Structure of influenza D virus NS1 reveals a novel Zn-binding site and ssRNA-binding activity

Influenza D virus (IDV) is an emerging pathogen associated with bovine respiratory disease and has potential for zoonotic transmission. Although nonstructural protein 1 (NS1) is a major virulence factor in influenza A and B viruses, the structure and function of IDV NS1 remain poorly defined. Here, we determined crystal structures of the IDV NS1 N-terminal RNA-binding domain (NTD) and C-terminal effector domain (CTD). The NTD retains the conserved homodimeric six-helix fold, whereas the CTD adopts a distinct architecture containing a novel Zn 2+ -binding site. IDV NS1 CTD preferentially binds single-stranded RNA, and mutagenesis identified lysine and arginine residues critical for this activity. Single-residue substitutions disrupting either the Zn 2+ -binding site or RNA-binding interface prevented recovery of infectious recombinant IDV. These findings reveal distinct structural and functional features of IDV NS1 and establish the importance of CTD integrity for viral replication. Conservation of key CTD residues in influenza C virus NS1 suggests shared structural features that distinguish influenza C and D virus NS1 proteins from their influenza A and B counterparts.