Differential Loss of OAS Genes Indicates Diversification of Antiviral Immunity in Mammals

Abstract

One of the main mechanisms of inducing an antiviral response depends on 2′-5′-oligoadenylate synthetases (OAS), which sense double-stranded RNA in the cytoplasm and activate RNase L. Mutations leading to the loss of functional OAS1 and OAS2 genes have been identified as important modifiers of the human immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we performed comparative genomics to search for inactivating mutations of OAS genes in other species of mammals and to establish a model for the diversifying evolution of the OAS gene family. We found that a recombination of the OAS and OAS-like (OASL) loci has led to the loss of OAS2 in camelids, which also lack OAS3. Both paralogs of OASL and OAS3 are absent in Asian pangolins. An evolutionarily ancient OAS paralog, which we tentatively name OAS4, has been lost in pangolins, bats and humans. A previously unknown OAS gene, tentatively named OAS5, is present in Yangochiroptera, a suborder of bats. These differences in the OAS gene repertoire may affect innate immune responses to coronaviruses and other RNA viruses.

1. Introduction

Mammalian cells have different mechanisms to sense viral infections and initiate an innate immune response. One of the main approaches is the binding of specific sensor proteins to nucleic acids with infection-associated features, such as cytoplasmic localization of DNA, presence of double-stranded RNA or presence of Z-nucleic acid structures [1,2,3]. These sensors trigger signaling cascades that lead to reactions of the infected cell, the tissue and the immune system aimed at stopping the replication and spread of viruses. 2′-5′ oligoadenylate synthetases (OASs) bind double-stranded (ds) RNA in the cytoplasm and subsequently catalyze the oligomerization of ATP to 2′-5′-oligoadenylate. This oligomer activates RNase L, which subsequently degrades viral RNA to suppress the replication of the virus [1]. RNA degradation also generates small self-RNA, which amplifies antiviral innate immunity by binding to other receptors [4]. Humans have three OAS proteins comprising either one, two or three repeats of a nucleotidyl-transferase (NT) and an OAS1 C-terminal (OAS1C) domain. Besides catalytically active OAS enzymes, an OAS-like (OASL) protein contributes to the control of signaling in response to dsRNA in the cytoplasm. OASL activates retinoic acid inducible gene I (RIG-I), a sensor of cytoplasmic dsRNA [5,6], and suppresses cyclic GMP-AMP synthase (cGAS), a sensor of cytoplasmic DNA, leading to reduced replication of RNA viruses and enhanced replication of DNA viruses [7] (Figure 1).

The OAS-RNase L pathway is involved in the defense against many viruses, some of which have evolved counteractive strategies [10,11,12,13]. Recent research has demonstrated that this pathway is also involved in the human immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [14]. Specifically, mutations inactivating OAS1, OAS2 or RNase L impair the normal immune response and lead to SARS-CoV-2–related multisystem inflammatory syndrome in children [14]. OAS1 was reported to inhibit SARS-CoV-2 through its prenylated isoform [15]. Interestingly, the antiviral activity of OAS1 differs among primates due to mutations of its amino acid sequence, suggesting that a reduction in or loss of OAS1 catalytic activity may have had advantages in evolution [16]. Another report linked the decay of OAS1 mRNAs with the risk of COVID-19 hospitalization [17].

OAS genes are evolutionarily ancient [9,18]. Species from the major phylogenetic metazoan clades contain different sets of OAS genes, and even within tetrapods, significant variation has been reported [9,19]. For instance, OAS1 has undergone duplications in rodents and cattle [15,20]. In contrast to humans, some other placental mammals and marsupials have two copies of OASL [18], indicating that two OASL genes were also present in the genome of evolutionary ancestors of humans, and one of these copies was lost in the lineage leading to humans. Recently, a new OAS paralog, hereafter referred to as OAS4, was reported to be present in some amphibian, sauropsid and mammalian species [9]. Phylogenetic analysis suggested that this paralog emerged earlier in evolution than OAS1, OAS2 and OAS3 and that it was lost in rodents and primates [9]. Likewise, OAS3 has been lost in cetartiodactyls and in the tree shrew [20,21].

Comparative genomic studies have revealed a high diversity of innate immune genes in mammals with striking cases of gene degeneration in bats and pangolins [22,23,24,25,26]. Genes involved in the sensing of cytoplasmic DNA, such as CGAS and STING1, and cytoplasmic RNA, such as IFIH1/MDA5 and ZBP1, have been lost during the evolution of pangolins [23,24]. As bats are considered a likely source of SARS-CoV-2 and pangolins possibly were intermediate hosts of this virus [27,28], we put forward the hypothesis that alterations in antiviral innate immunity may contribute to the differential persistence of viruses in populations of such species with the risk of virus spillover potentially causing pandemics.

Here, we extended the concept of gene loss as a driver of inter-species variation in innate immunity and screened a selected group of mammalian species for cases of gene loss in the OAS gene family. The results of this study have implications for comparative immunology, and the selection of animal models for studying host–virus interactions.

2. Materials and Methods

Comparative genomics was performed according to an approach reported previously [23,29]. Nucleotide and amino acid sequences were downloaded from GenBank. Accession numbers are indicated in the text. Genes were identified in the genome sequences of Homo sapiens, assembly: GRCh38.p14 (GCF_000001405.40); Camelus bactrianus, assembly: Ca_bactrianus_MBC_1.0 (GCF_000767855.1); Camelus dromedarius, assembly: CamDro3 (GCF_000803125.2); Vicugna pacos, assembly: VicPac3.1 (GCF_000164845.3); Canis familiaris, assembly: ROS_Cfam_1.0 (GCF_014441545.1); Manis javanica, assembly: YNU_ManJav_2.0 (GCF_014570535.1); Manis pentadactyla, assembly: YNU_ManPten_2.0 (GCF_014570555.1); Rhinolophus sinicus, assembly: ASM188883v1 (GCF_001888835.1); Molossus molossus, assembly: mMolMol1.p (GCF_014108415.1); Myotis myotis, assembly: mMyoMyo1.p (GCF_014108235.1); and Artibeus jamaicensis, assembly: WHU_Ajam_v2 (GCF_014825515.1).

The Basic Local Alignment Search Tool (BLAST) [30] was used to determine sequence similarities. Sequence alignments were made with Multalin (http://multalin.toulouse.inra.fr/multalin/, accessed on 30 December 2022) and MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/, accessed on 30 December 2022). Protein domains were identified with the NCBI Conserved Domain search tool [31]. Phylogenetic relationships and divergence times of phylogenetic lineages were obtained from the Timetree website (www.timetree.org, accessed on 30 December 2022) [32].

3. Results

3.1. Comparative Genomics Reveals Loss of OAS Genes during the Evolution of Humans and Camelids

We performed comparative genomics to determine the presence or absence of OAS genes in a selected subset of mammalian species. This study was focused on humans and clades of mammals (camelids, pangolins and bats), which were suspected or confirmed as reservoirs of coronaviruses with zoonotic potential [27,33,34,35]. An overview of the distribution of OAS family genes in the various species is provided in

Table 1. Conservation of OAS genes in mammalian species investigated in this study.

Species	Binomial Name	OAS1	OAS2	OAS3	OAS4	OAS5	OASL	OASL2
Human	Homo sapiens	+	+	+	–	–	+	–
Bactrian camel	Camelus bactrianus	+	–	–	+	–	+	–
Arabian camel	Camelus dromedarius	+	–	–	+	–	+	–
Alpaca	Vicugna pacos	+	–	–	+	–	+	–
Dog	Canis familiaris	+	+	+	+	–	+	+
Malayan pangolin	Manis javanica	+ *	+	–	–	–	–	–
Chinese pangolin	Manis pentadactyla	+	+	–	–	–	–	–
Chinese rufous horseshoe bat	Rhinolophus sinicus	+	+	+	–	–	+	+
Pallas’s mastiff bat	Molossus molossus	+ *	+	+	–	+	+	–
Greater mouse-eared bat	Myotis myotis	+	+	+	–	+	+	–
Jamaican fruit bat	Artibeus jamaicensis	+	+ *	+	–	+	+	–

Notes: +, presence of gene; –, absence of gene; *, frame shift or premature stop codon.

. The GenBank accession numbers of proteins encoded by these genes are listed in Table S1, and the corresponding amino acid sequences are documented in Figure S1. Together with the knowledge of phylogenetic relationships of mammals [32], the distribution of OAS paralogs in the various species allowed us to infer which genes were lost or gained during the evolutionary history of particular species or clades.

The gene loci of the OAS family share the same neighboring genes (synteny) in many but not all species. Comparative analysis indicated that chromosomal positions 12q24.13, 12q24.31 and 5q15 in the human genome correspond to the evolutionarily ancestral loci of OAS gene paralogs. The conservation of OAS4 in cetartiodactyls (Figure 2), African elephant and mouse lemur (Figure S1), and the absence of an OAS4 gene at human chromosome 5q15 indicated that, in agreement with a recent report [9], OAS4 was lost in the human lineage (Figure 2). Likewise, one of two ancestral OASL genes was lost during the evolution of humans (Figure 2).

Camelids, which are a subclade of cetartiodactyls, have lost OASL2 but retained OAS4 (Figure 2). In line with a previous report on artiodactyls [20], OAS3 is absent in camels and alpaca (Figure 2). Camelids are unique within terrestrial cetartiodactyls because they lack OAS2 (Figure 2). The unusual tandem arrangement of OAS1 and OASL in camels and alpacas indicates that a recombination event occurred in a common ancestor of camelids, and OAS2 was probably lost in the course of this chromosomal rearrangement.

3.2. Pangolins have Lost Multiple OAS Genes

Next, we investigated pangolins, which are carriers of SARS-CoV-2-like viruses [28] and have a degenerated set of innate immune genes [23,24,25]. Pangolins constitute the clade Pholidota, which is most closely related to Carnivora (dog-like and cat-like mammals). Therefore, we compared the genomes of the Malayan pangolin (Manis javanica), Chinese pangolin (Manis pentadactyla) and the dog. In contrast to the dog, which has the full set of ancestral OAS genes, the Asian pangolins lack OAS3, OAS4, and both OASL1 and OASL2 (Figure 3 and Figure S2). At present, gene annotations are not available for African pangolins. Due to the loss of four ancestral genes, the OAS gene family is massively degenerated in Malayan and Chinese pangolins (Figure 3).

3.3. The Evolution of Bats Was Associated with the Diversification of OAS Paralogs

Bats, comprising the phylogenetic clade Chiroptera, have special adaptations of the immune system that allow them to act as reservoirs of many viruses [36,37,38,39], most likely including the virus from which SARS-CoV-2 evolved [27]. We performed an exploratory analysis of OAS genes in a subset of bats and found that OAS2, OAS3 and at least one OASL gene are conserved in bats (Figure 4). OAS1 is also present in all species investigated; however, the OAS1 ortholog of Molossus molossus contains inactivating mutations. OAS3 of the same species is predicted to contain an extended number of domains (Figure S3). The sequence modifications and their impact on the gene function remain to be investigated because sequence confirmations and analyses of gene transcripts were not within the scope of the present study. OAS4 was absent in all bats investigated, suggesting that this gene has been lost (Figure 4). Unexpectedly, we identified an as-yet-uncharacterized paralog, tentatively named OAS5, by sequence similarity searches in the genomes of species of the suborder Yangochiroptera [40]. OAS5 is located between the TBCK and NPNT genes, a locus that does not contain an OAS paralog in any species investigated except those of the clade Yangochiroptera. OAS5 genes encode proteins with high sequence similarity to OAS1 (Figure 5). In contrast to all other known OAS paralogs, OAS5 does not have introns, so the open reading frame is entirely contained in a single exon, suggesting that OAS5 has arisen by reverse transcription of an OAS1 mRNA followed by insertion of the complementary DNA into the genome of a germ cell in an ancestor of Yangochiroptera (Figure 4).

Taken together, these data show that the OAS gene family underwent lineage-specific changes leading to significant differences in the repertoire of OAS paralogs in major clades of extant mammals (Figure 6).

4. Discussion

The OAS–RNase L pathway is one of the central mechanisms of antiviral defense. A large body of literature exists on the roles of OAS genes in human cells and cells of model organisms [2,42,43,44]. It was noted early on that the OAS gene complement differs between humans and rodents as the latter have an amplification of OAS1 genes [45]. Studies of other species, including many domestic and wild mammals, revealed further inter-species differences [18,20,21,46]. The present study extends the comparative analysis of the OAS family to a set of species implicated in the spread of coronaviruses and reveals previously unknown taxon-specific compositions of the OAS gene family.

Our results show that the OAS gene family is larger than the set of OAS1, OAS2, OAS3 and OASL genes in humans. Two copies of OASL genes have previously been identified in other species, and an as-yet-uncharacterized OAS paralog, which we name OAS4, was reported previously [9]. The present study identified another paralog, tentatively named OAS5, in a subgroup of bats. It will be important to investigate OAS4 and OAS5 proteins with regard to RNA-binding properties, catalytic activities and functions in antiviral defense and other processes.

Importantly, peculiar features of the OAS gene sets were found in camelids, pangolins and bats, which have been implicated in the origin of viral zoonoses [27,33,34]. These findings provide a basis for studying the impact of particular OAS gene combinations on the induction of anti-viral defense in follow-up studies. For a comprehensive evaluation of the significance of OAS genes in zoonoses, reports on other hosts of coronaviruses [27,47,48] and zoonoses involving other viruses should also be considered [35,49,50,51,52].

Vaccines play key roles in the fight against viral pathogens in human and veterinary medicine. Therefore, it is of special clinical interest to transfer basic immunological knowledge into the development of highly effective vaccines. OAS proteins are involved in early immune responses against RNA viruses and perhaps also against modified-live RNA virus vaccines [10,53]. Yellow fever vaccination induced upregulation of OAS1 among other innate antiviral molecules and also a strong acquired serological and cellular immune response [54]. A drawback in the development of vaccines directed against RNA viruses is their generally high rate of sequence mutations, as exemplified by one of the economically most important RNA viruses in veterinary medicine, the porcine reproductive and respiratory syndrome virus (PRRSV) [55,56]. Despite its high mutation rate, it is possible to design effective vaccines [57]. The PRRSV is an ssRNA virus, but dsRNA intermediates are formed during intracellular replication. Thus, veterinary species such as pigs could serve as models for investigating the role of OAS gene family members and other components of innate immunity in modulating the efficacy of vaccines.

This study has limitations that need to be considered in the interpretation of the data. First, most genes were predicted by automated computational analysis, which is used by GenBank to annotate genes of non-model species [58]. Although these predictions are useful for compiling orthologous genes, structural details of the predicted genes need to be corrected in some cases [59,60]. Second, the structure of the predicted mRNAs and proteins has not been experimentally confirmed yet. However, the mapping of RNA-seq reads onto the GenBank genome sequences confirms that the predicted exons are indeed present in mature mRNAs of many species investigated (Figure S3A). Finally, the proteins encoded by the predicted OAS family genes remain to be characterized with regard to their biochemical features and their roles in intracellular signaling in response to viral infections.

The differences in the conservation of established OAS family members, OAS1, OAS2, OAS3, OASL and OASL2, and possibly also the presence or absence of OAS4 and OAS5, may contribute to differences in responses to viral dsRNA in mammalian species. Studies are warranted to determine the impact of specific OAS gene sets on the control of viruses in phylogenetically diverse mammals [39,49,61,62].