Distinct domains of the influenza a virus M2 protein cytoplasmic tail mediate binding to the M1 protein and facilitate infectious virus production

Abstract

The cytoplasmic tail of the influenza A virus M2 protein is highly conserved among influenza A virus isolates. The cytoplasmic tail appears to be dispensable with respect to the ion channel activity associated with the protein but important for virus morphology and the production of infectious virus particles. Using reverse genetics and transcomplementation assays, we demonstrate that the M2 protein cytoplasmic tail is a crucial mediator of infectious virus production. Truncations of the M2 cytoplasmic tail result in a drastic decrease in infectious virus titers, a reduction in the amount of packaged viral RNA, a decrease in budding events, and a reduction in budding efficiency. The M1 protein binds to the M2 cytoplasmic tail, but the M1 binding site is distinct from the sequences that affect infectious virus particle formation. Influenza A virus strains A/Udorn/72 and A/WSN/33 differ in their requirements for M2 cytoplasmic tail sequences, and this requirement maps to the M1 protein. We conclude that the M2 protein is required for the formation of infectious virus particles, implicating the protein as important for influenza A virus assembly in addition to its well-documented role during virus entry and uncoating.

FIG. 1.

Stable expression of cytoplasmic tail-deleted and mutated forms of the M2 protein in cell lines and recombinant viruses. (A) Schematic depicting the truncated and alanine-substituted M2 proteins. Flow cytometry was used to quantify the percentage of cells expressing the M2 proteins at the cell surface. The relative intensity represents the mean channel fluorescence of cells incubated with an M2-specific monoclonal antibody that recognizes the M2 extracellular domain divided by the mean channel fluorescence of cells incubated with secondary antibody alone. A cell line expressing the M2Stop protein was not generated; the schematic depicts the protein encoded by the M2Stop recombinant viruses. NA, not applicable. (B) MDCK cells were infected at an MOI of 1.0, lysed at 18 h postinfection, and analyzed by Western blotting with monoclonal antibodies against the M1 and M2 proteins.

FIG. 2.

Truncation in the M2 protein cytoplasmic tail can reduce infectious virus production. MDCK cells stably expressing the indicated proteins were used to determine the TCID₅₀/ml of rWSN M2Stop (A), rUd M2Stop (B), and rWSN M1Ud M2Stop (C). (D and E) A multistep growth curve was performed on MDCK-M2Stop82 cells with rWSN and rWSN M2Stop (D) or rUd and rUd M2Stop (E). Infectious virus titers were determined on MDCK-M2 cells.

FIG. 3.

Production of filamentous influenza virus particles is dependent on the M2 cytoplasmic tail. MDCK-M2 (A and C) and MDCK-M2Stop70 (B and D) cells were infected at an MOI of 1.0, fixed at 12 h postinfection, and stained with antibodies specific for HA. Cells were infected with rWSN M1Ud M2Stop (A and B) or rUd M2Stop (C and D).

FIG. 4.

Thin-section transmission electron microscopy images of budding influenza particles. MDCK-M2 (A, B, C, G, and H) and MDCK-M2Stop70 (D, E, F, I, J, and K) cells were infected at an MOI of 5.0 with rWSN M2Stop or rWSN M1Ud M2Stop. The cells were infected for 12 h and then processed and imaged by TEM. MDCK-M2 cells were infected with rWSN M1Ud M2Stop (A and B) or rWSN M2Stop (G and H). MDCK-M2Stop70 cells were infected with rWSN M1Ud M2Stop (D and E) or rWSN M2Stop (I to K). →, elongated filamentous particles; ➞, linear spherical particles; ⇒, budding linear spherical particles; *, budding particles. Bars, 200 nm. (C and F) Mock-infected cells. The text in the figures indicates the virus (top line) and cell line (bottom line) used in that panel. Two panels for each condition are shown to demonstrate infected cells from independent experiments and multiple fields.

FIG. 5.

Effect of alanine substitution at M2 residues 82 to 89 on infectious virus production. MDCK cells stably expressing the indicated proteins were used to determine the TCID₅₀/ml of rUd M2Stop (A) and rWSN M1Ud M2Stop (B).

FIG. 6.

Effect of alanine substitution at M2 residues 70 to 81 on infectious virus production. MDCK cells stably expressing the indicated proteins were used to determine the TCID₅₀/ml of rWSN M2Stop (A), rWSN M1Ud M2Stop (B), and rUd M2Stop (C). A multistep growth curve was performed on MDCK-M2Stop90ala70-73 (D) and MDCK-M2Stop90ala74-77 (E) with rWSN and rWSN M2Stop.

FIG. 7.

Expression and incorporation of viral proteins and viral RNA into virions. The indicated cells were infected at an MOI of 5 with rWSN M2Stop (A and B) or an MOI of 1 with rUd M2Stop (C and D). At 12 h postinfection, the medium was collected and viral particles were purified by ultracentrifugation through a 20% sucrose cushion. An infected cell lysate was prepared by adding 1% SDS to the cells. (A and C) The protein composition of the virions and cells was analyzed by Western blotting with antibodies against HA, NP, and M1. (B and D) The viral RNA was extracted from purified particles, and the M segment was quantified by quantitative RT-PCR.

FIG. 8.

Ability of an M2-M1 fusion protein to support infectious virus production. MDCK cells stably expressing the indicated proteins were used to determine TCID₅₀/ml of rWSN M2Stop (A) and rUd M2Stop (B).

FIG. 9.

Interaction of M1 with the cytoplasmic tail of M2. A cell lysate from rWSN-infected MDCK cells metabolically labeled with [³⁵S]methionine and cysteine was incubated with the indicated GST fusion proteins. The bound M1 protein was resolved by SDS-PAGE and imaged using a phosphorimager. A Coomassie-stained gel (bottom panel) shows the amount of purified GST fusion protein used for each pull-down assay.