Influenza virus m2 ion channel protein is necessary for filamentous virion formation

Abstract

Influenza A virus buds from cells as spherical (approximately 100-nm diameter) and filamentous (approximately 100 nm x 2 to 20 microm) virions. Previous work has determined that the matrix protein (M1) confers the ability of the virus to form filaments; however, additional work has suggested that the influenza virus M2 integral membrane protein also plays a role in viral filament formation. In examining the role of the M2 protein in filament formation, we observed that the cytoplasmic tail of M2 contains several sites that are essential for filament formation. Additionally, whereas M2 is a nonraft protein, expression of other viral proteins in the context of influenza virus infection leads to the colocalization of M2 with sites of virus budding and lipid raft domains. We found that an amphipathic helix located within the M2 cytoplasmic tail is able to bind cholesterol, and we speculate that M2 cholesterol binding is essential for both filament formation and the stability of existing viral filaments.

FIG. 1.

Organization of the M2 cytoplasmic tail. (A) M2 cytoplasmic tail, with M2-Helix mutated residues in red (residues 47, 48, 51, 52, and 55), C50 in blue, and M2-Mut1 mutated residues in green (residues 71 to 73). The L/V-X_(1-5)-Y-X_(1-5)-R/K CRAC motif, ΦXXXXΦXXΦ caveolin-1 binding motif (CBD), and YXXΦ endocytosis motif are marked. (B) Helical wheel plot of M2 amphipathic helix, shown as generated at http://rzlab.ucr.edu/scripts/wheel/wheel.cgi. Hydrophilic residues are shown as circles, hydrophobic residues as diamonds, negatively charged residues as triangles, and positively charged residues as pentagons. Hydrophobicity is coded as green-yellow, hydrophilicity is coded as red-yellow, and charged residues are shown in blue. The red line separates the two faces of the helix. (C) MDCK cells were infected at an MOI of 0.001 PFU/cell with influenza virus A/Udorn/72 or mutant virus A/Udorn/72 M2-Helix, and virus yields were quantified at the indicated time points by plaque assay on M2-MDCK cells. Values shown are averages ± standard deviations for three repeats. Error bars are plotted for all points, but for small standard deviation values, the error bars are not always visible beyond the data point. (D) The wt influenza virus and the mutant virus M2-Helix were grown as described for panel C. At 64 h p.i., the medium was harvested and the virions were purified and concentrated to approximately equal protein levels. Virion polypeptides were then analyzed by SDS-PAGE and immunoblotting. The horizontal bar indicates separate blots.

FIG. 2.

The M2 cytoplasmic tail is necessary for influenza virus filament formation. MDCK cells were infected with wt and mutant influenza viruses (A/Udorn/72) at an MOI of 3 PFU/cell. At 18 h p.i., cells were fixed and stained for HA. (A) wt virus. (B) rA/Udorn/72-ΔM2. (C) rA/Udorn/72 M2-Mut1. (D) rA/Udorn/72 M2-F47A. (E) rA/Udorn/72 M2-F48A. (F) rA/Udorn/72 M2-I51A. (G) rA/Udorn/72 M2-Y52A. (H) rA/Udorn/72 M2-F55A. (I) rA/Udorn/72 M2-Helix. (J) wt influenza virus-infected cells were treated with 10 mM MβCD for 1 h before fixation. (K) rA/Udorn/72 M2-C50A. (L) wt influenza virus-infected cells were treated with 10 μM amantadine for the final 16 h of the infection. Bars, 10 μm.

FIG. 3.

M2 localization during virus budding. MDCK cells were infected at an MOI of 3 PFU/cell with wt influenza A/Udorn/72 virus for 18 h, fixed, stained for HA and M2, and shown as a maximal intensity projection. Bars, 10 μm. Arrows indicate M2 foci at the base of budding filamentous virions.

FIG. 4.

M2 associates with sites of budding. MDCK cells were infected at an MOI of 3 PFU/cell with wt influenza A/Udorn/72 virus (A) or A/Udorn/72 M2-Helix virus (B) for 18 h, fixed, and stained for HA and M2. Images are single z sections of infected cells. Bars, 10 μm. (C) Pearson's R correlation coefficients, calculated from z stacks of A/Udorn/72- and A/Udorn/72 M2-Helix-infected cells and depicted as means ± standard deviations, with filaments extending up from the cell excluded from analysis.

FIG. 5.

M2 associates with lipid rafts. (A) MDCK cells constitutively expressing M2 (M2-MDCK cells) or MDCK cells infected at an MOI of 3 PFU/cell with wt influenza A/Udorn/72 virus or rA/Udorn/72 M2-Helix virus were cross-linked with CTX-B and anti-CTX-B, fixed, and stained for CTX-B and M2. Bars, 10 μm. Insets are magnifications of 10-μm-square regions. (B) Quantification of M2-CTX-B colocalization data from panel A. Data are presented as means ± standard deviations for Pearson's R value for a minimum of 50 cells per sample. Asterisks indicate a significant correlation (P < 0.05). (C) Membranes from M2-MDCK cells or MDCK cells infected at an MOI of 1 with influenza A/Udorn/72 virus, rA/Udorn/72 M2-Mut1, or rA/Udorn/72 M2-Helix were extracted with 1% Triton X-100 at 4°C, separated into soluble (S) and insoluble (I) fractions, and analyzed by Western blotting for M2. Values indicate averages ± standard deviations for the percentage of insoluble M2/total M2 calculated for a minimum of three repeats. Vertical bars indicate separate blots.

FIG. 6.

M2 binds cholesterol. M2 was immunoprecipitated from mock-infected MDCK cells, M2-MDCK cells, and MDCK cells infected at an MOI of 1 PFU/cell with influenza A/Udorn/72 virus or rA/Udorn/72 M2-Helix for 18 h, and the associated cholesterol was quantified. For M2-containing samples, data were normalized by dividing the level measured by the amount of M2 immunoprecipitated in each reaction, as determined by Western blotting of the immunoprecipitated sample followed by quantification of band intensity. Data are depicted as means ± standard deviations, in arbitrary fluorescence units, for six samples. Statistical differences were determined by Student's t test. *, P < 0.001 compared to M2-MDCK; #, P < 0.001 compared to Udorn.

FIG. 7.

Cholesterol is important for filament stability. Supernatant from an influenza A/Udorn/72 virus infection was incubated with PBS (A) or 10 mM MβCD (B) for 1 h at 37°C and analyzed by electron microscopy. Bars, 100 nm.

FIG. 8.

M2 perturbation disrupts filament formation and stability. (a) MDCK cells were infected with influenza A/Udorn/72 virus at an MOI of 3 PFU/cell. At 18 h p.i., cells were washed with PBS, treated with DMEM (control), 15 μg/ml of MAb 5C4, or 15 μg/ml of MAb 14C2 for 1 h at 37°C, fixed, and stained for HA. Bars, 10 μm. (b) MDCK cells were infected with influenza A/Udorn/72 virus at an MOI of 0.001 PFU/cell, and at 48 h p.i., the medium was harvested. Virions were treated with DMEM (control), 15 μg/ml of MAb 5C4, or 15 μg/ml of MAb 14C2 for 1 h at 37°C before being processed for analysis by electron microscopy. (c) Additional fields of view of MAb 14C2-treated virions described above. Bars, 100 nm.

FIG. 9.

MAb 14C2 does not affect M2 ion channel activity. Membrane proton currents (downward deflection) were measured from oocytes expressing the wt M2 ion channel, bathed in neutral (blue bars) and low-pH (red bars) media in the absence of MAb 14C2 (first 2 min) to determine the expression in these particular oocytes. MAb 14C2 was then applied manually to the recording chamber to conserve antibody; manual application resulted in the baseline disturbance from 2 to ca. 4 min. Membrane proton currents were measured again to compare with the currents in the absence of antibody (second red bar, low pH; third blue bar, high pH). The trace shows one representative current recording from a total of 8 recordings.

FIG. 10.

MAb 14C2 does not alter the infectivity of filamentous virions. Influenza virions grown as described in the legend to Fig. 8 were treated with DMEM, 15 μg/ml of MAb 14C2, or DMEM buffered to pH 5.5 for 1 h at 37°C before titration in MDCK cells. Data indicate means ± standard deviations for three experiments. Statistical differences were determined by Student's t test. *, P < 0.05 compared to Udorn. No statistical difference was seen between Udorn and MAb 14C2-treated Udorn (P = 0.49).