Changes in RNA secondary structure affect NS1 protein expression during early stage influenza virus infection

Abstract

RNA secondary structures play a key role in splicing, gene expression, microRNA biogenesis, RNA editing, and other biological processes. The importance of RNA structures has been demonstrated in the life cycle of RNA-containing viruses, including the influenza virus. At least two regions of conserved secondary structure in NS segment (+) RNA are predicted to vary among influenza virus strains with respect to thermodynamic stability; both fall in the NS1 open reading frame. The NS1 protein is involved in multiple virus-host interaction processes, and its main function is to inhibit the cellular immune response to viral infection. Using a reverse genetics approach, four influenza virus strains were constructed featuring mutations that have different effects on RNA secondary structure. Growth curve experiments and ELISA data show that, at least in the first viral replication cycle, mutations G123A and A132G affecting RNA structure in the (82–148) NS RNA region influence NS1 protein expression.

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

“…Four virus variants with different combinations of secondary structures in the two NS RNA regions (82-148 and 497-564) were previously obtained by reverse genetics, followed by the virus production in Vero cells [26]. All four variants are based on the laboratory strain A/Puerto Rico/8/1934 (H1N1).…”

“…, based on the A/Puerto Rico/8/34 strain and with different combinations of NS (+)RNA secondary structure in the region 82-148 and region 497-564, were previously constructed [26]. We first compared the infectious activity of the viruses in Vero, MDCK, A549, and CK cells and in 10-day old embryonated chicken eggs.…”

“…Highly virulent avian influenza A (H5N1) strains that appeared after 2001 carry more stable RNA hairpins in the second region compared to previous strains [14]. Since the NS gene is closely associated with host adaptation, structural motifs may affect host specificity or viral pathogenicity through the regulation of NS1 expression [15,26]. However, the role of the observed hairpins is yet to be determined.…”

“…Here, we present the study results of 4 previously constructed viruses based on the A/Puerto Rico/8/1934 (H1N1) strain, featuring different combinations of secondary structures in the NS mRNA region 82-148 and region 497-564 [26]. 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.…”

Host–Virus Interaction: How Host Cells Defend against Influenza A Virus Infection

“…The mechanism underlying is revealed by determination of crystal structure of the NS1 and nuclear RNA export factor 1-nuclear transport factor 2-related export protein 1 (NXF1-NXT1) complex that NS1 could prevent mRNA export through the nuclear pore complex via binding of NXF1-NXT1 to nucleoporins [43]. Conserved RNA secondary structures at RNA positions 82-148 and 497-564 are essential for NS1 protein expression, thus affecting viral reproduction and virus-host interaction processes [44]. A detailed description of structure and function of the NS1 was reviewed by Han and his colleagues [45].…”