Single molecule studies of RNA secondary structure: AFM of TYMV viral RNA

2004 ◽  
Vol 65 (4-5) ◽  
pp. 235-245 ◽  
Author(s):  
Andrea Giro ◽  
Anna Bergia ◽  
Giampaolo Zuccheri ◽  
Hugo H.J. Bink ◽  
Cornelis W.A. Pleij ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Single Molecule ◽  
Rna Secondary Structure ◽  
Viral Rna ◽  
Single Molecule Studies

scholarly journals Functional Consequences of RNA 5′-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Nicolas Lévêque ◽  
Magali Garcia ◽  
Alexis Bouin ◽  
Joseph H. C. Nguyen ◽  
Genevieve P. Tran ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Secondary Structure ◽  
Rna Replication ◽  
Viral Proteins ◽  
Coxsackievirus B3 ◽  
Viral Rna ◽  
Stem Loop ◽  
Genomic Rnas ◽  
Ribonucleoprotein Complex ◽  
Genomic Deletions

ABSTRACT Group B coxsackieviruses are responsible for chronic cardiac infections. However, the molecular mechanisms by which the virus can persist in the human heart long after the signs of acute myocarditis have abated are still not completely understood. Recently, coxsackievirus B3 strains with 5′-terminal deletions in genomic RNAs were isolated from a patient suffering from idiopathic dilated cardiomyopathy, suggesting that such mutant viruses may be the forms responsible for persistent infection. These deletions lacked portions of 5′ stem-loop I, which is an RNA secondary structure required for viral RNA replication. In this study, we assessed the consequences of the genomic deletions observed in vivo for coxsackievirus B3 biology. Using cell extracts from HeLa cells, as well as transfection of luciferase replicons in two types of cardiomyocytes, we demonstrated that coxsackievirus RNAs harboring 5′ deletions ranging from 7 to 49 nucleotides in length can be translated nearly as efficiently as those of wild-type virus. However, these 5′ deletions greatly reduced the synthesis of viral RNA in vitro, which was detected only for the 7- and 21-nucleotide deletions. Since 5′ stem-loop I RNA forms a ribonucleoprotein complex with cellular and viral proteins involved in viral RNA replication, we investigated the binding of the host cell protein PCBP2, as well as viral protein 3CDpro, to deleted positive-strand RNAs corresponding to the 5′ end. We found that binding of these proteins was conserved but that ribonucleoprotein complex formation required higher PCBP2 and 3CDpro concentrations, depending on the size of the deletion. Overall, this study confirmed the characteristics of persistent CVB3 infection observed in heart tissues and provided a possible explanation for the low level of RNA replication observed for the 5′-deleted viral genomes—a less stable ribonucleoprotein complex formed with proteins involved in viral RNA replication. IMPORTANCE Dilated cardiomyopathy is the most common indication for heart transplantation worldwide, and coxsackie B viruses are detected in about one-third of idiopathic dilated cardiomyopathies. Terminal deletions at the 5′ end of the viral genome involving an RNA secondary structure required for RNA replication have been recently reported as a possible mechanism of virus persistence in the human heart. These mutations are likely to disrupt the correct folding of an RNA secondary structure required for viral RNA replication. In this report, we demonstrate that transfected RNAs harboring 5′-terminal sequence deletions are able to direct the synthesis of viral proteins, but not genomic RNAs, in human and murine cardiomyocytes. Moreover, we show that the binding of cellular and viral replication factors to viral RNA is conserved despite genomic deletions but that the impaired RNA synthesis associated with terminally deleted viruses could be due to destabilization of the ribonucleoprotein complexes formed.

scholarly journals RNA signals in the 3′ terminus of the genome of Equine arteritis virus are required for viral RNA synthesis

2006 ◽  
Vol 87 (7) ◽  
pp. 1977-1983 ◽  
Author(s):  
Nancy Beerens ◽  
Eric J. Snijder
Keyword(s):  
Secondary Structure ◽  
Virus Replication ◽  
Rna Secondary Structure ◽  
Rna Synthesis ◽  
Full Length ◽  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

RNA virus genomes contain cis-acting sequences and structural elements involved in virus replication. Both full-length and subgenomic negative-strand RNA synthesis are initiated at the 3′ terminus of the positive-strand genomic RNA of Equine arteritis virus (EAV). To investigate the molecular mechanism of EAV RNA synthesis, the RNA secondary structure of the 3′-proximal region of the genome was analysed by chemical and enzymic probing. Based on the RNA secondary structure model derived from this analysis, several deletions were engineered in a full-length cDNA copy of the viral genome. Two RNA domains were identified that are essential for virus replication and most likely play a key role in viral RNA synthesis. The first domain, located directly upstream of the 3′ untranslated region (UTR) (nt 12610–12654 of the genome), is mainly single-stranded but contains one small stem–loop structure. The second domain is located within the 3′ UTR (nt 12661–12690) and folds into a prominent stem–loop structure with a large loop region. The location of this stem–loop structure near the 3′ terminus of the genome suggests that it may act as a recognition signal during the initiation of minus-strand RNA synthesis.

Single molecule studies of surface-induced secondary structure in a model peptide

2004 ◽  
Author(s):  
Douglas S. English ◽  
Joy A. Cunningham ◽  
Sarah C. Wehri ◽  
Amy F. Petrik ◽  
Kenji Okamoto
Keyword(s):  
Secondary Structure ◽  
Single Molecule ◽  
Model Peptide ◽  
Single Molecule Studies

scholarly journals Amino Acid Stabilization of Nucleic Acid Secondary Structure: Kinetic Insights from Single-Molecule Studies

2018 ◽  
Vol 122 (43) ◽  
pp. 9869-9876 ◽  
Author(s):  
David A. Nicholson ◽  
Abhigyan Sengupta ◽  
Hsuan-Lei Sung ◽  
David J. Nesbitt
Keyword(s):  
Amino Acid ◽  
Nucleic Acid ◽  
Secondary Structure ◽  
Single Molecule ◽  
Single Molecule Studies

scholarly journals bpRNA: Large-scale Automated Annotation and Analysis of RNA Secondary Structure

2018 ◽  
Author(s):  
Padideh Danaee ◽  
Mason Rouches ◽  
Michelle Wiley ◽  
Dezhong Deng ◽  
Liang Huang ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Single Molecule ◽  
Structure Prediction ◽  
Rna Secondary Structure ◽  
Large Scale ◽  
Secondary Structure Prediction ◽  
Sequence Data ◽  
Secondary Structures ◽  
Base Pairs ◽  
Statistical Trends

ABSTRACTWhile RNA secondary structure prediction from sequence data has made remarkable progress, there is a need for improved strategies for annotating the features of RNA secondary structures. Here we present bpRNA, a novel annotation tool capable of parsing RNA structures, including complex pseudoknot-containing RNAs, to yield an objective, precise, compact, unambiguous, easily-interpretable description of all loops, stems, and pseudoknots, along with the positions, sequence, and flanking base pairs of each such structural feature. We also introduce several new informative representations of RNA structure types to improve structure visualization and interpretation. We have further used bpRNA to generate a web-accessible meta-database, “bpRNA-1m”, of over 100,000 single-molecule, known secondary structures; this is both more fully and accurately annotated and over 20-times larger than existing databases. We use a subset of the database with highly similar (≥90% identical) sequences filtered out to report on statistical trends in sequence, flanking base pairs, and length. Both the bpRNA method and the bpRNA-1m database will be valuable resources both for specific analysis of individual RNA molecules and large-scale analyses such as are useful for updating RNA energy parameters for computational thermodynamic predictions, improving machine learning models for structure prediction, and for benchmarking structure-prediction algorithms.

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