scholarly journals Opium Poppy Mosaic Virus Has an Xrn-Resistant, Translated Subgenomic RNA and a BTE 3′ CITE

2021 ◽  
Vol 95 (9) ◽  
Author(s):  
Muhammad Ilyas ◽  
Zhiyou Du ◽  
Anne E. Simon
Keyword(s):  
Mosaic Virus ◽  
Structural Features ◽  
Opium Poppy ◽  
Luciferase Reporter ◽  
Long Distance ◽  
Subgenomic Rna ◽  
The Family ◽  
Rna Interaction ◽  
Independent Translation

ABSTRACT Opium poppy mosaic virus (OPMV) is a recently discovered umbravirus in the family Tombusviridae. OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNAD). sgRNA1 codes for a 30-kDa protein in vitro that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3′ untranslated region of OPMV contains two 3′ cap-independent translation enhancers (3′ CITEs), a T-shaped structure (TSS) near its 3′ end, and a Barley yellow dwarf virus-like translation element (BTE) in the central region. Only the BTE is functional in luciferase reporter constructs containing gRNA or sgRNA2 5′ sequences in vivo, which differs from how umbravirus 3′ CITEs were used in a previous study. Similarly to most 3′ CITEs, the OPMV BTE links to the 5′ end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence. IMPORTANCE Opium poppy mosaic virus (OPMV) is an umbravirus in the family Tombusviridae. We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5′ end just upstream from a previously predicted xrRNAD site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNAD sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation in vivo. We also characterized the OPMV BTE structure, a 3′ cap-independent translation enhancer (3′ CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3′ CITEs.

scholarly journals A Ribosome-Binding, 3′ Translational Enhancer Has a T-Shaped Structure and Engages in a Long-Distance RNA-RNA Interaction

2012 ◽  
Vol 86 (18) ◽  
pp. 9828-9842 ◽  
Author(s):  
Feng Gao ◽  
Wojciech Kasprzak ◽  
Vera A. Stupina ◽  
Bruce A. Shapiro ◽  
Anne E. Simon
Keyword(s):  
Mosaic Virus ◽  
Structural Features ◽  
Initiation Factor ◽  
Long Distance ◽  
Content Type ◽  
Ribosome Binding ◽  
Translational Enhancer ◽  
Rna Interaction ◽  
Kissing Loop

Many plant RNA viruses contain elements in their 3′ untranslated regions (3′ UTRs) that enhance translation. The PTE (Panicum mosaic virus-like translational enhancer) ofPea enation mosaic virus(PEMV) binds to eukaryotic initiation factor 4E (eIF4E), but how this affects translation from the 5′ end is unknown. We have discovered a three-way branched element just upstream of the PEMV PTE that engages in a long-distance kissing-loop interaction with a coding sequence hairpin that is critical for the translation of a reporter construct and the accumulation of the viral genomein vivo. Loss of the long-distance interaction was more detrimental than elimination of the adjacent PTE, indicating that the RNA-RNA interaction supports additional translation functions besides relocating the PTE to the 5′ end. The branched element is predicted by molecular modeling and molecular dynamics to form a T-shaped structure (TSS) similar to the ribosome-binding TSS ofTurnip crinkle virus(TCV). The PEMV element binds to plant 80S ribosomes with aKd(dissociation constant) of 0.52 μM and to 60S subunits with aKdof 0.30 μM. Unlike the TCV TSS, the PEMV element also binds 40S subunits (Kd, 0.36 μM). Mutations in the element that suppressed translation reduced either ribosome binding or the RNA-RNA interaction, suggesting that ribosome binding is important for function. This novel, multifunctional element is designated a kl-TSS (kissing-loop T-shaped structure) to distinguish it from the TCV TSS. The kl-TSS has sequence and structural features conserved with the upper portion of most PTE-type elements, which, with the exception of the PEMV PTE, can engage in similar long-distance RNA-RNA interactions.

scholarly journals Cap-Independent Translational Enhancement by the 3′ Untranslated Region of Red Clover Necrotic Mosaic Virus RNA1

2003 ◽  
Vol 77 (22) ◽  
pp. 12113-12121 ◽  
Author(s):  
Hiroyuki Mizumoto ◽  
Masahiro Tatsuta ◽  
Masanori Kaido ◽  
Kazuyuki Mise ◽  
Tetsuro Okuno
Keyword(s):  
Mosaic Virus ◽  
Untranslated Region ◽  
Red Clover ◽  
Luciferase Reporter ◽  
Base Substitution ◽  
Luciferase Reporter Gene ◽  
Stem Loop ◽  
Genomic Rnas ◽  
Translational Activity ◽  
Independent Translation

ABSTRACT Red clover necrotic mosaic virus (RCNMV) is a member of the genus Dianthovirus and has a bipartite positive-sense genomic RNA with 3′ ends that are not polyadenylated. In this study, we show that both genomic RNA1 and RNA2 lack a 5′ cap structure and that uncapped in vitro transcripts of RCNMV RNA1 replicated to a level comparable to that for capped transcripts in cowpea protoplasts. Because the 5′ cap and 3′ poly(A) tail play important roles in the translation of many eukaryotic mRNAs, genomic RNAs of RCNMV should contain an element(s) responsible for 5′ cap- and poly(A) tail-independent translation of viral protein. By using a luciferase reporter assay system in vivo, we showed that the 3′ untranslated region (UTR) of RNA1 alone significantly enhanced translation of the luciferase reporter gene in the absence of the 5′ cap structure. Deletion studies revealed that the middle region (between nucleotides 3596 and 3732) in the 3′ UTR, designated the 3′ translation element of Dianthovirus RNA1 (3′TE-DR1), plays an important role in cap-independent translation. This region contained a stem-loop structure conserved among members of the genera Dianthovirus and Luteovirus. A five-base substitution in the loop abolished cap-independent translational activity, as reported for a luteovirus, indicating that this stem-loop is one of the functional structures in the 3′TE-DR1 involved in cap-independent translation. Finally, we suggest that cap-independent translational activity is required for RCNMV RNA1 replication in protoplasts.

scholarly journals A unique internal ribosome entry site representing a dynamic equilibrium state of RNA tertiary structure in the 5′-UTR of Wheat yellow mosaic virus RNA1

2019 ◽  
Author(s):  
Guowei Geng ◽  
Chengming Yu ◽  
Xiangdong Li ◽  
Xuefeng Yuan
Keyword(s):  
Equilibrium State ◽  
Mosaic Virus ◽  
Dynamic Equilibrium ◽  
Yellow Mosaic Virus ◽  
Wheat Yellow Mosaic Virus ◽  
Long Distance ◽  
Internal Ribosome Entry ◽  
Interaction Sites ◽  
Dynamic Equilibrium State ◽  
Rna Interaction

Abstract Internal ribosome entry sites (IRESes) were first reported in RNA viruses and subsequently identified in cellular mRNAs. In this study, IRES activity of the 5′-UTR in Wheat yellow mosaic virus (WYMV) RNA1 was identified, and the 3′-UTR synergistically enhanced this IRES activity via long-distance RNA–RNA interaction between C80U81and A7574G7575. Within the 5′-UTR, the hairpin 1(H1), flexible hairpin 2 (H2) and linker region (LR1) between H1 and H2 played an essential role in cap-independent translation, which is associated with the structural stability of H1, length of discontinuous stems and nucleotide specificity of the H2 upper loop and the long-distance RNA–RNA interaction sites in LR1. The H2 upper loop is a target region of the eIF4E. Cytosines (C55, C66, C105 and C108) in H1 and H2 and guanines (G73, G79 and G85) in LR1 form discontinuous and alternative base pairing to maintain the dynamic equilibrium state, which is used to elaborately regulate translation at a suitable level. The WYMV RNA1 5′-UTR contains a novel IRES, which is different from reported IRESes because of the dynamic equilibrium state. It is also suggested that robustness not at the maximum level of translation is the selection target during evolution of WYMV RNA1.

scholarly journals Beet necrotic yellow vein virus subgenomic RNA3 is a cleavage product leading to stable non-coding RNA required for long-distance movement

2012 ◽  
Vol 93 (5) ◽  
pp. 1093-1102 ◽  
Author(s):  
Claire Peltier ◽  
Elodie Klein ◽  
Kamal Hleibieh ◽  
Massimiliano D’Alonzo ◽  
Philippe Hammann ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rna Virus ◽  
Cleavage Product ◽  
Yellow Vein ◽  
35S Promoter ◽  
Long Distance ◽  
Non Coding Rna ◽  
Long Distance Movement

Beet necrotic yellow vein virus (BNYVV) is a multipartite RNA virus. BNYVV RNA3 does not accumulate in non-host transgenic Arabidopsis thaliana plants when expressed using a 35S promoter. However, a 3′-derivative species has been detected in transgenic plants and in transient expression assays conducted in Nicotiana benthamiana and Beta macrocarpa. The 3′-derivative species is similar to the previously reported subgenomic RNA3 produced during virus infection. 5′ RACE revealed that the truncated forms had identical 5′ ends. The 5′ termini carried the coremin motif also present on BNYVV RNA5, beet soil-borne mosaic virus RNA3 and 4, and cucumber mosaic virus group 2 RNAs. This RNA3 species lacks a m7Gppp at the 5′ end of the cleavage products, whether expressed transiently or virally. Mutagenesis revealed the importance of the coremin sequence for both long-distance movement and stabilization of the cleavage product in vivo and in vitro. The isolation of various RNA3 5′-end products suggests the existence of a cleavage between nt 212 and 1234 and subsequent exonucleolytic degradation, leading to the accumulation of a non-coding RNA. When RNA3 was incubated in wheatgerm extracts, truncated forms appeared rapidly and their appearance was protein- and divalent ion-dependent.

scholarly journals Mechanism of Stimulation of Plus-Strand Synthesis by an RNA Replication Enhancer in a Tombusvirus

2005 ◽  
Vol 79 (15) ◽  
pp. 9777-9785 ◽  
Author(s):  
Tadas Panavas ◽  
Peter D. Nagy
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Replication ◽  
Dual Function ◽  
Long Distance ◽  
Replication Assay ◽  
Rna Interaction ◽  
Stimulation Of

ABSTRACT Replication of RNA viruses is regulated by cis-acting RNA elements, including promoters, replication silencers, and replication enhancers (REN). To dissect the function of an REN element involved in plus-strand RNA synthesis, we developed an in vitro trans-replication assay for tombusviruses, which are small plus-strand RNA viruses. In this assay, two RNA strands were tethered together via short complementary regions with the REN present in the nontemplate RNA, whereas the promoter was located in the template RNA. We found that the template activity of the tombusvirus replicase preparation was stimulated in trans by the REN, suggesting that the REN is a functional enhancer when located in the vicinity of the promoter. In addition, this study revealed that the REN has dual function during RNA synthesis. (i) It binds to the viral replicase. (ii) It interacts with the core plus-strand initiation promoter via a long-distance RNA-RNA interaction, which leads to stimulation of initiation of plus-strand RNA synthesis by the replicase in vitro. We also observed that this RNA-RNA interaction increased the in vivo accumulation and competitiveness of defective interfering RNA, a model template. We propose that REN is important for asymmetrical viral RNA replication that leads to more abundant plus-strand RNA progeny than the minus-strand intermediate, a hallmark of replication of plus-strand RNA viruses.

scholarly journals The short- and long-range RNA-RNA Interactome of SARS-CoV-2

2020 ◽  
Author(s):  
Omer Ziv ◽  
Jonathan Price ◽  
Lyudmila Shalamova ◽  
Tsveta Kamenova ◽  
Ian Goodfellow ◽  
...  
Keyword(s):  
Long Range ◽  
Rna Structure ◽  
Purifying Selection ◽  
Etiologic Agent ◽  
Long Distance ◽  
Rna Interaction ◽  
Positive Strand Rna ◽  
Discontinuous Transcription ◽  
Transcription And Replication

SUMMARYThe Coronaviridae is a family of positive-strand RNA viruses that includes SARS-CoV-2, the etiologic agent of the COVID-19 pandemic. Bearing the largest single-stranded RNA genomes in nature, coronaviruses are critically dependent on long-distance RNA-RNA interactions to regulate the viral transcription and replication pathways. Here we experimentally mapped the in vivo RNA-RNA interactome of the full-length SARS-CoV-2 genome and subgenomic mRNAs. We uncovered a network of RNA-RNA interactions spanning tens of thousands of nucleotides. These interactions reveal that the viral genome and subgenomes adopt alternative topologies inside cells, and engage in different interactions with host RNAs. Notably, we discovered a long-range RNA-RNA interaction - the FSE-arch - that encircles the programmed ribosomal frameshifting element. The FSE-arch is conserved in the related MERS-CoV and is under purifying selection. Our findings illuminate RNA structure based mechanisms governing replication, discontinuous transcription, and translation of coronaviruses, and will aid future efforts to develop antiviral strategies.

Mechanism of synthesis of turnip yellow mosaic virus coat protein subgenomic RNA in vivo

Virology ◽  
1989 ◽  
Vol 171 (2) ◽  
pp. 386-393 ◽  
Author(s):  
Radhia Gargouri ◽  
Rajiv L. Joshi ◽  
John F. Bol ◽  
Suzanne Astier-Manifacier ◽  
Anne-Lise Haenni
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Yellow Mosaic Virus ◽  
Turnip Yellow Mosaic Virus ◽  
Subgenomic Rna ◽  
Virus Coat Protein ◽  
Virus Coat

scholarly journals Similarities and Differences between the Subgenomic and Minus-Strand Promoters of an RNA Plant Virus

2004 ◽  
Vol 78 (8) ◽  
pp. 4048-4053 ◽  
Author(s):  
René C. L. Olsthoorn ◽  
P. C. Joost Haasnoot ◽  
John F. Bol
Keyword(s):  
Plant Virus ◽  
Alfalfa Mosaic Virus ◽  
Structural Features ◽  
Minus Strand ◽  
Promoter Regions ◽  
Subgenomic Rna ◽  
Live Virus ◽  
The Family ◽  
Subgenomic Promoter

ABSTRACT Promoter regions required for minus-strand and subgenomic RNA synthesis have been mapped for several plus-strand RNA viruses. In general, the two types of promoters do not share structural features even though they are recognized by the same viral polymerase. The minus-strand promoter of Alfalfa mosaic virus (AMV), a plant virus of the family Bromoviridae, consists of a triloop hairpin (hpE) which is attached to a 3′ tRNA-like structure (TLS). In contrast, the AMV subgenomic promoter consists of a single triloop hairpin that bears no sequence homology with hpE. Here we show that hpE, when detached from its TLS, can function as a subgenomic promoter in vitro and can replace the authentic subgenomic promoter in the live virus. Thus, the AMV subgenomic and minus-strand promoters are basically the same, but the minus-strand promoter is linked to a 3′ TLS to force the polymerase to initiate at the very 3′end.

scholarly journals Requirements for Brome Mosaic Virus Subgenomic RNA Synthesis In Vivo and Replicase-Core Promoter Interactions In Vitro

2004 ◽  
Vol 78 (12) ◽  
pp. 6091-6101 ◽  
Author(s):  
K. Sivakumaran ◽  
Seung-Kook Choi ◽  
Masarapu Hema ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Wild Type ◽  
Subgenomic Rna ◽  
The Core ◽  
Rna Hairpin

ABSTRACT Based solely on in vitro results, two contrasting models have been proposed for the recognition of the brome mosaic virus (BMV) subgenomic core promoter by the replicase. The first posits that the replicase recognizes at least four key nucleotides in the core promoter, followed by an induced fit, wherein some of the nucleotides base pair prior to the initiation of RNA synthesis (S. Adkins and C. C. Kao, Virology 252:1-8, 1998). The second model posits that a short RNA hairpin in the core promoter serves as a landing pad for the replicase and that at least some of the key nucleotides help form a stable hairpin (P. C. J. Haasnoot, F. Brederode, R. C. L. Olsthoorn, and J. Bol, RNA 6:708-716, 2000; P. C. J. Haasnoot, R. C. L. Olsthoorn, and J. Bol, RNA 8:110-122, 2002). We used transfected barley protoplasts to examine the recognition of the subgenomic core promoter by the BMV replicase. Key nucleotides required for subgenomic initiation in vitro were found to be important for RNA4 levels in protoplasts. In addition, additional residues not required in vitro and the formation of an RNA hairpin within the core promoter were correlated with wild-type RNA4 levels in cells. Using a template competition assay, the core promoter of ca. 20 nucleotides was found to be sufficient for replicase binding. Mutations of the key residues in the core promoter reduced replicase binding, but deletions that disrupt the predicted base pairing in the proposed stem retained binding at wild-type levels. Together, these results indicate that key nucleotides in the BMV subgenomic core promoter direct replicase recognition but that the formation of a stem-loop is required at a step after binding. Additional functional characterization of the subgenomic core promoter was performed. A portion of the promoter for BMV minus-strand RNA synthesis could substitute for the subgenomic core promoter in transfected cells. The comparable sequence from Cowpea Chlorotic Mottle Virus (CCMV) could also substitute for the BMV subgenomic core promoter. However, nucleotides in the CCMV core required for RNA synthesis are not identical to those in BMV, suggesting that the subgenomic core promoter can induce the BMV replicase in interactions needed for subgenomic RNA transcription in vivo.

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