Secondary Structure of Influenza A Virus Genomic Segment 8 RNA Folded in a Cellular Environment.

Barbara Szutkowska,Klaudia Wieczorek,Walter N Moss,Elzbieta Kierzek,Ryszard Kierzek,Pawel Zmora,Jake M Peterson,David H Mathews

doi:10.3390/ijms23052452

Abstract

Influenza A virus (IAV) is a member of the single-stranded RNA (ssRNA) family of viruses. The most recent global pandemic caused by the SARS-CoV-2 virus has shown the major threat that RNA viruses can pose to humanity. In comparison, influenza has an even higher pandemic potential as a result of its high rate of mutations within its relatively short (<13 kbp) genome, as well as its capability to undergo genetic reassortment. In light of this threat, and the fact that RNA structure is connected to a broad range of known biological functions, deeper investigation of viral RNA (vRNA) structures is of high interest. Here, for the first time, we propose a secondary structure for segment 8 vRNA (vRNA8) of A/California/04/2009 (H1N1) formed in the presence of cellular and viral components. This structure shows similarities with prior in vitro experiments. Additionally, we determined the location of several well-defined, conserved structural motifs of vRNA8 within IAV strains with possible functionality. These RNA motifs appear to fold independently of regional nucleoprotein (NP)-binding affinity, but a low or uneven distribution of NP in each motif region is noted. This research also highlights several accessible sites for oligonucleotide tools and small molecules in vRNA8 in a cellular environment that might be a target for influenza A virus inhibition on the RNA level.

Highlights

The influenza viruses are classified as types A, B, C, and D (IAV, IBV, ICV, and IDV) and belong to the Orthomyxoviridae family of viruses [1]
Two different chemical reagents were used to probe vRNA8 in cell lysates: DMS, which can react with adenosine and cytosine (N1 or N3 position, respectively), and 1M7, which is one of the SHAPE (2 -hydroxyl acylation analyzed by primer extension) reagents that react at the 2 OH position of all accessible nucleotides
Before reverse transcription, the isolated RNA was checked for integrity on a non-denaturing agarose gel which verified an intact total RNA and single vRNA8 product (890 nt long)

Summary

Introduction

The influenza viruses are classified as types A, B, C, and D (IAV, IBV, ICV, and IDV) and belong to the Orthomyxoviridae family of viruses [1]. Vaccines are developed annually and some of them contain strains of inactive influenza virus that are predicted as the most probable to occur each year [5,6]. This prediction may be incorrect, leading to seasonal outbreaks. The A/California/04/2009 (H1N1) pandemic strain wreaked havoc and very quickly affected 88 million people worldwide [7]. It is considered very threatening because humans have a lack of immunity to it from previous viral infections [4,8]. With these challenges in mind, the World Health Organization (WHO) started the Global Influenza Programme (2019–2030), focusing on prevention, control, and preparation for future influenza outbreaks

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Conclusion