The Coding Region of the HCV Genome Contains a Network of Regulatory RNA Structures

ABSTRACT Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

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Search for 5′-leader regulatory RNA structures based on gene annotation aided by the RiboGap database

Methods ◽

10.1016/j.ymeth.2017.02.009 ◽

2017 ◽

Vol 117 ◽

pp. 3-13 ◽

Cited By ~ 2

Author(s):

Mohammad Reza Naghdi ◽

Katia Smail ◽

Joy X. Wang ◽

Fallou Wade ◽

Ronald R. Breaker ◽

...

Keyword(s):

Gene Annotation ◽

Regulatory Rna ◽

Rna Structures

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Identification of a cis-Acting Replication Element within the Poliovirus Coding Region

Journal of Virology ◽

10.1128/jvi.74.10.4590-4600.2000 ◽

2000 ◽

Vol 74 (10) ◽

pp. 4590-4600 ◽

Cited By ~ 173

Author(s):

Ian Goodfellow ◽

Yasmin Chaudhry ◽

Andrew Richardson ◽

Janet Meredith ◽

Jeffrey W. Almond ◽

...

Keyword(s):

Rna Virus ◽

Site Directed Mutagenesis ◽

Evolutionary Significance ◽

Rna Structures ◽

Coding Region ◽

Rna Sequences ◽

Stem Loop ◽

Cis Acting ◽

Function Recovery ◽

Positive Sense

ABSTRACT The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel polioviruscis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560–11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.

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Identification of conserved regulatory RNA structures in prokaryotic metabolic pathway genes

Biosystems ◽

10.1016/j.biosystems.2004.11.002 ◽

2005 ◽

Vol 80 (2) ◽

pp. 145-154 ◽

Cited By ~ 11

Author(s):

Elena A. Lesnik ◽

Gary B. Fogel ◽

Dana Weekes ◽

Timothy J. Henderson ◽

Harold B. Levene ◽

...

Keyword(s):

Metabolic Pathway ◽

Regulatory Rna ◽

Rna Structures

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Pausing guides RNA folding to populate transiently stable RNA structures for riboswitch-based transcription regulation

eLife ◽

10.7554/elife.21297 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 29

Author(s):

Hannah Steinert ◽

Florian Sochor ◽

Anna Wacker ◽

Janina Buck ◽

Christina Helmling ◽

...

Keyword(s):

Ligand Binding ◽

Messenger Rna ◽

Regulation Of Gene Expression ◽

Untranslated Regions ◽

Structural Description ◽

Regulatory Rna ◽

Structural Elements ◽

Rna Structures ◽

Cis Acting ◽

Aptamer Domain

In bacteria, the regulation of gene expression by cis-acting transcriptional riboswitches located in the 5'-untranslated regions of messenger RNA requires the temporal synchronization of RNA synthesis and ligand binding-dependent conformational refolding. Ligand binding to the aptamer domain of the riboswitch induces premature termination of the mRNA synthesis of ligand-associated genes due to the coupled formation of 3'-structural elements acting as terminators. To date, there has been no high resolution structural description of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA element. Here, we show that for the guanine-sensing xpt-pbuX riboswitch from Bacillus subtilis, the conformation of the full-length transcripts is static: it exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand. We show that only the combined matching of transcription rates and ligand binding enables transcription intermediates to undergo ligand-dependent conformational refolding.

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Structure and function of RNA elements present in enteroviral genomes

Acta Biochimica Polonica ◽

10.18388/abp.2016_1337 ◽

2017 ◽

Vol 63 (4) ◽

Cited By ~ 5

Author(s):

Mariola Dutkiewicz ◽

Aleksandra Stachowiak ◽

Agata Swiatkowska ◽

Jerzy Ciesiołka

Keyword(s):

Regulatory Elements ◽

Structure And Function ◽

Host Cells ◽

Rna Structures ◽

Coding Region ◽

Protein Coding ◽

Reading Frame ◽

Noncoding Regions ◽

Long Time ◽

And Function

Enteroviruses are small RNA(+) viruses that encode one open reading frame flanked by two extensive noncoding regions carrying structural RNA regulatory elements that control replication and translation processes. For a long time the central, coding region was thought to remain single-stranded and its only function was supposed to be as the template for polyprotein synthesis. It turned out, however, that the protein coding region also encodes important RNA structures crucial for the viral life cycle and virus persistence in host cells. This review considers the RNA structures in enteroviral genomes identified and characterized to date.

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Bacterial cis-regulatory RNA structures

Molecular Biology ◽

10.1134/s0026893306040066 ◽

2006 ◽

Vol 40 (4) ◽

pp. 541-550 ◽

Cited By ~ 2

Author(s):

M. S. Gelfand

Keyword(s):

Regulatory Rna ◽

Rna Structures

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Nucleotide sequence of a single-stranded RNA phage from Pseudomonas aeruginosa:Kinship to coliphages and conservation of regulatory RNA structures

Virology ◽

10.1016/s0042-6822(95)80078-6 ◽

1995 ◽

Vol 206 (1) ◽

pp. 611-625 ◽

Cited By ~ 39

Author(s):

R.C.L. Olsthoorn ◽

G. Garde ◽

T. Dayhuff ◽

J.F. Atkins ◽

J. Van Duin

Keyword(s):

Nucleotide Sequence ◽

Regulatory Rna ◽

Rna Structures ◽

Rna Phage

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Genome-wide discovery of local RNA structural elements in Zika virus

10.7287/peerj.preprints.27101 ◽

2018 ◽

Author(s):

Ryan J Andrews ◽

Julien Roche ◽

Walter N Moss

Keyword(s):

Zika Virus ◽

Genome Replication ◽

Rna Structures ◽

Coding Region ◽

Step Size ◽

Base Pairs ◽

Local Structures ◽

Genome Wide ◽

Viral Polyprotein ◽

Functional Rna

In addition to encoding RNA primary structures, genomes also encode RNA secondary and tertiary structures that play roles in gene regulation and, in the case of RNA viruses, genome replication. Methods for the identification of functional RNA structures in genomes typically rely on scanning analysis windows, where multiple partially-overlapping windows are used to predict RNA structures and folding metrics to deduce regions likely to form functional structure. Separate structural models are produced for each window, where the step size can greatly affect the returned model. This makes deducing unique local structures challenging, as the same nucleotides in each window can be alternatively base paired. In the presented approach, all base pairs from all analysis windows are considered and weighted by favorable folding metrics throughout all windows. This results in unique base pairing throughout the genome and the generation of local regions/structures that can be ranked by their propensity to form unusually thermodynamically stable folds. This approach was applied to the Zika virus (ZIKV) genome. ZIKV is linked to a variety of neurological ailments including microcephaly and Guillain-Barré syndrome and its (+)-sense RNA genome encodes two, previously described, functionally essential structured RNA regions. Our approach is able to successfully identify and model the structures of these regions, while also finding additional regions likely to form functional RNA structures throughout the viral polyprotein coding region. All data for the ZIKV genome have been archived at the RNAStructuromeDB, a repository of RNA folding data for humans and their pathogens.

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