TY - JOUR
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
N1 - Funding Information:
This work was supported by Hatch Equipment Grants from the University of Nebraska —Lincoln (2013-2014; Blair Siegfried, Etsuko Moriyama, Hideaki Moiryma; bought the Two-electrode voltage clamp tool; The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript), https://ard.unl.edu/funding-programrfa-title/hatch-equipment-grants; Nebraska Corn Board (1759080; PI, Etsuko Moriyama; Hideaki Moriyama Co-PI; partly support supplies and partly support for Evan Kesinger; The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript), http://www.nebraskacorn.org; Central States Center for Agricultural Safety and Health (CS-CASH) Pilot Project, College of Public Health, Nebraska Medical Center (Hideaki Moriyama; partly support supplies and publication fee; The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript), https://www.unmc.edu/publichealth/ cscash/; Undergraduate Creative Activities and Research Experience (UCARE) at University of Nebraska-Lincoln (2015-2016; Aaron Jensen; support for Aaron Jensen in part; The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript), https://ucare.unl.edu; Graduate Student Special Funds, School of Biological Sciences, University of Nebraska-Lincoln (2017-2018; Evan Kesinger; support for supply in part; The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript), https://biosci.unl. edu/financial-aid-current-graduate-students. We are very grateful to Drs. Kiran Sapkota and Daniel Monaghan at the University of Nebraska Medical Center, USA, for cross-checking the DM2 ion channel activity. We thank Drs. Terri Fangman and You “Joe” Zhou at Morrison Microscopy Core Research Facility and Drs. Mike Naldrett and Sophie Alvarez at Proteomics & Metabolomics Facility, Center for Biotechnology, University of Nebraska—Lincoln, USA (UNL), for their technical support. We thank Drs. Eric Weaver, Biological Sciences (SBS), UNL; Feng Li at South Dakota State University, USA, for interesting discussions. We are also indebted to Eloy De La O, Kari Heck, Angelica Kallenberg, Car Reen Kok, Remy Sass, McKayla Ann Wieczorek, Karen Goeschel, and Kamryn Koziol from UNL for sharing their research experiences. Finally, we greatly appreciate the help of Drs. Etsuko Moriyama, SBS, UNL; Blair D. Siegfried, Entomology and Nematology Department, University of Florida; and Ana Maria Velez Arango, Department of Entomology, UNL, for their collaboration and support.
Publisher Copyright:
© 2018 Kesinger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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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U2 - 10.1371/journal.pone.0199227
DO - 10.1371/journal.pone.0199227
M3 - Article
C2 - 29927982
AN - SCOPUS:85048899040
VL - 13
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 6
M1 - e0199227
ER -