Secondary structure model of the naked segment 7 influenza A virus genomic RNA

2016 ◽  
Vol 473 (23) ◽  
pp. 4327-4348 ◽  
Author(s):  
Agnieszka Ruszkowska ◽  
Elzbieta Lenartowicz ◽  
Walter N. Moss ◽  
Ryszard Kierzek ◽  
Elzbieta Kierzek
Keyword(s):  
Secondary Structure ◽  
Influenza A Virus ◽  
Rna Structure ◽  
Influenza A ◽  
Comparative Sequence Analysis ◽  
Rnase H ◽  
Structure Model ◽  
Chemical Mapping ◽  
Secondary Structure Model ◽  
Negative Sense

The influenza A virus (IAV) genome comprises eight negative-sense viral (v)RNA segments. The seventh segment of the genome encodes two essential viral proteins and is specifically packaged alongside the other seven vRNAs. To gain insights into the possible roles of RNA structure both within and without virions, a secondary structure model of a naked (protein-free) segment 7 vRNA (vRNA7) has been determined using chemical mapping and thermodynamic energy minimization. The proposed structure model was validated using microarray mapping, RNase H cleavage and comparative sequence analysis. Additionally, the detailed structures of three vRNA7 fragment constructs — comprising independently folded subdomains — were determined. Much of the proposed vRNA7 structure is preserved between IAV strains, suggesting their importance in the influenza replication cycle. Possible structure rearrangements, which allow or preclude long-range RNA interactions, are also proposed.

scholarly journals Mutational Analysis of the Influenza Virus cRNA Promoter and Identification of Nucleotides Critical for Replication

2004 ◽  
Vol 78 (12) ◽  
pp. 6263-6270 ◽  
Author(s):  
Mandy Crow ◽  
Tao Deng ◽  
Mark Addley ◽  
George G. Brownlee
Keyword(s):  
Secondary Structure ◽  
Influenza A Virus ◽  
Influenza A ◽  
Promoter Activity ◽  
Mrna Levels ◽  
Structure Model ◽  
Hairpin Loop ◽  
Base Pairing ◽  
Secondary Structure Model

ABSTRACT Replication of the influenza A virus virion RNA (vRNA) requires the synthesis of full-length cRNA, which in turn is used as a template for the synthesis of more vRNA. A “corkscrew” secondary-structure model of the cRNA promoter has been proposed recently. However the data in support of that model were indirect, since they were derived from measurement, by use of a chloramphenicol acetyltransferase (CAT) reporter in 293T cells, of mRNA levels from a modified cRNA promoter rather than the authentic cRNA promoter found in influenza A viruses. Here we measured steady-state cRNA and vRNA levels from a CAT reporter in 293T cells, directly measuring the replication of the authentic influenza A virus wild-type cRNA promoter. We found that (i) base pairing between the 5′ and 3′ ends and (ii) base pairing in the stems of both the 5′ and 3′ hairpin loops of the cRNA promoter were required for in vivo replication. Moreover, nucleotides in the tetraloop at positions 4, 5, and 7 and nucleotides forming the 2-9 base pair of the 3′ hairpin loop were crucial for promoter activity in vivo. However, the 3′ hairpin loop was not required for polymerase binding in vitro. Overall, our results suggest that the corkscrew secondary-structure model is required for authentic cRNA promoter activity in vivo, although the precise role of the 3′ hairpin loop remains unknown.

scholarly journals Isoenergetic penta- and hexanucleotide microarray probing and chemical mapping provide a secondary structure model for an RNA element orchestrating R2 retrotransposon protein function

2008 ◽  
Vol 36 (6) ◽  
pp. 1770-1782 ◽  
Author(s):  
Elzbieta Kierzek ◽  
Ryszard Kierzek ◽  
Walter N. Moss ◽  
Shawn M. Christensen ◽  
Thomas H. Eickbush ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Protein Function ◽  
Structure Model ◽  
Chemical Mapping ◽  
Secondary Structure Model

scholarly journals A functional RNA structure in the influenza A virus ribonucleoprotein complex for segment bundling

2020 ◽  
Author(s):  
Naoki Takizawa ◽  
Koichi Higashi ◽  
Risa Karakida Kawaguchi ◽  
Yasuhiro Gotoh ◽  
Yutaka Suzuki ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Influenza A Virus ◽  
Rna Structure ◽  
Recombinant Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Virus Genome ◽  
Pseudoknot Structure ◽  
Functional Rna

AbstractThe influenza A virus genome is segmented into eight viral RNAs (vRNA). Intersegment interactions are necessary for segment bundling, and secondary structures on vRNA are assumed to be involved in the process. However, the RNA structure required for segment bundling remains unidentified because the secondary structure of vRNA in virion was partially unwound by binding viral non-specific RNA binding proteins. Here, we revealed the global intersegment interactions and the secondary structure of the vRNA in virion. We demonstrated that a pseudoknot structure was formed on a segment in the virion and the impairment of replication and packaging of the other specific segment was observed in cells infected with recombinant virus which had mutations in the pseudoknot structure. Moreover, we showed that the intersegment interactions were reconstituted in the recombinant virus. Our data provides the first evidence that the functional RNA structure on the influenza A virus genome affects segment bundling.

scholarly journals Conserved Structural Motifs of Two Distant IAV Subtypes in Genomic Segment 5 RNA

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 525
Author(s):  
Paula Michalak ◽  
Julita Piasecka ◽  
Barbara Szutkowska ◽  
Ryszard Kierzek ◽  
Ewa Biala ◽  
...  
Keyword(s):  
Experimental Data ◽  
Secondary Structure ◽  
Influenza A Virus ◽  
Influenza A ◽  
Chemical Mapping ◽  
Rna Structures ◽  
Structural Motifs ◽  
2009 H1n1

The functionality of RNA is fully dependent on its structure. For the influenza A virus (IAV), there are confirmed structural motifs mediating processes which are important for the viral replication cycle, including genome assembly and viral packaging. Although the RNA of strains originating from distant IAV subtypes might fold differently, some structural motifs are conserved, and thus, are functionally important. Nowadays, NGS-based structure modeling is a source of new in vivo data helping to understand RNA biology. However, for accurate modeling of in vivo RNA structures, these high-throughput methods should be supported with other analyses facilitating data interpretation. In vitro RNA structural models complement such approaches and offer RNA structures based on experimental data obtained in a simplified environment, which are needed for proper optimization and analysis. Herein, we present the secondary structure of the influenza A virus segment 5 vRNA of A/California/04/2009 (H1N1) strain, based on experimental data from DMS chemical mapping and SHAPE using NMIA, supported by base-pairing probability calculations and bioinformatic analyses. A comparison of the available vRNA5 structures among distant IAV strains revealed that a number of motifs present in the A/California/04/2009 (H1N1) vRNA5 model are highly conserved despite sequence differences, located within previously identified packaging signals, and the formation of which in in virio conditions has been confirmed. These results support functional roles of the RNA secondary structure motifs, which may serve as candidates for universal RNA-targeting inhibitory methods.

Phylogeny of Criconematina Siddiqi, 1980 (Nematoda: Tylenchida) based on morphology and D2-D3 expansion segments of the 28S-rRNA gene sequences with application of a secondary structure model

Nematology ◽  
2005 ◽  
Vol 7 (6) ◽  
pp. 927-944 ◽  
Author(s):  
Renato Crozzoli ◽  
Franco Lamberti ◽  
Nicola Vovlas ◽  
James Baldwin ◽  
Sergei Subbotin ◽  
...  
Keyword(s):  
Maximum Likelihood ◽  
Secondary Structure ◽  
Sequence Divergence ◽  
Complex Model ◽  
Rrna Gene ◽  
28S Rrna ◽  
Structure Model ◽  
Secondary Structure Model ◽  
Loop Region ◽  
Unidentified Species

AbstractThe suborder Criconematina is a large group of ecto- and endoparasitic nematodes, including several species of major agricultural importance. The D2-D3 expansion segments of the 28S nuclear ribosomal RNA gene were amplified and sequenced from 23 nominal and six unidentified species from the genera Mesocriconema, Criconemoides, Ogma, Criconema, Xenocriconemella, Hemicriconemoides, Hemicycliophora, Paratylenchus, Tylenchulus, Trophonema and Sphaeronema, together with outgroup taxa from Tylenchidae (Aglenchus) and Atylenchidae (Eutylenchus). A sequence alignment optimised using the secondary structure model was analysed using maximum parsimony, maximum likelihood and Bayesian inference approaches under two models. All analyses yielded a similar topology with differences primarily in the position of poorly supported clades. Although some molecular trees differ from the previous morphologically based hypotheses of criconematid phylogeny, maximum likelihood tests did not yield statistically significant differences between some of the tested classical morphological and molecular topologies. DNA data support monophyly for the genera Mesocriconema, Hemicriconemoides and Criconema and reject the hypothesis of a single origin of criconematids with a cuticular sheath or 'double cuticle'. Application of the complex model of rRNA evolution, considering paired nucleotides for the stem and unpaired nucleotides for the loop region, resulted in a majority rule consensus Bayesian tree with unresolved relationships between main clades. This lack of resolution is expected by the low number of independently evolving nucleotides. Sequence divergence in this DNA segment between populations of Mesocriconema xenoplax, M. sphaerocephalum and Hemicriconemoides cocophillus suggest the presence of several sibling species under these taxa names.

scholarly journals RNA Secondary Structure as a First Step for Rational Design of the Oligonucleotides towards Inhibition of Influenza A Virus Replication

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 925 ◽  
Author(s):  
Marta Szabat ◽  
Dagny Lorent ◽  
Tomasz Czapik ◽  
Maria Tomaszewska ◽  
Elzbieta Kierzek ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rational Design ◽  
Rna Virus ◽  
Viral Rna ◽  
Structure Information ◽  
A Genome ◽  
Function Relationship ◽  
Interfering Rna

Influenza is an important research subject around the world because of its threat to humanity. Influenza A virus (IAV) causes seasonal epidemics and sporadic, but dangerous pandemics. A rapid antigen changes and recombination of the viral RNA genome contribute to the reduced effectiveness of vaccination and anti-influenza drugs. Hence, there is a necessity to develop new antiviral drugs and strategies to limit the influenza spread. IAV is a single-stranded negative sense RNA virus with a genome (viral RNA—vRNA) consisting of eight segments. Segments within influenza virion are assembled into viral ribonucleoprotein (vRNP) complexes that are independent transcription-replication units. Each step in the influenza life cycle is regulated by the RNA and is dependent on its interplay and dynamics. Therefore, viral RNA can be a proper target to design novel therapeutics. Here, we briefly described examples of anti-influenza strategies based on the antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA) and catalytic nucleic acids. In particular we focused on the vRNA structure-function relationship as well as presented the advantages of using secondary structure information in predicting therapeutic targets and the potential future of this field.

scholarly journals Organization of the Influenza A Virus Genomic RNA in the Viral Replication Cycle—Structure, Interactions, and Implications for the Emergence of New Strains

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 951
Author(s):  
Julita Piasecka ◽  
Aleksandra Jarmolowicz ◽  
Elzbieta Kierzek
Keyword(s):  
Viral Replication ◽  
Influenza A Virus ◽  
Rna Structure ◽  
Influenza A ◽  
Respiratory Infections ◽  
Specific Interaction ◽  
Replication Cycle ◽  
Genome Packaging ◽  
Functional Roles ◽  
Interaction Sites

The influenza A virus is a human pathogen causing respiratory infections. The ability of this virus to trigger seasonal epidemics and sporadic pandemics is a result of its high genetic variability, leading to the ineffectiveness of vaccinations and current therapies. The source of this variability is the accumulation of mutations in viral genes and reassortment enabled by its segmented genome. The latter process can induce major changes and the production of new strains with pandemic potential. However, not all genetic combinations are tolerated and lead to the assembly of complete infectious virions. Reports have shown that viral RNA segments co-segregate in particular circumstances. This tendency is a consequence of the complex and selective genome packaging process, which takes place in the final stages of the viral replication cycle. It has been shown that genome packaging is governed by RNA–RNA interactions. Intersegment contacts create a network, characterized by the presence of common and strain-specific interaction sites. Recent studies have revealed certain RNA regions, and conserved secondary structure motifs within them, which may play functional roles in virion assembly. Growing knowledge on RNA structure and interactions facilitates our understanding of the appearance of new genome variants, and may allow for the prediction of potential reassortment outcomes and the emergence of new strains in the future.

scholarly journals Mismatches in the Influenza A Virus RNA Panhandle Prevent Retinoic Acid-Inducible Gene I (RIG-I) Sensing by Impairing RNA/RIG-I Complex Formation

2015 ◽  
Vol 90 (1) ◽  
pp. 586-590 ◽  
Author(s):  
Stéphanie Anchisi ◽  
Jessica Guerra ◽  
Geneviève Mottet-Osman ◽  
Dominique Garcin
Keyword(s):  
Influenza Virus ◽  
Retinoic Acid ◽  
Complex Formation ◽  
Influenza A Virus ◽  
Rna Structure ◽  
Influenza A ◽  
Double Stranded Rna ◽  
Virus Rna ◽  
Inducible Gene

Influenza virus RNA (vRNA) promoter panhandle structures are believed to be sensed by retinoic acid-inducible gene I (RIG-I). The occurrence of mismatches in this double-stranded RNA structure raises questions about their effect on innate sensing. Our results suggest that mismatches in vRNA promoters decrease binding to RIG-Iin vivo, affecting RNA/RIG-I complex formation and preventing RIG-I activation. These results can be inferred to apply to other viruses and suggest that mismatches may represent a general viral strategy to escape RIG-I sensing.

scholarly journals In vivo analysis of influenza A mRNA secondary structures identifies critical regulatory motifs

2019 ◽  
Vol 47 (13) ◽  
pp. 7003-7017 ◽  
Author(s):  
Lisa Marie Simon ◽  
Edoardo Morandi ◽  
Anna Luganini ◽  
Giorgio Gribaudo ◽  
Luis Martinez-Sobrido ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Influenza A ◽  
Host Cells ◽  
Messenger Rnas ◽  
Infected Cells ◽  
In Vivo Analysis ◽  
First Time ◽  
Negative Sense

AbstractThe influenza A virus (IAV) is a continuous health threat to humans as well as animals due to its recurring epidemics and pandemics. The IAV genome is segmented and the eight negative-sense viral RNAs (vRNAs) are transcribed into positive sense complementary RNAs (cRNAs) and viral messenger RNAs (mRNAs) inside infected host cells. A role for the secondary structure of IAV mRNAs has been hypothesized and debated for many years, but knowledge on the structure mRNAs adopt in vivo is currently missing. Here we solve, for the first time, the in vivo secondary structure of IAV mRNAs in living infected cells. We demonstrate that, compared to the in vitro refolded structure, in vivo IAV mRNAs are less structured but exhibit specific locally stable elements. Moreover, we show that the targeted disruption of these high-confidence structured domains results in an extraordinary attenuation of IAV replicative capacity. Collectively, our data provide the first comprehensive map of the in vivo structural landscape of IAV mRNAs, hence providing the means for the development of new RNA-targeted antivirals.

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