Introduction
The family
Polyomaviridae is composed of small, non-enveloped, double-stranded DNA viruses with a circular genome of approximately 4,000–7,000 nucleotides (nts) in length [
1,
2]. These viruses exhibit a conserved organization with an early region and a late region separated by a non-coding regulatory region (NCRR) [
3], which contains the early and late promoters and the origin of replication. The early region encodes up to five non-structural tumor antigen proteins, which are involved in viral replication and oncogenesis. The large and small tumor antigen proteins (LT and sT, respectively) are universally expressed by polyomaviruses [
1,
3]. The LT is a multiregulatory protein that is required for the initiation of viral replication and activation of the late region promoter, but also for the suppression of its own promoter, thus regulating early gene expression (reviewed in [
4]). The precise function of the sT protein is unclear, but it has been suggested to play a role in the regulation of the viral replication cycle (reviewed in [
4]). The late region encodes the capsid proteins VP1, VP2 and VP3, which are important for virion assembly and nuclear egress [
2,
3]. In addition, the polyomavirus simian virus 40 (SV40) also produces a late VP4 protein [
5]. The early and late regions of polyomavirus (PyVs) genomes may further encode alternatively spliced forms of the encoded proteins, such as the alternative large T antigen open reading frame (ALTO; [
6] or the agnoprotein [
7]). The agnoprotein is a regulatory protein essential for sustaining a productive viral life cycle, being involved in viral DNA replication, viral transcription, virion maturation and release (reviewed in [
4]). The ALTO protein has been shown to be expressed, but not being essential, during replication, most likely playing an accessory role [
6]. More recently, the DUO protein has also been identified in different polyomavirus (
https://ccrod.cancer.gov/confluence/display/LCOTF/Polyomavirus), however, there are no studies addressing its function.
PyVs have been identified in several mammals [
8‐
11], birds [
12], fishes [
13,
14] and arthropods [
15]. In fish, PyVs have been described in perciform fish such as the black sea bass (
Centropristis striata) [
16], the gilt-head sea bream (
Sparus aurata) [
17], the sharp-spined notothen (
Trematomus pennellii) and emerald notothen (
Trematomus bernacchii) [
1,
14], but also in cartilaginous fish taxa such as the giant guitarfish (
Rhynchobatus djiddensis) [
1,
13]. Chimeric viral genomes that encode proteins related to those of PyVs have been reported in the eel species
Anguilla japonica and
A. marmorata [
18‐
20]. Fish PyVs belong to at least two distinct evolutionary lineages, one comprising perciform-fish PyVs and the other encompassing cartilaginous fish PyVs [
14], which only includes a single PyV detected in the giant guitarfish (GfPyV1; [
13].
Cartilaginous fishes are the oldest group of extant vertebrates, being the most basal living jawed vertebrates. Cartilaginous fishes can be divided into two very distinct subclasses, Elasmobranchii (sharks, rays and skates) and Holocephali (chimaeras), which branched off from each other almost 420 million years ago [
21]. They present a complex immune system, exhibiting one of the greatest functional diversities when compared to other vertebrates [
22]. Screening for pathogen communities in cartilaginous fish hosts has been mostly opportunistic and descriptive, and has not covered the taxonomic and ecological diversity of the group. While only a few viruses have been isolated from cartilaginous fish taxa, the few data available show extensive retroviral diversity in the elephant shark (
Callorhinchus milii) genome [
23], and newly identified RNA and double stranded DNA (dsDNA) viruses in shark and ray species [
13,
24‐
30].
Here, as part of an ongoing study aiming to provide a baseline data of the viruses associated with various cartilaginous fish with different ecologies, we report the identification of a complete polyomavirus genome from the thornback skate, Raja clavata, a coastal benthic elasmobranch from the order Rajiformes.
Discussion
Polyomavirus genomes range from 3962 to 7369 nts, with the smallest PyV being identified in a cartilaginous fish, the giant guitarfish (
Rhynchobatus djiddensis) [
13]. The
R. clavata polyomavirus reported here, RcPyV1, has a 4,195 nt-long genome, being the second smallest polyomavirus described to date.
Similar to other polyomaviruses, the RcPyV1 genome contains CDS homologous to sT, LT, VP1, VP2 and DUO encoding proteins (Fig.
1; Table
1). When compared with GfPyV1 [
1], RcPyV1 lacks the Agnoprotein. The predicted sT antigen seems to be smaller (132 bp; 44aa) when compared to other vertebrates and, like in GfPyV1 [
13], it does not contain any conserved motif. Previous studies on fish polyomaviruses indicates that sT usually presents the DnaJ motif while the remaining ORF show no similarity among sequences [
14]. This is in agreement with our BLAST search results that revealed that RcPyV1 VP1 has no sequence identity to known proteins. In turn, the LT antigen, known to be crucial for viral replication [
8], presents all the other conserved motifs described for polyomaviruses, with the exception of the retinoblastoma protein binding motif (pRB) which is specific to amniote polyomaviruses, and the protein phosphate 2 A (Fig.
1; Table
2). Within LT, we identified the CR1 motif (LQKLL), important for transcriptional regulation [
42], the hexapeptide (HPDKGG) involved in protein interactions, the putative nuclear localization signal (PRRSIN), the zinc-finger motif (CVLCKEDKVHSETH) and ATPase motifs (GPYNSGKT and GLCPVGLE), which are important to recruit cellular proteins involved in replication [
43].
Additional ORFs of potential interest were detected. These include the putative ORF1 that presents the LxCxE motif (LHCYE), the putative ORF2 and DUO protein. DUO protein has been identified in mammals, birds, fishes and aracnids (
https://ccrod.cancer.gov/confluence/display/LCOTF/Polyomavirus), however its function and importance is still unkown.
According to ICTV Polyomavirdae study group recommendations for the classification of polyomaviruses, polyomaviruses that share < 85% pairwise identity in the LT antigen should be considered as a separate species [
40]. The LT antigen sequences from the two cartilaginous fish share 56% identity and thus GfPyV1 and RcPyV1 represent members of two different species of polyomaviruses. However, fish polyomaviruses have not yet been assigned to a genus [
15].
No signs of infection were detected in the collected samples, however, since this virus was detected in the spleen, it is likely that RcPyV1 is not a contaminant from the environment, but rather a virus infecting the thornback skate. This is also supported by the previous identification of GfPyV1 in the giant guitarfish [
13], which is phylogenetically related to RcPyV1.
It has been proposed that polyomaviruses evolution is driven by their hosts, although they often show higher among-sequence divergence levels compared to those of their hosts suggesting that other factors contribute to their evolution [
1,
9]. Previous analysis [
14] suggested that fish polyomaviruses are divided in two lineages: one lineage clustering viral genomes isolated from perciform fish and lacking the DnaJ domain, and the other lineage grouping viral genomes isolated from cartilaginous fish exhibiting the DnaJ domain. Our results are in line with these observations as in both phylogenetic trees, GfPyV1 and RcPyV1 cluster together, but separately from the remaining perciform fish PyVs. Recombination, which plays an important role in the evolution of PyVs [
1,
14], was not found in the GfPyV1 and RcPyV1.
In conclusion, we identified a novel polyomavirus from the cartilaginous fish Raja clavata, which presents the typical features of polyomaviruses: LT with conserved motifs, sT, VP1 and VP2 proteins. RcPyV1 belongs to the same evolutionary lineage as the previously identified GfPyV1, reinforcing that Perciform and cartilaginous fish PyVs are not monophyletic, but rather represent two divergent groups. The pairwise comparisons between RcPyV1 and GfPyV1 and the remaining fish PyVs are in line with this.
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