https://www.cell.com/cell/pdf/0092-8674(92)90452-I.pdf

Influenza virus M2 protein has ion channel activity.

Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface

The influenza A virus M2 protein is an integral membrane protein of 97 amino acids that is expressed at the surface of infected cells with an extracellular N-terminal domain of 18 to 23 amino acid residues, an internal hydrophobic domain of approximately 19 residues, and a C-terminal cytoplasmic domain of 54 residues. To gain an understanding of the M2 protein function in the influenza virus replicative pathway, we produced and characterized a monoclonal antibody to M2. The antibody-binding site was located to the extracellular N terminus of M2 as shown by the loss of recognition after proteolysis at the infected-cell surface, which removes 18 N-terminal residues, and by the finding that the antibody recognizes M2 in cell surface fluorescence. The epitope was further defined to involve residues 11 and 14 by comparing the predicted amino acid sequences of M2 from several avian and human strains and the ability of the M2 protein to be recognized by the antibody. The M2-specific monoclonal antibody was used in a sensitive immunoblot assay to show that M2 protein could be detected in virion preparations. Quantitation of the amount of M2 associated with virions by two unrelated methods indicated that in the virion preparations used there are 14 to 68 molecules of M2 per virion. The monoclonal antibody, when included in a plaque assay overlay, considerably showed the growth of some influenza virus strains. This plaque size reduction is a specific effect for the M2 antibody as determined by an analysis of recombinants with defined genome composition and by the observation that competition by an N-terminal peptide prevents the antibody restriction of virus growth.

Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions.

Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex.

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The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins.

Four major acidic polypeptides, with molecular weights of 88, 72, 71, and 23 thousand, and minor polypeptides with molecular weights of 110, 50, 38, and 30 thousand rapidly accumulated in cultured chick embryo (CE) cells which were exposed for three hours to the arginine analogue canavanine. P110, P88, P71,72, and P23 had unique peptide maps. Evidence of a 27,000 dalton precursor to P23 was obtained. The analogue-stimulated proteins were not related to another set of inducible avian polypeptides known as the glucose-regulated proteins. In mammalian cells, the rate of accumulation of several polypeptides, which were similar in size to the avian proteins, sharply increased after canavanine treatment. Proteins with the same electrophoretic mobilities, isoelectric points, and peptide maps as the analogue-stimulated proteins were expressed at low levels in untreated cultures. To determine the time courses of the canavanine-mediated increases in protein accumulation and the recovery of protein metabolism after analogue treatment, radioactively labeled proteins were extracted from CE cells and analyzed on SDS-polyacrylamide gels. In cultures exposed to canavanine, the rates of accumulation of P88 and P71,72 increased from basal to new plateau levels in about 1.5 hours, while P23 required about 2.5 hours. When added with the analogue, actinomycin D and cordycepin blocked the increases in protein accumulation. These inhibitors also blocked the rapid decline in the rates of accumulation of the enhanced proteins which occurred after removal of canavanine. Studies of the matabolic stability of the enhanced proteins indicated that the changes in their accumulation were caused by alterations in their rates of synthesis. Thus, the analogue-mediated response fulfilled several of the criteria for inducible eucaryotic gene expression. The amino acid analogue p-fluorophenylalanine and the chain-terminating analogue of amino acyl-tRNA puromycin stimulated the synthesis of the same set of proteins induced by canavanine. The enhanced synthesis of these proteins appeared to be a cellular response to either the presence or catabolism of abnormal proteins and puromycyl peptides.

Polly R. Etkind

Papers

Evidence for a ninth influenza viral polypeptide

In vitro synthesis of structural and nonstructural proteins of Sendai and SV5 viruses.