Atypical RNA Elements Modulate Translational Readthrough in Tobacco Necrosis Virus D

Newburn, Laura R.; White, K. Andrew

doi:10.1128/jvi.02443-16

Cited by 15 publications

(23 citation statements)

References 47 publications

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“…The 6-nt DRTE base pairs to a bulge in a very large stem-loop structure (called the proximal readthrough element [PRTE]) immediately adjacent to the leaky stop codon. Betanecroviruses (Tombusviridae) contain an additional readthrough-enhancing stem-loop adjacent to the PRTE (43). Alternative base pairings adjacent to the DRTE appear to regulate the readthrough efficiency of tombusviruses (13).…”

Section: Discussionmentioning

confidence: 99%

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream

DeBlasio

et al. 2018

J Virol

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Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein. Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strat...

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Section: Discussionmentioning

confidence: 99%

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream

DeBlasio

et al. 2018

J Virol

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“…In vitro SHAPE RNA structure probing Selective 2 0 -hydroxyl acylation analyzed by primer extension (SHAPE) was completed as described previously [34]. Briefly, full-length in vitro transcribed RNA1 genomic RNA was refolded and then treated with 1-methyl-7-nitroisatoic anhydride.…”

Section: Rna Secondary Structure Predictionmentioning

confidence: 99%

A trans-activator-like structure in RCNMV RNA1 evokes the origin of the trans-activator in RNA2

Newburn

White

2020

PLoS Pathog

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The Red clover necrotic mosaic virus (RCNMV) genome consists of two plus-strand RNA genome segments, RNA1 and RNA2. RNA2 contains a multifunctional RNA structure known as the trans-activator (TA) that (i) promotes subgenomic mRNA transcription from RNA1, (ii) facilitates replication of RNA2, and (iii) mediates particle assembly and copackaging of genome segments. The TA has long been considered a unique RNA element in RCNMV. However, by examining results from RCNMV genome analyses in the ViRAD virus (re-)annotation database, a putative functional RNA element in the polymerase-coding region of RNA1 was identified. Structural and functional analyses revealed that the novel RNA element adopts a TA-like structure (TALS) and, similar to the requirement of the TA for RNA2 replication, the TALS is necessary for the replication of RNA1. Both the TA and TALS possess near-identical asymmetrical internal loops that are critical for efficient replication of their corresponding genome segments, and these structural motifs were found to be functionally interchangeable. Moreover, replacement of the TA in RNA2 with a stabilized form of the TALS directed both RNA2 replication and packaging of both genome segments. Based on their comparable properties and considering evolutionary factors, we propose that the TALS appeared de novo in RNA1 first and, subsequently, the TA arose de novo in RNA2 as a functional mimic of the TALS. This and other related information were used to formulate a plausible evolutionary pathway to describe the genesis of the bi-segmented RCNMV genome. The resulting scenario provides an evolutionary framework to further explore and test possible origins of this segmented RNA plant virus.

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“…A stable bulged readthrough stem-loop (RTSL) immediately downstream of the leaky stop codon contains a G-rich bulge which must base pair to a distant readthrough element (DRTE) located 3 kb downstream in the structure required for replication initiation (Newburn et al, 2014). A pseudoknot immediately 3 ′ to the RTSL, and a stem-loop adjacent 5 ′ to the DRTE in the 3 ′ -UTR are also necessary for optimal readthrough (Newburn and White, 2017). The long-distance interactions within the viral genome required for frameshifting and readthrough may play a regulatory role as switch between translation and replication (Cimino et al, 2011), by allowing replicase entering at the 3 ′ end of the genome to stop its own translation 3-4 kb upstream, as it disrupts this essential long-distance interaction (Miller and White, 2006).…”

Section: Stop-codon Readthroughmentioning

confidence: 99%

Non-canonical Translation in Plant RNA Viruses

et al. 2017

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Viral protein synthesis is completely dependent upon the host cell's translational machinery. Canonical translation of host mRNAs depends on structural elements such as the 5′ cap structure and/or the 3′ poly(A) tail of the mRNAs. Although many viral mRNAs are devoid of one or both of these structures, they can still translate efficiently using non-canonical mechanisms. Here, we review the tools utilized by positive-sense single-stranded (+ss) RNA plant viruses to initiate non-canonical translation, focusing on cis-acting sequences present in viral mRNAs. We highlight how these elements may interact with host translation factors and speculate on their contribution for achieving translational control. We also describe other translation strategies used by plant viruses to optimize the usage of the coding capacity of their very compact genomes, including leaky scanning initiation, ribosomal frameshifting and stop-codon readthrough. Finally, future research perspectives on the unusual translational strategies of +ssRNA viruses are discussed, including parallelisms between viral and host mRNAs mechanisms of translation, particularly for host mRNAs which are translated under stress conditions.

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Atypical RNA Elements Modulate Translational Readthrough in Tobacco Necrosis Virus D

Cited by 15 publications

References 47 publications

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream

A trans-activator-like structure in RCNMV RNA1 evokes the origin of the trans-activator in RNA2

Non-canonical Translation in Plant RNA Viruses

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