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

Irina L Baranovskaya,Mariia V Sergeeva,Alexey A Lozhkov,Aleksandr S Taraskin,Andrey V Vasin

doi:10.18527/2500-2236-2021-8-1-1-9

Abstract

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.

Highlights

It is well known that RNA secondary structures are vitally important for the life cycle of many DNA and RNA containing viruses
We have shown that certain NS (+)RNA secondary structure combinations regulate NS1 and NEP production in vitro and can affect the level of viral pathogenicity in vivo
497-564 and absence of a hairpin structure at position 82-148) leads to decreased NEP expression in infected A549 cells (V0-1 compared to V0-0) (Fig. 3A)

Summary

INTRODUCTION

It is well known that RNA secondary structures are vitally important for the life cycle of many DNA and RNA containing viruses. The most precise secondary structure of (+)RNA has been determined for the NS segment of influenza A virus [13,14,15,16,17] This genomic segment encodes at least two important non-structural proteins (NS and NEP), and contains a splicing regulated switch between the corresponding open reading frames (ORFs). The NS gene sequences, providing stable RNA hairpins in the corresponding regions, were adopted from the aforementioned influenza strains, while the sequences with no stable secondary structures were taken from their closest “counterparts” with a minimum number of mismatches (Fig. 1). Virus variants bearing different secondary structure combinations were named according to the presence (1) or absence (0) of an RNA hairpin in the NS region 82-148 and/or region 497-564. The variants were named as: V1-1, V1-0, V0-1, or V0-0

MATERIALS AND METHODS

DISCUSSION