Avian Influenza NS1 Proteins Inhibit Human, but Not Duck, RIG-I Ubiquitination and Interferon Signaling.

The nonstructural protein 1 (NS1) of influenza A viruses is an important virulence factor that controls host cell immune responses. In human cells, NS1 proteins inhibit the induction of type I interferon by several mechanisms, including potentially, by preventing the activation of the retinoic acid-inducible gene I (RIG-I) receptor by the ubiquitin ligase tripartite motif-containing protein 25 (TRIM25). It is unclear whether the inhibition of human TRIM25 is a universal function of all influenza A NS1 proteins or is strain dependent. It is also unclear if NS1 proteins similarly target the TRIM25 of mallard ducks, a natural reservoir host of avian influenza viruses with a long coevolutionary history and unique disease dynamics. To answer these questions, we compared the ability of five different NS1 proteins to interact with human and duck TRIM25 using coimmunoprecipitation and microscopy and assessed the consequence of this on RIG-I ubiquitination and signaling in both species. We show that NS1 proteins from low-pathogenic and highly pathogenic avian influenza viruses potently inhibit RIG-I ubiquitination and reduce interferon promoter activity and interferon-beta protein secretion in transfected human cells, while the NS1 of the mouse-adapted PR8 strain does not. However, all the NS1 proteins, when cloned into recombinant viruses, suppress interferon in infected alveolar cells. In contrast, avian NS1 proteins do not suppress duck RIG-I ubiquitination and interferon promoter activity, despite interacting with duck TRIM25. IMPORTANCE Influenza A viruses are a major cause of human and animal disease. Periodically, avian influenza viruses from wild waterfowl, such as ducks, pass through intermediate agricultural hosts and emerge into the human population as zoonotic diseases with high mortality rates and epidemic potential. Because of their coevolution with influenza A viruses, ducks are uniquely resistant to influenza disease compared to other birds, animals, and humans. Here, we investigate a mechanism of influenza A virus interference in an important antiviral signaling pathway that is orthologous in humans and ducks. We show that NS1 proteins from four avian influenza strains can block the coactivation and signaling of the human RIG-I antiviral receptor, while none block the coactivation and signaling of duck RIG-I. Understanding host-pathogen dynamics in the natural reservoir will contribute to our understanding of viral disease mechanisms, viral evolution, and the pressures that drive it, which benefits global surveillance and outbreak prevention.