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

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.