Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates

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1. A subset of the virons for X31-HA is already arrested prior to the pH drop. Some speculation about the origin of this phenomenon might be warranted. Perhaps some fusion peptides have been released for this variant even at higher pH?

We have added to the Results and Materials and methods statements to address this observation and speculate about its cause. We have also added two additional movies (Video 9 and Video 10), which speak to this point. In summary, while it is possible and likely that some fusion peptides are released and inserted into the target membrane even at neutral pH (we occasionally observe hemifusion events at neutral pH – see Video 9), we believe that this is a rare occurrence and that most of the pre-arrested events in the context of X31-HA virions at pH 5.5 and above (∼20%) result from imperfections in either the bilayer or the virions themselves.

2. What is the efficiency of arrest upon the pH drop for the X31-HA variant? In other words, how many virions transiently dock to the bilayer but do arrest and hemifuse (Movie 1)? Some rough estimation would be sufficient.

3. Likewise, what is the estimated efficiency of hemifusion for those virions that are arrested?

Response to points 2 & 3: Virion arrest preceded hemifusion in all cases and hemifusion followed virion arrest in 75% (X31-HA virions) and 65% (UdornS4G virions) of the cases at pH 5.5. We had originally included this measurement in Table 1, and we have now added a sentence in the Materials and methods further discussing these percentages. We also added a sentence about the relative efficiency of arrest, which was nearly 100% up to the highest pH analyzed (pH 5.65). At pH 5.65 we observe a small subset of virions that continue to move under hydrodynamic force well after the pH drop, but in this case also nearly 100% seem to arrest within a few minutes of pH drop. We cannot estimate the exact percentage of those virions that never arrest or that arrest very late, because we do not have a way to automatically detect and track the virions that never arrest, but careful observation of the movies in slow motion allowed us to conclude that ∼100% of virions do arrest at all the pHs we analyzed.

From the subset of virions that were arrested preceding pH drop, 82% (X31-HA virions) and 81% (UdornS4G virions) hemifused at pH 5.5. We have now added these percentages to Table 1.

4. […] Could the observed lag-time distributions be reproduced with a simple simulation? For example, one could start with a triangular lattice of a certain size and simulate the entire process based on the proposed model. Each trimer could release peptide with a rate of k, and if N of them release peptide, one calls it an arrest; and then if N of them accumulate next to each other, one calls it hemi-fusion. The simulation could then be repeated with different values for N. Such a simulation would be more directly related to the model that is proposed here.

This was an astute point and an excellent suggestion. We have done the simulation, added a new figure (now Figure 5), and we have added a new section to the Results (titled ‘Simulation of molecular events in the contact patch between virion and target membrane'). Briefly, we have generated a computer simulation of our predicted model that virion arrest results from independent triggering and irreversible membrane insertion from several HA trimers anywhere at the target membrane interface, and that hemifusion is limited by the same molecular rearrangements, fusion peptide release, and membrane insertion, but requires participation of neighboring HA trimers, and is thus related to a different probability. This exercise not only supported our original contention but narrowed down our interpretation of results, allowing us to state that virion arrest results from 3–4 trimers inserting into the target membrane anywhere in the contact patch and hemifusion from 3–4 neighboring HA trimers undergoing the same molecular rearrangement. In other words, our observed data are inconsistent with participation of as few as 2 or as many as 5 neighboring HA trimers in hemifusion under our current experimental conditions for a range of relevant contact–patch sizes.

Optional: 5. It would be nice (but not required) to investigate the effect of the same mutations on content mixing using the content marker that was used in the previous PNAS study. In that previous work, it was concluded that the transition from hemifusion to full fusion is a one step process. One would then expect that the step from hemifusion to full fusion would not be influenced by the HA mutations studied here unless an additional HA trimer is actively engaged to achieve complete fusion.

This is a good suggestion and one that should be addressed with future work, but we believe it is beyond the scope of the current manuscript.