Poliovirus‐induced intracellular alkalinization involves a proton ATPase and protein phosphorylation

Abstract

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.

Intracellular Vesicle Acidification Promotes Maturation of Infectious Poliovirus Particles

“…We think these explanations are unlikely due to the important role autophagosomes play in virus production and the observed presence of virus particles inside autophagosomes [2]. A second possibility is that autophagosomes act as “sponges” for ions, increasing the pH of the cytosol to promote virion maturation [67]. We believe this is unlikely because PV is an enteric virus, capable of remaining infectious in the low pH environment of the human gut, and should not be adversely affected by low pH.…”

Human immunodeficiency virus infection of T-lymphoblastoid cells reduces intracellular pH

“…Reduction in pH i potentially could be important in several aspects of the HIV-1 replication cycle. In other viral systems, changes in pH of various cellular compartments trigger conformational changes in virion proteins during uncoating and/or penetration, permit effective virion assembly, budding and maturation, or alter expression or processing of viral proteins (5,7,10,31,33,41,43). The M 2 protein of influenza virus and the E2 protein of Semliki Forest virus are both thought to form functional ion channels that can transport protons and other ions (43,47).…”

Tyrosine phosphorylation events during coxsackievirus B3 replication

“…Holsey et al have reported that poliovirus infection of HeLa cells led to an increase in intracellular pH which promoted viral replication (16). Protein phosphorylation inhibitors of PKC inhibited virus-specific alkalinization, suggesting that PKC-mediated phosphorylation on serine and/or threonine residues activates the mechanism responsible for the increase in intracellular pH in poliovirus-infected cells (15). A further signal transduction system has also been implicated to be important during poliovirus infection of host cells.…”