RESEARCH ARTICLE Characterization of Potent Fusion Inhibitors of Influenza Virus

New inhibitors of influenza viruses are needed to combat the potential emergence of novel human influenza viruses. We have identified a class of small molecules that inhibit replica-tion of influenza virus at picomolar concentrations in plaque reduction assays. The com-pound also inhibits replication of vesicular stomatitis virus. Time of addition and dilution experiments with influenza virus indicated that an early time point of infection was blocked and that inhibitor 136 tightly bound to virions. Using fluorescently labeled influenza virus, in-hibition of viral fusion to cellular membranes by blocked lipid mixing was established as the mechanism of action for this class of inhibitors. Stabilization of the neutral pH form of hem-agglutinin (HA) was ruled out by trypsin digestion studies in vitro and with conformation spe-cific HA antibodies within cells. Direct visualization of 136 treated influenza virions at pH 7.5 or acidified to pH 5.0 showed that virions remain intact and that glycoproteins become disor-ganized as expected when HA undergoes a conformational change. This suggests that ex-posure of the fusion peptide at low pH is not inhibited but lipid mixing is inhibited, a different mechanism than previously reported fusion inhibitors. We hypothesize that this new class of inhibitors intercalate into the virus envelope altering the structure of the viral envelope re-quired for fusion to cellular membranes