The evolution of S100A7: an unusual gene expansion in Myotis bats.

BACKGROUND: The S100A7 gene, also called psoriasin, was first described as an upregulated protein in psoriatic skin. For the past years, the importance of this protein as a key effector of innate immunity has been clearly established, not only due to its importance protecting against bacteria skin insult in humans, but also because of its important role in amplifying inflammatory processes. Given the importance of S100A7 in host defense, S100A7 genes have been mostly studied in humans. Here we provide a detailed analysis of the evolution of the gene family encoding for the S100A7 protein in mammals. RESULTS: Examination of several mammalian genomes revealed an unexpected variation in the copy number of S100A7. Among the most representative mammalian groups, we report that multiple events of duplication, gene loss and high mutation rates are shaping the evolution of this gene family. An unexpected result comes from Myotis species (order Chiroptera), where we found an outstanding S100A7 gene radiation, resulting in more than 10 copies in M. lucifugus and 5 copies in M. brandtii. These findings suggest a unique adaptive road in these species and are suggestive of special role of this protein in their immune system. CONCLUSIONS: We found different evolutionary histories among different mammalian groups. Overall, our results suggest that this gene family is evolving under the birth-and-death model of evolution. To our knowledge, this work represents the first detailed analysis of phylogenetic relationships of S100A7 within mammals and therefore will pave the way to further clarify their unique function in the immune system.

Analysis of substitution rates showed that TLR5 is evolving at different rates among mammalian groups.

BACKGROUND: Toll-like receptors (TLRs) are the most widely studied innate immunity receptors responsible for recognition of invading pathogens. Among the TLR family, TLR5 is the only that senses and recognizes flagellin, the major protein of bacterial flagella. TLR5 has been reported to be under overall purifying selection in mammals, with a small proportion of codons under positive selection. However, the variation of substitution rates among major mammalian groups has been neglected. Here, we studied the evolution of TLR5 in mammals, comparing the substitution rates among groups. RESULTS: In this study we analysed the TLR5 substitution rates in Euungulata, Carnivora, Chiroptera, Primata, Rodentia and Lagomorpha, groups. For that, Tajima's relative rate test, Bayesian inference of evolutionary rates and genetic distances were estimated with CODEML's branch model and RELAX. The combined results showed that in the Lagomorpha, Rodentia, Carnivora and Chiroptera lineages TLR5 is evolving at a higher substitution rate. The RELAX analysis further suggested a significant relaxation of selective pressures for the Lagomorpha (K = 0.22, p < 0.01), Rodentia (K = 0.58, p < 0.01) and Chiroptera (K = 0.65, p < 0.01) lineages and for the Carnivora ancestral branches (K = 0.13, p < 0.01). CONCLUSIONS: Our results show that the TLR5 substitution rate is not uniform among mammals. In fact, among the different mammal groups studied, the Lagomorpha, Rodentia, Carnivora and Chiroptera are evolving faster. This evolutionary pattern could be explained by 1) the acquisition of new functions of TLR5 in the groups with higher substitution rate, i.e. TLR5 neofunctionalization, 2) by the beginning of a TLR5 pseudogenization in these groups due to some redundancy between the TLRs genes, or 3) an arms race between TLR5 and species-specific parasites.

The evolution of S100A7 in primates: a model of concerted and birth-and-death evolution.

The human S100A7 resides in the epidermal differentiation complex (EDC) and has been described as a key effector of innate immunity. In humans, there are five S100A7 genes located in tandem-S100A7A, S100A7P1, S100AL2, S100A7, and S100AP2. The presence of several retroelements in the S100A7A/S100A7P1 and S100A7/S100A7P2 clusters suggests that these genes were originated from a duplication around ~ 35 million years ago, during or after the divergence of Platyrrhini and Catarrhini primates. To test this hypothesis, and taking advantage of the high number of genomic sequences available in the public databases, we retrieved S100A7 gene sequences of 12 primates belonging to the Cercopithecoidea and Hominoidea (Catarrhini species). Our results support the duplication theory, with at least one gene of each cluster being identified in both Cercopithecoidea and Hominoidea species. Moreover, given the presence of an ongoing gene conversion event between S100A7 and S100A7A, a high rate of mutation in S100A7L2 and the presence of pseudogenes, we proposed a model of concerted and birth-and-death evolution to explain the evolution of S100A7 gene family. Indeed, our results suggest that S100A7L2 most likely suffered a neofunctionalization in the Catarrhini group. Being S100A7 a major protein in innate defense, we believe that our findings could open new doors in the study of this gene family in immune system.

Strong selection of the TLR2 coding region among the Lagomorpha suggests an evolutionary history that differs from other mammals.

Toll-like receptors (TLRs) are one of the first lines of defense against pathogens and are crucial for triggering an appropriate immune response. Among TLRs, TLR2 is functional in all vertebrates and has high ability in detecting bacterial and viral pathogen ligands. The mammals' phylogenetic tree of TLR2 showed longer branches for the Lagomorpha clade, raising the hypothesis that lagomorphs experienced an acceleration of the mutation rate. This hypothesis was confirmed by (i) Tajima's test of neutrality that revealed different evolutionary rates between lagomorphs and the remaining mammals with lagomorphs presenting higher nucleotide diversity; (ii) genetic distances were similar among lagomorphs and between lagomorphs and other mammals; and (iii) branch models reinforced the existence of an acceleration of the mutation rate in lagomorphs. These results suggest that the lagomorph TLR2 has been strongly involved in pathogen recognition, which probably caused a host-pathogen arms race that led to the observed acceleration of the mutation rate.

Evolution of CCR5 and CCR2 Genes in Bats Showed Multiple Independent Gene Conversion Events.

Chemokine receptors are an important determinant for the infectiousness of different pathogens, which are able to target the host cells by binding to the extracellular domains of these proteins. This is the mechanism of infection of HIV-1, among other concerning human diseases. Over the past years, it has been shown that two chemokine receptors, CCR2 and CCR5, have been shaped by events of gene conversion in different mammalian lineages, which has been linked to a possible selective advantage against pathogens. Here, by taking advantage of available bat genomes, we present the first insight of CCR2 and CCR5 evolution within the Chiroptera order. In total, four independent events of recombination between CCR2 and CCR5 were detected: two in a single species, Miniopterus natalensis; one in two species from the Rhinolophoidea superfamily; and one in four species from the Pteropodidae family. The regions affected by the gene conversions were generally extensive and always encompassed extracellular domains. Overall, we demonstrate that CCR2 and CCR5 have been subject to extensive gene conversion in multiple species of bats. Considering that bats are known to be large reservoirs of virus in nature, these results might indicate that chimeric CCR2-CCR5 genes might grant some bat species a selective advantage against viruses that rely in the extracellular portions of either CCR2 or CCR5 as gateways into the cell.

Evolution of TRIM5 and TRIM22 in Bats Reveals a Complex Duplication Process.

The innate immunological response in mammals involves a diverse and complex network of many proteins. Over the last years, the tripartite motif-containing protein 5 (TRIM5) and 22 (TRIM22) have shown promise as restriction factors of a plethora of viruses that infect primates. Although there have been studies describing the evolution of these proteins in a wide range of mammals, no prior studies of the TRIM6/34/5/22 gene cluster have been performed in the Chiroptera order. Here, we provide a detailed analysis of the evolution of this gene cluster in several bat genomes. Examination of different yangochiroptera and yinpterochiroptera bat species revealed a dynamic history of gene expansion occurring in TRIM5 and TRIM22 genes. Multiple copies of TRIM5 were found in the genomes of several bats, demonstrating a very low degree of conservation in the synteny of this gene among species of the Chiroptera order. Our findings also reveal that TRIM22 is often found duplicated in yangochiroptera bat species, an evolutionary phenomenon not yet observed in any other lineages of mammals. In total, we identified 31 TRIM5 and 19 TRIM22 amino acids to be evolving under positive selection, with most of the residues being placed in the PRYSPRY domain, known to be responsible for binding to the viral capsid during restriction in the primate orthologous TRIM proteins. Altogether, our results help to shed light on the distinctive role of bats in nature as reservoirs of viruses, many of which have become threatening zoonotic diseases through virus spillover in the last decades.

Identification of a Novel Myxoma Virus C7-Like Host Range Factor That Enabled a Species Leap from Rabbits to Hares.

Myxoma virus (MYXV) is naturally found in rabbit Sylvilagus species and is known to cause lethal myxomatosis in European rabbits (Oryctolagus cuniculus). In 2019, an MYXV strain (MYXV strain Toledo [MYXV-Tol]) causing myxomatosis-like disease in Iberian hares (Lepus granatensis) was identified. MYXV-Tol acquired a recombinant region of ∼2.8 kb harboring several new genes, including a novel host range gene (M159) that we show to be an orthologous member of the vaccinia virus C7 host range family. Here, to test whether M159 alone has enabled MYXV to alter its host range to Iberian hares, several recombinant viruses were generated, including an MYXV-Tol ΔM159 (knockout) strain. While MYXV-Tol underwent fully productive infection in hare HN-R cells, neither the wild-type MYXV-Lau strain (lacking M159) nor vMyxTol-ΔM159 (deleted for M159) was able to infect and replicate, showing that the ability of MYXV-Tol to infect these cells and replicate depends on the presence of M159. Similar to other C7L family members, M159 was shown to be expressed as an early/late gene but was translocated into the nucleus at later time points, indicating that further studies are needed to elucidate its role in the nucleus. Finally, in rabbit cells, the M159 protein did not contribute to increased replication but was able to upregulate the replication levels of MYXV in nonpermissive and semipermissive human cancer cells, suggesting that the M159-targeted pathway is conserved across mammalian species. Altogether, these observations demonstrate that the M159 protein plays a critical role in determining the host specificity of MYXV-Tol in hare and human cells by imparting new host range functions. IMPORTANCE The coevolution of European rabbit populations and MYXV is a textbook example of an arms race between a pathogen and a host. Recently, a recombinant MYXV (MYXV-Tol) crossed the species barrier by jumping from leporid species to another species, causing lethal myxomatosis-like disease. Given the highly pathogenic nature of this new virus in hares and the incidences of other poxvirus cross-species spillovers into other animals, including humans, it is important to understand how and why MYXV-Tol was able to become virulent in a new host species. The results presented clearly demonstrate that M159 is the key factor allowing MYXV-Tol replication in hare cells by imparting new host range functions. These results have the potential to improve current knowledge about the virulence of poxviruses and provide a platform to better understand the new MYXV-Tol, rendering the virus capable of leaping into a new host species.

Convergent Loss of the Necroptosis Pathway in Disparate Mammalian Lineages Shapes Viruses Countermeasures.

Programmed cell death is a vital process in the life cycle of organisms. Necroptosis, an evolutionary form of programmed necrosis, contributes to the innate immune response by killing pathogen-infected cells. This virus-host interaction pathway is organized around two components: the receptor-interacting protein kinase 3 (RIPK3), which recruits and phosphorylates the mixed lineage kinase-like protein (MLKL), inducing cellular plasma membrane rupture and cell death. Critically, the presence of necroptotic inhibitors in viral genomes validates necroptosis as an important host defense mechanism. Here, we show, counterintuitively, that in different mammalian lineages, central components of necroptosis, such as RIPK3 and MLKL, are deleted or display inactivating mutations. Frameshifts or premature stop codons are observed in all the studied species of cetaceans and leporids. In carnivores' genomes, the MLKL gene is deleted, while in a small number of species from afrotheria and rodentia premature stop codons are observed in RIPK3 and/or MLKL. Interestingly, we also found a strong correlation between the disruption of necroptosis in leporids and cetaceans and the absence of the N-terminal domain of E3-like homologs (responsible for necroptosis inhibition) in their naturally infecting poxviruses. Overall, our study provides the first comprehensive picture of the molecular evolution of necroptosis in mammals. The loss of necroptosis multiple times during mammalian evolution highlights the importance of gene/pathway loss for species adaptation and suggests that necroptosis is not required for normal mammalian development. Moreover, this study highlights a co-evolutionary relationship between poxviruses and their hosts, emphasizing the role of host adaptation in shaping virus evolution.

Coinfections of Novel Polyomavirus, Anelloviruses and a Recombinant Strain of Myxoma Virus-MYXV-Tol Identified in Iberian Hares.

Viruses are ubiquitous in nature; however, very few have been identified in the Leporid species. In the fall of 2018, an outbreak of myxomatosis in Iberian hares (Lepus granatensis) was reported in Spain and a novel recombinant myxoma virus strain (MYXV-Tol) was identified. To investigate variability within the recombinant region of the MYXV-Tol and identify any potential viral coinfections, samples (ear, eyelid or vaginal) of Iberian hares were collected from Spain and analyzed. The presence of the recombinant region of the MYXV-Tol was confirmed in six out of eleven samples analyzed. Additionally, a polyomavirus (family Polyomaviridae), representing a putative new species, and anelloviruses (family Anelloviridae) belonging to two putative species were identified, some as coinfection with the recombinant MYXV-Tol. The two polyomavirus genomes were identified in two hares and share >99% genome-wide identity. Based on the analysis of their large T-antigen, the new polyomavirus clusters in a distant clade from other mammals sharing <64% amino acid identity. A total of 14 anelloviruses were identified, which share 63-99% genome-wide identity. Overall, our results show a coinfection of different DNA viruses in the studied samples and raise awareness regarding the extensive unsampled diversity of viruses in hares.

Not so unique to Primates: The independent adaptive evolution of TRIM5 in Lagomorpha lineage.

The plethora of restriction factors with the ability to inhibit the replication of retroviruses have been widely studied and genetic hallmarks of evolutionary selective pressures in Primates have been well documented. One example is the tripartite motif-containing protein 5 alpha (TRIM5α), a cytoplasmic factor that restricts retroviral infection in a species-specific fashion. In Lagomorphs, similarly to what has been observed in Primates, the specificity of TRIM5 restriction has been assigned to the PRYSPRY domain. In this study, we present the first insight of an intra-genus variability within the Lagomorpha TRIM5 PRYSPRY domain. Remarkably, and considering just the 32 residue-long v1 region of this domain, the deduced amino acid sequences of Daurian pika (Ochotona dauurica) and steppe pika (O. pusilla) evidenced a high divergence when compared to the remaining Ochotona species, presenting values of 44% and 66% of amino acid differences, respectively. The same evolutionary pattern was also observed when comparing the v1 region of two Sylvilagus species members (47% divergence). However, and unexpectedly, the PRYSPRY domain of Lepus species exhibited a great conservation. Our results show a high level of variation in the PRYSPRY domain of Lagomorpha species that belong to the same genus. This suggests that, throughout evolution, the Lagomorpha TRIM5 should have been influenced by constant selective pressures, likely as a result of multiple different retroviral infections.

Genetic Characterization of a Recombinant Myxoma Virus in the Iberian Hare (Lepus granatensis).

Myxomatosis is a lethal disease in wild European and domestic rabbits (Oryctolagus cuniculus), which is caused by a Myxoma virus (MYXV) infection-a leporipoxvirus that is found naturally in some Sylvilagus rabbit species in South America and California. The introduction of MYXV into feral European rabbit populations of Australia and Europe, in the early 1950s, demonstrated the best-documented field example of host-virus coevolution, following a cross-species transmission. Recently, a new cross-species jump of MYXV has been suggested in both Great Britain and Spain, where European brown hares (Lepus europaeus) and Iberian hares (Lepus granatensis) were found dead with lesions consistent with those observed in myxomatosis. To investigate the possibility of a new cross-species transmission event by MYXV, tissue samples collected from a wild Iberian hare found dead in Spain (Toledo region) were analyzed and deep sequenced. Our results reported a new MYXV isolate (MYXV Toledo) in the tissues of this species. The genome of this new virus was found to encode three disruptive genes (M009L, M036L, and M152R) and a novel ~2.8 kb recombinant region, which resulted from an insertion of four novel poxviral genes towards the 3' end of the negative strand of its genome. From the open reading frames inserted into the MYXV Toledo virus, a new orthologue of a poxvirus host range gene family member was identified, which was related to the MYXV gene M064R. Overall, we confirmed the identity of a new MYXV isolate in Iberian hares, which, we hypothesized, was able to more effectively counteract the host defenses in hares and start an infectious process in this new host.