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. 2018 Aug 9;174(4):926-937.e12.
doi: 10.1016/j.cell.2018.05.050. Epub 2018 Jun 28.

Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers

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Free PMC article

Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers

Dibyendu Kumar Das et al. Cell. .
Free PMC article

Abstract

Influenza hemagglutinin (HA) is the canonical type I viral envelope glycoprotein and provides a template for the membrane-fusion mechanisms of numerous viruses. The current model of HA-mediated membrane fusion describes a static "spring-loaded" fusion domain (HA2) at neutral pH. Acidic pH triggers a singular irreversible conformational rearrangement in HA2 that fuses viral and cellular membranes. Here, using single-molecule Förster resonance energy transfer (smFRET)-imaging, we directly visualized pH-triggered conformational changes of HA trimers on the viral surface. Our analyses reveal reversible exchange between the pre-fusion and two intermediate conformations of HA2. Acidification of pH and receptor binding shifts the dynamic equilibrium of HA2 in favor of forward progression along the membrane-fusion reaction coordinate. Interaction with the target membrane promotes irreversible transition of HA2 to the post-fusion state. The reversibility of HA2 conformation may protect against transition to the post-fusion state prior to arrival at the target membrane.

Keywords: membrane fusion; protein dynamics; single-molecule fluorescence; smFRET; virus entry.

Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1
Figure 1. smFRET assay for direct visualization of HA conformational dynamics
(A) Viral particles containing a single HA*-Cy3/Cy5 protomer, labeled at positions 17 and 127 in the HA2 domain, within an HA trimer were immobilized on a quartz microscope slide and imaged at room temperature with TIRF microscopy (STAR Methods). (B) Molecular models of HA*-Cy3/Cy5 predict that fluorophores placed at positions 17 and 127 in the HA2 domain will report on the transition from the pre-fusion to the coiled-coil conformation of HA2 via changes in FRET efficiency (models were based on PDB IDs 2FK0 and 1HTM, STAR Methods) (Bullough et al., 1994a; Stevens et al., 2006). See also Figure S4.
Figure 2
Figure 2. Direct visualization of pH-triggered conformational changes in HA
(A) (left) FRET contour plots constructed from the compilation of the population of smFRET trajectories. The contour plots were summed over time, generating FRET histograms, which are shown with three Gaussian distributions overlaid (red) with means ± standard deviations of 0.20 ± 0.08, 0.53 ± 0.09 and 0.95 ± 0.06 that represent the states identified through HMM analysis (black). The number of smFRET traces (n) compiled into each histogram is indicated. (right) TDPs displaying the distributions of initial and final FRET values for every observed transition in FRET. The number of observed transitions (n) and the total transitions per second (t/s) are indicated. (B–E) The same data acquired at the indicated pH. See also Figure S3 and Table S1.
Figure 3
Figure 3. Pre-steady state smFRET imaging indicates an intermediate conformation adopted during triggering of HA by acidic pH
(A) Representative fluorescence (donor, green; acceptor, red) and FRET trajectories (blue) obtained from a single HA*-Cy3/Cy5 protomer within an HA trimer on the surface of a viral particle. At each time point FRET efficiency was calculated as the ratio of acceptor fluorescence intensity to total fluorescence intensity. Overlaid on the FRET trajectory in red is an idealized trace generated through HMM analysis. (B) Contour plot of smFRET trajectories synchronized to the transition out of high FRET. Transition through the intermediate-FRET state is observed in route to low FRET. (C) As in Figure 2, the TDP displays the distribution in initial and final FRET values for every transition observed. The TDP indicates that trajectories predominantly transition from high to intermediate FRET, and from intermediate to low FRET. A minority of transitions occurs directly from high to low FRET with no observation of intermediate FRET. This can be explained by the instability of the intermediate-FRET state and the limited time resolution of our imaging.
Figure 4
Figure 4. Restoration of the pre-fusion conformation after return to neutral pH
FRET contour plot, FRET histogram and TDP acquired immediately after exposure of HA to (A) pH 5.6 (repeated from Figure 2), (B) pH 5.3, or (C) pH 5.2. The same data were acquired after returning to pH 7.5 following (D–F) 5 min, (G–I) 15 min, or (J–L) 30 min. Data are displayed as in Figure 2. See also Table S1.
Figure 5
Figure 5. Interaction with a sialic acid-containing target membrane regulates HA conformational dynamics
(A) Schematic of the smFRET-imaging assay in the presence of liposomes containing GD1a, which contains sialic acid (STAR Methods). (B) Representative fluorescence and FRET traces at pH 6.1 displayed as in Figure 2. (C) FRET contour plots (left), FRET histograms (middle) and TDPs (right) displayed as in Figure 2. Quantification of the occupancies in the (D) high-FRET pre-fusion and (E) low-FRET conformations in the absence (blue) and presence (red) of liposomes, which was determined through HMM analysis. See also Table S1.
Figure 6
Figure 6. A sialic acid-containing target membrane promotes transition of HA to the irreversible coiled-coil conformation
(A) FRET contour plot, histograms and TDPs acquired in the presence of GD1a-containing liposome immediately after exposure to pH 5.6. The same data were acquired after return to pH 7.5 following (B) 5 min, or (C) 30 min. Data are displayed as in Figure 2. See also Table S1.
Figure 7
Figure 7. Kinetic model of HA2 conformational dynamics
(A) smFRET analysis indicates a sequence of conformational changes in HA during conversion from the pre-fusion to the coiled-coil conformation. The HA2 domain reversibly interconverts between three conformations (pre-fusion, intermediate state I, and intermediate state II). Low pH stalls HA2 in intermediate state II in the absence of a receptor-containing membrane, from which it can return to the pre-fusion state by reneutralizing the pH. Interaction with the receptor-containing target membrane promotes formation of the coiled-coil conformation. (B) The rate constants k1, k−1, k2, are k−2 were determined by exponential fitting of the dwell time histograms obtained through HMM analysis (STAR Methods). Rates of transition between the observed states are shown as a function of pH in the (C–D) absence, or (E–F) presence of a receptor-containing target membrane. Error bars represent the 95% confidence intervals obtained during fitting of the dwell time histograms. The plotted lines represent linear fits. See also Table S3.

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