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The hidden diversity of ancient bornaviral sequences from X and P genes in 1 vertebrate genomes 2

25 Endogenous bornavirus-like elements (EBLs) are heritable sequences derived from 26 bornaviruses in vertebrate genomes that originate from transcripts of ancient 27 bornaviruses. EBLs have been detected using sequence similarity searches such as 28 tBLASTn, whose technical limitations may hinder the detection of EBLs derived from 29 small and/or rapidly evolving viral X and P genes. Indeed, no EBLs derived from the X 30 and P genes of orthobornaviruses have been detected to date in vertebrate genomes. 31 Here, our aim was to develop a novel strategy to detect such "hidden" EBLs. To that 32 aim, we focused on the 1.9 kb readthrough transcript of orthobornaviruses, which 33 encodes a well-conserved N gene and small and rapidly evolving X and P genes. We 34 show a series of evidence supporting the existence of EBLs derived from 35 orthobornaviral X and P genes (EBLX/Ps) in mammalian genomes. Furthermore, we 36 found that an EBLX/P is expressed as a fusion transcript with the cellular gene, 37 ZNF451 , which potentially encodes the ZNF451EBLP-4 fusion protein in miniopterid 38 bat cells. This study contributes to a deeper understanding of ancient bornaviruses and 39 co-evolution between bornaviruses and their hosts. Furthermore, our data suggest that 40 endogenous viral elements detected thus far are just the tip of the iceberg, and further 41 studies are required to understand ancient viruses more accurately.

transcription signal sequences S1, T1/S2, and T2 (Fig. 2b) [12]. Therefore, we 119 investigated the presence of these signal-like sequences in the 1.9-kb miEBLN-1 120 insertion. We found transcription signal-like sequences at reasonable positions with 121 respect to extant N/X/P transcripts (Fig. 2c). A poly-A stretch was also observed 122 immediately downstream of the T2 signal, indicating that the insertion originated from 123 an mRNA transcript ( Fig. 2c and Fig. S2). Furthermore, a tandem duplication sequence, 124 TTGTGCTA/TTCTTGTA, was observed immediately upstream and downstream of S1 125 and T2-poly-A sequences, respectively. This is a potential target site duplication (TSD), 126 which is a hallmark of LINE-1-mediated mRNA integration [13]. These results 127 suggested that the 1.9-kb insertion, including miEBLN-1, is derived from an ancient 128 N/X/P transcript integrated by LINE-1. Therefore, we tentatively refer to this locus as 129 miEBLN/X/P-1. 130 131 Additional EBLs possibly derived from N/X/P transcripts in miniopterid bat genomes 132 As described above, orthobornaviruses express mRNAs encoding N (1.2 kb), X and P 133 (X/P mRNA, 0.7 kb), or N, X, and P (N/X/P mRNA, 1.9 kb). If miEBLN/X/P-1 is truly 134 derived from N/X/P mRNA, there may be additional EBLs derived from N/X/P or X/P 135 mRNA. To examine this possibility, we conducted a BLASTn search against bat 136 genomes using the nucleotide sequence of miEBLN/X/P-1 as a query (Fig. 3a). We 137 obtained six hits from the genomes of M. natalensis and M. schreibersii. While four hits 138 were aligned in the entire N/X/P region of miEBLN/X/P-1, interestingly, two other hits 139 were aligned only to the putative X/P region ( Fig. 3a and Table S1). 140 We first assessed hits that aligned to the entire region of miEBLN/X/P-1. Four hits 141 showed 1.9 kb alignment lengths and high sequence similarities (76-84% nucleotide 142 identity) to miEBLN/X/P-1 (Table S1). The flanking regions of each pair of hit 143 sequences were well aligned between M. natalensis and M. schreibersii, indicating that 144 they are orthologous between these species (Table S7). We tentatively refer to these loci 145 as miEBLN/X/P-2 and miEBLN/X/P-3. We also confirmed the presence of orthologs of 146 these EBLN/X/Ps in the genome of M. fuliginosus by PCR and Sanger sequencing of 147 genomic DNA isolated from M. fuliginosus (Table S7). 148 Next, we examined the presence of miEBLN/X/P-2 or miEBLN/X/P-3-empty loci 149 by comparative genomics. We observed that the miEBLN/X/P-3 empty locus is present 150 in genomes of non-miniopterid bats such as Tadarida brasiliensis ( Fig. S1B and Table  151 S7). We did not obtain an alignment of the flanking region of miEBLN/X/P-2 with the 152 other bat genomes and therefore could not identify its empty locus. 153 We then analyzed the nucleotide sequences of miEBLN/X/P-2 and miEBLN/X/P-154 3. Similar to miEBLN/X/P-1, both EBLN/X/Ps contained transcription signal-like 155 sequences, and poly-A stretches were observed downstream of T2-like sequences (Fig.  156 S2). Furthermore, tandem duplication sequences, TGTTCA/[T/C]GTTCA and 157 GAGGAACG/GAGGGATG, were also observed immediately upstream of S1 and 158 downstream of T2/poly-A in miEBLN/X/P-2 and miEBLN/X/P-3, respectively. These 159 results suggest that miEBLN/X/P-2 and miEBLN/X/P-3 are also derived from ancient 160 N/X/P transcripts and were integrated into the genome of a miniopterid bat ancestor 161 through LINE-1 activity. 162 163

EBLs derived from X/P-like transcripts exist in miniopterid bat genomes 164
Next, we assessed the hits aligned to the 0.7-kb putative X/P region of miEBLN/X/P-1. 165 75% nucleotide identity to the 0.7-kb X/P region (Table S1). The flanking regions of the 167 hit sequences were well aligned, indicating that the loci are orthologous between these 168 species (Table S7). We also found an orthologous locus in the genome of M. fuliginosus 169 using PCR and Sanger sequencing (Table S7). Furthermore, we detected empty loci in 170 genomes of non-miniopterid bats, genomes such as Myotis lucifugus ( Fig. 3b and Fig.  171 S3a). 172 The orthobornaviral X/P mRNA contains the transcription signal sequences S2/T1 173 in the 5'-UTR and T2 in the 3'-UTR. We observed that S2/T1 and T2-like sequences are 174 present and located at positions similar to those in extant X/P mRNA. Additionally, 175 poly-A stretches were observed downstream of the T2 sequences. Furthermore, the S2 176 and poly-A stretches were flanked by TSDs, CTTAAGGC/CTTAAGGC (Fig. 3a, b and 177 Fig. S5). Taken together, these results indicated that the locus may have been derived 178 from an ancient X/P transcript and integrated into the genome of a miniopterid bat 179 ancestor through LINE-1 activity. We tentatively refer to it as the miEBLX/P-4. 180 181

Preservation of intact ORFs in miEBLX/P-4 and characterization of the putatively 182 encoded proteins 183
Most EBLs do not retain viral ORFs because of the accumulation of mutations after 184 integration. Remarkably, however, miEBLX/P-4 retains intact X-and P-like ORFs in 185 different reading frames, which is consistent with the extant orthobornaviral X/P mRNA 186 ( Fig. 3c and Fig. S4a). Therefore, we named these ORFs as miEBLX-4 and miEBLP-4. 187 Notably, miEBLP-4 in M. natalensis is disrupted by a premature stop codon. 188 Although there is very low sequence similarity between the amino acid sequences 189 of these ORFs and extant orthobornaviral proteins, further analyses showed that 190 miEBLX-4 and miEBLP-4 are intrinsically similar to extant orthobornaviral X and P 191 proteins. 192 First, we examined the miEBLX-4 ORF. The ORF consists of 109 codons, which is 193 longer than the that of extant orthobornaviral X (86-90 codons). We then predicted the 194 secondary structure and analyzed the hydrophobicity of the putative miEBLX-4 protein 195 with respect to extant X proteins. Both extant X and miEBLX-4 were predicted to 196 contain an alpha helix in the N-terminal region ( Fig. 4a and Fig. S6a). The hydropathy 197 plot showed similar hydrophobicity patterns for these proteins (Fig. S7a). 198 We also determined whether the functional domains are conserved between extant 199 X and miEBLX-4. Amino acid sequence alignment showed that the N-terminal region is Next, we examined the miEBLP-4 ORF. The ORF consists of 233 codons; it is 208 therefore longer than those of extant orthobornaviral P (201-208 codons). Extant P and 209 putative miEBLP-4 proteins also showed similar predicted secondary structures (Fig. 4b  210 and Fig. S6b) and hydrophobicity patterns (Fig. S7b). The predicted secondary 211 structures of miEBLP-4p were similar to those of the modern counterparts ( Fig. 4b and  212 Fig . S6b). Alignment of the amino acid sequences revealed that some of the functional 213 domains previously characterized in BoDV-1 P may be shared between extant P and 214 miEBLP-4 (Fig. 4d). Proline residues (Fig. 4d, blue) in the N-terminal and C-terminal 215 nuclear localization signals were prominent in both proteins [17]. 216 We also explored the interaction between miEBLX-4 and miEBLP-4, which is a 217 notable characteristic of the orthobornaviral X and P proteins [18]. We expressed Myc-218 tagged miEBLX-4 (miEBLX-4-Myc) and FLAG-tagged miEBLP-4 (miEBLP-4-FLAG) 219 in 293T cells and then immunoprecipitated them using an anti-FLAG antibody. 220 miEBLX-4-Myc was co-immunoprecipitated with miEBLP-4-FLAG, indicating the 221 interaction between these proteins (Fig. 4e). 222 These results showed that miEBLX-4 and miEBLP-4 share similar intrinsic 223 properties with the extant X and P proteins. Furthermore, together with the above data, 224 they strongly suggest that miEBLN/X/P-1-3 and miEBLX/4 are indeed derived from 225 ancient viral N/X/P and X/P mRNAs, respectively. 226 227

Diverse EBLX/Ps are found in vertebrate genomes 228
To further identify the presence of other EBLX/Ps, we conducted TFASTX and 229 TFASTY searches against vertebrate genomes using the amino acid sequences of 230 miEBLX-4 and miEBLP-4 as queries. TFASTX and TFASTY are sequence similarity 231 search programs that allow frameshifts for alignment [19]. As a result, 65 unique hits 232 (Table S2- Interestingly, the insertion sizes of bat mpEBLX/P and ctEBLX/P were both 0.8 249 kb, which is similar to that of miEBLX/P-4. In contrast, that of rodent ctEBLX/P is 0.7 250 kb, which is closer to the size of extant orthobornaviral X/P transcripts (see discussion). 251 252

Possible co-option of EBLX/P in miniopterid bats 253
Some EVEs, including EBLs, retain intact ORFs that encode proteins that play roles in 254 host-related functions [20]. As described above, miEBLX/P-4 contains intact ORFs 255 corresponding to orthobornaviral X and P genes. Therefore, we analyzed miEBLX/P-4 256 in detail. We first examined the location of miEBLX/P-4 in the M. natalensis genome. 257 miEBLX/P-4 is located within the intron region of ZNF451 gene (Fig. 6). 258 Next, we analyzed the expression of miEBLX/P-4 by mRNA sequencing of M. 259 schreibersii-derived SubK12-08 cells. The read coverage of the miEBLX/P-4 locus was 260 comparable to that of ZNF451 (Fig. 6A). Interestingly, the mapping pattern revealed the 261 existence of an alternative splicing site for ZNF451 that could produce a chimeric 262 transcript comprising ZNF451 exons 1-4 and the miEBLX/P-4 locus (Fig. 6A). 263 The putative chimeric transcript contains two ORFs consisting of more than 100 264 codons in different reading frames: a chimeric ORF of ZNF451 in-frame with miEBLP-265 4 or that of the miEBLX-4. The chimeric ORF potentially encodes a fusion protein 266 comprising the N-terminal region of ZNF451, which contains a SUMO-interacting 267 motif (SIM) domain, a short intronic region of ZNF451, and miEBLP-4 (Fig. 6). 268 These results suggest that miEBLX/P-4 has been co-opted by miniopterid bats as a 269 chimeric gene with ZNF451. We previously demonstrated that the X/P mRNA of a modern orthobornavirus can be 273 reverse-transcribed and integrated into the host genome [7]; however, no EBLX and 274 only a few EBLPs have been reported [6]. We hypothesized that the technical 275 limitations of the sequence similarity search hinder the detection of EBLs derived from 276 the X and P genes. EBLs have been routinely searched using tBLASTn, but this can be 277 an unsuitable method for detecting small and rapidly evolving genes such as the X and P 278 genes [8]. In this study, our aim was to develop a novel strategy to detect these hidden 279 EBLs. To that aim, we focused on the 1.9-kb readthrough transcript of 280 orthobornaviruses, consisting of a well-conserved N gene and small and rapidly 281 evolving X and P genes [12], to detect hidden EBLX/Ps. We show a series of evidence 282 implying that EBLN/X/Ps and EBLX/Ps are present in vertebrate genomes but are not 283 detectable by BLAST searches. Interestingly, miEBLX/P-4 has maintained intact ORFs 284 for more than 17 million years and is expressed as a fusion transcript with the cellular 285 Therefore, this study revealed the presence of hidden EBLs, which could contribute to a 287 deeper understanding of ancient bornaviruses and the co-evolution between 288 bornaviruses and their hosts. 289 Here, we uncovered the potential co-option of miEBLX/P-4 by miniopterid bats, as 290 indicated by the expression of the ZNF451/miEBLP-4 chimeric transcript, which 291 potentially encodes a fusion protein consisting of the N-terminal region of ZNF451 and 292 miEBLP-4. To the best of our knowledge, this is the first report of a non-retroviral EVE 293 being expressed as a chimeric transcript with a known host gene. Although we could not 294 confirm the expression of the ZNF451/miEBLP-4 fusion protein due to the lack of 295 antibodies, our data suggest that miEBLX/P-4 co-opted by miniopterid bats. Further 296 studies are needed to understand the biological significance of EBLs. 297 Our data strongly suggest that the genome organization and expression patterns of 298 the N, X, and P genes have not substantially changed over the past million years. Here, 299 we show that T1-readthrough transcription occurred in ancient orthobornaviruses 300 present at least 17.3 MYA (Fig. 2). Furthermore, the ORF positions of the X and P genes 301 of ancient orthobornaviruses were highly similar to those of modern orthobornaviruses 302 (Fig. 3). In addition, the sequences and order of transcription signals are highly 303 conserved among ancient and modern orthobornaviruses (Figs. 2 and 3). In BoDV-1, 304 readthrough transcription has been suggested to be involved in the regulation of viral 305 gene expression [12]. This hypothetical gene regulation mechanism has been 306 maintained for more than 17.3 million years. Interestingly, our previous study showed 307 that the order of S2-T1 is also conserved in some EBLNs shared among Old World 308 monkeys, which were estimated to be integrated into the host genomes more than 43 309 orthobornaviruses earlier than 17.3 MYA. 311 The fundamental functions of viral X and P proteins may also be evolutionarily 312 conserved between ancient and modern orthobornaviruses. We observed similarities in 313 the intrinsic properties of extant orthobornaviral X and P proteins with miEBLX-4p and 314 miEBLP-4p despite their very low amino acid identities (Fig. 4). Furthermore, the 315 interaction between X and P may have been conserved in ancient orthobornaviruses 316 (Fig. 4). Although the evolutionary rates of X and P genes are high, the fundamental 317 features and functions of X and P proteins may have been maintained for at least 17.3 318 million years. Some orthobornaviruses are known to be fetal zoonotic pathogens [21]. 319 Thus, although the biological functions of the X protein and the interaction between X 320 and P proteins are not fully understood, these proteins can be good targets for the 321 development of antivirals. 322 For the EVE search, TFASTX/Y, a conventional sequence similarity search for the 323 detection of EVEs, may be more suitable than tBLASTn. After integration into the host 324 genome, many EVEs have acquired random mutations, which caused frameshifts in the 325 ORFs. Such frameshifted EVEs are a kind of "fragmented" EVEs and are thus less 326 detectable by tBLASTn. In contrast, TFASTX/Y takes frameshifts into account and can 327 thus detect such fragmented EVEs more sensitively. We are currently conducting a 328 benchmark analysis of tBLASTn and TFASTX/Y in detail, showing that some of the 329 EBLX/Ps detected by TFASTX/Y cannot be detected by tBLASTn or other BLAST 330 programs (data not shown). Therefore, TFASTX/Y can detect fragmented EVEs more 331 sensitively than tBLASTn, which may contribute to identifying other hidden EVEs. 332 The EBLX/Ps identified in this study would represent only the tip of the iceberg on 333 the hidden EVEs that could exist in the genome. In this study, the majority of EBLX/Ps 334 were detected in bat and rodent genomes. Note that we failed to detect any EBLX/Ps in 335 primate genomes, which contain numerous EBLs. Considering the number of EBLs in 336 the genomes of primates (i.e., 24 orthobornaviral EBLs were detected in the human 337 genome [6]), it is plausible that hidden EBLX/Ps exist in primates that are undetectable 338 because of the inherent low amino acid similarities in X and P. 339 We may be able to detect such hidden EBLX/Ps by identifying other EBLN/X/P 340 integrations. However, searching for other EBLN/X/Ps by manual curation is time-341 consuming. Thus, the development of an efficient searching method to identify 342 approximately 1.9-kb insertions with EBLN would facilitate further exploration of 343 EBLX/Ps. This is probably not limited to bornaviruses; there may be many hidden 344 EVEs that are derived from small and/or rapidly evolving viral genes. Readthrough 345 transcription has also been observed for other mononegaviruses. Our search strategy can 346 be applied to the detection of such EVEs. 347 Taken together, we identified EBLs that were undetectable by conventional 348 BLAST searches. Our findings provide novel insights into ancient bornaviruses. 349 Furthermore, our search strategy will contribute to paleovirological research.

Construction of miEBLX-4 and miEBLP-4 expressing plasmids 370
Expression vectors with C-terminal FLAG or Myc tag were first constructed. pcDNA3 371 was digested with EcoRI and XhoI, which was assembled with oligo DNA containing 372 Myc or FLAG tag sequence using NEBuilder HiFi DNA Assembly Cloning Kit (New 373 England Biolabs). The resultant plasmids were named pcDNA3-C-Myc or pcDNA3-C-374 FLAG. miEBLX-4 and miEBLP-4 sequences were then PCR-amplified from the 375 YubFKT1 DNA sample, and were subsequently assembled with pcDNA3-C-Myc or 376 pcDNA3-C-FLAG linearized with KpnI and EcoRI using NEBuilder. The oligo DNA 377 sequences are available in Table S6.