Mutations of the segment-specific nucleotides at the 3’ end of influenza virus NS segment control viral replication

The vRNAs of influenza A viruses contain 12 and 13 nucleotide-long sequences at their 3′ and 5′ termini respectively that are highly conserved and constitute the vRNA promoter. These sequences and the next three segment-specific nucleotides show inverted partial complementarity and are followed by several unpaired nucleotides of poorly characterized function at the 3′ end. We have performed systematic point-mutations at the segment-specific nucleotides 15–18 of the 3′-end of a NS-like vRNA segment. All NS-like vRNAs containing mutations at position 15, and some at positions 16–18 showed reduced transcription/replication efficiency in a transfection/infection system. In addition, the replication of recombinant viruses containing mutations at position 15 was impaired both in single and multi-cycle experiments. This reduction was the consequence of a decreased expression of the NS segment. The data indicate that NS1 plays a role in the transcription/replication of its own segment, which elicits a global defect on virus replication. •Influenza A viruses contain segment-specific nucleotides at the 3′ and 5′ ends.•Pairing and unpairing segment-specific nucleotides modulate genome expression.•Mutations at nucleotide 15 of the 3′ end have the highest effect on NS segment expression.•These mutations reduce NS1 expression.•NS1 plays a role in the transcription/replication of its own segment.