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3â•Ž untranslated regions of Marburg and Ebola virus mRNAs possess negative regulators of translation that are modulated by ADAR1 editing

The filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5' and 3' untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3'UTRs are prone to editing by ADAR1. A reporter mRNA approach, in which different 5' or 3'UTRs were inserted into luciferase encoding mRNAs, demonstrates that MARV 3'UTRs yield different levels of reporter gene expression suggesting modulation of translation. The modulation occurs in cells unable to produce miRNAs and can be recapitulated in a MARV minigenome assay. Deletion mutants identified negative regulatory regions at end of the MARV NP and L 3'UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3'UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3'UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3'UTR, but not the edited or deletion mutant NP 3'UTRs, induce a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the VP40 3'UTR, VP40 3'UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3'UTR edits also enhanced translation but neither the wildtype nor the edited 3'UTRs induced IFN. These findings implicate filoviral mRNA 3'UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1. Importance UTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and of ADAR1 editing have been uncertain. This study demonstrates that MARV and EBOV 3'UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3'UTRs, relieves the negative effects of the UTRs. These data indicate that filovirus 3'UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity.

The filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus 23 (MARV). A substantial portion of filovirus genomes encode 5' and 3' untranslated regions 24 (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3'UTRs are 25 prone to editing by ADAR1. A reporter mRNA approach, in which different 5' or 3'UTRs were 26 inserted into luciferase encoding mRNAs, demonstrates that MARV 3'UTRs yield different levels 27 of reporter gene expression suggesting modulation of translation. The modulation occurs in cells 28 unable to produce miRNAs and can be recapitulated in a MARV minigenome assay. Deletion 29 mutants identified negative regulatory regions at end of the MARV NP and L 3'UTRs. Apparent 30 ADAR1 editing mutants were previously identified within the MARV NP 3'UTR. Introduction of 31 these changes into the MARV nucleoprotein (NP) 3'UTR or deletion of the region targeted for 32 editing enhances translation, as indicated by reporter assays and polysome analysis. In 33 addition, the parental NP 3'UTR, but not the edited or deletion mutant NP 3'UTRs, induce a type 34

I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the 35
West Africa outbreak exhibited ADAR1 editing of the VP40 3'UTR, VP40 3'UTRs with parental 36 and edited sequences were similarly assayed. The EBOV VP40 3'UTR edits also enhanced 37 translation but neither the wildtype nor the edited 3'UTRs induced IFN. These findings implicate 38 filoviral mRNA 3'UTRs as negative regulators of translation that can be inactivated by innate 39 immune responses that induce ADAR1. 40

Introduction 51
Since it appears that ADAR1 editing occurs on the MARV negative sense genomic RNA, we 246 constructed a bicistronic minigenome system that incorporates the NP 3'UTR. The bicistronic 247 minigenome was designed to code for model NP and L mRNAs where the coding sequences 248 were replaced with Renilla and firefly luciferase coding sequences, respectively (Fig. 7A). 249 Negative strand minigenome RNA was in vitro transcribed, purified and then transfected into the 250 cells 24 hours after transfection with helper plasmids encoding MARV NP, VP35, VP30 and L, 251 which transcribe and replicate the minigenome RNA. A further 24 hours after transfection of the 252 minigenome RNA, Renilla luciferase activity was measured to assess the impact of changes to 253 the NP 3'UTR. As was observed with the mRNA transfections, the minigenome encoding a 254 mRNA that has mutations in the NP 3'UTR had higher luciferase activity compared to the one 255 encoding the WT NP 3'UTR ( Fig. 7B). The controls lacking the VP35 or the L helper plasmids 256 did not yield any luciferase activity, demonstrating that active replication and transcription from 257 transfected minigenome RNA was necessary for expression of the reporter gene. 258

mRNAs with mutations in the 3'UTR better associate with the polysomes 259
To further assess how mutations in the NP 3'UTR modulate translation, polysome analysis was 260 performed. HEK293T cells were transfected with purified mRNA possessing WT or mutant NP 261 3'UTRs. The cells were harvested 4 hours post transfection after a brief treatment with 262 cycloheximide to immobilize the ribosomes on actively translating mRNAs. The cleared lysate 263 was then separated on a continuous sucrose density gradient by ultracentrifugation and 264 fractions were collected. Absorbance profiles (260 nm) of the flow during fractionation were 265 taken to identify the polysome containing fractions. RNA was then extracted from all the 266 fractions and analyzed by qRT-PCR to quantify the amount of transfected RNA present. 267 Compared to the mRNA with WT 3'UTR mRNA (Fig. 8A), a larger proportion of the mRNA with 268 mutated 3'UTR associated with the polysome fractions (Fig. 8B). Increased association of the 269 edited NP 3'UTR containing mRNA with polysomes is in agreement with our observation that 270 the 3'UTR mutations enhance translation efficiency. 271

Apparent ADAR1 editing of the VP40 3'UTR from EBOV Makona isolates results in 272 increased translation 273
During the 2014-2016 West Africa EBOV outbreak varied AG changes were identified in the 274 negative-sense viral RNAs of different isolates, with sequences encoding the VP40 3'UTR being 275 a hotspot for such changes (13)(14)(15)(16)(17)(18)(19). A comprehensive analysis of 1086 publicly available full-276 length EBOV-Makona genome sequences identified 49 with clusters of AG substitutions (25). 277 Of these 30 had AG clusters in sequences corresponding to 3'UTRs, and fifteen of these had clusters in sequences corresponding to the VP40 3'UTR. Within this group, eleven isolates had 279 12-13 AG changes (25). Analysis of intra-patient virus sequences documented conversion of 280 13 AG changes during the course of disease in two different patients, demonstrating that 281 these mutations can arise independently in different individuals (25). In total, 15 different 282 ADAR1 mediated mutations were described, with fourteen of these from T to C and one change 283 from G to A (Fig. 9A). We incorporated these changes into the EBOV VP40 3'UTR and 284 compared expression to that of the WT VP40 3'UTR sequence using our reporter system. 285 Similar to what was observed for the MARV NP 3'UTR, the edited EBOV VP40 3'UTRs yielded 286 higher translation efficiency at both early (2h) and later (18h) time points (Fig. 9B). To examine 287 RNA stability, the ratio of RNA in the cells at earlier and later timepoints was calculated. The 288 ratios were similar for the WT and edited 3'UTR containing constructs, suggesting a very similar 289 decay rate for both the RNA species (Fig. 9C). This suggests that the increased translation is 290 not attributable to the stability of the RNAs. 291 To directly assess effects of 3'UTR mutations on translation, polysome analysis was performed. 292 A higher abundance of the edited 3'UTR mRNA was present in the polysome fractions as 293 compared to the WT 3'UTR mRNA ( Fig 9D). This suggests that like the MARV 3'UTR, the 294 EBOV VP40 mRNA 3'UTR contains elements that impair translation and that the impairment 295 can be relieved by ADAR1 editing. We also assessed activation of the IFN-β reporter gene with 296 these mRNAs in reporter cells. Unlike the MARV NP 3'UTRs, we did not observe differential 297 induction of the IFN-β promoter by the edited mRNAs ( Fig 9E). The positive control Sendai virus 298 infection robustly activated the promoter at both 14 and 24 hours post infection. 299 300

301
Given the minimal information available regarding the functional role of filovirus UTRs, this study 302 sought to determine how filovirus UTRs modulate the translation of mRNAs. In order to make 303 comparisons between different MARV UTRs, the model mRNAs encoding Renilla luciferase 304 were flanked by MARV 5'UTRs, 3' UTRs or both 5' and 3'UTRs. After in vitro transcription, 305 capping and polyadenylation, the mRNAs were purified and transfected into cells. Using 306 luciferase activity as a measure of gene expression at different time points post-transfection, we 307 characterized the impact of both the 5' and 3' UTRs on expression. Comparing the translation 308 efficiency of the model MARV mRNAs with both 5' and 3'UTRs identified differences in 309 expression for each model mRNA. Expression increased in the following order: L, GP, VP40, we observed that the expression levels for all mRNAs were comparable although the NP and 312 the VP35 5'UTR mRNAs yielded slightly higher levels than the rest. This suggested that the 313 variation in translation efficiency in the MARV mRNAs was mostly due to the 3'UTRs. When 314 constructs with only the MARV 3'UTRs were compared, varying effects on translation were 315 obtained. The NP, GP, VP24 and L 3' UTR containing mRNAs showed lower expression 316 compared to the rest. Interestingly, the L 3'UTR yielded the least signal, suggesting limited 317 expression of the L protein in infected cells. Low expression of protein translated from mRNAs 318 with MARV L UTRs parallels the observation that EBOV L mRNA translation is maintained at 319 low levels by cis-acting elements present in the L mRNA 5'UTR (8). Notably, translation 320 efficiency did not correspond to the lengths of either the 5' or 3'UTRs. To further refine where 321 regulatory elements reside, model mRNAs with 3'UTRs truncated from the 3' end were 322 produced. Significant enhancement was demonstrated following transfection of truncated NP 323 and L 3'UTRs. To further address the possibility that the 5'UTRs impact the effects of the 324 3'UTRs, swaps of MARV 5'UTRs with unrelated human 5'UTRs of varying lengths were made. 325 These changes did not substantially alter the modulating effects of the 3'UTRs. 326 UTRs can act as determinants of mRNA stability with various sequences imparting longer or 327 shorter half-lives for the mRNAs. Short AU rich elements (AREs) present in 3'UTRs are well 328 documented to regulate mRNA stability in the eukaryotic cells (26). Structural elements present 329 within the 3'UTRs also play a role in mRNA stability. For example, the constitutive decay 330 element present in the tumor necrosis factor 3 (TNF3) mRNA forms a stem-loop structure that is 331 recognized by Roquin and Roquin2 proteins which then recruit Ccr4-Caf1-Not deadenylase 332 complex for initiation of mRNA decay (27). The Zc3h12a endonuclease recognizes a stem-loop 333 structure in the 3'UTR of Interleukin (IL)-6 mRNA and regulates its decay (28). To address 334 possible impacts of the 3'UTRs on the stability of the transfected mRNAs, we harvested cells at 335 early and later time points, determined the amount of each transfected mRNA present in the 336 cells by qRT-PCR and the ratio of transfected mRNA in at early and late time points was then 337 calculated to determine the rate of decay. Comparable rates of decay were observed for the 338 mRNAs tested, suggesting they have very similar stabilities, regardless of whether the 3'UTR 339 sequence was wild type or edited. Therefore, mRNA stability is unlikely to explain differences 340 observed in translation. 341 A major mechanism regulating mRNA translation in eukaryotic cells is by the action of miRNAs. 342 While coding sequences and both the 5' and 3' UTRs in host and viral mRNAs are known to harbor miRNA target sites, it is the 3'UTRs where such target sites are most abundant (29)(30)(31). 344 The complex relationship between the host miRNAs and the viral RNA is well documented in  In these cells, no difference in the translation regulation by either the 5' or the 3' UTR was 350 detected, ruling out an essential role for miRNAs in regulation of translation mediated by the 351 UTRs in our assays. 352 The negative regulatory elements in the NP 3'UTR correspond to sites in the MARV genome 353 that appear to be edited by ADAR1 (10). ADAR1 enzymes catalyze the deamination of 354 adenosine (A) to inosine (I) in dsRNA structures (9). A-to-I RNA editing in the context of 355 cytoplasmic RNA viruses has been attributed to ADAR1, which has two isoforms, p110 and 356 p150. p110 is constitutively expressed and mostly nuclear, whereas p150 is IFN-inducible and 357 mainly cytoplasmic. ADAR1 prevents inappropriate activation of the innate immune system by 358 editing cytoplasmic RNAs, primarily pol II-transcribed Alu elements, that otherwise activate 359 MDA5 (36-41). ADAR1 also suppresses activation of the IFN-induced, dsRNA-activated antiviral 360 kinase, PKR; edits RNA viruses and can act in a pro-viral manner (42-44). 361 within sequences corresponding to the 600 nucleotide long 3'UTR of the GP mRNA (12). It will 374 be of interest to determine if these editing events also impact translation of the targeted mRNAs 375 and whether this facilitates adaptation to the mouse. 376 ADAR1 also appears to act on EBOV. During the 2014-2016 West Africa EBOV outbreak varied 377 AG changes were identified in the negative-sense viral RNAs of different isolates, with 378 sequences encoding the VP40 3'UTR being a hotspot for such changes (13)(14)(15)(16)(17)(18)(19)25). Of these 379 30 had AG clusters in sequences corresponding to 3'UTRs, and fifteen of these had clusters 380 in sequences corresponding to the VP40 3'UTR. Within this group, eleven isolates had 12-13 381 AG changes (25). Analysis of intra-patient virus sequences documented conversion of 13 382 AG changes during the course of disease in two different patients, demonstrating that these 383 mutations can arise independently in different individuals (25). Building these mutations into 384 model mRNAs with EBOV VP40 3'UTRs yielded a similar outcome as with the MARV NP 3'UTR 385 mRNAs, suggesting negative-regulatory elements in EBOV mRNAs as well. Given that ADAR1 386 targets dsRNA, these data suggest the presence of secondary structures that contribute to the 387 negative effects of the 3'UTR on translation. Analysis of MARV NP and EBOV VP40 3'UTRs 388 with the program RNAfold indicates the presence of potential secondary structures that are 389 altered by the RNA editing mutations (data not shown)(45). Experimental characterization of the 390 structures of these sequences should be pursued in future studies. These data also suggest the 391 possibility that activation of innate antiviral responses which upregulate ADAR1 p150 may 392 increase translation efficiency of some filovirus mRNAs. A caveat to these analyses is that not 393 all sites are edited at the same efficiency. For edits identified in MARV infected Vero cells, at 24 394 hours post-infection, frequencies of UC changes in the NP 3'UTR ranged from 8-24 percent. 395 In infected THP-1 cells at the same time point, frequencies of mutations ranged from 9-35 396 percent (10). Further, the deep sequencing approaches that identified the ADAR1 edits do not 397 determine which of the various changes are present on the same RNA molecule. 398 The potential for RNA secondary structures suggests that these might be recognized by other 399 pattern recognition receptors. We addressed this by asking whether wildtype and edited 400 3'UTRs, which would be complementary to the edited genomic RNA, might activate the 401 interferon β (IFNβ) promoter or an IFN-inducible ISRE promoter. Our data demonstrate that 5'-402 capped, 3'-polyadenylated and phosphatase-treated mRNAs possessing the MARV WT NP 403 3'UTR but not the edited 3'UTR induced IFN responses, suggesting this UTR may contribute to 404 IFN responses. Interestingly, a similar effect was not detected with the EBOV VP40 3'UTR. Why 405 the MARV NP and EBOV VP40 3'UTRs differ in this respect is unclear. It could reflect stability 406 or structure of any stem-loop structures but this will require further study. There is precedent for 407 the activation of IFN responses by capped RNAs, as 3'UTRs from influenza virus and some 408 RNA aptamers have been described as 5'-triphosphate-independent RIG-I activators (46, 47).
There is also substantial evidence that ADAR1 editing serves as a mechanism to prevent 410 induction of IFN responses and activation of PKR by self and viral RNAs (36,37,39,41,43,411 48). Therefore, ADAR1 may be hypothesized to relieve the IFN response triggered by NP 412

3'UTRs during MARV infection. To what extent such an effect occurs in infected cells remains to 413
be determined. The VP35 proteins of EBOV and MARV inhibit RIG-I mediated activation of IFN 414 responses, so it is possible these activities would mask the effects of 3'UTR (49, 50). 415 The finding that filovirus 3'UTRs include elements that decrease translation efficiency is 416 intriguing. That such elements can also activate innate immune responses, as in the case of the 417 NP 3'UTR containing mRNAs, and correspond to genome RNA sequences susceptible to 418 modification by ADAR1 is also notable. These findings suggest that possible costs of these 419 features are offset by other, yet to be identified, benefits. A previous study identified an 420 upstream open reading frame in the EBOV L 5' UTR as a suppressor of L translation, but the 421 suppression could be relieved when phosphorylation of translation initiation factor eIF2a was 422 induced. This was proposed as a mechanism by which L protein levels could be maintained at a 423 low but consistent level, even when innate immune responses are activated (8)  Total RNA was prepared from transfected samples using Trizol (Invitrogen). cDNA synthesis 488 was performed using oligo-dT primers for mRNA or random hexamers for total RNA using the 489 SuperScript IV Reverse Transcriptase kit (ThermoFisher Scientific) as per the manufacturer's 490 protocol. Quantitative PCR (qPCR) was carried out following the manufacturer's protocol using 491 SYBR Green Master Mix (ThermoFisher) on a Biorad CFX real time PCR system (51). GAPDH 492 mRNA, levels were quantified using specific primers (Fwd-CACCCACTCCTCCTACTTT, Rev-493 CCCTGTTGCTGTAGCCAAAT). Comparisons were made by the ΔΔCT method. For rate of 494 decay assays, total RNA was extracted at early (2h-4h) and late time points (18h-24h). 495 Following cDNA synthesis using oligo dT primers, reporter mRNA was quantified was by using 496 region encoding wildtype or edited MARV NP 3'UTRs was PCR amplified from constructs used 526 in the comparative expression assays. cDNA obtained from infected THP1 cells was used to 527 amplify the MARV L 5'UTR. A 'middle fragment' was assembled by PCR and was comprised of 528 the MARV NP 3'UTRthe intergenic sequence TCAAT-the L 5'UTRand firefly luciferase. 529 Similarly, a 'front fragment' comprised of T7 terminator-ribozyme-MARV leader-NP 5'UTR-RLuc 530 and an 'end fragment' comprised of L 3'UTR-trailer-T7 promoter were PCR amplified from a 531 monocistronic MARV minigenome template. Next, the front, middle and end fragments were 532 assembled by overlapping PCR amplification. All PCRs were carried out using Q5 High-Fidelity 533 polymerase (NEB). The resulting construct was designed to produce a genomic-sense RNA 534 upon in vitro transcription. The PCR amplicons were gel purified, sequenced and used as a 535 template for in vitro transcription. Transcribed bicistronic minigenome RNA was purified by LiCl 4 536 precipitation and size verified by running on a formaldehyde agarose gel. 537 To confirm transcription of mRNAs containing the wild type and edited NP 3'UTRs in cells 538 transfected with the bicistronic minigenome and helper plasmids, total RNA was harvested from 539 transfected cells. cDNA was generated by using oligo-dT primers and quantitative PCR and 540 sequencing was performed. 541

Sequence alignments 542
Conserved sequences at the 3' ends of the MARV UTRs were identified with WebLogo (53). 543 544

Sucrose gradients 545
Continuous sucrose gradients were manually prepared. 50% and 15% sucrose solutions were 546 prepared in RNAse free buffer (10 mM HEPES-KOH pH7.5, 150 mM KCl, 10 mM MgCl 2 , 547 100ug/mL cycloheximide and 1 mM DTT in RNAse free H 2 O). 1 mL each of decreasing 548 concentrations of sucrose buffers, starting with 50% and topping off with 15% for a total of 11 549 mL was added to thin wall polypropylene ultracentrifuge tubes (Beckman Coulter). The tubes 550 were immediately frozen and stored at -80 °C. On the day of use, the tubes were thawed at 551 room temperature, resulting in the formation of a continuous gradient and immediately placed 552 on ice. Formation of a continuous gradient was confirmed by adding different color dyes to the 553 50% and 15% starter sucrose solutions in a test preparation. 554 555

Polysome fractionation 556
Polysome fractionation was performed as outlined previously (54). Briefly, roughly 80% 557 confluent 10 mm plates of HEK293T cells were transfected with the in vitro transcribed test 558 mRNAs. Two hours post transfection the cells were treated with media containing 100 ug/ml 559 cycloheximide for 10 minutes to immobilize the ribosomes. The treated cells were then washed 560 once with PBS containing 100 µg/ml cycloheximide. Cells were lysed on the plates and 561 harvested by adding 1 mL ice cold lysis buffer (10 mM HEPES-KOH pH7.4, 150 mM KCl, 10 562 mM MgCl 2 , 1mM DTT, 100 µg/mL cycloheximide, 2%NP-40, protease inhibitor, 6 U/mL RNase 563 inhibitor). Lysates were centrifuged at 13000 rpm for 10 min at 4C to remove cellular debris. 800 564 µl of the lysate was loaded onto the sucrose gradient. The gradient tubes were ultracentrifuged 565 in a Beckman Coulter SW41 Ti swinging-bucket rotor at 40,000 rpm for 2 h at 4C. Samples 566 were fractionated by pumping into the tubes a chase solution of 60% sucrose in RNAse-free 567 water using a BR-188 Density Gradient Fractionation System (Brandel). Real time concentration 568 of RNA in the flow was measured by reading absorbance at 260 nm. 1 mL fractions were 569 collected and set aside for RNA extraction. RNA was extracted from 200 µl of each fraction with 570 Trizol reagent, and the quantity of transfected RNA in each fraction was determined by qRT-571 PCR. Absorbance data was analyzed by Peak Chart Data Acquisition software (Brandel) and 572 overlaid with qRT-PCR data using Prism software (Graphpad  The experiment was performed as described for Fig. 6A T7  RLUC  3'UTR   T5512C  T5519C  T5522C  T5523C  G5548A  T5553C  T5566C  T5587C  T5594C  T5595C  T5596C  T5604C  T5616C  T5631C  T5849C  C5880T   RLUC  3'