A Naturally Occurring NS1 Variant with Effector Domain Deletion Gains Growth Advantages in Influenza Virus Infection.

High pathogenicity avian influenza virus (HPAIV) poses major threats to both poultry health and public safety. The viral nonstructural protein 1 (NS1) plays a crucial role in counteracting innate immunity. NS1 typically consists of approximately 230 amino acids with two domains: an RNA-binding domain (RBD) and the effector domain (ED). A novel naturally occurring NS1 variant, termed NS100, was reported herein, isolated during propagation of a recombinant H5N2 virus. NS100 encodes only the 1-95 residues of NS1, lacking the entire ED. Surprisingly, the NS100 virus replicated as efficiently as the wild-type (WT) virus in both cell and animal models. Cells infected with NS100 exhibited reduced interferon and cytokine expression, indicating preserved immune evasion capacity. Biochemical analyses revealed that NS100 retains key NS1 functions mediated by the RBD, including dsRNA binding, PKR interaction, and homodimer formation. In addition, NS100 cooperated with the viral RNA polymerase complex in a mini-genome assay, indicating functional synergy. Interestingly, the NS100 virus showed markedly reduced NS2 protein expression, likely due to impaired splicing caused by a GA transversion at a polypyrimidine tract. Transcriptome analysis further confirmed reduced splicing efficiency of NS transcripts and lower expression of antiviral host genes in NS100-infected cells. In conclusion, our findings demonstrate that NS1 can maintain essential pro-viral functions despite the loss of its ED, and that NS100 provides new insights into the modularity of NS1 and its broader role in regulating host-virus interactions.