Abstract
Eurasian avian-like H1N1 influenza A viruses that have reassorted with the 2009 pandemic H1N1 virus pose a potential public health threat. We previously reported that synergistic mutations in HA and NS genes enhanced the virulence of a mouse-adapted EA H1N1 canine influenza virus (CIV), yet the underlying mechanisms remained unclear. Here, we demonstrate that the adaptive mutations NS1-A53D and NEP-R42K synergistically promote viral replication by enhancing viral ribonucleoprotein (vRNP) nuclear export, while NEP-R42K alone boosts polymerase activity. Critically, we uncover a novel synergy between HA mutations (N198D and A227E) and NS1-A53D, but not NEP-R42K, in facilitating early viral entry, including attachment and internalization. Mechanistically, NS1-A53D significantly enhances HA protein stability without altering receptor binding specificity, thereby compensating for the intrinsic antagonism between the two HA mutations that impairs receptor binding affinity. These findings reveal that HA and NS genes cooperate through distinct but complementary mechanisms: NS1-A53D stabilizes HA to promote entry, while NS1-A53D and NEP-R42K collaboratively enhance vRNP export, collectively driving mammalian adaptation and pathogenicity.