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

Lisa Marie Simon,Edoardo Morandi,Giorgio Gribaudo,Danny Incarnato,Anna Luganini,Salvatore Oliviero,Luis Martinez-Sobrido,Douglas H Turner

doi:10.1093/nar/gkz318

Abstract

The 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.

Highlights

Influenza A virus (IAV) represents a considerable public health threat due to its epidemic, pandemic, and zoonotic potential, causing high costs every year [1], exemplified by the tragic “Spanish” flu in 1918 which led to the death of millions of people [2]
RAPiD-MaPseq enables targeted in vivo RNA structure mapping of low-abundance influenza A virus (IAV) messenger RNAs (mRNAs)
To investigate IAV mRNA structures in the context of infected cells, we here developed RNA antisense purification combined with dimethyl sulfate (DMS) mutational profiling (RAPiD-MaPseq), an approach that couples in vivo DMS probing with consecutive oligo-dT and biotinylated antisense oligonucleotide-mediated capture of target mRNAs, exploiting the high affinity and specificity of the biotin-streptavidin interaction to achieve over 13-fold enrichment of IAV mRNAs (Supplementary Figure S1A)

Summary

Introduction

Influenza A virus (IAV) represents a considerable public health threat due to its epidemic, pandemic, and zoonotic potential, causing high costs every year [1], exemplified by the tragic “Spanish” flu in 1918 which led to the death of millions of people [2]. According to the World Health Organization (WHO), annual influenza epidemics cause worldwide 3–5 million cases of severe disease and up to half million deaths among high-risk groups, such as children, elderly and immunocompromised patients, with an even greater impact in developing countries. VRNAs are associated with the viral polymerase (consisting of PA, PB1, PB2) via the panhandle structure (which poises them to be transcribed as soon as they enter the nucleus of the host cell) to form the viral ribonucleoproteins (vRNP) complexes [5]. The correct packaging of all eight segments into one viral particle is believed to be conferred at least in part via structured RNA domains of vRNAs protruding from vRNPs [7,8,9]

Methods

Results

Conclusion