The 1-127 HA2 construct of influenza virus hemagglutinin induces cell-cell hemifusion.

Conformational changes in the HA2 subunit of influenza hemagglutinin (HA) are coupled to membrane fusion. We investigated the fusogenic activity of the polypeptide FHA2 representing 127 amino-terminal residues of the ectodomain of HA2. While the conformation of FHA2 both at neutral and at low pH is nearly identical to the final low-pH conformation of HA2, FHA2 still induces lipid mixing between liposomes in a low-pH-dependent manner. Here, we found that FHA2 induces lipid mixing between bound cells, indicating that the "spring-loaded" energy is not required for FHA2-mediated membrane merger. Although, unlike HA, FHA2 did not form an expanding fusion pore, both acidic pH and membrane concentrations of FHA2, required for lipid mixing, have been close to those required for HA-mediated fusion. Similar to what is observed for HA, FHA2-induced lipid mixing was reversibly blocked by lysophosphatidylcholine and low temperature, 4 degrees C. The same genetic modification of the fusion peptide inhibits both HA- and FHA2-fusogenic activities. The kink region of FHA2, critical for FHA2-mediated lipid mixing, was exposed in the low-pH conformation of the whole HA prior to fusion. The ability of FHA2 to mediate lipid mixing very similar to HA-mediated lipid mixing is consistent with the hypothesis that hemifusion requires just a portion of the energy released in the conformational change of HA at acidic pH.

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 the structural morphology of two fragments of the influenza hemagglutinin protein 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 fragment of the HA2 subunit of the hemagglutinin protein. While BHA is water soluble at neutral pH and is known to bind to membranes via specific interactions with a viral receptor, FHA2 can only be solubilized in water with an appropriate detergent. Furthermore, FHA2 is known to readily bind to membranes at neutral pH in the absence of a receptor. Our in situ AFM studies demonstrated that, when bound to supported bilayers at neutral pH, both these proteins are self-assembled as single trimeric molecules. In situ acidification resulted in further lateral association of the FHA2 without a large perturbation of the bilayer. In contrast, BHA remained largely unaffected by acidification, except in areas of exposed mica where it is aggregated. Remarkably, these results are consistent with previous observations that FHA2 promotes membrane fusion while BHA only induces liposome leakage at low pH. The results presented here are the first example of in situ imaging of the ectodomain of a viral envelope protein allowing characterization of the real-time self-assembly of a membrane fusion protein.

Factors determining vesicular lipid mixing induced by shortened constructs of influenza hemagglutinin.

The HA2 subunit of influenza hemagglutinin is responsible for fusion of the viral and host-cell membranes during infection. An N-terminal 127 amino acid construct of HA2, FHA2-127, is shown to induce lipid mixing of large unilamellar vesicles under endosomal low pH conditions. Thus, FHA2 could serve as a good model system for biophysical studies of membrane fusion. With FHA2, we began to develop a mechanistic model which could explain how this short construct facilitates membrane fusion. In this endeavor, we studied the possible role of the kinked loop region (amino acids 105-113). A construct missing this loop, FHA2-90, although able to induce lipid mixing, has lost the sharp pH-dependent transition seen with FHA2-127 and native HA. In addition, FHA2-127 promotes extensive vesicle aggregation more effectively than FHA2-90 upon acidification. These data suggest that the kinked loop may play a pH-dependent regulatory role. To test this, we compared bis-ANS binding to the two constructs and observed that binding to FHA2-127 increases at a faster rate than FHA2-90 as the pH is decreased, indicating that the kinked loop not only is an ANS-binding site, but that it binds better at low pH. The pH dependence of this transition directly correlates with that observed in lipid mixing. Further, cysteine mutations of acidic residues in the kinked region are both fusion inactive and bind much less ANS, whereas a similar mutation of a threonine residue had little effect on fusion activity or ANS binding. This evidence lends further support to our idea that the kinked loop serves a regulatory role. To test the physiological relevance of the FHA2-127 fusion mechanism, we studied the effects of a G1E mutation, known to abolish fusion in native HA. We found that G1E-127 is fusion inactive as expected. This evidence indirectly suggests that the mechanism of FHA2-127 is perhaps physiologically relevant and from its study, we can learn much about the mechanism of native HA.

Insights into a structure-based mechanism of viral membrane fusion.

A number of different viral spike proteins, responsible for membrane fusion, show striking similarities in their core structures. The prospect of developing a general structure-based mechanism seems plausible in light of these newly determined structures. Influenza hemagglutinin (HA) is the best-studied fusion machine, whose action has previously been described by a hypothetical "spring-loaded" model. This model has recently been extended to explain the mechanism of other systems, such as HIV gp120-gp41. However, evidence supporting this idea is insufficient, requiring re-examination of the mechanism of HA-induced membrane fusion. Recent experiments with a shortened construct of HA, which is able to induce lipid mixing, have provided evidence for an alternative scenario for HA-induced membrane fusion and perhaps that of other viral systems.