Influenza A viral nucleoprotein interacts with cytoskeleton scaffolding protein α-actinin-4 for viral replication.

Influenza A virus (IAV), similar to other viruses, exploits the machinery of human host cells for its survival and replication. We identified α-actinin-4, a host cytoskeletal protein, as an interacting partner of IAV nucleoprotein (NP). We confirmed this interaction using co-immunoprecipitation studies, first in a coupled in vitro transcription-translation assay and then in cells either transiently co-expressing the two proteins or infected with whole IAV. Importantly, the NP–actinin-4 interaction was observed in several IAV subtypes, including the 2009 H1N1 pandemic virus. Moreover, immunofluorescence studies revealed that both NP and actinin-4 co-localized largely around the nucleus and also in the cytoplasmic region of virus-infected A549 cells. Silencing of actinin-4 expression resulted in not only a significant decrease in NP, M2 and NS1 viral protein expression, but also a reduction of both NP mRNA and viral RNA levels, as well as viral titers, 24 h post-infection with IAV, suggesting that actinin-4 was critical for viral replication. Furthermore, actinin-4 depletion reduced the amount of NP localized in the nucleus. Treatment of infected cells with wortmannin, a known inhibitor of actinin-4, led to a decrease in NP mRNA levels and also caused the nuclear retention of NP, further strengthening our previous observations. Taken together, the results of the present study indicate that actinin-4, a novel interacting partner of IAV NP, plays a crucial role in viral replication and this interaction may participate in nuclear localization of NP and/or viral ribonucleoproteins. Structured digital abstract •NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (1, 2)•NP and actnin-4 colocalize by fluorescence microscopy (View interaction)•NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction)•NP binds to actinin-4 by anti tag coimmunoprecipitation (1, 2)•NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction)•NP physically interacts with actinin-4 by anti tag coimmunoprecipitation (View interaction)•NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction) •NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction) •NP physically interacts with actinin-4 by two hybrid (1, 2) •NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction)