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

Abstract

Influenza virus causes seasonal epidemics and dangerous pandemic outbreaks. It is a single stranded (−)RNA virus with a segmented genome. Eight segments of genomic viral RNA (vRNA) form the virion, which are then transcribed and replicated in host cells. The secondary structure of vRNA is an important regulator of virus biology and can be a target for finding new therapeutics. In this paper, the secondary structure of segment 5 vRNA is determined based on chemical mapping data, free energy minimization and structure-sequence conservation analysis for type A influenza. The revealed secondary structure has circular folding with a previously reported panhandle motif and distinct novel domains. Conservations of base pairs is 87% on average with many structural motifs that are highly conserved. Isoenergetic microarray mapping was used to additionally validate secondary structure and to discover regions that easy bind short oligonucleotides. Antisense oligonucleotides, which were designed based on modeled secondary structure and microarray mapping, inhibit influenza A virus proliferation in MDCK cells. The most potent oligonucleotides lowered virus titer by ~90%. These results define universal for type A structured regions that could be important for virus function, as well as new targets for antisense therapeutics.

Anti-Influenza Strategies Based on Nanoparticle Applications

“…The group of silencers includes antisense oligonucleotides (ASO), ribozymes, DNAzymes, short interfering RNAs (siRNA), microRNAs (miRNAs), short hairpin RNA (shRNAs), or peptide nucleic acids (PNAs). The evidence for their effectiveness in vivo has been provided in many, including those performed in our group [87][88][89][90][91][92]. However, the use of synthetic nucleic acids is limited due to imperfect systems of their introduction into cells.…”

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

“…Numerous reports have shown the biological importance of the viral RNA secondary structure in key stages of the pathogen replication cycle. Influenza RNA secondary structure motifs have been thoroughly studied in vitro [ 24 , 25 , 26 ]. Based on complex experimental studies and bioinformatic analyses, structural models have been proposed for full-length naked vRNA5, 7, and 8, so far [ 24 , 25 , 26 ].…”

“…Influenza RNA secondary structure motifs have been thoroughly studied in vitro [ 24 , 25 , 26 ]. Based on complex experimental studies and bioinformatic analyses, structural models have been proposed for full-length naked vRNA5, 7, and 8, so far [ 24 , 25 , 26 ]. These analyses included a number of methods, such as chemical mapping, isoenergetic microarrays, RNase H cleavage in the presence of DNA oligonucleotides, evaluation of base-pairing probability, and preservation of canonical base-pairs in multiple strains.…”

Mutagenesis Mapping of RNA Structures within the Foot-and-Mouth Disease Virus Genome Reveals Functional Elements Localised in the Polymerase (3Dpol) Encoding Region

“…The genomes of RNA viruses not only encode proteins but also contain non-templated functional elements in both the coding and untranslated regions (UTRs). These can be secondary or higher order RNA structures such as simple stem-loops or more complex structures which include pseudoknots and so-called kissing-loops that mediate long-range RNA-RNA interactions (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) . The function, shape and number of such RNA functional elements is often characteristic for a particular group of viruses, where they play important roles in processes such as the initiation of viral RNA translation and replication, subgenomic mRNA transcription, frame shift events, viral RNA encapsidation and modulation of host's antiviral responses (reviewed in (13)).…”