Poliovirus-induced intracellular alkalinization involves a proton ATPase and protein phosphorylation.
We reported previously that poliovirus infection induces alkalinization in HeLa cells and that an alkaline intracellular pH (pHi) promoted viral replication. Additional experiments were carried out to understand the underlying mechanism. Virus-infected or control monolayer cultures were incubated with nominally bicarbonate-free Eagle's minimal essential medium (MEM) buffered with N-2-hydroxyethylpiperazine-N-3-ethanesulfonic acid (HEPES), and immediately following preincubations, changes in pHi were monitored via benzoic acid uptake around 2 h postinfection. The absence of pH increase in cells infected with ultraviolet light-inactivated virus (UV-virus) indicated that viral gene expression was required for this effect. On the other hand, lack of effect of 3 mM guanidine, an inhibitor of poliovirus-specific RNA but not protein synthesis, suggested that translation of input viral genome RNA is sufficient for the pH increase. Activation of Na+/H+ exchange, Cl(-)HCO3- exchange, or H(+)-ATPase was considered as possible mechanisms by which alkalinization occurs in virus-infected cells. Na+/H+ exchange was excluded because the pH effect occurred in a Na+/H+ exchange deficient HeLa cell mutant. Similarly, Cl-/HCO3- exchange was excluded because virus-specific alkalinization was evident in the presence of Cl- or bicarbonate deficient medium and was not associated with an increase in HCO3- uptake or a decrease in Cl- uptake. Lack of dependence on Na+, abrogation by 10 microM 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), and resistance to 1 mM vandate suggested that this effect was due to the activation of a vacuolar-type (V) proton ATPase. Studies using protein kinase inhibitors indicated that activation of the ATPase in virus-infected cells probably involved protein kinase C-mediated phosphorylation.
Holsey
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Department of Immunology and Microbiology, Medical College of Georgia, Augusta 30912-2400.
Nair
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D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D001565:Benzoates / Q000378:metabolism; D019817:Benzoic Acid; D001639:Bicarbonates / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D015870:Gene Expression; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017955:Poliovirus / Q000235:genetics / Q000502:physiology*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D001565:Benzoates; D001639:Bicarbonates; D002712:Chlorides; D010750:Phosphoproteins; D014638:Vanadates; D006859:Hydrogen; D019817:Benzoic Acid; D012964:Sodium; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan