scholarly journals Atypical RNA Elements Modulate Translational Readthrough in Tobacco Necrosis Virus D

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
Laura R. Newburn ◽  
K. Andrew White
Keyword(s):  
Rna Structure ◽  
Viral Genome ◽  
Tobacco Necrosis Virus ◽  
Virus Infectivity ◽  
Necrosis Virus ◽  
Stem Loop ◽  
Content Type ◽  
Translational Readthrough ◽  
Rna Elements ◽  
Positive Strand Rna

ABSTRACT Tobacco necrosis virus, strain D (TNV-D), is a positive-strand RNA virus in the genus Betanecrovirus and family Tombusviridae. The production of its RNA-dependent RNA polymerase, p82, is achieved by translational readthrough. This process is stimulated by an RNA structure that is positioned immediately downstream of the recoding site, termed the readthrough stem-loop (RTSL), and a sequence in the 3′ untranslated region of the TNV-D genome, called the distal readthrough element (DRTE). Notably, a base pairing interaction between the RTSL and the DRTE, spanning ∼3,000 nucleotides, is required for enhancement of readthrough. Here, some of the structural features of the RTSL, as well as RNA sequences and structures that flank either the RTSL or DRTE, were investigated for their involvement in translational readthrough and virus infectivity. The results revealed that (i) the RTSL-DRTE interaction cannot be functionally replaced by stabilizing the RTSL structure, (ii) a novel tertiary RNA structure positioned just 3′ to the RTSL is required for optimal translational readthrough and virus infectivity, and (iii) these same activities also rely on an RNA stem-loop located immediately upstream of the DRTE. Functional counterparts for the RTSL-proximal structure may also be present in other tombusvirids. The identification of additional distinct RNA structures that modulate readthrough suggests that regulation of this process by genomic features may be more complex than previously appreciated. Possible roles for these novel RNA elements are discussed. IMPORTANCE The analysis of factors that affect recoding events in viruses is leading to an ever more complex picture of this important process. In this study, two new atypical RNA elements were shown to contribute to efficient translational readthrough of the TNV-D polymerase and to mediate robust viral genome accumulation in infections. One of the structures, located close to the recoding site, could have functional equivalents in related genera, while the other structure, positioned 3′ proximally in the viral genome, is likely limited to betanecroviruses. Irrespective of their prevalence, the identification of these novel RNA elements adds to the current repertoire of viral genome-based modulators of translational readthrough and provides a notable example of the complexity of regulation of this process.

scholarly journals Investigation of Novel RNA Elements in the 3′UTR of Tobacco Necrosis Virus-D

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 856
Author(s):  
Laura R. Newburn ◽  
Baodong Wu ◽  
K. Andrew White
Keyword(s):  
Tobacco Necrosis Virus ◽  
Regulatory Rna ◽  
Necrosis Virus ◽  
D Genome ◽  
Translational Readthrough ◽  
Rna Elements ◽  
Rna Interaction ◽  
Rna Hairpin ◽  
Virus Survival ◽  
Independent Translation

RNA elements in the untranslated regions of plus-strand RNA viruses can control a variety of viral processes including translation, replication, packaging, and subgenomic mRNA production. The 3′ untranslated region (3′UTR) of Tobacco necrosis virus strain D (TNV-D; genus Betanecrovirus, family Tombusviridae) contains several well studied regulatory RNA elements. Here, we explore a previously unexamined region of the viral 3′UTR, the sequence located upstream of the 3′-cap independent translation enhancer (3′CITE). Our results indicate that (i) a long-range RNA–RNA interaction between an internal RNA element and the 3′UTR facilitates translational readthrough, and may also promote viral RNA synthesis; (ii) a conserved RNA hairpin, SLX, is required for efficient genome accumulation; and (iii) an adenine-rich region upstream of the 3′CITE is dispensable, but can modulate genome accumulation. These findings identified novel regulatory RNA elements in the 3′UTR of the TNV-D genome that are important for virus survival.

scholarly journals A Comprehensive Analysis of cis-Acting RNA Elements in the SARS-CoV-2 Genome by a Bioinformatics Approach

2020 ◽  
Vol 11 ◽  
Author(s):  
Firoz Ahmed ◽  
Monika Sharma ◽  
Abdulsalam Abdullah Al-Ghamdi ◽  
Sultan Muhammad Al-Yami ◽  
Abdulaziz Musa Al-Salami ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Rna Structure ◽  
Viral Genome ◽  
Genomic Sequence ◽  
Evolutionary Relationship ◽  
Cis Acting ◽  
Full Genomic Sequence ◽  
Rna Elements ◽  
Global Threat ◽  
Genomic Regions

The emergence of a new coronavirus (CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for severe respiratory disease in humans termed coronavirus disease of 2019 (COVID-19), became a new global threat for health and the economy. The SARS-CoV-2 genome is about a 29,800-nucleotide-long plus-strand RNA that can form functionally important secondary and higher-order structures called cis-acting RNA elements. These elements can interact with viral proteins, host proteins, or other RNAs and be involved in regulating translation and replication processes of the viral genome and encapsidation of the virus. However, the cis-acting RNA elements and their biological roles in SARS-CoV-2 as well as their comparative analysis in the closely related viral genome have not been well explored, which is very important to understand the molecular mechanism of viral infection and pathogenies. In this study, we used a bioinformatics approach to identify the cis-acting RNA elements in the SARS-CoV-2 genome. Initially, we aligned the full genomic sequence of six different CoVs, and a phylogenetic analysis was performed to understand their evolutionary relationship. Next, we predicted the cis-acting RNA elements in the SARS-CoV-2 genome using the structRNAfinder tool. Then, we annotated the location of these cis-acting RNA elements in different genomic regions of SARS-CoV-2. After that, we analyzed the sequence conservation patterns of each cis-acting RNA element among the six CoVs. Finally, the presence of cis-acting RNA elements across different CoV genomes and their comparative analysis was performed. Our study identified 12 important cis-acting RNA elements in the SARS-CoV-2 genome; among them, Corona_FSE, Corona_pk3, and s2m are highly conserved across most of the studied CoVs, and Thr_leader, MAT2A_D, and MS2 are uniquely present in SARS-CoV-2. These RNA structure elements can be involved in viral translation, replication, and encapsidation and, therefore, can be potential targets for better treatment of COVID-19. It is imperative to further characterize these cis-acting RNA elements experimentally for a better mechanistic understanding of SARS-CoV-2 infection and therapeutic intervention.

scholarly journals Structural and Functional Elements of the Promoter Encoded by the 5′ Untranslated Region of the Venezuelan Equine Encephalitis Virus Genome

2009 ◽  
Vol 83 (17) ◽  
pp. 8327-8339 ◽  
Author(s):  
Raghavendran Kulasegaran-Shylini ◽  
Svetlana Atasheva ◽  
David G. Gorenstein ◽  
Ilya Frolov
Keyword(s):  
Viral Genome ◽  
Untranslated Region ◽  
Rna Replication ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Venezuelan Equine Encephalitis ◽  
Functional Elements ◽  
Equine Encephalitis Virus ◽  
Rna Elements ◽  
Positive Strand Rna

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. The pathogenesis of this virus depends strongly on the sequences of the structural proteins and on the mutations in the RNA promoter encoded by the 5′ untranslated region (5′UTR) of the viral genome. In this study, we performed a detailed investigation of the structural and functional elements of the 5′-terminal promoter and analyzed the effect of multiple mutations introduced into the VEEV 5′UTR on virus and RNA replication. The results of this study demonstrate that RNA replication is determined by two synergistically functioning RNA elements. One of them is a very 5′-terminal AU dinucleotide, which is not involved in the stable RNA secondary structure, and the second is a short, G-C-rich RNA stem. An increase or decrease in the stem's stability has deleterious effects on virus and RNA replication. In response to mutations in these RNA elements, VEEV replicative machinery was capable of developing new, compensatory sequences in the 5′UTR either containing 5′-terminal AUG or AU repeats or leading to the formation of new, heterologous stem-loops. Analysis of the numerous compensatory mutations suggested that at least two different mechanisms are involved in their generation. Some of the modifications introduced into the 5′ terminus of the viral genome led to an accumulation of the mutations in the VEEV nsPs, which suggested to us that there is a direct involvement of these proteins in promoter recognition. Furthermore, our data provide new evidence that the 3′ terminus of the negative-strand viral genome in the double-stranded RNA replicative intermediate is represented by a single-stranded RNA. Both the overall folding and the sequence determine its efficient function as a promoter for VEEV positive-strand RNA genome synthesis.

scholarly journals Translational readthrough in Tobacco necrosis virus-D

Virology ◽  
2014 ◽  
Vol 450-451 ◽  
pp. 258-265 ◽  
Author(s):  
Laura R. Newburn ◽  
Beth L. Nicholson ◽  
Michael Yosefi ◽  
Peter A. Cimino ◽  
K. Andrew White
Keyword(s):  
Tobacco Necrosis Virus ◽  
Necrosis Virus ◽  
Translational Readthrough

scholarly journals A 212-nt long RNA structure in the Tobacco necrosis virus-D RNA genome is resistant to Xrn degradation

2019 ◽  
Vol 47 (17) ◽  
pp. 9329-9342 ◽  
Author(s):  
Chaminda D Gunawardene ◽  
Laura R Newburn ◽  
K Andrew White
Keyword(s):  
Rna Structure ◽  
Rna Viruses ◽  
Degradation Products ◽  
Rna Degradation ◽  
Viral Rna ◽  
Tobacco Necrosis Virus ◽  
Rna Structures ◽  
Necrosis Virus

Abstract Plus-strand RNA viruses can accumulate viral RNA degradation products during infections. Some of these decay intermediates are generated by the cytosolic 5′-to-3′ exoribonuclease Xrn1 (mammals and yeast) or Xrn4 (plants) and are formed when the enzyme stalls on substrate RNAs upon encountering inhibitory RNA structures. Many Xrn-generated RNAs correspond to 3′-terminal segments within the 3′-UTR of viral genomes and perform important functions during infections. Here we have investigated a 3′-terminal small viral RNA (svRNA) generated by Xrn during infections with Tobacco necrosis virus-D (family Tombusviridae). Our results indicate that (i) unlike known stalling RNA structures that are compact and modular, the TNV-D structure encompasses the entire 212 nt of the svRNA and is not functionally transposable, (ii) at least two tertiary interactions within the RNA structure are required for effective Xrn blocking and (iii) most of the svRNA generated in infections is derived from viral polymerase-generated subgenomic mRNA1. In vitro and in vivo analyses allowed for inferences on roles for the svRNA. Our findings provide a new and distinct addition to the growing list of Xrn-resistant viral RNAs and stalling structures found associated with different plant and animal RNA viruses.

scholarly journals Structural Properties of a Multifunctional T-Shaped RNA Domain That Mediate Efficient Tomato Bushy Stunt Virus RNA Replication

2004 ◽  
Vol 78 (19) ◽  
pp. 10490-10500 ◽  
Author(s):  
Debashish Ray ◽  
Hong Na ◽  
K. Andrew White
Keyword(s):  
Rna Structure ◽  
Function Analysis ◽  
Rna Replication ◽  
Tomato Bushy Stunt Virus ◽  
Viral Rna ◽  
Genome Replication ◽  
Dependent Manner ◽  
Stem Loop ◽  
Virus Rna ◽  
Positive Strand Rna

ABSTRACT In positive-strand RNA viruses, 5′ untranslated regions (5′ UTRs) mediate many essential viral processes, including genome replication. Previously, we proposed that the 5′-terminal portion of the genomic leader sequence of Tomato bushy stunt virus (TBSV) forms an RNA structure containing a 3-helix junction, termed the T-shaped domain (TSD). In the present study, we have carried out structure-function analysis of the proposed TSD and have confirmed an important role for this domain in mediating efficient viral RNA amplification. Using a model TBSV defective interfering RNA replicon and a protoplast system, we demonstrated that various TSD subelements contribute to the efficiency of viral RNA replication. In particular, the stabilities of all three stems (S1, S2, and S4) forming the 3-helix junction are important, while stem-loop 3—a terminal extension of S2—is largely dispensable. Additionally, some of the sequences forming the 3-helix junction are required in an identity-dependent manner. Thus, both secondary structure and nucleotide identity are important for TSD-mediated viral RNA replication. Importantly, these results are fully consistent with the dual functions we defined previously for the sequences corresponding to loops 3 and 4, respectively, in facilitating 5′ cap- and 3′ poly(A) tail-independent translation of the genome by forming a loop-loop interaction with the 3′-proximal translational enhancer and in mediating viral RNA replication through formation of a pseudoknot with the adjacent downstream RNA domain. Also, since comparable TSDs and associated interactions are predicted in the 5′ UTRs of all sequenced Aureusvirus genomes, members of at least one other genus in the family Tombusviridae appear to utilize this type of multifunctional RNA domain.

scholarly journals A Stem-Loop Structure inPotato Leafroll VirusOpen Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream

2018 ◽  
Vol 92 (11) ◽  
Author(s):  
Yi Xu ◽  
Ho-Jong Ju ◽  
Stacy DeBlasio ◽  
Elizabeth J. Carino ◽  
Richard Johnson ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Rna Structure ◽  
Stop Codon ◽  
Loop Structure ◽  
Long Distance ◽  
Stem Loop ◽  
Content Type ◽  
Stem Loop Structure ◽  
Rna Domains ◽  
Stop Codon Readthrough

ABSTRACTTranslational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of theLuteoviridaeto 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 ofPotato leafroll virus(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 ofCereal yellow dwarf viruswas 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.IMPORTANCEProgrammed 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 theLuteoviridaeby identifying the amino acids inserted at the stop codon and RNA structures that facilitate this “leakiness” of the stop codon. Members of theLuteoviridaeencode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains inPotato leafroll virus(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. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed.

scholarly journals The Host Factor AUF1 p45 Supports Flavivirus Propagation by Triggering the RNA Switch Required for Viral Genome Cyclization

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
Susann Friedrich ◽  
Susanne Engelmann ◽  
Tobias Schmidt ◽  
Grit Szczepankiewicz ◽  
Sandra Bergs ◽  
...  
Keyword(s):  
Viral Replication ◽  
Viral Genome ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Viral Rna ◽  
West Nile ◽  
Rna Chaperone ◽  
Stem Loop ◽  
Content Type ◽  
Rna Switch

ABSTRACTIn previous studies, we showed that the cellular RNA-binding protein AUF1 supports the replication process of the flavivirus West Nile virus. Here we demonstrate that the protein also enables effective proliferation of dengue virus and Zika virus, indicating that AUF1 is a general flavivirus host factor. Further studies demonstrated that the AUF1 isoform p45 significantly stimulates the initiation of viral RNA replication and that the protein's RNA chaperone activity enhances the interactions of the viral 5′UAR and 3′UAR genome cyclization sequences. Most interestingly, we observed that AUF1 p45 destabilizes not only the 3′-terminal stem-loop (3′SL) but also 5′-terminal stem-loop B (SLB) of the viral genome. RNA structure analyses revealed that AUF1 p45 increases the accessibility of defined nucleotides within the 3′SL and SLB and, in this way, exposes both UAR cyclization elements. Conversely, AUF1 p45 does not modulate the fold of stem-loop A (SLA) at the immediate genomic 5′ end, which is proposed to function as a promoter of the viral RNA-dependent RNA polymerase (RdRp). These findings suggest that AUF1 p45, by destabilizing specific stem-loop structures within the 5′ and 3′ ends of the flaviviral genome, assists genome cyclization and concurrently enables the RdRp to initiate RNA synthesis. Our study thus highlights the role of a cellular RNA-binding protein inducing a flaviviral RNA switch that is crucial for viral replication.IMPORTANCEThe genusFlaviviruswithin theFlaviviridaefamily includes important human pathogens, such as dengue, West Nile, and Zika viruses. The initiation of replication of the flaviviral RNA genome requires a transformation from a linear to a cyclized form. This involves considerable structural reorganization of several RNA motifs at the genomic 5′ and 3′ ends. Specifically, it needs a melting of stem structures to expose complementary 5′ and 3′ cyclization elements to enable their annealing during cyclization. Here we show that a cellular RNA chaperone, AUF1 p45, which supports the replication of all three aforementioned flaviviruses, specifically rearranges stem structures at both ends of the viral genome and in this way permits 5′-3′ interactions of cyclization elements. Thus, AUF1 p45 triggers the RNA switch in the flaviviral genome that is crucial for viral replication. These findings represent an important example of how cellular (host) factors promote the propagation of RNA viruses.

scholarly journals Interplay of RNA Elements in the Dengue Virus 5′ and 3′ Ends Required for Viral RNA Replication

2010 ◽  
Vol 84 (12) ◽  
pp. 6103-6118 ◽  
Author(s):  
Peter Friebe ◽  
Eva Harris
Keyword(s):  
Dengue Virus ◽  
Rna Structure ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Viral Rna ◽  
Coding Region ◽  
Stem Loop ◽  
Complementary Sequences ◽  
Translation Start ◽  
Rna Elements

ABSTRACT Dengue virus (DENV) is a member of the Flavivirus genus of positive-sense RNA viruses. DENV RNA replication requires cyclization of the viral genome mediated by two pairs of complementary sequences in the 5′ and 3′ ends, designated 5′ and 3′ cyclization sequences (5′-3′ CS) and the 5′ and 3′ upstream of AUG region (5′-3′ UAR). Here, we demonstrate that another stretch of six nucleotides in the 5′ end is involved in DENV replication and possibly genome cyclization. This new sequence is located downstream of the AUG, designated the 5′ downstream AUG region (5′ DAR); the motif predicted to be complementary in the 3′ end is termed the 3′ DAR. In addition to the UAR, CS and DAR motifs, two other RNA elements are located at the 5′ end of the viral RNA: the 5′ stem-loop A (5′ SLA) interacts with the viral RNA-dependent RNA polymerase and promotes RNA synthesis, and a stem-loop in the coding region named cHP is involved in translation start site selection as well as RNA replication. We analyzed the interplay of these 5′ RNA elements in relation to RNA replication, and our data indicate that two separate functional units are formed; one consists of the SLA, and the other includes the UAR, DAR, cHP, and CS elements. The SLA must be located at the 5′ end of the genome, whereas the position of the second unit is more flexible. We also show that the UAR, DAR, cHP, and CS must act in concert and therefore likely function together to form the tertiary RNA structure of the circularized DENV genome.

scholarly journals Poliovirus CRE-Dependent VPg Uridylylation Is Required for Positive-Strand RNA Synthesis but Not for Negative-Strand RNA Synthesis

2003 ◽  
Vol 77 (8) ◽  
pp. 4739-4750 ◽  
Author(s):  
Kenneth E. Murray ◽  
David J. Barton
Keyword(s):  
Rna Structure ◽  
Rna Synthesis ◽  
Critical Role ◽  
Rna Replication ◽  
Stem Loop ◽  
Positive Strand ◽  
Negative Strand ◽  
Step Model ◽  
Positive Strand Rna

ABSTRACT The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpUOH, the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpUOH, however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpUOH synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpUOH were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpUOH in the asymmetric replication of poliovirus RNA.

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