The influenza A virus M2 protein (A/M2) is a homotetrameric pH-activated proton transporter/channel that mediates acidification of the interior of endosomally encapsulated virus. This 97-residue protein has a single transmembrane (TM) helix, which associates to form homotetramers that bind the anti-influenza drug amantadine. However, the minimal fragment required for assembly and proton transport in cellular membranes has not been defined. Therefore, the conductance properties of truncation mutants expressed in Xenopus oocytes were examined. A short fragment spanning residues 21-61, M2(21-61), was inserted into the cytoplasmic membrane and had specific, amantadine-sensitive proton transport activity indistinguishable from that of full-length A/M2; an epitope-tagged version of an even shorter fragment, M2(21-51)-FLAG, had specific activity within a factor of 2 of the full-length protein. Furthermore, synthetic fragments including a peptide spanning residues 22-46 were found to transport protons into liposomes in an amantadine-sensitive manner. In addition, the functionally important His-37 residue pKa values are highly perturbed in the tetrameric form of the protein, a property conserved in the TM peptide and full-length A/M2 in both micelles and bilayers. These data demonstrate that the determinants for folding, drug binding, and proton translocation are packaged in a remarkably small peptide that can now be studied with confidence.liposomes ͉ oocyte ͉ H ϩ ͉ intracellular pH ͉ Xenopus laevis

with a polar hydrogen bond between rimantadine and aspartic acid residue 44 (D44) that appears to be important. These two distinct drug-binding sites led to two incompatible drug inhibition mechanisms. We mutagenized D44 and R45 to alanine as these mutations are likely to interfere with rimantadine binding and lead to a drug insensitive channel. However, the D44A channel was found to be sensitive to amantadine when measured by electrophysiological recordings in oocytes of Xenopus laevis and in mammalian cells, and when the D44 and R45 mutations were introduced into the influenza virus genome. Furthermore, transplanting A/M2 pore residues 24 -36 into BM2, yielded a pHactivated chimeric ion channel that was partially inhibited by amantadine. Thus, taken together our functional data suggest that amantadine/rimantadine binding outside of the channel pore is not the primary site associated with the pharmacological inhibition of the A/M2 ion channel.amantadine inhibition of influenza virus ͉ amantadine resistance ͉ drug-binding site ͉ influenza reverse genetics

The A/M2 proton channel of influenza A virus is a target for the anti-influenza drugs amantadine and rimantadine, whose effectiveness was diminished by the appearance of naturally occurring point mutants in the A/M2 channel pore, among which the most common are S31N, V27A and L26F. We have synthesized and characterized the properties of a series of compounds, originally derived from the A/M2 inhibitor BL-1743. A lead compound emerging from these investigations, spiro[5.5]undecan-3-amine, is an effective inhibitor of wild type A/M2 channels and L26F and V27A mutant ion channels in vitro, and also inhibits replication of recombinant mutant viruses bearing these mutations in plaque reduction assays. Differences in the inhibition kinetics between BL-1743, known to bind inside the A/M2 channel pore, and amantadine were exploited to demonstrate competition between these compounds; consistent with the conclusion that amantadine binds inside the channel pore. Inhibition by all of these compounds was shown to be voltage-independent, suggesting that their charged groups within the N-terminal half of the pore, prior to the selectivity filter that defines the region over which the transmembrane potential occurs. These findings not only help define the location and mechanism of binding of M2 channel-blocking drugs, but also demonstrate the feasibility of discovering new inhibitors that target this binding site in a number of amantadine-resistant mutants.

Successful uncoating of the influenza B virus in endosomes is predicted to require acidification of the interior of the virus particle. We report that a virion component, the BM2 integral membrane protein, when expressed in Xenopus oocytes or in mammalian cells, causes acidification of the cells and possesses ion channel activity consistent with proton conduction. Furthermore, coexpression of BM2 with hemagglutinin (HA) glycoprotein prevents HA from adopting its low-pH-induced conformation during transport to the cell surface, and overexpression of BM2 causes a delay in intracellular transport in the exocytic pathway and causes morphological changes in the Golgi. These data are consistent with BM2 equilibrating the pH gradient between the Golgi and the cytoplasm. The transmembrane domain of BM2 protein and the influenza A virus A/M2 ion channel protein both contain the motif HXXXW, and, for both proteins, the His and Trp residues are important for channel function.

The M2 protein from influenza A is a pH-activated proton channel that plays an essential role in the viral life cycle and serves as a drug target. Using spin labeling EPR spectroscopy we studied a 38-residue M2 peptide spanning the transmembrane region and its C-terminal extension. We obtained residue-specific environmental parameters under both high and low pH conditions for nine consecutive C-terminal sites. The region forms a membrane surface helix at both high and low pH although the arrangement of the monomers within the tetramer changes with pH. Both electrophysiology and EPR data point to a critical role for residue Lys 49.M2 is a 96-residue homotetrameric integral membrane protein with a small N-terminal ectodomain, a single transmembrane helix and a C-terminal cytoplasmic tail. Despite data from solid state NMR (1), x-ray crystallography (2) and solution NMR (3), a detailed understanding of how the M2 protein works continues to puzzle investigators and generate sharp controversy.The majority of published studies on the proton channel function of M2 have focused on the transmembrane (TM) 1 domain. However, truncation studies indicate that the cytoplasmic domain also plays a role in channel stability (4). Proteolysis of micelle-bound full length M2 revealed that a 15-20 residue segment C-terminal to the TM helix was highly protected from cleavage by proteases (5). Helical wheel analysis of the protected region (5) suggested that the segment could form an amphiphilic helix, consistent with later findings from solid state NMR on M2 protein in lipid bilayers (6). In order to further test the proposed models, we probed the conformation of the segment C-terminal to the TM domain at both high and low pH using sitedirected spin-labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy.EPR studies were performed on a series of 38-residue synthetic M2 peptides (residues 23-60; M2TMC) spanning the TM region and the beginning of the C-terminal domain. We spin- † This research was supported by R01AI57363 (LHP), GM56423 (WFD), a Henry Dreyfus Teacher Scholar Award (KPH) and R15AI074033 (KPH).