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Background  The non-structural protein 1 (NS1) of influenza A virus (IAV) is a key player in inhibiting antiviral response in host cells, thereby facilitating its replication. However, other roles of NS1, which are independent of antagonising host cells’ antiviral response, are less characterised.    Methods  To investigate these unidentified roles, we used a recombinant virus, which lacks NS1 expression, and observed its phenotypes during the infection of antiviral defective cells (RIG-I KO cells) in the presence or absence of exogeneous NS1. Moreover, we used virus-like particle (VLP) production system to further support our findings.    Results  Our experiments demonstrated that IAV deficient in NS1 replicates less efficiently than wild-type IAV in RIG-I KO cells and this replication defect was complemented by ectopic expression of NS1. As suggested previously, NS1 is incorporated in the virion and participates in the regulation of viral transcription and translation. Using the VLP production system, in which minigenome transcription or viral protein production was unaffected by NS1, we demonstrated that NS1 facilitates viral genome packaging into VLP, leading to efficient minigenome transfer by VLP. Furthermore, the incorporation of NS1 and the minigenome into VLP were impaired by introducing a point mutation (R38A) in the double stranded RNA-binding domain of NS1.    Conclusion  These results suggest a novel function of NS1 in improving genome packaging in a dsRNA binding-dependent manner. Taken together, NS1 acts as an essential pro-viral regulator, not only by antagonizing host immunity but also by facilitating viral replication and genome packaging.

Influenza A virus NS1 optimises virus infectivity by enhancing genome packaging in a dsRNA-binding dependent manner