Influenza A Virus M₂Ion Channel Activity Is Essential for Efficient Replication in Tissue Culture

Abstract

The discovery that the influenza A virus M 2 protein has proton-selective ion channel activity stemmed from an understanding of the life cycle of influenza virus and the two steps in the life cycle that are inhibited by the antiviral drug amantadine (reviewed in reference 14). Direct evidence that the M 2 protein has a low-pH-activated, proton-selective conductance was obtained by expressing the M 2 protein in oocytes of Xenopus laevis (20,21,47,53,64,65,67) or mammalian cells (6,40,41,66). M 2 -specific cell surface currents were measured, and they were found to be specifically blocked by amantadine. Furthermore, when either peptides corresponding to the M 2 transmembrane (TM) domain or purified M 2 protein was incorporated into planar bilayers, an amantadine-sensitive current was measured (11,36,52,63).Amantadine inhibits the early step of uncoating of influenza virus in endosomes (reviewed in references 14, 31, 32, and 34). When a virion has entered the cell by receptor-mediated endocytosis and the virus particle is in the acidic environment of the endosomal lumen, the M 2 ion channel is activated and conducts protons across the viral membrane. The lowered internal virion pH is thought to weaken protein-protein interactions between the viral matrix protein (M 1 ) and the ribonucleoprotein (RNP) core (4,5,38,72,73; reviewed in reference 18

Potassium Ion Channels: Could They Have Evolved from Viruses?

“…Their functional importance in the viral life cycle was further substantiated by experiments in which channelencoding genes were deleted or mutated in a viral genome. Typically, these procedures did not prevent viral replication, but they reduced formation of viral progeny and viral pathogenicity, thus reducing the competitiveness and fitness of the virus (Takeda et al, 2002;Gonzalez and Carrasco, 2003). The conclusion from these experiments is that there was strong selection pressure on the evolution of viral channels.…”

“…After the discovery of virusencoded channel proteins, many of the channels were tested to determine their role(s) in the viral life cycle. In those cases where it has been examined, it turned out that functional viral channels, including the viral K + channels, benefited viral replication (Takeda et al, 2002;Hsu et al, 2004;Greiner et al, 2009). Their functional importance in the viral life cycle was further substantiated by experiments in which channelencoding genes were deleted or mutated in a viral genome.…”

Influenza A virus NS1 protein binds p85β and activates phosphatidylinositol-3-kinase signaling

“…Influenza A virus strains PR8, A͞Udorn͞72, and A͞WSN͞33 were propagated in 10-day-old chicken eggs and titrated by plaque assay in MDCK cells as described (41). WT and mutant influenza A viruses (Ud and WSN genetic backbones) were generated by reverse genetics from cDNAs as described (41,42). Tyrosine 89 in the NS1 proteins of both WSN and Ud was changed to phenylalanine by PCR mutagenesis of the relevant NS segment cDNA (pHH21 vector).…”

“…The WT parainfluenza virus-5 strain (PIV-5) and the IFNsensitive strain (CPIϪ) were propagated and titrated in Vero cells. Influenza A virus strains PR8, A͞Udorn͞72, and A͞WSN͞33 were propagated in 10-day-old chicken eggs and titrated by plaque assay in MDCK cells as described (41). WT and mutant influenza A viruses (Ud and WSN genetic backbones) were generated by reverse genetics from cDNAs as described (41,42).…”

Modulation of cell function by small transmembrane proteins modeled on the bovine papillomavirus E5 protein

“…Many viruses encode transmembrane proteins that are required for a variety of functions including transit across the host cell membrane or assembly of essential transmembrane protein complexes such as the M2 ion channel of influenza virus (e.g. Takeda et al, 2002). It may be possible to screen for small transmembrane proteins that can block virus replication by inhibiting the activity of these viral proteins.…”