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.
Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Henju
H
Marjuki
Alex
A
Gornitzky
Bindumadhav M
BM
Marathe
Natalia A
NA
Ilyushina
Jerry R
JR
Aldridge
Gururao
G
Desai
Richard J
RJ
Webby
Robert G
RG
Webster
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization; D048490:raf Kinases / Q000378:metabolism
D020033:Protein Isoforms; D021122:Protein Subunits; D034741:RNA, Small Interfering; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D048490:raf Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases; D025262:Vacuolar Proton-Translocating ATPases