T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) contains the canonical phosphoprotein phosphatase 1 (PPP1) binding motif, composed by the amino acid sequence RVSF. We identified and validated the binding of TCTEX1D4 to PPP1 and demonstrated that indeed this protein is a novel PPP1 interacting protein. Analyses of twenty-one mammalian species available in public databases and seven Lagomorpha sequences obtained in this work showed that the PPP1 binding motif 90RVSF93 is present in all of them and is flanked by a palindromic sequence, PLGS, except in three species of pikas (Ochotona princeps, O. dauurica and O. pusilla). Furthermore, for the Ochotona species an extra glycosylation site, motif 96NLS98, and the loss of the palindromic sequence were observed. Comparison with other lagomorphs suggests that this event happened before the Ochotona radiation. The dN/dS for the sequence region comprising the PPP1 binding motif and the flanking palindrome highly supports the hypothesis that for Ochotona species this region has been evolving under positive selection. In addition, mutational screening shows that the ability of pikas TCTEX1D4 to bind to PPP1 is maintained, although the PPP1 binding motif is disrupted, and the N- and C-terminal surrounding residues are also abrogated. These observations suggest pika as an ideal model to study novel PPP1 complexes regulatory mechanisms.

The mammalian RIG-I-like receptors, RIG-I, MDA5 and LGP2, are a family of DExD/H box RNA helicases responsible for the cytoplasmic detection of viral RNA. These receptors detect a variety of RNA viruses, or DNA viruses that express unusual RNA species, many of which are responsible for a great number of severe and lethal diseases. Host innate sentinel proteins involved in pathogen recognition must rapidly evolve in a dynamic arms race with pathogens, and thus are subjected to long-term positive selection pressures to avoid potential infections. Using six codon-based Maximum Likelihood methods, we were able to identify specific codons under positive selection in each of these three genes. The highest number of positively selected codons was detected in MDA5, but a great percentage of these codons were located outside of the currently defined protein domains for MDA5, which likely reflects the imposition of both functional and structural constraints. Additionally, our results support LGP2 as being the least prone to evolutionary change, since the lowest number of codons under selection was observed for this gene. On the other hand, the preponderance of positively selected codons for RIG-I were detected in known protein functional domains, suggesting that pressure has been imposed by the vast number of viruses that are recognized by this RNA helicase. Furthermore, the RIG-I repressor domain, the region responsible for recognizing and binding to its RNA substrates, exhibited the strongest evidence of selective pressures. Branch-site analyses were performed and several species branches on the three receptor gene trees showed evidence of episodic positive selection. In conclusion, by looking for evidence of positive evolutionary selection on mammalian RIG-I-like receptor genes, we propose that a multitude of viruses have crafted the receptors biological function in host defense, specifically for the RIG-I gene, contributing to the innate species-specific resistance/susceptibility to diverse viral pathogens.

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.