Pharmacological Relevant Amantadine Binding Site is in the Pore of Influenza a Virus M2 Proton Channel

Abstract

The influenza A virus M2 protein (A/M2) and the influenza B virus BM2 protein are both homotetrameric pH-activated proton channel that facilitates viral uncoating by acidification the interior of endosomally encapsulated virus. Anti-viral drugs amantadine and its derivative rimantadine inhibit A/M2 channel of influenza A virus, but not BM2 channel of influenza B virus. The atomic structure of the pore-transmembrane (TM) domain peptide has been determined by X-ray crystallography (Stouffer et al., Nature 451, 596-599 [2008]) and of a larger M2 peptide by NMR methods (Schnell and Chou, Nature 451, 591-595 [2008]). The crystallographic data shows electron density (at 3.5 A resolution) in the channel pore, consistent with amantadine blocking the pore of the channel. In contrast, the NMR data show four rimantadine molecules bound on the outside of the helices towards the cytoplasmic side of the membrane. Drug binding includes interactions with residues 40-45 and a hydrogen bond between rimantadine and Asp44. These two distinct drug-binding sites led to two incompatible drug inhibition mechanisms. The cytoplasmic binding site predicts that D44 and R45 to alanine mutations would 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 TEVC recordings in oocytes of Xenopus laevis, and when the D44 and R45 mutations were introduced into the influenza virus genome. Furthermore, two chimeras containing 5 residues of the A/M2 ectodomain and residues 24-36 of the A/M2 TM domain show 85% amantadine/rimantadine sensitivity and specific activity comparable to wt BM2. These functional data suggest the pharmacological relevant amantadine/rimantadine binding site is in the pore of the M2 channel.