How influenza virus is locked out of the cell.

E nveloped viruses acquire a lipid membrane when they bud across a cellular membrane during virus assembly. In cell entry, the viral membrane must be fused to the host-cell membrane to deliver the viral genome into the cytoplasm for replication. Membrane fusion is therefore an essential step in the life cycle of enveloped viruses, and a great deal of research in recent years has been directed at identifying inhibitors of viral membrane fusion. One notable success in this area is the HIV fusion inhibitor enfurvirtide (T-20, Fuzeon) (1), which has become part of the standard treatment for patients who have detectable viral loads after treatment with protease and reverse transcriptase inhibitors. Although influenza remains a primary global health problem, there are no clinically useful fusion inhibitors available against influenza virus. In a recent issue of PNAS, Russell et al. (2) report the crystal structure of the influenza virus hemagglutinin (HA) envelope protein bound to a compound that was reported to inhibit membrane fusion and infectivity of certain strains of influenza. The structure provides an excellent starting framework for the rational design of more effective membrane fusion inhibitors for use as therapeutics against influenza. For 2 membranes to fuse, they must be bent toward each other until they are separated by only a fraction of a nanometer. Bending membranes requires energy, which in viral membrane fusion is provided by envelope proteins anchored in the viral membrane as they undergo a large, spontaneous, ‘‘fusogenic’’ conformational change (3). The fusogenic conformational change of HA is well understood from numerous biophysical and biochemical studies, making HA the prototype of viral fusion proteins (4). Moreover, various small hydrophobic molecules such as tert-butyl hydroquinone (TBHQ) have been found to block influenza virus infectivity in cell culture by inhibiting (or prematurely inducing) the fusogenic conformational change in HA (5–7). The development of more effective fusion inhibitors has, however, been limited by the lack of crystal structures of relevant HA complexes and the failure of known fusion inhibitors to neutralize all influenza subtypes. Russell et al. (2) report the crystal structures of HA from 2 subtypes of influenza, H14 and H3, in complex with TBHQ. TBHQ specifically inhibits the fusogenic conformational change of HA and reduces viral infectivity of group 2 influenza subtypes such as H3 and H14 (6). The structures show that TBHQ does not bind near the fusion peptide of HA as predicted by in silico docking analyses (6). Instead, TBHQ binds in a hydrophobic pocket at the interface between monomers in the prefusion HA trimer (Fig. 1) . This binding pocket is present only in group 2 HAs, which explains the failure of TBHQ to inhibit membrane fusion and the infectivity of group 1 viruses. The apparent mechanism for fusion inhibition is that TBHQ stabilizes the prefusion conformation of HA, thus increasing the energy barrier for the fusogenic conformational change to the point that it no longer responds to the acidic environment of the endosomal lumen (2). Specifically, TBHQ locks HA in its prefusion conformation by extending the hydrophobic core between the ‘‘outer layer’’ and ‘‘inner layer’’ -helices (Fig. 1). In other words, TBHQ acts as a sort of molecular glue. To exclude water and other solvent molecules, hydrophobic compounds must fit tightly into their binding pockets, with a high degree of shape complementarity. The snug fit of TBHQ into its binding pocket leaves little room for additional hydrophobic substitutions. However, the structure shows that space for additional polar substituents on TBHQ exists in