Influenza A viruses (IAVs) have multiple mechanisms for altering the host immune response to aid in virus survival and propagation. While both type I and II interferons (IFNs) have been associated with increased bacterial superinfection (BSI) susceptibility, we found that in some cases type I IFNs can be beneficial for BSI outcome. Specifically, we have shown that antagonism of the type I IFN response during infection by some IAVs can lead to the development of deadly BSI. The nonstructural protein 1 (NS1) from IAV is well known for manipulating host type I IFN responses, but the viral proteins mediating BSI severity remain unknown. In this study, we demonstrate that the PDZ-binding motif (PDZ-bm) of the NS1 C-terminal region from mouse-adapted A/Puerto Rico/8/34-H1N1 (PR8) IAV dictates BSI susceptibility through regulation of IFN-a/b production. Deletion of the NS1 PDZ-bm from PR8 IAV (PR8-TRUNC) resulted in 100% survival and decreased bacterial burden in superinfected mice compared with 0% survival in mice superinfected after PR8 infection. This reduction in BSI susceptibility after infection with PR8-TRUNC was due to the presence of IFN-b, as protection from BSI was lost in Ifn-b-/mice, resembling BSI during PR8 infection. PDZ-bm in PR8-infected mice inhibited the production of IFN-b posttranscriptionally, and both delayed and reduced expression of the tunable interferon-stimulated genes. Finally, a similar lack of BSI susceptibility, due to the presence of IFN-b on day 7 post-IAV infection, was also observed after infection of mice with A/TX98-H3N2 virus that naturally lacks a PDZ-bm in NS1, indicating that this mechanism of BSI regulation by NS1 PDZ-bm may not be restricted to PR8 IAV. These results demonstrate that the NS1 C-terminal PDZ-bm, like the one present in PR8 IAV, is involved in controlling susceptibility to BSI through the regulation of IFN-b, providing new mechanisms for NS1-mediated manipulation of host immunity and BSI severity.

Highlights An HA-based vaccine candidate, created by DNA shuffling (HA-113), can be immunogenic when the recombinant antigen is expressed by PIV5 (PIV5-113). Immunity induced by the PIV5-113 vaccine can protect mice against infection with 4 of 5 parental HAs used to create the vaccine. Immunity induced by PIV5-113 can protect pigs against infection with an influenza virus isolate that is known to be infectious in pigs.

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