The influenza A virus M2 cytoplasmic tail is required for infectious virus production and efficient genome packaging

Abstract

The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.

FIG. 1.

Effect of M₂ cytoplasmic tail truncations on protein stability and ion channel activity. (A) 293T cells expressing the indicated M₂ protein were metabolically labeled, and cell lysates were immunoprecipitated with an MAb to M₂. The proteins were separated by SDS-PAGE and visualized by using a phosphorimager. (B) 293T cells transfected with pC FPV HA and pC, pC M₂, or pC M₂Stop70 were metabolically labeled, cell lysates were generated at the indicated times, and immunoprecipitations were performed with polyclonal sera against FPV HA. The proteins were separated by SDS-PAGE and visualized with a phosphorimager. The amount of labeled HA2, for each sample, after a 120-min chase was normalized to the pC sample without amantadine.

FIG. 2.

Effect of M₂ cytoplasmic tail truncations on influenza A virus replication. (A) MDCK-M₂ cells were analyzed by flow cytometry for expression of the M₂ protein. The thin line represents cells stained with only secondary antibody, and the thick line represents cells stained with a primary antibody against M₂ and a fluorescein isothiocyanate-conjugated secondary antibody. (B) A plaque assay on the virus rWSN M-Ud M₂Stop70 was performed with MDCK and MDCK-M₂ cells. (C) A multistep growth curve was performed on MDCK and MDCK-M₂ cells with the viruses rWSN M-Ud, rWSN M-Ud M₂Stop90, and rWSN M-Ud M₂Stop70.

FIG. 3.

The M₂Stop70 protein is unable to support influenza A virus replication. (A) 293T cells were infected at an MOI of 1.0 with the indicated virus and metabolically labeled, and cell lysates were immunoprecipitated with an MAb to M₂. The proteins were separated by SDS-PAGE and visualized with a phosphorimager. (B and C) 293T (B) or MDCK (C) cells were infected at an MOI of 1.0 with the indicated virus. At 3, 6, 9, and 12 h postinfection the cells were lysed, and the polypeptides were separated by SDS-PAGE and analyzed by Western blotting for the M₁ and M₂ proteins. (D) MDCK-M₂Stop70 cells were analyzed by flow cytometry for expression of the M₂ protein. The thin line represents cells stained with only secondary antibody, and the thick line represents cells stained with a primary antibody against M₂ and a fluorescein isothiocyanate-conjugated secondary antibody. (E) A multistep growth curve was performed on MDCK and MDCK-M₂Stop70 cells with rWSN M-Ud and rWSN M-Ud M₂Stop70.

FIG. 4.

The effect of M₂ protein trans-complementation on rWSN M-Udorn M₂Stop70 virus morphology and polypeptide composition. (A) Purified virions were negatively stained with uranyl acetate and examined with a JEOL 1200EX transmission electron microscope. Scale bar, 100 nm. (B and C) Purified virions from MDCK, MDCK-M₂, and MDCK-M₂Stop70 infected cells were analyzed by Western blotting. In panel B an MAb to M₂ was used, and in panel C antibodies to HA, NP, and M₁ were used. The band migrating slightly faster than full-length M₂ is a proteolytic fragment of M₂.

FIG. 5.

RNA incorporation into rWSN M-Ud and rWSN M-Ud M₂Stop70 virions. RNA was extracted from the indicated virions, and a semiquantitative RT-PCR was performed with primers specific for the NS segment (A), the M segment (B), and the NP segment (C). (D) RNA was extracted from the indicated virions, reverse transcribed with primers specific for the M segment, and quantitated by real-time PCR.

FIG. 6.

Characterization of MDCK cells with inducible M₂ expression. MDCK-Tet M₂ cells were incubated in the absence or presence of increasing concentrations of doxycycline for 48 h. At 48 h the cells were lysed, the polypeptides were separated by SDS-PAGE and analyzed by Western blotting with an antibody to the M₂ protein.

FIG. 7.

Analysis of rWSN M-Ud M₂Stop70 Revertant. (A) Western blot of virus-infected cell lysates with MAbs to M₂ and M₁. (B) A multistep growth curve was performed on MDCK cells infected with rWSN M-Ud, rWSN M-Ud M₂Stop70, or rWSN M-Ud M₂Stop70 revertant.