Identification of potential conserved RNA secondary structure throughout influenza A coding regions

Moss, William C.; Priore, Salvatore F.; Turner, Douglas H.

doi:10.1261/rna.2619511

Cited by 88 publications

(193 citation statements)

References 84 publications

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“…1A), suggesting that functional constraints operate at the nucleotide sequence level. These observations are consistent with Moss et al 28 Consensus secondary structures that may be formed in these regions were predicted using RNAalifold. 33 Analyses were undertaken 4 times separately for human, swine, and avian IAVs by randomly choosing 10 strains from each host species (Table S3).…”

Section: Identification and Prediction Of Rna Secondary Structuressupporting

confidence: 91%

“…This structure has been reported to be involved in the splicing of M segment mRNAs. 28,29,31 In addition, we newly identified stemloop structures (named SL3-10(C) in CRNA and SL3-10(¡) in -RNA) at positions 219-240 in M segment sequences from all 3 host species (Figs. 1, 2A and 3A; Figs.…”

Section: Identification and Prediction Of Rna Secondary Structuresmentioning

confidence: 99%

“…26 Incorporation of the M segment vRNA into the IAV virion has been suggested to be critical to the packaging process 18,27 and in silico analyses have predicted secondary RNA structures in the positive and negative sense RNA sequences of the M segment. [28][29][30][31] Here, we use evolutionary and bioinformatic methods to identify stem loop structures in the M segment that are highly conserved among 1,884 IAV strains and that are located in regions previously proposed to contain packaging signals. 18,20 These in silico findings were then followed up with reverse genetics experiments in order to investigate the functional significance of these regions.…”

Section: Introductionmentioning

confidence: 99%

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Computational and molecular analysis of conserved influenza A virus RNA secondary structures involved in infectious virion production

et al. 2016

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As well as encoding viral proteins, genomes of RNA viruses harbor secondary and tertiary RNA structures that have been associated with functions essential for successful replication and propagation. Here, we identified stem-loop structures that are extremely conserved among 1,884 M segment sequences of influenza A virus (IAV) strains from various subtypes and host species using computational and evolutionary methods. These structures were predicted within the 3 0 and 5 0 ends of the coding regions of M1 and M2, respectively, where packaging signals have been previously proposed to exist. These signals are thought to be required for the incorporation of a single copy of 8 different negative-strand RNA segments (vRNAs) into an IAV particle. To directly test the functionality of conserved stem-loop structures, we undertook reverse genetic experiments to introduce synonymous mutations designed to disrupt secondary structures predicted at 3 locations and found them to attenuate infectivity of recombinant virus. In one mutant, predicted to disrupt stem loop structure at nucleotide positions 219-240, attenuation was more evident at increased temperature and was accompanied by an increase in the production of defective virus particles. Our results suggest that the conserved secondary structures predicted in the M segment are involved in the production of infectious viral particles during IAV replication.

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Section: Identification and Prediction Of Rna Secondary Structuressupporting

confidence: 91%

Section: Identification and Prediction Of Rna Secondary Structuresmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Computational and molecular analysis of conserved influenza A virus RNA secondary structures involved in infectious virion production

et al. 2016

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“…9,10 However, apart from well-studied universally conserved structures at the ends of each of the RNA segments, only few conserved structures were revealed in the influenza A virus RNA genome. [11][12][13][14][15][16] Here, we describe the analysis of potential folding patterns in the NP segment (segment 5) of the influenza A virus genome, using the available sequences from various strains. The evolution of this segment resulted in several distinct host-specific clusters.…”

Section: Introductionmentioning

confidence: 99%

RNA structural constraints in the evolution of the influenza A virus genome NP segment

Gultyaev

Tsyganov-Bodounov

Spronken³

et al. 2014

RNA Biology

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Conserved RNA secondary structures were predicted in the nucleoprotein (NP) segment of the influenza A virus genome using comparative sequence and structure analysis. A number of structural elements exhibiting nucleotide covariations were identified over the whole segment length, including protein-coding regions. Calculations of mutual information values at the paired nucleotide positions demonstrate that these structures impose considerable constraints on the virus genome evolution. Functional importance of a pseudoknot structure, predicted in the NP packaging signal region, was confirmed by plaque assays of the mutant viruses with disrupted structure and those with restored folding using compensatory substitutions. Possible functions of the conserved RNA folding patterns in the influenza A virus genome are discussed.

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“…This analysis allowed us to determine the number of mutations required to be introduced into the prototype virus to change its codon bias from human influenza virus-like to avian influenza virus-like (Table 1; see also Supplemental Data S1 in the supplemental material). In order to avoid affecting critical viral RNA signals essential for virus replication, mutations were introduced into the regions that are known to not be involved in viral RNP (vRNP) packaging and splicing (27)(28)(29). Sequence regions with out-of-frame ORFs (e.g., PB1 and PB1-F2 ORFs) were also excluded from mutagenesis.…”

Section: Methodsmentioning

confidence: 99%

Generation of Live Attenuated Influenza Virus by Using Codon Usage Bias

Fan

Valkenburg

Wong

et al. 2015

J Virol

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Seasonal influenza epidemics and occasional pandemics threaten public health worldwide. New alternative strategies for generating recombinant viruses with vaccine potential are needed. Interestingly, influenza viruses circulating in different hosts have been found to have distinct codon usage patterns, which may reflect host adaptation. We therefore hypothesized that it is possible to make a human seasonal influenza virus that is specifically attenuated in human cells but not in eggs by converting its codon usage so that it is similar to that observed from avian influenza viruses. This approach might help to generate human live attenuated viruses without affecting their yield in eggs. To test this hypothesis, over 300 silent mutations were introduced into the genome of a seasonal H1N1 influenza virus. The resultant mutant was significantly attenuated in mammalian cells and mice, yet it grew well in embryonated eggs. A single dose of intranasal vaccination induced potent innate, humoral, and cellular immune responses, and the mutant could protect mice against homologous and heterologous viral challenges. The attenuated mutant could also be used as a vaccine master donor strain by introducing hemagglutinin and neuraminidase genes derived from other strains. Thus, our approach is a successful strategy to generate attenuated viruses for future application as vaccines. IMPORTANCE Vaccination has been one of the best protective measures in combating influenza virus infection. Current licensed influenza vaccines and their production have various limitations. Our virus attenuation strategy makes use of the codon usage biases of human and avian influenza viruses to generate a human-derived influenza virus that is attenuated in mammalian hosts. This method, however, does not affect virus replication in eggs. This makes the resultant mutants highly compatible with existing egg-based vaccine production pipelines. The viral proteins generated from the codon bias mutants are identical to the wild-type viral proteins. In addition, our massive genome-wide mutational approach further minimizes the concern over reverse mutations. The potential use of this kind of codon bias mutant as a master donor strain to generate other live attenuated viruses is also demonstrated. These findings put forward a promising live attenuated influenza vaccine generation strategy to control influenza. Seasonal influenza strikes every year, and the threat of avian influenza outbreaks and worldwide pandemics together make influenza a significant health risk to the general public, particularly young children, pregnant women, the elderly, and patients with underlying medical conditions (1). Vaccination remains one of the best control measures against influenza. However, currently licensed inactivated and live attenuated influenza vaccines have their limitations. Therefore, new options for vaccine development are needed.Vaccination with an updated virus strain is required every year in response to the frequent occurrence of antigenic drift. Even w...

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Identification of potential conserved RNA secondary structure throughout influenza A coding regions

Cited by 88 publications

References 84 publications

Computational and molecular analysis of conserved influenza A virus RNA secondary structures involved in infectious virion production

Computational and molecular analysis of conserved influenza A virus RNA secondary structures involved in infectious virion production

RNA structural constraints in the evolution of the influenza A virus genome NP segment

Generation of Live Attenuated Influenza Virus by Using Codon Usage Bias

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