Mechanism and structural diversity of exoribonuclease-resistant RNA structures in flaviviral RNAs

MacFadden, Andrea; O'Donoghue, Zoe Gonzales; Silva, Patrícia A. G. C.; Chapman, Erich G.; Olsthoorn, René C. L.; Sterken, M.G.; Pijlman, Gorben P.; Bredenbeek, Peter J.; Kieft, Jeffrey

doi:10.1038/s41467-017-02604-y

Cited by 113 publications

(221 citation statements)

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“…Additionally, viral genomes encode many conserved structural RNA elements [26,27] that are typically not annotated in GenBank. In this work, the four dengue virus RefSeqs were updated to include annotation of a subgenomic flavivirus RNA (sfRNA) and associated stem loop structures, that are relevant in pathogenicity and evasion of the host immune system in at least some flaviviruses [5,28,29]. Incoming dengue virus sequence submissions will now include these RNA annotations because of VADR, which employs covariance models of both the conserved sequence and secondary structure of the RNA elements.…”

Section: Discussionmentioning

confidence: 99%

VADR: validation and annotation of virus sequence submissions to GenBank

Schäffer

Hatcher

Yankie

et al. 2019

Preprint

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Background: GenBank contains over 3 million viral sequences. The National Center for Biotechnology Information (NCBI) previously made available a tool for validating and annotating influenza virus sequences that is used to check submissions to GenBank. Before this project, there was no analogous tool in use for non-influenza viral sequence submissions. Results: We developed a system called VADR (Viral Annotation DefineR) that validates and annotates viral sequences in GenBank submissions. The annotation system is based on analysis of the input nucleotide sequence using models built from curated RefSeqs. Hidden Markov models are used to classify sequences by determining the RefSeq they are most similar to, and feature annotation from the RefSeq is mapped based on a nucleotide alignment of the full sequence to a covariance model. Predicted proteins encoded by the sequence are validated with nucleotide-to-protein alignments using BLAST. The system identifies 43 types of "alerts" that (unlike the previous BLAST-based system) provide deterministic and rigorous feedback to researchers who submit sequences with unexpected characteristics. VADR has been integrated into GenBank's submission processing pipeline allowing for viral submissions passing all tests to be accepted and annotated automatically, without the need for any human (GenBank indexer) intervention. Unlike the previous submission-checking system, VADR is freely available (https://github.com/nawrockie/vadr) for local installation and use. VADR has been used for Norovirus submissions since May 2018 and for Dengue virus submissions since January 2019. Other viruses with high numbers of submissions will be added incrementally. Conclusion: VADR improves the speed with which non-flu virus submissions to GenBank can be checked and improves the content and quality of the GenBank annotations. The availability and portability of the software allows researchers to run the GenBank checks prior to submitting their viral sequences, and thereby gain confidence that their submissions will be accepted immediately without the need to correspond with GenBank staff. Reciprocally, the adoption of VADR frees GenBank staff to spend more time on services other than checking routine viral sequence submissions.

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Section: Discussionmentioning

confidence: 99%

VADR: validation and annotation of virus sequence submissions to GenBank

Schäffer

Hatcher

Yankie

et al. 2019

Preprint

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“…Previous studies on the 3' UTR of both flavi-and dianthoviruses have indicated that elaborate structures are formed by the xrRNAs they utilize 19,21,35,36 . For instance, the crystal structure of Murray Valley Encephalitis Virus (MVE) flaviviral xrRNA revealed a ring-like conformation through tertiary interactions between its 5' end and a downstream hairpin, which itself forms a pseudoknot with nucleotides even more downstream 36 .…”

Section: Discussionmentioning

confidence: 99%

“…While elaborate tertiary structures are required to block Xrn1 progression in flavivirus and dianthovirus RNAs 19,21 , the role of RNA structure in the production of beny-and cucumovirus subgenomic RNAs has remained enigmatic. During infection of Beta macrocarpa by Beet necrotic yellow vein virus (BNYVV), a member of the Benyviridae family and Benyvirus genus 22 , a non-coding RNA is produced from BNYVV RNA3 23 .…”

Section: Introductionmentioning

confidence: 99%

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A widespread Xrn1-resistant RNA motif composed of two short hairpins

2019

Preprint

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Xrn1 is a major 5'-3' exoribonuclease involved in the RNA metabolism of many eukaryotic species.RNA viruses have evolved ways to thwart Xrn1 in order to produce subgenomic non-coding RNA that affects the hosts RNA metabolism. The 3' untranslated region of several beny-and cucumovirus RNAs harbors a so-called 'coremin' motif that is required for Xrn1 stalling. The structural features of this motif have not been studied in detail yet. Here, by using in vitro Xrn1 degradation assays, we tested over 50 different RNA constructs based on the Beet necrotic yellow vein virus sequence, to deduce putative structural features responsible for Xrn1-stalling. We demonstrated that the minimal benyvirus stalling site consists of two hairpins of 3 and 4 base pairs respectively. The 5' proximal hairpin requires a YGAD (Y = U/C, D = G/A/U) consensus loop sequence, whereas the 3' proximal hairpin loop sequence is variable. The sequence of the 9-nucleotide spacer that separates the hairpins is highly conserved and potentially involved in tertiary interactions. Similar coremin motifs were identified in plant virus isolates from other families including Betaflexiviridae, Virgaviridae and Secoviridae (order of the Picornavirales). We conclude that Xrn-stalling motifs are more widespread among RNA viruses than previously realized.

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“…Xrn1-resistant RNAs and sfRNAs appear to be ubiquitously present in many flaviviruses. They have been described in MBFVs, including DENV 29 , YFV 30 , JEV 31 , and ZIKV 32 , TBFVs 23,33 , and recently in ISFVs and NKVs 34,35 . There is typically more than one xrRNA, given the diverse molecular architecture of different flavivirus 3'UTRs.…”

Section: Flavivirus Genome Organizationmentioning

confidence: 99%

Musashi binding elements in Zika and related Flavivirus 3’UTRs: A comparative studyin silico

Schneider

Wolfinger

2018

Preprint

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Zika virus (ZIKV) belongs to a class of neurotropic viruses that have the ability to cause congenital infection, which can result in microcephaly or fetal demise. Recently, the RNA-binding protein Musashi-1 (Msi1), which mediates the maintenance and self-renewal of stem cells and acts as a translational regulator, has been associated with promoting ZIKV replication, neurotropism, and pathology. Msi1 predominantly binds to single-stranded motifs in the 3' untranslated region (UTR) of RNA that contain a UAG trinucleotide in their core. We systematically analyzed the properties of Musashi binding elements (MBEs) in the 3'UTR of flaviviruses with a thermodynamic model for RNA folding. Our results indicate that MBEs in ZIKV 3'UTRs occur predominantly in unpaired, single-stranded structural context, thus corroborating experimental observations by a biophysical model of RNA structure formation. Statistical analysis and comparison with related viruses show that ZIKV MBEs are maximally accessible among mosquito-borne flaviviruses. Our study addresses the broader question of whether other emerging arboviruses can cause similar neurotropic effects through the same mechanism in the developing fetus by establishing a link between the biophysical properties of viral RNA and teratogenicity. Moreover, our thermodynamic model can explain recent experimental findings and predict the Msi1-related neurotropic potential of other viruses. 2/15

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Mechanism and structural diversity of exoribonuclease-resistant RNA structures in flaviviral RNAs

Cited by 113 publications

References 50 publications

VADR: validation and annotation of virus sequence submissions to GenBank

VADR: validation and annotation of virus sequence submissions to GenBank

A widespread Xrn1-resistant RNA motif composed of two short hairpins

Musashi binding elements in Zika and related Flavivirus 3’UTRs: A comparative studyin silico

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