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.A productive virus infection begins with the release of the viral genome into a permissive host cell. The viral genome can then be replicated, transcribed, and translated. To complete a productive life cycle, a virus must temporarily package its genome into a particle that facilitates the transport and release of the viral genetic material into another permissive cell. For most human viral pathogens, the mechanisms responsible for genome incorporation and virus assembly are yet to be clearly defined.Budding of enveloped viruses requires the action of a viral protein capable of mediating curvature of the lipid bilayer, followed by fission of the new viral membrane from the cellular membrane (43, 61). The Gag protein of human immunodeficiency virus (12,19); the matrix proteins of influenza A virus (20, 31), vesicular stomatitis virus (VSV) (33), and simian virus 5 (63); and the VP40 proteins of Ebola (36,51) and Marburg viruses (71) have been shown to mediate budding of virus-like particles (VLPs) in the absence of other viral proteins.While matrix proteins alone can drive the process of VLP formation, coexpression of other viral proteins can improve the efficiency of VLP formation. In the case of Ebola and Marburg viruses, coexpression of the viral glycoprotein with VP40 enhances the efficiency of VLP formation over that observed with VP40 alone (3,35,71). The extracellular, membrane-proximal region of the VSV G protein has been shown to increase the budding efficiency of VSV (58). The budding of a fully infectious viral particle requires the coordinated action of multiple viral proteins with the lipid bilayer, viral genome, and most likely host proteins. This concerted event increases the efficiency of particle release over that demonstrated in most VLP systems (35,58).Influenza A viruses are classified...

Improvement of H5N1 influenza vaccine viruses: Influence of internal gene segments of avian and human origin on production and hemagglutinin content

“…This interaction is thought to be essential for assembly and budding of virus particles [22]. Moreover, the cytoplasmic tail of the M2 protein was found to be involved in the production of new virus particles and the efficient packaging of virus genomes [24] and [25]. As the PR8-M proteins may not interact optimally with the HA and NA proteins of H5N1 strains we hypothesized that M segments from other strains could influence the growth and possibly antigen yield of H5N1 influenza vaccine viruses.…”

XFEL structures of the influenza M2 proton channel: Room temperature water networks and insights into proton conduction

“…The regions C-terminal to the TM helices play different important functional roles. An amphiphilic helix formed by residues 46-60 induces membrane curvature and is involved in viral budding and scission (21)(22)(23), and an intrinsically disordered C-terminal tail (residues 62-97) interacts with the matrix 1 protein during the packing and budding of new virus particles (24,25).…”

Structural basis for proton conduction and inhibition by the influenza M2 protein

“…8 Finally, the Cterminal tail of the protein interacts with the matrix protein M1 and is essential for virus packaging and budding. 9 In some strains of the influenza A virus, M2 is also important for equilibrating the pH of the lumen of the Golgi apparatus with the cytoplasm, preventing premature conformational change of the viral hemagglutinin. 10 The M2 protein has been extensively studied because of its medical importance, particularly following the emergence of widespread resistance to M2-blocking drugs amantadine and rimantadine.…”