Three amino acid substitutions in the NS1 protein change the virus replication of H5N1 influenza virus in human cells

Highlights: • All three H5N1 AIV display differential virus replication in human cells. • HN01 virus reveal poor replication and defective ability to suppress IFN response. • Residues 55/66/133 of NS1 in HN01 alter IFN response and viral titer. Influenza A viruses have sophisticated strategies to promote their own replication. Here, we found that three H5N1 influenza viruses display different replication patterns in human A549 and macrophage cells. The HN01 virus displayed poor replication compared to HN021 and JS01. In addition, the HN01 virus was unable to counteract the interferon response and block general gene expression. This capability was restored by three amino acid substitutions on the NS1 protein: K55E, K66E, and C133F, resulting in recovered binding to CPSF30 and decreased interferon response activity. Furthermore, a recombinant HN01 virus expressing either NS1-C133F or the triple mutation replicate with higher titers in human A549 cells and macrophages compared to the parent virus. These three amino acid mutations reveal a new pathway to alter H5N1 virus replication.