pH-dependent changes in photoaffinity labeling patterns of the H1 influenza virus hemagglutinin by using an inhibitor of viral fusion

The hemagglutinin (HA) protein undergoes a low-pH-induced conformational ch ange in the acidic milieu of the endosome, resulting in fusion of viral and cellular membranes. A class of compounds that specifically interact with t he HA protein of H1 and H2 subtype viruses and inhibit this conformational change was recently described (G. X. Luo et al., Virology 226:66-76, 1996, and J. Virol. 71:4062-4070, 1997), In this study, purified HA trimers (brom elain-cleaved HA [BHA]) are used to examine the properties and binding char acteristics of these inhibitors. Compounds were able to inhibit the low-pH- induced change of isolated trimers, as detected by resistance to digestion with trypsin. Protection from digestion was extremely stable, as BHA-inhibi tor complexes could be incubated for 24 h in low pH with almost no change i n BHA structure. One inhibitor was prepared as a radiolabeled photoaffinity analog and used to probe for specific drug interactions with the HA protei n. Analysis of BHA after photoaffinity analog binding and UV cross-linking revealed that the HA2 subunit of the HA was specifically radiolabeled, Cros s-linking of the photoaffinity analog to BHA under neutral (native) pH cond itions identified a stretch of amino acids within the alpha-helix of HA2 th at interact with the inhibitor. Interestingly, cross-linking of the analog under acidic conditions identified a different region within the HA2 N term inus which interacts with the photoaffinity compound. These attachment site s help to delineate a potential binding pocket and suggest a model whereby the BHA is able to undergo a partial, reversible structural change in the p resence of inhibitor compound.