Abstract

The non-structural protein NS1 of influenza A viruses is an RNA-binding protein of which its activities in the infected cell contribute to the success of the viral cycle, notably through interferon antagonism. We have previously shown that NS1 strongly binds RNA aptamers harbouring virus-specific sequence motifs (Marc et al., Nucleic Acids Res. 41, 434–449). Here, we started out investigating the putative role of one particular virus-specific motif through the phenotypic characterization of mutant viruses that were genetically engineered from the parental strain WSN. Unexpectedly, our data did not evidence biological importance of the putative binding of NS1 to this specific motif (UGAUUGAAG) in the 3′-untranslated region of its own mRNA. Next, we sought to identify specificity determinants in the NS1-RNA interaction through interaction assays in vitro with several RNA ligands and through solving by X-ray diffraction the 3D structure of several complexes associating NS1′s RBD with RNAs of various affinities. Our data show that the RBD binds the GUAAC motif within double-stranded RNA helices with an apparent specificity that may rely on the sequence-encoded ability of the RNA to bend its axis. On the other hand, we showed that the RBD binds to the virus-specific AGCAAAAG motif when it is exposed in the apical loop of a high-affinity RNA aptamer, probably through a distinct mode of interaction that still requires structural characterization. Our data are consistent with more than one mode of interaction of NS1′s RBD with RNAs, recognizing both structure and sequence determinants.

Highlights

  • With 3–5 million severe cases and an excess mortality of 290–650 thousand deaths per year [1,2], influenza viruses remain one of the major infectious threats to human health

  • RNA is part of the U12 sequence that makes up the viral polymerase promoter, is strictly conserved at the 50 end of all influenza A viruses complementary RNAs

  • We set out to investigate the importance of three distinct sequence and structure motifs in the interaction of influenza A virus NS1 with RNAs

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Summary

Introduction

With 3–5 million severe cases and an excess mortality of 290–650 thousand deaths per year [1,2], influenza viruses remain one of the major infectious threats to human health. In addition to this seasonal disease burden, the sporadic human cases of infection with avian influenza viruses There is a pressing need to enrich our assortment of antivirals, both to improve our preparedness against future pandemics and to design combination therapies that are less prone to the emergence of resistances. One of the most promising targets is the influenza virus non-structural protein

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