Influenza virus-membrane fusion triggered by proton uncaging for single particle studies of fusion kinetics

Abstract

We report a method for studying membrane fusion, focusing on influenza virus fusion to lipid bilayers, which provides high temporal resolution through the rapid and coordinated initiation of individual virus fusion events. Each fusion event proceeds through a series of steps, much like multistep chemical reaction. Fusion is initiated by a rapid decrease in pH that accompanies the "uncaging" of an effector molecule from o-nitrobenzaldehyde, a photoisomerizable compound that releases a proton to the surrounding solution within microseconds of long-wave ultraviolet irradiation. In order to quantify pH values upon UV irradiation and uncaging, we introduce a simple silica nanoparticle pH sensor, useful for reporting the pH in homogeneous nanoliter volumes under conditions where traditional organic dye-type pH probes fail. Subsequent single-virion fusion events are monitored using total internal reflection fluorescence microscopy. Statistical analysis of these stochastic events uncovers kinetic information about the fusion reaction. This approach reveals that the kinetic parameters obtained from the data are sensitive to the rate at which protons are delivered to the bound viruses. Higher resolution measurements can enhance fundamental fusion studies and aid antiviral antifusogenic drug development.

Figure 1

(A) An illustration of the microfluidic device coupled to a TIRF microscope for imaging individual virion fusion events. The purple arrow entering the top of the device represents a UV laser that is aligned directly with the microscope objective beneath the device. Note that the dimensions of this drawing are not to scale. The actual channel is about 1mm wide by 70 μm high and the diameter of the UV laser beam is about 100 μm. (B) An inset of the region within the field of view of the camera, drawn as the black rectangle in (A), prior to UV irradiation at a neutral pH. This illustration shows that the glass surface comprising the fourth wall of the microchannel is coated with a solid supported lipid bilayer (gray). Virus labeled with a quenching concentration of fluorophore is colored light green with a red interior. The dark pink boxes represent proton cages (o NBA) that release protons when illuminated with 355 nm light. Note that this drawing is also not to scale; influenza virus is typically 100 nm in diameter and the bilayer is ~ 4 nm thick. (C) Immediately following UV irradiation the caged protons are released (denoted as free H+ in the diagram), acidifying the surrounding solution. Fusing viruses are now colored bright green to denote the dequenching of green fluorophores and the escape of the internal red dye upon pore formation. (D) The photochemistry of uncaging: the conversion of o-nitrobenzaldehyde to o-nitrosobenzoic acid and a proton upon irradiation with UV light. (Adapted from ref ).

Figure 2

(A) Virus fusion initiated by acidic buffer flow exchange. Green and red fluorescence images of a single fusing virus, marked by the arrows. After acidification, the green channel shows the hemifusion of the membranes; the spike in fluorescence is observed in the plot to the right. The red channel shows the radial diffusion of the internal red fluorophore after pore formation. The drop in red signal can be observed in the plot to the right; here it takes ~ 20 seconds between hemifusion and pore formation. (B) Virus fusion initiated by proton uncaging. Here it takes ~ 15 seconds between hemifusion and pore formation.

Figure 3

(A) Frequency of hemifusion events plotted as a function of time for initiation pH 4.5 obtained either by acidic buffer exchange (open black circles) or proton uncaging using 14 mM o-NBA (open green diamonds). The lines are the best fits to gamma function equation shown in the inset, and described in detail in the Supporting Information. The rate of hemifusion, k_H, was 0.20 ± 0.01 s⁻¹ and 0.17 ± 0.01 s⁻¹ for acidic buffer exchange and proton uncaging, respectively. N values for acid exchange and uncaging are 3.2 ± 0.1 s⁻¹ and 1.51 ± 0.05 s⁻¹, respectively. B) Histograms of lag times between the onset of hemifusion and the onset of pore formation. (Top) acidic buffer exchange; (Bottom) proton uncaging. The rate of transition from hemifusion to pore formation (k_H→P) using the acid flow and uncaging methods was found to be 0.08 ± 0.02s⁻¹ and 0.09 ± 0.05s⁻¹ respectively. N was less than 1 in both cases (0.7 ± 0.1 for acid flow and 0.5 ± 0.1 for uncaging), which agrees with previous findings that there is a single step transition between hemifusion and pore formation.

Figure 4

(A) Hemifusion rate constants, k_H, and (B) N parameters for a range of fusion initiation pH values.

Figure 5

(A) Comparison of photobleaching between Oregon green C dots sensor and free Oregon green after exposure to UV light for 200 ms. Note that the error bars in the free OG case are within the data point. All values are normalized to the intensity value before the 200 ms UV bleach to obtain a fractional photostability at each pH. (B) Calibration curve for Oregon green C dot sensor fluorescence intensity at various pH values. All data were normalized to the pH 7.0 value so that intensities post UV irradiation could be compared directly. Note that these data were taken after irradiating the samples with UV light to account for photobleaching in the uncaging runs. (Inset) Structure of the Oregon green C dot.

Figure 6

Fusion data at an initiation pH of 4.7. As the flow rate increases, the data trends shift closer to the uncaging data.