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Abstract

Influenza A viruses (IAV) are a common cause of acute upper respiratory tract infections in humans. As an intracellular pathogen, IAV utilizes host proteins and processes to complete its life-cycle. The host RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1), which deaminates adenosine to inosine in dsRNA, has been implicated as either a proviral or antiviral factor for viruses from diverse families; however, the role of ADAR1 in IAV infection has yet to be completely understood. ADAR1 is expressed as two isoforms; the constitutively expressed p110 isoform and the interferon inducible p150 isoform. Using the CRISPR/Cas9 gene disruption strategy, we generated ADAR1 knockouts (KO) in human lung epithelial cells (A549) and observed decreased IAV replication, suggesting that ADAR1 is a proviral factor for IAV. By generating isoform specific KO cell lines, we determined that the p150 isoform was critical for IAV replication. As p150 has been implicated in regulating MDA5-mediated signaling, we generated p150/MDA5 double KOs (DKOs) and assessed IAV replication. IAV replication was reduced in the p150/MDA5 DKOs, demonstrating that p150 is critical for IAV replication independent of MDA5 suppression. During IAV infection, IFN-β expression and PARP cleavage were increased in the p150/MDA5 DKOs, suggesting that p150 suppresses IFN-β expression and apoptosis. To determine if RIG-I mediated signaling contributes to these processes, we generated p150/MDA5/RIG-I triple knockouts (TKOs). IAV replication was increased in the p150/MDA5/RIG-I TKOs while PARP cleavage and IFN-β expression was reduced. We also demonstrate that p150 and the IAV nonstructural protein 1 (NS1) colocalize in cytoplasmic puncta during IAV infection. In addition, overexpression of p150 and NS1 showed increased suppression of IFN-β luciferase reporter activity. Together, this study demonstrates that p150 suppresses the activation of RIG-I during IAV infection, thereby reducing the induction of IFN-β expression and apoptosis in order to generate a more hospitable environment for IAV replication.

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