Influenza D virus M2 protein exhibits ion channel activity in Xenopus laevis oocytes

Evan Kesinger, Jianing Liu, Aaron Jensen, Catherine P. Chia, Andrew Demers, Hideaki Moriyama

Research output: Contribution to journalArticle

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Abstract

Background A new type of influenza virus, known as type D, has recently been identified in cattle and pigs. Influenza D virus infection in cattle is typically asymptomatic; however, its infection in swine can result in clinical disease. Swine can also be infected with all other types of influenza viruses, namely A, B, and C. Consequently, swine can serve as a “mixing vessel” for highly pathogenic influenza viruses, including those with zoonotic potential. Currently, the only antiviral drug available targets influenza M2 protein ion channel is not completely effective. Thus, it is necessary to develop an M2 ion channel blocker capable of suppressing the induction of resistance to the genetic shift. To provide a basis for developing novel ion channel-blocking compounds, we investigated the properties of influenza D virus M2 protein (DM2) as a drug target. Results To test the ion channel activity of DM2, the DNA corresponding to DM2 with cMyc-tag conjugated to its carboxyl end was cloned into the shuttle vector pNCB1. The mRNA of the DM2–cMyc gene was synthesized and injected into Xenopus oocytes. The translation products of DM2–cMyc mRNA were confirmed by immunofluorescence and mass spectrometry analy-ses. The DM2–cMyc mRNA-injected oocytes were subjected to the two-electrode voltage-clamp (TEVC) method, and the induced inward current was observed. The midpoint (V mid ) values in Boltzmann modeling for oocytes injected with DM2–cMyc RNA or a buffer were −152 and −200 mV, respectively. Assuming the same expression level in the Xenopus oocytes, DM2 without tag and influenza C virus M2 protein (CM2) were subjected to the TEVC method. DM2 exhibited ion channel activity under the condition that CM2 ion channel activity was reproduced. The gating voltages represented by V mid for CM2 and DM2 were – 141 and –146 mV, respectively. The reversal potentials observed in ND96 for CM2 and DM2 were −21 and −22 mV, respectively. Compared with intact DM2, DM2 variants with mutation in the YxxxK motif, namely Y72A and K76A DM2, showed lower V mid values while showing no change in reversal potential. Conclusion The M2 protein from newly isolated influenza D virus showed ion channel activity similar to that of CM2. The gating voltage was shown to be affected by the YxxxK motif and by the hydrophobicity and bulkiness of the carboxyl end of the molecule.

Original languageEnglish (US)
Article numbere0199227
JournalPloS one
Volume13
Issue number6
DOIs
StatePublished - Jun 2018

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Thogotovirus
Xenopus laevis
ion channels
Ion Channels
Viruses
Oocytes
oocytes
Swine
Proteins
proteins
swine
Influenza A virus
Clamping devices
Xenopus
Electric potential
Orthomyxoviridae
electrodes
Messenger RNA
Influenza C virus
Electrodes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

Cite this

Influenza D virus M2 protein exhibits ion channel activity in Xenopus laevis oocytes. / Kesinger, Evan; Liu, Jianing; Jensen, Aaron; Chia, Catherine P.; Demers, Andrew; Moriyama, Hideaki.

In: PloS one, Vol. 13, No. 6, e0199227, 06.2018.

Research output: Contribution to journalArticle

Kesinger, Evan ; Liu, Jianing ; Jensen, Aaron ; Chia, Catherine P. ; Demers, Andrew ; Moriyama, Hideaki. / Influenza D virus M2 protein exhibits ion channel activity in Xenopus laevis oocytes. In: PloS one. 2018 ; Vol. 13, No. 6.
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abstract = "Background A new type of influenza virus, known as type D, has recently been identified in cattle and pigs. Influenza D virus infection in cattle is typically asymptomatic; however, its infection in swine can result in clinical disease. Swine can also be infected with all other types of influenza viruses, namely A, B, and C. Consequently, swine can serve as a “mixing vessel” for highly pathogenic influenza viruses, including those with zoonotic potential. Currently, the only antiviral drug available targets influenza M2 protein ion channel is not completely effective. Thus, it is necessary to develop an M2 ion channel blocker capable of suppressing the induction of resistance to the genetic shift. To provide a basis for developing novel ion channel-blocking compounds, we investigated the properties of influenza D virus M2 protein (DM2) as a drug target. Results To test the ion channel activity of DM2, the DNA corresponding to DM2 with cMyc-tag conjugated to its carboxyl end was cloned into the shuttle vector pNCB1. The mRNA of the DM2–cMyc gene was synthesized and injected into Xenopus oocytes. The translation products of DM2–cMyc mRNA were confirmed by immunofluorescence and mass spectrometry analy-ses. The DM2–cMyc mRNA-injected oocytes were subjected to the two-electrode voltage-clamp (TEVC) method, and the induced inward current was observed. The midpoint (V mid ) values in Boltzmann modeling for oocytes injected with DM2–cMyc RNA or a buffer were −152 and −200 mV, respectively. Assuming the same expression level in the Xenopus oocytes, DM2 without tag and influenza C virus M2 protein (CM2) were subjected to the TEVC method. DM2 exhibited ion channel activity under the condition that CM2 ion channel activity was reproduced. The gating voltages represented by V mid for CM2 and DM2 were – 141 and –146 mV, respectively. The reversal potentials observed in ND96 for CM2 and DM2 were −21 and −22 mV, respectively. Compared with intact DM2, DM2 variants with mutation in the YxxxK motif, namely Y72A and K76A DM2, showed lower V mid values while showing no change in reversal potential. Conclusion The M2 protein from newly isolated influenza D virus showed ion channel activity similar to that of CM2. The gating voltage was shown to be affected by the YxxxK motif and by the hydrophobicity and bulkiness of the carboxyl end of the molecule.",
author = "Evan Kesinger and Jianing Liu and Aaron Jensen and Chia, {Catherine P.} and Andrew Demers and Hideaki Moriyama",
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T1 - Influenza D virus M2 protein exhibits ion channel activity in Xenopus laevis oocytes

AU - Kesinger, Evan

AU - Liu, Jianing

AU - Jensen, Aaron

AU - Chia, Catherine P.

AU - Demers, Andrew

AU - Moriyama, Hideaki

PY - 2018/6

Y1 - 2018/6

N2 - Background A new type of influenza virus, known as type D, has recently been identified in cattle and pigs. Influenza D virus infection in cattle is typically asymptomatic; however, its infection in swine can result in clinical disease. Swine can also be infected with all other types of influenza viruses, namely A, B, and C. Consequently, swine can serve as a “mixing vessel” for highly pathogenic influenza viruses, including those with zoonotic potential. Currently, the only antiviral drug available targets influenza M2 protein ion channel is not completely effective. Thus, it is necessary to develop an M2 ion channel blocker capable of suppressing the induction of resistance to the genetic shift. To provide a basis for developing novel ion channel-blocking compounds, we investigated the properties of influenza D virus M2 protein (DM2) as a drug target. Results To test the ion channel activity of DM2, the DNA corresponding to DM2 with cMyc-tag conjugated to its carboxyl end was cloned into the shuttle vector pNCB1. The mRNA of the DM2–cMyc gene was synthesized and injected into Xenopus oocytes. The translation products of DM2–cMyc mRNA were confirmed by immunofluorescence and mass spectrometry analy-ses. The DM2–cMyc mRNA-injected oocytes were subjected to the two-electrode voltage-clamp (TEVC) method, and the induced inward current was observed. The midpoint (V mid ) values in Boltzmann modeling for oocytes injected with DM2–cMyc RNA or a buffer were −152 and −200 mV, respectively. Assuming the same expression level in the Xenopus oocytes, DM2 without tag and influenza C virus M2 protein (CM2) were subjected to the TEVC method. DM2 exhibited ion channel activity under the condition that CM2 ion channel activity was reproduced. The gating voltages represented by V mid for CM2 and DM2 were – 141 and –146 mV, respectively. The reversal potentials observed in ND96 for CM2 and DM2 were −21 and −22 mV, respectively. Compared with intact DM2, DM2 variants with mutation in the YxxxK motif, namely Y72A and K76A DM2, showed lower V mid values while showing no change in reversal potential. Conclusion The M2 protein from newly isolated influenza D virus showed ion channel activity similar to that of CM2. The gating voltage was shown to be affected by the YxxxK motif and by the hydrophobicity and bulkiness of the carboxyl end of the molecule.

AB - Background A new type of influenza virus, known as type D, has recently been identified in cattle and pigs. Influenza D virus infection in cattle is typically asymptomatic; however, its infection in swine can result in clinical disease. Swine can also be infected with all other types of influenza viruses, namely A, B, and C. Consequently, swine can serve as a “mixing vessel” for highly pathogenic influenza viruses, including those with zoonotic potential. Currently, the only antiviral drug available targets influenza M2 protein ion channel is not completely effective. Thus, it is necessary to develop an M2 ion channel blocker capable of suppressing the induction of resistance to the genetic shift. To provide a basis for developing novel ion channel-blocking compounds, we investigated the properties of influenza D virus M2 protein (DM2) as a drug target. Results To test the ion channel activity of DM2, the DNA corresponding to DM2 with cMyc-tag conjugated to its carboxyl end was cloned into the shuttle vector pNCB1. The mRNA of the DM2–cMyc gene was synthesized and injected into Xenopus oocytes. The translation products of DM2–cMyc mRNA were confirmed by immunofluorescence and mass spectrometry analy-ses. The DM2–cMyc mRNA-injected oocytes were subjected to the two-electrode voltage-clamp (TEVC) method, and the induced inward current was observed. The midpoint (V mid ) values in Boltzmann modeling for oocytes injected with DM2–cMyc RNA or a buffer were −152 and −200 mV, respectively. Assuming the same expression level in the Xenopus oocytes, DM2 without tag and influenza C virus M2 protein (CM2) were subjected to the TEVC method. DM2 exhibited ion channel activity under the condition that CM2 ion channel activity was reproduced. The gating voltages represented by V mid for CM2 and DM2 were – 141 and –146 mV, respectively. The reversal potentials observed in ND96 for CM2 and DM2 were −21 and −22 mV, respectively. Compared with intact DM2, DM2 variants with mutation in the YxxxK motif, namely Y72A and K76A DM2, showed lower V mid values while showing no change in reversal potential. Conclusion The M2 protein from newly isolated influenza D virus showed ion channel activity similar to that of CM2. The gating voltage was shown to be affected by the YxxxK motif and by the hydrophobicity and bulkiness of the carboxyl end of the molecule.

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