DNA, which triggers the inflammosome pathway through PYCARD resulting in caspase1-mediated cleavage of IL-1b (Schattgen and Fitzgerald 2011). Conversely, IFI16 has a mainly nuclear activity (in analogy to MNDA and PYHIN1), although it can also sense dsDNA in the cytoplasm, as its nuclear-cytoplasmic shuttling is regulated by a multipartite nuclear localization signal (Li et al. 2012). IFI16 signals through STING-dependent pathways. PYHIN proteins, according to their function as innate DNA sensors, are also termed AIM2-like receptors (ALRs) (Unterholzner et al. 2010). A recent analysis of ALR genes in mammals indicated that the cluster is extremely dynamic: Distinct species carry diverse sets of functional genes, suggesting that strong selective pressures have been acting on these loci (Brunette et al. 2012). Indeed, evolutionary analysis of other genes involved in nucleic acid sensing or in the downstream signaling pathways identified signatures of natural selection. Thus, MAVS evolved adaptively in primates, the underlying pressure being accounted for by hepaciviruses (Patel et al. 2012). Likewise, analysis of RIG-I-like receptors in human populations revealed signatures of local adaptation at the IFIH1 and DHX58 genes (Fumagalli et al. 2010; Vasseur et al. 2011; Quintana-Murci and Clark 2013). These observations are in line with viruses, and pathogens, in general, being a major determinant of molecular evolution in mammals and human populations (Kosiol et al. 2008; Fumagalli et al. 2011). Herein, we performed an evolutionary study of the ALR cluster by analyzing both inter- and intraspecies diversity. Materials and Methods Evolutionary Analysis in Mammals Primate sequences for MNDA, PYHIN1, IFI16, and AIM2 were retrieved from the Ens (...truncated)