We have investigated the effect of a monoclonal antibody on influenza virus release and the cell surface expression of M2, comparing virus strains which were observed previously to be sensitive (A/Udorn) or resistant (A/WSN and A/Udorn variants) to growth inhibition by M2 antibody 14C2. Incubation of A/Udorn virus-infected cells in the presence of the inhibitory M2 antibody resulted in a significant reduction in the yield of virus, as measured by infectivity assays as well as by the release of purified virions. The release of A/Udorn virus was not inhibited by the presence of monovalent 14C2 Fab, in contrast to IgG, indicating that a bivalent structure is essential for 14C2 antibody-mediated viral growth restriction. The level of M2 surface expression in A/Udorn virus-infected MDCK cells was found to be reduced to approximately 60% of control levels in cells incubated with the 14C2 antibody. In contrast, M2 surface expression levels in A/WSN virus-infected cells were decreased by only approximately 5-15%, and A/WSN virus assembly appeared to be unaffected by the M2 antibody treatment. M2 antigen associated with cell membranes and virus particles was redistributed into clusters after M2 antibody treatment in infected cells. Incubation in the presence of the 14C2 antibody also reduced M2 surface expression by approximately 40-50% in cells infected with a recombinant vaccinia virus that expresses the M2 A/Udorn protein. These results demonstrate that M2 antibody reduces the level of influenza virus particle formation in a single cycle of infection and suggest that inhibition of A/Udorn virus replication by the 14C2 antibody is related to the reduced cell surface expression and redistribution of the M2 protein induced by the antibody treatment.

The M2 protein of influenza A virus is a small, nonglycosylated transmembrane protein that is expressed on surfaces of virus-infected cells. A monoclonal antibody specific for the M2 protein was used to investigate its expression in polarized epithelial cells infected with influenza virus or a recombinant vaccinia virus that expresses M2. The expression of M2 on the surfaces of influenza virus-infected cells was found to be restricted to the apical surface, closely paralleling that of the influenza virus hemagglutinin (HA). Membrane domain-specific immunoprecipitation indicated that the M2 protein was inserted directly into the apical membrane with transport kinetics similar to those of HA. In polarized cells infected with a recombinant vaccinia virus that expresses M2, we found that 86 to 93% of surface M2 was restricted to the apical domain compared with 88 to 90% of HA in a similar assay. These results indicate that the M2 protein undergoes directional transport in the absence of other influenza virus proteins and that M2 contains the structural features required for apical transport in polarized epithelial cells. The ultrastructural localization of the M2 protein in influenza virus-infected MDCK cells was investigated by immunoelectron microscopy using M2 antibody and a gold conjugate. In cells in which extensive virus budding was occurring, the apical cell membrane was labeled with gold particles evenly distributed between microvilli and the surrounding membrane. In addition, a significant fraction of the M2 label was apparently associated with virions. A monoclonal antibody specific for HA demonstrated a similar labeling pattern. These results indicate that M2 is localized in close proximity to budding and assembled virions.