scholarly journals Requirement for Host RNA-Silencing Components and the Virus-Silencing Suppressor when Second-Site Mutations Compensate for Structural Defects in the 3′ Untranslated Region

2015 ◽  
Vol 89 (22) ◽  
pp. 11603-11618 ◽  
Author(s):  
Maitreyi Chattopadhyay ◽  
Vera A. Stupina ◽  
Feng Gao ◽  
Christine R. Szarko ◽  
Micki M. Kuhlmann ◽  
...  
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Untranslated Region ◽  
Higher Order ◽  
Order Structure ◽  
Silencing Suppressor ◽  
Turnip Crinkle Virus ◽  
Site Mutation ◽  
Primary Mutation ◽  
Positive Strand Rna

ABSTRACTTurnip crinkle virus (TCV) contains a structured 3′ region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3′ untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE (selective 2′-hydroxylacylation analyzed byprimerextension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3′UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or indcl-2/dcl4orago1/ago2backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCV-rev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs.IMPORTANCEGenomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through reestablishment of canonical base pairing with the altered region. In this study, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3′ UTR hairpin in the genomic RNA of turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3′ UTR primary-site mutation proposed to disrupt higher-order structure and the RNA-silencing machinery.

scholarly journals Stem-Loop IV in the 5′ Untranslated Region Is a cis-Acting Element in Bovine Coronavirus Defective Interfering RNA Replication

2005 ◽  
Vol 79 (19) ◽  
pp. 12434-12446 ◽  
Author(s):  
Sharmila Raman ◽  
David A. Brian
Keyword(s):  
Untranslated Region ◽  
Rna Replication ◽  
Higher Order ◽  
Cellular Proteins ◽  
Order Structure ◽  
Sars Coronavirus ◽  
Bovine Coronavirus ◽  
Stem Loop ◽  
Group 2 ◽  
Group 1

ABSTRACT The 210-nucleotide (nt) 5′ untranslated region (UTR) in the positive-strand bovine coronavirus (BCoV) genome is predicted to contain four higher-order structures identified as stem-loops I to IV, which may function as cis-acting elements in genomic RNA replication. Here, we describe evidence that stem-loop IV, a bulged stem-loop mapping at nt 186 through 215, (i) is phylogenetically conserved among group 2 coronaviruses and may have a homolog in groups 1 and 3, (ii) exists as a higher-order structure on the basis of enzyme probing, (iii) is required as a higher-order element for replication of a BCoV defective interfering (DI) RNA in the positive but not the negative strand, and (iv) as a higher-order structure in wild-type (wt) and mutant molecules that replicate, specifically binds six cellular proteins in the molecular mass range of 25 to 58 kDa as determined by electrophoretic mobility shift and UV cross-linking assays; binding to viral proteins was not detected. Interestingly, the predicted stem-loop IV homolog in the severe acute respiratory syndrome (SARS) coronavirus appears to be group 1-like in that it is in part duplicated with a group 1-like conserved loop sequence and is not group 2-like, as would be expected by the SARS coronavirus group 2-like 3′ UTR structure. These results together indicate that stem-loop IV in the BCoV 5′ UTR is a cis-acting element for DI RNA replication and that it might function through interactions with cellular proteins. It is postulated that stem-loop IV functions similarly in the virus genome.

scholarly journals Structural Domains within the 3′ Untranslated Region of Turnip Crinkle Virus

2008 ◽  
Vol 82 (17) ◽  
pp. 8706-8720 ◽  
Author(s):  
John C. McCormack ◽  
Xuefeng Yuan ◽  
Yaroslava G. Yingling ◽  
Wojciech Kasprzak ◽  
Rodolfo E. Zamora ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Turnip Crinkle Virus ◽  
Parallel Genetic Algorithm ◽  
Satellite Rna ◽  
Structural Domains ◽  
Structure Probing ◽  
Simultaneous Existence ◽  
Positive Strand Rna ◽  
Viral Accumulation

ABSTRACT The genomes of positive-strand RNA viruses undergo conformational shifts that complicate efforts to equate structures with function. We have initiated a detailed analysis of secondary and tertiary elements within the 3′ end of Turnip crinkle virus (TCV) that are required for viral accumulation in vivo. MPGAfold, a massively parallel genetic algorithm, suggested the presence of five hairpins (H4a, H4b, and previously identified hairpins H4, H5, and Pr) and one H-type pseudoknot (Ψ3) within the 3′-terminal 194 nucleotides (nt). In vivo compensatory mutagenesis analyses confirmed the existence of H4a, H4b, Ψ3 and a second pseudoknot (Ψ2) previously identified in a TCV satellite RNA. In-line structure probing of the 194-nt fragment supported the coexistence of H4, H4a, H4b, Ψ3 and a pseudoknot that connects H5 and the 3′ end (Ψ1). Stepwise replacements of TCV elements with the comparable elements from Cardamine chlorotic fleck virus indicated that the complete 142-nt 3′ end, and subsets containing Ψ3, H4a, and H4b or Ψ3, H4a, H4b, H5, and Ψ2, form functional domains for virus accumulation in vivo. A new 3-D molecular modeling protocol (RNA2D3D) predicted that H4a, H4b, H5, Ψ3, and Ψ2 are capable of simultaneous existence and bears some resemblance to a tRNA. The related Japanese iris necrotic ring virus does not have comparable domains. These results provide a framework for determining how interconnected elements participate in processes that require 3′ untranslated region sequences such as translation and replication.

scholarly journals A Structured Viroid RNA Serves as a Substrate for Dicer-Like Cleavage To Produce Biologically Active Small RNAs but Is Resistant to RNA-Induced Silencing Complex-Mediated Degradation

2007 ◽  
Vol 81 (6) ◽  
pp. 2980-2994 ◽  
Author(s):  
Asuka Itaya ◽  
Xuehua Zhong ◽  
Ralf Bundschuh ◽  
Yijun Qi ◽  
Ying Wang ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Small Rnas ◽  
Rna Silencing ◽  
Effective Means ◽  
Critical Role ◽  
Potato Spindle Tuber Viroid ◽  
Biologically Active ◽  
Antiviral Defense ◽  
Silencing Suppressor ◽  
Rna Structures

ABSTRACT RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.

scholarly journals Modification of Small RNAs Associated with Suppression of RNA Silencing by Tobamovirus Replicase Protein

2007 ◽  
Vol 81 (19) ◽  
pp. 10379-10388 ◽  
Author(s):  
Hannes Vogler ◽  
Rashid Akbergenov ◽  
Padubidri V. Shivaprasad ◽  
Vy Dang ◽  
Monika Fasler ◽  
...  
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Fluorescent Protein ◽  
Plant Viruses ◽  
Micro Rna ◽  
Silencing Suppressor ◽  
Small Interfering Rnas ◽  
Suppressor Activity ◽  
Virus Family ◽  
Green Fluorescent

ABSTRACT Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.

scholarly journals Cucumber Mosaic Virus 2b Protein Subcellular Targets and Interactions: Their Significance to RNA Silencing Suppressor Activity

2010 ◽  
Vol 23 (3) ◽  
pp. 294-303 ◽  
Author(s):  
Inmaculada González ◽  
Llucia Martínez ◽  
Daria V. Rakitina ◽  
Mathew G. Lewsey ◽  
Félix A. Atencio ◽  
...  
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Small Rnas ◽  
Rna Silencing ◽  
Silencing Suppressor ◽  
Suppressor Activity ◽  
Rna Silencing Suppressor ◽  
2B Protein

The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been variously attributed to its nuclear targeting, its interaction with and inhibition of Argonaute 1 (AGO1), or its ability to bind small RNAs in vitro. In addition, the 2b ortholog of Tomato aspermy virus forms aggregates and binds RNAs in vitro. We have further studied the relationships between CMV 2b protein silencing suppressor activity and its subcellular distribution, protein-protein interactions in vivo, and interactions with small interfering RNAs in vitro. To do this, we tagged the protein with fluorescent markers and showed that it retained suppressor activity. We showed that the 2b protein is present in the nucleolus and that it self-interacts and interacts with AGO1 and AGO4 in vivo. Using a battery of mutants, we showed that the putative nuclear localization signals and phosphorylation motif of the 2b protein are not required for self-interaction or for interaction with AGO proteins. The occurrence of neither of these interactions or of nucleolar targeting was sufficient to provide local silencing-suppression activity. In contrast, the ability of the 2b protein to bind small RNAs appears to be indispensable for silencing suppressor function.

scholarly journals RNA silencing-suppressor function of Turnip crinkle virus coat protein cannot be attributed to its interaction with the Arabidopsis protein TIP

2004 ◽  
Vol 85 (11) ◽  
pp. 3415-3420 ◽  
Author(s):  
Chang Won Choi ◽  
Feng Qu ◽  
Tao Ren ◽  
Xiaohong Ye ◽  
T. Jack Morris
Keyword(s):  
Amino Acid ◽  
Coat Protein ◽  
Amino Acid Substitution ◽  
Rna Silencing ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Silencing Suppressor ◽  
Turnip Crinkle Virus ◽  
Suppressor Activity ◽  
Rna Silencing Suppressor

The interaction of the coat protein (CP) of Turnip crinkle virus (TCV) with a host protein, TCV-interacting protein (TIP), from Arabidopsis thaliana has been reported previously. This interaction correlates with the ability of TCV CP to elicit the resistance response that is mediated by the resistance gene HRT in Arabidopsis ecotype Di-17. It has also been established that TCV CP is a suppressor of RNA silencing, a process by which the host plant targets viral RNA for degradation. These results have led to the speculation that TIP might be a component of the RNA-silencing pathway and that TCV CP suppresses RNA silencing through its interaction with TIP. In the current report, a number of TCV CP mutants have been investigated for their ability to suppress RNA silencing. These mutants include single amino acid substitution mutants that are known to have lost their ability to interact with TIP, as well as deletion mutants of TCV CP that are of different sizes and from different regions of the protein. Results showed that each of the single amino acid substitution mutants tested retained high levels of RNA silencing-suppressor activity. In addition, a mutant containing a 5 aa deletion in the region that is known to be critical for TIP interaction retained the ability to suppress RNA silencing significantly. Larger deletions in all regions of TCV CP abolished silencing-suppressor activity. It can be concluded from these results that the RNA silencing-suppressor activity of TCV CP cannot be attributed to its ability to interact directly with TIP.

scholarly journals Genetic evidence for the involvement of Dicer-like 2 and 4 as well as Argonaute 2 in the Nicotiana benthamiana response against Pelargonium line pattern virus

2021 ◽  
Vol 102 (10) ◽  
Author(s):  
Miryam Pérez-Cañamás ◽  
Elizabeth Hevia ◽  
Konstantina Katsarou ◽  
Carmen Hernández
Keyword(s):  
Nicotiana Benthamiana ◽  
Small Rnas ◽  
Rna Silencing ◽  
Rna Degradation ◽  
Host Race ◽  
Silencing Suppressor ◽  
Line Pattern ◽  
Argonaute 2 ◽  
Guide Sequence ◽  
Antiviral Mechanism

In plants, RNA silencing functions as a potent antiviral mechanism. Virus-derived double-stranded RNAs (dsRNAs) trigger this mechanism, being cleaved by Dicer-like (DCL) enzymes into virus small RNAs (vsRNAs). These vsRNAs guide sequence-specific RNA degradation upon their incorporation into an RNA-induced silencing complex (RISC) that contains a slicer of the Argonaute (AGO) family. Host RNA dependent-RNA polymerases, particularly RDR6, strengthen antiviral silencing by generating more dsRNA templates from RISC-cleavage products that, in turn, are converted into secondary vsRNAs by DCLs. Previous work showed that Pelargonium line pattern virus (PLPV) is a very efficient inducer and target of RNA silencing as PLPV-infected Nicotiana benthamiana plants accumulate extraordinarily high amounts of vsRNAs that, strikingly, are independent of RDR6 activity. Several scenarios may explain these observations including a major contribution of dicing versus slicing for defence against PLPV, as the dicing step would not be affected by the RNA silencing suppressor encoded by the virus, a protein that acts via vsRNA sequestration. Taking advantage of the availability of lines of N. benthamiana with DCL or AGO2 functions impaired, here we have tried to get further insights into the components of the silencing machinery that are involved in anti-PLPV-silencing. Results have shown that DCL4 and, to lesser extent, DCL2 contribute to restrict viral infection. Interestingly, AGO2 apparently makes even a higher contribution in the defence against PLPV, extending the number of viruses that are affected by this particular slicer. The data support that both dicing and slicing activities participate in the host race against PLPV.

Macrocycles of Higher Ortho-Phenylenes: Assembly and Folding

2019 ◽  
Author(s):  
Zacharias Kinney ◽  
Viraj Kirinda ◽  
Scott Hartley
Keyword(s):  
Chemical Shift ◽  
Higher Order ◽  
Order Structure ◽  
Complex Geometries ◽  
Spatial Relationships ◽  
Predominant Species ◽  
Bite Angle ◽  
Direct Assembly

<p>Higher-order structure in abiotic foldamer systems represents an important but largely unrealized goal. As one approach to this challenge, covalent assembly can be used to assemble macrocycles with foldamer subunits in well-defined spatial relationships. Such systems have previously been shown to exhibit self-sorting, new folding motifs, and dynamic stereoisomerism, yet there remain important questions about the interplay between folding and macrocyclization and the effect of structural confinement on folding behavior. Here, we explore the dynamic covalent assembly of extended <i>ortho</i>-phenylenes (hexamer and decamer) with rod-shaped linkers. Characteristic <sup>1</sup>H chemical shift differences between cyclic and acyclic systems can be compared with computational conformer libraries to determine the folding states of the macrocycles. We show that the bite angle provides a measure of the fit of an <i>o</i>-phenylene conformer within a shape-persistent macrocycle, affecting both assembly and ultimate folding behavior. For the <i>o</i>-phenylene hexamer, the bite angle and conformer stability work synergistically to direct assembly toward triangular [3+3] macrocycles of well-folded oligomers. For the decamer, the energetic accessibility of conformers with small bite angles allows [2+2] macrocycles to be formed as the predominant species. In these systems, the <i>o</i>-phenylenes are forced into unusual folding states, preferentially adopting a backbone geometry with distinct helical blocks of opposite handedness. The results show that simple geometric restrictions can be used to direct foldamers toward increasingly complex geometries.</p>

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