The influenza A virus NS genome segment displays lineage-specific patterns in predicted RNA secondary structure

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.

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pH-dependent secondary structure propensity of the influenza A virus M2 cytoplasmic tail

Biomolecular NMR Assignments ◽

10.1007/s12104-020-09937-8 ◽

2020 ◽

Vol 14 (1) ◽

pp. 157-161

Author(s):

Jolyon K. Claridge ◽

Faiz Mohd-Kipli ◽

Andrei Florea ◽

Thomas Gate ◽

Jason R. Schnell

Keyword(s):

Secondary Structure ◽

Influenza A Virus ◽

Influenza A ◽

Cytoplasmic Tail ◽

Ph Dependent

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

Journal of Virology ◽

10.1128/jvi.78.12.6263-6270.2004 ◽

2004 ◽

Vol 78 (12) ◽

pp. 6263-6270 ◽

Cited By ~ 37

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.

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

Biochemical Journal ◽

10.1042/bcj20160651 ◽

2016 ◽

Vol 473 (23) ◽

pp. 4327-4348 ◽

Cited By ~ 12

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.

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Mutations designed to modify the NS gene mRNA secondary structure affect influenza A pathogenicity in vivo

Microbiology Independent Research journal ◽

10.18527/2500-2236-2021-8-1-1-9 ◽

2021 ◽

Vol 8 (1) ◽

pp. 1-9

Author(s):

I. L. Baranovskaya ◽

M. V. Sergeeva ◽

A. S. Taraskin ◽

A. A. Lozhkov ◽

A. V. Vasin

Keyword(s):

Secondary Structure ◽

Viral Replication ◽

Influenza A Virus ◽

Messenger Rna ◽

Influenza A ◽

Early Stage ◽

Secondary Structures ◽

Mrna Secondary Structure ◽

Splice Sites ◽

Ns Gene

The influenza A virus genome consists of eight segments of negative-sense RNA that encode up to 18 proteins. During the process of viral replication, positive-sense (+)RNA (cRNA) or messenger RNA (mRNA) is synthesized. Today, there is only a partial understanding of the function of several secondary structures within vRNA and cRNA promoters, and splice sites in the M and NS genes. The most precise secondary structure of (+)RNA has been determined for the NS segment of influenza A virus. The influenza A virus NS gene features two regions with a conserved mRNA secondary structure located near splice sites. Here, we compared 4 variants of the A/Puerto Rico/8/1934 strain featuring different combinations of secondary structures at the NS segment (+)RNA regions 82-148 and 497-564. We found that RNA structures did not affect viral replication in cell culture. However, one of the viruses demonstrated lower NS1 and NEP expression levels during early stage cell infection as well as reduced pathogenicity in mice compared to other variants. In particular, this virus is characterized by an RNA hairpin in the 82-148 region and a stable hairpin in the 497-564 region.

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Secondary structure of the segment 5 genomic RNA of influenza A virus and its application for designing antisense oligonucleotides

Scientific Reports ◽

10.1038/s41598-019-40443-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

Author(s):

Paula Michalak ◽

Marta Soszynska-Jozwiak ◽

Ewa Biala ◽

Walter N. Moss ◽

Julita Kesy ◽

...

Keyword(s):

Secondary Structure ◽

Influenza A Virus ◽

Antisense Oligonucleotides ◽

Influenza A ◽

Genomic Rna

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

10.1101/2020.03.05.975870 ◽

2020 ◽

Cited By ~ 1

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.

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

Viruses ◽

10.3390/v13030525 ◽

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.

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