Influenza A virus‐induced early activation of ERK and PI3K mediates V‐ATPase‐dependent intracellular pH change required for fusion

Summary The vacuolar (H+)‐ATPases (V‐ATPases) facilitate the release of influenza A virus (IAV) genome into the cytoplasm by acidifying the endosomal interior. The regulation of V‐ATPases by signalling pathways has been demonstrated in various model systems. However, little is known about signalling‐regulated V‐ATPase activation during IAV infection. Here we show that V‐ATPase activity is elevated during infection of cell monolayers with IAV, as measured by intracellular pH change, via a mechanism mediated by extracellular signal‐regulated kinase (ERK) and phosphatidylinositol 3‐kinase (PI3K). Inhibition of IAV‐induced early activation of these kinases reduced V‐ATPase activity and the acidification of intracellular compartments in infected cells. IAV‐activated ERK and PI3K appear to interact directly, and they colocalize with the E subunit of V‐ATPase V1 domain. Further, siRNAs targeting the E2 subunit isoform significantly reduced virus titres. Interestingly, suppression of PI3K early activation, but not that of ERK or V‐ATPase, negatively affected virus internalization, suggesting the involvement of the pathway in earlier, V‐ATPase‐independent infection‐promoting events. Cell treatment with a V‐ATPase‐specific inhibitor impaired the nuclear localization of incoming viral ribonucleoproteins, inhibiting replication/transcription of viral RNAs. These findings highlight the importance of IAV‐induced ERK and PI3K early activation as signalling mediators in V‐ATPase‐stimulated endosomal acidification required for fusion.