Self-assembly of influenza hemagglutinin: studies of ectodomain aggregation by in situ atomic force microscopy

We have used in situ tapping mode atomic force microscopy (AFM) to study th e structural morphology of two fragments of the influenza hemagglutinin pro tein bound to supported bilayers. The two proteins that we studied are the bromelain-cleaved hemagglutinin (BHA), corresponding to the full ectodomain of the hemagglutinin protein. and FHA2, the 127 amino acid N-terminal frag ment of the HA2 subunit of the hemagglutinin protein. While BHA is water so luble at neutral pH and is known to bind to membranes via specific interact ions with a viral receptor, FHA2 can only be solubilized in water with an a ppropriate detergent. Furthermore, FHA2 is known to readily bind to membran es at neutral pH in the absence of a receptor. Our in situ AFM studies demo nstrated that, when bound to supported bilayers at neutral pH, both these p roteins are self-assembled as single trimeric molecules. In situ acidificat ion resulted in further lateral association of the FHA2 without a large per turbation of the bilayer. In contrast, BHA remained largely unaffected by a cidification, except in areas of exposed mica where it is aggregated. Remar kably, these results are consistent with previous observations that FHA2 pr omotes membrane fusion while BHA only induces liposome leakage at low pH. T he results presented here are the first example of in situ imaging of the e ctodomain of a viral envelope protein allowing characterization of the real -time self-assembly of a membrane fusion protein. (C) 2001 Elsevier Science BN. All rights reserved.