key: cord-328695-nptfd6c2
authors: Tengs, Torstein; Delwiche, Charles F.; Jonassen, Christine Monceyron
title: A mobile genetic element in the SARS-CoV-2 genome is shared with multiple insect species
date: 2020-06-29
journal: bioRxiv
DOI: 10.1101/2020.06.29.177030
sha: 
doc_id: 328695
cord_uid: nptfd6c2

Unprecedented quantities of sequence data have been generated from the newly emergent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative agent of COVID-19. We document here the presence of s2m, a highly conserved, mobile genetic element with unknown function, in both the SARS-CoV-2 genome and a large number of insect genomes. Although s2m is not universally present among coronaviruses and appears to undergo horizontal transfer, the high sequence conservation and universal presence of s2m among isolates of SARS-CoV-2 indicate that, when present, the element is essential for viral function.

Single-stranded RNA viruses, such as coronaviruses, are known to have genomes with strong secondary structural features, such as stem-loop regions and pseudoknots. We have previously reported the presence of a 41-43 nucleotide long hairpin-forming element, referred to as stemloop II-like motif (s2m) (Jonassen, et al. 1998) , in several families of positive-sense singlestranded RNA ((+)ssRNA virus)) viruses (Tengs, et al. 2013) . The molecular structure has been mapped in great detail for SARS-CoV (Robertson, et al. 2005) . Its sequence and secondary structure are highly conserved, but the phylogenetic distribution among viral genomes is very patchy. These properties indicate that the sequence is mobile, and, to our knowledge, s2m represents the only known example of a genetic element with the ability to move between distantly related viruses. The function and evolutionary origin of s2m remains unknown, but the high level of conservation seen even in distantly related viruses despite relatively high overall mutation rates suggests that s2m is under strong selection. When comparing s2m motifs from different virus species, there are conserved residues both in loopand base-pairing regions, indicating that there is selective pressure to maintain both the primary and secondary structure. The element is always present near the 3' end of the genome, and in all virus families where s2m has been reported, there are examples of species carrying two (non-identical) back-to-back copies (Quan, et al. 2010; Tengs, et al. 2013 ). As noted above, related viruses may lack s2m entirely, but when present, its sequence is always highly conserved. SARS-CoV-2 (Gorbalenya, et al. 2020 ) is a member of the SARS-related Sarbecovirus subgenus (Andersen, et al. 2020 ) and this group of coronaviruses is known to contain s2m (Tengs and Jonassen 2016) . The presence of s2m in the SARS-CoV-2 genome (GenBank accession MN908947, position 29727-29768) and other members of this group is probably the result of a single horizontal transfer event, predating the divergence of the SARS-related viruses (Tengs, et al. 2013; Tengs and Jonassen 2016) .

To characterize the specific genotype of s2m found in SARS-CoV-2, BLASTN (Altschul, et al. 1990 ) was used to search the entire virus section of GenBank using all s2m sequence genotypes reported in the literature (n = 97) (Jonassen, et al. 1998; Robertson, et al. 2005; Tengs, et al. 2013; Tengs and Jonassen 2016) as query sequences. To check for the presence of s2m motifs in insects, both the TSA and the Whole genome shotgun contigs (wgs) databases were mined using the same approach.

For the phylogenetic analyses, sequences were aligned using the Clustal W algorithm (Thompson, et al. 1994 ) and maximum likelihood analysis were performed using MEGA X (Kumar, et al. 2018) . The James-Taylor-Thornton (JTT) substitution model was used with gamma distribution (5 categories) and invariable sites. Branch swapping was done using the subtree-pruning-regrafting method with 'very strong' filter.

A total of 5553 s2m-containing accessions were identified, representing at least four virus families (Supplementary table 1 Two of the s2m virus accessions identified were from a recently published RNA-based invertebrate virosphere project (Shi, et al. 2016 ). These two highly similar sequences, derived from the virome of the spider species Tetragnatha maxillosa, encode a hypothetical protein immediately upstream of s2m that could not readily be identified using protein sequence similarity searches. Protein-protein BLAST searches against the non-redundant (nr) GenBank protein database revealed the two best matching non-viral proteins to be from the insect species winter moth (Operophtera brumata) and bagworm moth (Eumeta japonica). In the O. A phylogenetic analysis focusing on s2m accessions and using the longest and most similar amino sequences obtained from the TSA, wgs and nr databases (Supplementary table 2) gave a topology that was biased towards lepidopteran species ( Figure 2B ). Translation of both genomic and transcriptomic data in some cases gave reading frames containing several internal stop codons, but amino acid sequences that could still be aligned to (near) full length ( Figure 2B, Supplementary table 2) .

As SARS-CoV-2 is embedded within the Sarbecoviruses, it is likely that the unique G > U mutation has occurred specifically during the evolution of the current pandemic strain of SARS-CoV ( Figure 2C ). An Australian isolate with a 10 base deletion in s2m was also discovered (accession MT007544). Intriguingly, the deletion occurred after passaging isolates in Vero cells (Caly, et al. 2020) . This could represent an attenuated SARS-CoV-2 strain, as culturing in permissive cell lines may have altered the selective pressure and alleviated the need to maintain a functional version of the motif.

In the four virus families where s2m has previously been described, the flanking protein is easily recognizable. The protein found in insect species, as well as the two T. maxillosa viruses, does not appear to have homologs in any of the better-characterized virus groups, nor does it appear to contain any recognizable motifs. A protein secondary structure homology search (Drozdetskiy, et al. 2015) indicated that the O. brumata protein may contain an integrase catalytic domain, which would be consistent with the mobile nature of s2m. We were unable to identify any signature of a retroviral origin of the s2m loci in insect genomes, or any other indication of the insect s2m contigs being of viral origin. In several of the genomic contigs, the open reading frame (ORF) covering the uncharacterized protein also appeared to contain introns, generally considered a hallmark of eukaryotic genes. In addition, PCR and Sanger sequencing was performed to confirm the presence of s2m and the upstream ORF in the O. brumata genome (Supplementary figure 2) , making it very unlikely that the downloaded sequence data stem from (RNA) viruses and do not represent bona fide insect sequences.

The insect species that contain s2m (and the associated protein) are distantly related, indicating either a deep evolutionary origin with multiple losses or that this genetic construct is also a mobile element, perhaps using viruses as a vector . The T. maxillosa virus could represent such a vector, albeit no s2m sequences or proteins similar to the O. brumata protein could be found in any arachnid species using sequence similarity searches. Mobility of genetic elements such as transposable elements (TEs) has previously been reported in insects (Peccoud, et al. 2017) , and an analysis of the genomic s2m contigs using the Dfam portal (Hubley, et al. 2016) revealed that several of the accessions had regions with a significant degree of similarity with the long interspersed nuclear element (LINE) L2-1_Ldor_D, previously reported from butterflies (Ray, et al. 2019) . The exact evolutionary link between the xenologs of s2m and the unknown protein found in insects and viruses can not be established based on our data. The s2m genotypes found in insects and viruses have similar primary sequence profiles, albeit there appear to be some subtle differences (Figure 3 ).

We believe that the most likely mode of transfer for s2m in viruses is through nonhomologous recombination between RNA molecules. Outside the astroviruses, SARS-CoV and SARS-CoV-2 represent the only known examples of s2m-carrying viruses that infect humans, but it seems probable that s2m is still evolutionary active and that this element will continue to affect the evolution of (+)ssRNA viruses. Because the clade of s2m-containing coronaviruses that includes SARS-CoV and SARS-Cov-2 also includes viruses isolated from bat and pangolin, it is unlikely that s2m has played a direct role in zoonotic transfer, but its high degree of sequence conservation suggests that it may present a target for therapy, and the presence of closely related systems in insect hosts provides an opportunity to study the biology of this apparent mobile element in relatively tractable experimental systems.

Basic local alignment search tool

The proximal origin of SARS-CoV-2

Isolation and rapid sharing of the 2019 novel coronavirus (SAR-CoV-2) from the first patient diagnosed with COVID-19 in Australia

WebLogo: a sequence logo generator

JPred4: a protein secondary structure prediction server

Viruses as vectors of horizontal transfer of genetic material in eukaryotes

The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

The Dfam database of repetitive DNA families

A common RNA motif in the 3 ' end of the genomes of astroviruses, avian infectious bronchitis virus and an equine rhinovirus

MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms

Massive horizontal transfer of transposable elements in insects

Identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in Nigeria

Simultaneous TE Analysis of 19 Heliconiine Butterflies Yields Novel Insights into Rapid TE-Based Genome Diversification and Multiple SINE Births and Deaths

The structure of a rigorously conserved RNA element within the SARS virus genome

Redefining the invertebrate RNA virosphere

Distribution and Evolutionary History of the Mobile Genetic Element s2m in Coronaviruses

A mobile genetic element with unknown function found in distantly related viruses

CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice

The authors would like to thank M.Sc Anbjørg Rangberg (Østfold Hospital Trust) for help with PCR and sequencing and Dr. Snorre Hagen (Norwegian Institute of Bioeconomy Research) for providing the Operophtera brumata DNA. This work was supported by the

Figure 1. Secondary structure of s2m in SARS-CoV-2 based on SARS-CoV model (Robertson, et al. 2005) .

analysis was performed on ORF1ab polyprotein sequences from selected coronavirus species.s2m-containing accessions have been highlighted and bootstrap values > 90 % indicated (100 psedoreplicates). B) Maximum likelihood analysis using data from the s2m-associated hypothetical protein (see main text for details). Sequences in boldface stem from reading frames with (multiple) internal stop codons. * -genomic data, ** -accessions without s2m. C) s2m sequences corresponding to operational taxonomic units in the phylogenetic trees. Lines above alignment show (non-canonical) base-pairing residues (Robertson, et al. 2005 ) and the position with the unique G > U mutation in SARS-CoV-2 has been indicated. (bottom panel). Generated using WebLogo (Crooks, et al. 2004 ).

Operophtera brumata and related insect species. Identical sequences from the same species were removed and a maximum likelihood analysis was performed using MEGA X (Kumar, et al. 2018 ) after aligning amino acid sequences using the Clustal W algorithm (Thompson, et al. 1994 ). The James-Taylor-Thornton (JTT) substitution model was used with gamma distribution (5 categories) and invariable sites. Branch swapping was done using the subtreepruning-regrafting method with 'very strong' filter. s2m-containing accessions have been indicated (red: insect species, blue: invertebrate viruses) and bootstrap values > 90 % are shown (100 psedoreplicates).Supplementary figure 2. PCR amplification and Sanger sequencing of the s2m locus in the Operophtera brumata genome. PCR was performed using the AmpliTaq Gold 360 PCR Master Mix (Thermo Fisher Scientific, Oslo, Norway). Primer sequences, ORF stop codon (TAA) and s2m sequence have been underlined.