The active oligomeric state of the minimalistic influenza virus M2 ion channel is a tetramer

Abstract

The influenza A virus M2 integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes and also modulates the pH of the trans-Golgi network in virus-infected cells. The M2 protein is a homo-oligomer of 97 residues, and analysis by chemical cross-linking and SDS/PAGE indicates M2 forms a tetramer. However, a higher order molecular form is sometimes observed and, thus, it is necessary to determine the active form of the molecule. This was done by studying the currents of oocytes that expressed mixtures of the wild-type M2 protein (epitope tagged) and the mutant protein M2-V27S, which is resistant to the inhibitor amantadine. The composition of mixed oligomers of the two proteins expressed at the plasma membrane of individual oocytes was quantified after antibody capture of the cell surface expressed molecules and it was found that the subunits mixed freely. When the ratio of wild-type to mutant protein subunits was 0. 85:0.15, the amantadine sensitivity was reduced to 50% and for a ratio of 0.71:0.29 to 20%. These results are consistent with the amantadine-resistant mutant being dominant and the oligomeric state being a tetramer.

Figure 1

M₂tag and M₂-V₂₇S are similarly activated by low pH and yield similar whole cell surface currents, but their amantadine sensitivities differ. Whole cell membrane currents of oocytes expressing M₂tag (open bars) or M₂-V₂₇S (hatched bars) were measured in Barth’s solution at pH 7.5, after 30 s of incubation in Barth’s solution at pH 6.2, and again after 2 min of incubation at pH 6.2 with 100 μM amantadine. Currents are plotted as a multiple of current at pH 7.5. Values are given as mean ± SEM, n ≥ 5. Note that M₂tag was fully sensitive to amantadine and that M₂-V₂₇S was fully resistant to amantadine.

Figure 2

M₂tag and M₂-V₂₇S freely form-mixed oligomers. HeLa-T4 cells were infected with vaccinia virus vTF7.3 for 30 min and then transfected with various amount (0–2.5 μg) of pTM3-M₂tag or pTM3-M₂-V₂₇S DNA, with the total amount of DNA adjusted to 5 μg per dish by adding pTM3 vector DNA. At 3 h posttransfection cells were labeled with [³⁵S]-Pro-mix (100 μCi/ml) for 30 min (A and B). RIPA buffer lysates were prepared in the presence of 50 mM iodoacetamide and immunoprecipitated with M₂-specific mAb 14C2. Polypeptides were analyzed on SDS/PAGE under (A) reducing or (B) nonreducing conditions. In A, the amount of M₂tag as a percentage of total M₂ species was: lane 1, 0%; lane 2, 15%; lane 3, 35%; lane 4, 50%; lane 5, 72%; lane 6, 87%, and lane 7, 100%. (C) Cells were lysed in 1% Nonidet P-40, 50 mM Tris⋅HCl (pH 8.0), 100 mM NaCl, and 50 mM iodoacetamide and treated with dithiobis(succinimidyl proprionate) prior to immunoprecipitation with mAb 14C2 and analysis by SDS/PAGE under nonreducing conditions. D, disulfide-linked dimers of M₂; T, disulfide-linked tetramers of M₂; M, multimer of M₂.

Figure 3

Cell surface expression of M₂tag and M₂-V₂₇S oligomers in oocytes of X. laevis. Synthetic mRNAs encoding M₂tag, M₂-V₂₇S, or a mixture of the two RNAs and using water as a control were microinjected (50 nl of RNA of 0.85 μg/μl for M₂tag, 50 nl of 0.25 μg/μl for M₂-V₂₇S, and 50 nl of RNA containing 0.75 μg/μl M₂tag mixed with 0.25 μg/μl M₂-V₂₇S) into oocytes of X. laevis. At 24 h after injection, oocytes were labeled with [³⁵S]methionine for 18 h and surface-expressed M₂ protein captured with M₂-specific mAb (14C2). Oocytes were washed, homogenized, and detergent lysates prepared in the presence of 50 mM iodoacetamide as described. Antibody was recovered by adding protein A-Sepharose beads and immune complexes were analyzed by SDS/PAGE under nonreducing conditions.

Figure 4

Principle for determining active M₂ protein oligomers. The cartoon depicts possible oligomers resulting from coexpression of amantadine-sensitive and epitope-tagged M₂ subunits (○) with an amantadine-insensitive (V₂₇S) mutant subunit () assuming that the active oligomer is a tetramer. All possible combinations of the two subunits in a tetrameric channel are shown. As shown in Fig. 2, all five species can be identified.

Figure 5

Amantadine sensitivity of ion channel activities of mixed oligomers of known stoichiometry of M₂tag and M₂-V₂₇S. Oocytes were injected with mRNA encoding M₂tag, M₂-V₂₇S, or mixtures of these two mRNAs. After incubation for 2 days, the membrane currents of oocytes were measured in Barth’s solution at pH 6.2 with or without 20 μM of amantadine. The time course of the fraction of M₂ ion channel current that was inhibited by amantadine, Inh_mix, is plotted after addition of amantadine for M₂tag protein (•), M₂-V₂₇S (♦), and for mixtures of M₂tag protein and M₂-V₂₇S of defined stoichiometry (open symbols). The fraction of M₂tag expressed in each batch of cells is given by the number above the curves and was measured by metabolically labeling a subset of the oocytes as described in Fig. 3.