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

Date Published: June 21, 2018

Publisher: Public Library of Science

Author(s): Evan Kesinger, Jianing Liu, Aaron Jensen, Catherine P. Chia, Andrew Demers, Hideaki Moriyama, Claude Prigent.


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.

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 analyses. 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 (Vmid) 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 Vmid 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 Vmid values while showing no change in reversal potential.

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.

Partial Text

Influenza virus can infect various animal species, including humans [1, 2]. Although most influenza virus infections result in mild disease, genetic shift, drift, and reassortment events have been shown to result in highly pathogenic strains [3]. To date, four influenza virus species have been identified, namely A, B, C, and D [2, 4]. Type A infects several species, including humans as well as porcine, bovine, and canine species [5]. Types B and C infect humans and pigs [6]. Type D is a relatively newly identified type of influenza virus, which has been found to infect cattle and pigs [4]; it was recognized as a new virus type by the International Committee of Taxonomy in 2016 (

The gating activity of M2 proteins involves amino acid side chains, namely through cation—pi interactions [41]. In the channel activation of AM2, lowering the pH gradually opens the Trp41 gate first, followed by a decrease in the deprotonation barrier of the His37 tetrad [21]. Trp41Ala substitution in AM2 was reported to result in the complete loss of ion channel activity [42]. In the case of CM2, which lacks Trp41 in AM2, Hongo et al. [25] reported a modest acid activation. A change of pH from 8.5 to 5.5 increases the relative current by approximately 1.3-fold in CM2. In DM2, we recorded the induced current at pH 8.5, 6.5, and 5.5. Nevertheless, there weren’t significant change in the induced current.

This research was performed under the supervision of the Institutional Biosafety Committee at the University of Nebraska—Lincoln (Protocol Number 174).




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