RNA structural constraints in the evolution of the influenza A virus genome NP segment

Abstract

Conserved RNA secondary structures were predicted in the nucleoprotein (NP) segment of the influenza A virus genome using comparative sequence and structure analysis. A number of structural elements exhibiting nucleotide covariations were identified over the whole segment length, including protein-coding regions. Calculations of mutual information values at the paired nucleotide positions demonstrate that these structures impose considerable constraints on the virus genome evolution. Functional importance of a pseudoknot structure, predicted in the NP packaging signal region, was confirmed by plaque assays of the mutant viruses with disrupted structure and those with restored folding using compensatory substitutions. Possible functions of the conserved RNA folding patterns in the influenza A virus genome are discussed.

Subtype-specific structural constraints in the evolution of influenza A virus hemagglutinin genes

“…From previous work in the search for structured regions in the influenza virus RNA segments81517 it was anticipated that the conserved structural elements in the HA segment, if any, could be relatively small domains with moderate thermodynamic stability. The deviations from thermodynamically optimal structures, common in any large RNA molecule, are expected to be even more significant in the native influenza vRNA due to its functioning in the structure-destabilizing complex with multiple NP molecules181920.…”

“…On the other hand, functional significance of predicted local structural elements, irrespectively of their thermodynamic properties, can be estimated using analysis of nucleotide covariations that indicate significant evolutionary constraints and thus functional importance. Thanks to the availability of a large number of sequences from various influenza virus strains, mutual information calculations can help to identify significant covariations15.…”

“…Some of the conserved structures in segments 7 and 8 were shown to affect splicing14. Conserved structures were also suggested to fold in several domains of segment 51516.…”

“…This may be attributed to the functioning of vRNA in ribonucleoprotein (vRNP) complexes where most of the vRNA self-organized structure is melted upon nucleoprotein (NP) binding181920. However, the absence of a global ordered RNA structure does not mean the absence of local structural motifs, and some predicted structured regions could be functional in both polarities815. Protein-free segment 8 vRNA has been shown to form stable conserved structures in vitro 21.…”

“…Protein-free segment 8 vRNA has been shown to form stable conserved structures in vitro 21. The functional importance of a pseudoknot structure folded in the packaging signal region of segment 5 vRNA has been demonstrated by compensatory mutagenesis using reverse genetics15.…”