The influenza virus hemagglutinin (HA) fusion protein has long been viewed as a “spring-loaded” fusion machine whereby activation at low pH initiates a rapid and irreversible cascade of conformational changes that drives the membrane fusion reaction. This mechanism has shaped our understanding of how type 1 viral fusion proteins function as a whole. Experimental limitations have hindered efforts to expand our mechanistic and structural understanding of viral membrane fusion. Here, we used pulse-labeling hydrogen/deuterium exchange mass spectrometry and cryo–electron tomography to monitor and characterize the structural dynamics of HA during fusion activation on intact virions. Our data reveal how concurrent reorganizations at the HA1 receptor binding domain interface and HA2 fusion subunit produce a dynamic fusion intermediate ensemble in full-length HA. The soluble HA ectodomain transitions directly to the postfusion state with no observable intermediate.
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