Distinct functional determinants of influenza hemagglutinin-mediated membrane fusion
- University of Colorado, United States
- Harvard Medical School, United States
- Brandeis University, United States
- Howard Hughes Medical Institute, Harvard Medical School, United States
Figures
Figure 1
Productive and non-productive HA refolding, and membrane fusion by cooperative action of multiple, stochastically triggered HAs.
(A) Proton binding increases the relative time HA spends in the ‘open’ conformation allowing fusion peptides to project toward the target membrane. HA1 is shown in green and HA2 in magenta (fusion …
Figure 2 with 1 supplement
The functional variables of influenza membrane fusion modeled in this work.
We modeled the kinetics and the extent of membrane fusion with the following parameters: (A) the number of HAs in contact with the target membrane (patch size, PS), (B) the rate (ksim) of stochastic …
Figure 2—figure supplement 1
Definition of six-mers (Nh = 6) in the simulation.
Since multiple HAs must fold back cooperatively, possible six-mers have distinct properties: two groups of three HAs that can join around either two adjacent or one wider target-membrane deformation …
Figure 3 with 3 supplements
Effects of fnp on hemifusion yield and kinetics for Nh = 3–6 (PS = 121).
(A) Illustration of simulated contact patches. (B) Hemifusion yield as a function of fnp. (C) Mean hemifusion-delay times normalized to fnp = 0. (D) Parameter N derived from fitting hemifusion delay …
Figure 3—figure supplement 1
Effects of fnp on hemifusion yield and kinetics for Nh = 3–6 (PS = 55).
Refer to the main Figure 3 legend, which shows results of an analogous set of simulations using PS = 121 instead of PS = 55.
Figure 3—figure supplement 2
Effects of fnp on hemifusion yield and kinetics for Nh = 2.
Simulation results for both PS = 55 and PS = 121 are shown. Hemifusion yield (top row), mean hemifusion-delay times normalized to fnp = 0 (second row), parameter N derived from fitting hemifusion …
Figure 3—figure supplement 3
Ngamma for pH-drop-to-hemifusion frequency distributions from previously published experiment data (Ivanovic et al., 2013).
Udorn and X31-G4S are previously published values. X31 and Udorn-S4G are newly determined values from those published datasets. (A) Ngamma values with 95% confidence intervals (error bars) for the …
Figure 4 with 1 supplement
Complete processing of virion-associated HAs and complete conformational change at low pH.
We show WT UdornHA-Udorn and X31HA-Udorn virions used in our previous single-virion fusion experiments (Ivanovic et al., 2013). SDS-PAGE and western blot of virions probed with HA1-specific antibody …
Figure 4—figure supplement 1
Complete processing of virion-associated HAs and complete conformational change at low pH.
See main Figure 4, panels (B,C) legend, showing an analogous experiment performed with a different set of UdornHA-Udorn and X31HA-Udorn clones.
Figure 5 with 1 supplement
Hemifusion yield as a function of the fraction of unproductive HAs (fun) for virions with no bound antibody and for those with 261 or 493 bound Fabs (PS = 55).
(A) Illustrations of simulated contact patches. The frequency of Fab-bound HAs (fFab) and fun were combined in the parameter fnp as described in Materials and methods. (B–D) The results for Nh = 3 (B…
Figure 5—figure supplement 1
Hemifusion yield as a function of the fraction of unproductive HAs (fun) for virions with no bound antibody and for those with 261 or 493 bound Fabs (PS = 121).
See main Figure 5 legend which shows results of an analogous set of simulations using PS = 121 instead of PS = 55.
Figure 6 with 3 supplements
Effects of Fab binding on hemifusion yield and kinetics for given pairs of Nh and fun (PS = 121).
(A) Illustrations of simulated contact patches at the time of hemifusion for several fnp values (fun was kept constant while fFab was increased). (B–E) Comparison of simulation-derived results …
Figure 6—figure supplement 1
Effects of Fab binding on hemifusion yield and kinetics for given pairs of Nh and fun (PS = 55).
See main Figure 6 legend, which shows results of an analogous set of simulations using PS = 121 instead of PS = 55. For the adjusted values of fun, PS = 55 data are indistinguishable from PS = 121 …
Figure 6—figure supplement 2
Effect of sample size on variability in Ngamma.
We derived Ngamma values for 6 small datasets and a single large data set for both PS = 55 and PS = 121 and for each pair of Nh and fun identified for H3 X31 and H1 PR8 HAs in Figure 5 and 7, …
Figure 6—figure supplement 3
Effects on our conclusions of potential error in the measurement of the number of Fabs needed for 50% hemifusion inhibition (#Fab1/2hemi) for H3N2 X31 influenza virions.
For the simulation results shown, we included several #Fab1/2hemi values covering the entire 95% confidence interval for this measurement (Otterstrom et al., 2014). (A) fun required to give …
Figure 7 with 1 supplement
Hemifusion yield as a function of fun for virions with no bound antibody or those with 74 or 248 bound Fabs (PS = 121).
(A) Illustrations of simulated contact patches. (B–E) The results for Nh = 3 (B), Nh = 4 (C), Nh = 5 (D), and Nh = 6 (E) were derived from simulations that yielded 1000 to 3000 hemifusion events. …
Figure 7—figure supplement 1
Hemifusion yield as a function of fun for virions with no bound antibody or those with 74 or 248 bound Fabs (PS = 55).
See main Figure 7 legend, which shows results of an analogous set of simulations using PS = 121 instead of PS = 55.
Figure 8 with 2 supplements
Effects of Fab binding on hemifusion yield and kinetics for given pairs of Nh and fun (PS = 121)
(A) Illustrations of simulated contact patches at the time of hemifusion for several fnp values (fun was kept constant while fFab was increased). (B–E) Comparison of simulation-derived results (1000–…
Figure 8—figure supplement 1
Effects of Fab binding on hemifusion yield and kinetics for given pairs of Nh and fun (PS = 55).
See main Figure 8 legend, which shows results of an analogous set of simulations using PS = 121 instead of PS = 55. For the adjusted values of fun, PS = 55 data are indistinguishable from PS = 121 …
Figure 8—figure supplement 2
Effects on our conclusions of potential error in the measurement of the number of Fabs needed for 50% hemifusion inhibition (#Fab1/2hemi) for H1N1 PR8 influenza virions.
For the simulation results shown, we included several #Fab1/2hemi values covering the entire 95% confidence interval for this measurement (Otterstrom et al., 2014). (A) fun required to give …
Figure 9 with 1 supplement
Independent functional determinants of HA-mediated membrane fusion and their effects on the influenza virus susceptibility to neutralization.
Conclusions are presented in the context of the PS = 121 contact patch. (A) The rate of irreversible HA extension (ke) and the frequency of unproductive or inactive HAs determine the rate of target …
Figure 9—figure supplement 1
Independent functional determinants of HA-mediated membrane fusion and their effects on the influenza virus susceptibility to neutralization.
Conclusions are presented in the context of the PS = 55 contact patch. See main Figure 9 legend.
Additional files
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Source code 1
The simulation model of fusogenic molecular events at the virus target-membrane interface.
The code consists of the main code text and four functions written in MATLAB (version R2015a). The main code script is titled s_arrest_hemifusion_simulation_eLife2015resubmission.m, and the functions are generate_patch.m, s_randomdist.m, isaN2tuplet6AllGeos.m, and findFlippedNeighbors.m. The simulation process is outlined within the main code text and in the Computer Simulation subsection of the Materials and methods. The code was adapted from Ivanovic et al (2013) to include a possibility of Nh=6 and the unproductive HA population, and to measure hemifusion delay from the start of the simulation rather than from the arrest intermediate.
- https://doi.org/10.7554/eLife.11009.025
