Abstract
Influenza A virus and influenza B virus particles both contain small integral membrane proteins (A/M2 and BM2, respectively) that function as a pH-sensitive proton channel and are essential for virus replication. The mechanism of action of the M2 channels is a subject of scientific interest particularly as A/M2 channel was shown to be a target for the action of the antiviral drug amantadine. Unfortunately, an inhibitor of the BM2 channel activity is not known. Thus, knowledge of the structural and functional properties of the BM2 channel is essential for the development of potent antiviral drugs. The characterization of the oligomeric state of the BM2 channel is an essential first step in the understanding of channel function. Here we describe determination of the stoichiometry of the BM2 proton channel by utilizing three different approaches. 1) We demonstrated that BM2 monomers can be chemically cross-linked to yield species consistent with dimers, trimers, and tetramers. 2) We studied electrophysiological and biochemical properties of mixed oligomers consisting of wild-type and mutated BM2 subunits and related these data to predicted binomial distribution models. 3) We used fluorescence resonance energy transfer (FRET) in combination with biochemical measurements to estimate the relationships between BM2 channel subunits expressed in the plasma membrane. Our experimental data are consistent with a tetrameric structure of the BM2 channel. Finally, we demonstrated that BM2 transmembrane domain is responsible for the channel oligomerization.
Highlights
Functional properties, the only homology between their amino acid sequences is found in the HXXXW motif of the membrane-spanning region
As knowledge of the active oligomeric state of a channel is an essential first step in understanding the mechanism of action of a channel, we determined the stoichiometry of the influenza B virus proton channel by utilizing three different approaches
This finding is consistent with our knowledge about the A/M2 oligomeric state [15] and confirms suggestions regarding the BM2 oligomeric state made by Paterson et al [5], the data do not prove that BM2 channels function as tetramers
Summary
Molecular Biology, in Vitro cRNA Transcription, and Transient Expression of Plasmids in HEK293-cultured Cells—The cDNAs encoding influenza virus A/Udorn/72 A/M2 protein, the B/Lee/40 BM2 protein and chimeric cDNAs were inserted into pGEMHJ All other protein samples from HEK293 cells or oocytes which were subject to Western blot analysis were homogenized in the appropriate solubilization buffers described above. Primary antibodies and their respective working dilutions were: 14C2 (anti-A/M2) monoclonal antibody 1:1000 [20], anti-BM2 polyclonal antibody 1:600 [21], anti-FLAG monoclonal antibody 1:2000 (Sigma), anti-Myc polyclonal antibody 1:1000 (Abcam, Cambridge, MA). If we assume that FRET efficiency measured from cells expressing 9 BM2-CFP to 1 FLAG-BM2-YFP protein ratio is mainly contributed by channels with three donors and one acceptor subunit configuration, the total efficiency for this configuration can be presented as Equation 12. The relation between Radj and Rdiag was compared with that expected for a quadratic arrangement of subunits by using the Pythagorean theorem in Equation 14
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