Conformational changes in intact dengue virus reveal serotype-specific expansion

Abstract

Dengue virus serotype 2 (DENV2) alone undergoes structural expansion at 37 °C (associated with host entry), despite high sequence and structural homology among the four known serotypes. The basis for this differential expansion across strains and serotypes is unknown and necessitates mapping of the dynamics of dengue whole viral particles to describe their coordinated motions and conformational changes when exposed to host-like environments. Here we capture the dynamics of intact viral particles of two serotypes, DENV1 and DENV2, by amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and time resolved Förster Resonance Energy Transfer. Our results show temperature-dependent dynamics hotspots on DENV2 and DENV1 particles with DENV1 showing expansion at 40 °C but not at 37 °C. HDXMS measurement of virion dynamics in solution offers a powerful approach to identify potential epitopes, map virus-antibody complex structure and dynamics, and test effects of multiple host-specific perturbations on viruses and virus-antibody complexes.

Figure 1. Temperature-dependent changes in DENV1 and 2 accompanying transmission from mosquito vector (28 °C) to human host (37 °C).

Cryo-EM structures of DENV1 and DENV2 icosahedral particles (Triangulation, T=3) showing symmetry units of E-proteins straddling the five-fold (pentamer), two-fold (dimer) and three-fold (trimer) vertices in dark, medium and light blue respectively. The triangle highlights a single symmetry unit and the numbers indicate the five-fold, three-fold vertices and two-fold midpoint. Left, cryo-EM structure of smooth, compact DENV2 (PDB ID: 3J27, 3.5 Å resolution) and DENV1 (PDB ID: 4CCT, 4.5 Å resolution) at 28 °C (environment inside the mosquito vector). Right, cryo-EM structure of expanded DENV2 (PDB ID: 3ZKO, 13 Å resolution) and unexpanded DENV1 (PDB ID: 4CCT, 4.5 Å resolution) at 37 °C (environment within human host following transmission). White spaces at three-fold icosahedral vertices indicate exposed regions of the lipid bilayer upon expansion. Inset 1: DENV structural proteome. Cryo-EM structures of DENV E (blue) (PDB ID: 3J27) and M (yellow) (PDB ID: 3J27) proteins shown in the context of the viral structure. The lipid bilayer is highlighted in dark grey. Since C-proteins are not observable in the cryo-EM structures of intact DENVs, an approximate position (region coloured red) based on its proposed role in bridging the RNA genome (not shown) beneath the lipid bilayer is represented. Inset 2: NMR structure of C (red) (PDB ID: 1R6R) protein dimer. The N and C-termini of one C-monomer are labelled.

Figure 2. Deuterium exchange of E-protein in unexpanded DENV2 and DENV1 at 28 °C in solution.

(a) Representative mass spectra of peptides from E-protein of DENV2 and DENV1 for undeuterated and t=1 min of deuterium exchange at 28 °C. Differences in sequence between DENV2 and DENV1 are indicated in black. Dashed line represents centroid of the mass envelope. Average number of deuterons exchanged in each peptide is determined by subtracting the centroid of the deuterium exchanged peptide from the undeuterated peptide. Relative deuterium exchange (RFU) is the fraction of average deuterons exchanged relative to the maximum number of exchangeable amides in each peptide. (b,c) RFU values for each E-protein pepsin fragmentation peptide listed from the N-to-C-terminus after 1 min of deuterium exchange are shown for DENV2 and DENV1 at 28 °C in a modified mirror plot respectively. Each dot represents one pepsin fragment peptide, Y-axis-Relative fractional uptake of deuterium (RFU), X-axis-pepsin fragment peptides, listed from N to C terminus. (d) RFU after 1 min of deuterium exchange at 28 °C is mapped onto whole viral particles in a colour coded gradient scale on DENV2 (PDB ID: 1OAN) and DENV1 (PDB ID: 4CCT). Regions with no peptide coverage are white. Each E-protein monomer is outlined (consisting of 6 monomers). E-protein units adjacent to the five-fold, two-fold and three-fold vertices are labelled A, B and C respectively. Insets: RFU after 1 min of deuterium exchange at 28 °C mapped onto a unit of E-protein dimer from DENV2 and DENV1. S.e. for each peptide is shown as shaded regions along the X-axis. The s.e. for a given peptide represents a single sigma s.d.. of all (minimum three) the independent HDXMS measurements.

Figure 3. Non-uniform temperature-specific changes in DENV2 and DENV1 at 37 and 40 °C by HDXMS.

Temperature-induced differences in deuterium exchange (t=1 min) in E-protein and M-protein from DENV2 (a,b, respectively) and DENV1 (e,f, respectively) between 28 and 37 °C (Orange line) and between 28 and 40 °C (Green line) are represented on a difference plot. Difference refers to the difference in average number of deuterons exchanged between two conditions. The difference plot displays the differences in exchange protein-wide where each dot represents a pepsin fragment peptide, listed from the N- to C-terminus. Y-axis-difference in deuterons, X-axis-pepsin fragment peptides. Differences in deuterium exchange above 0.5 D are considered significant (red dash line). Domain organisation of E-protein is indicated below the X-axis. S.e. for each peptide is shown as overlapping shaded regions along the X-axis and coloured according to the conditions in the difference plots. The individual s.e. in each condition is calculated as s.d.'s observed across all the HDXMS measurements (from at least three independent HDXMS experiments). The s.e. for a given peptide in the difference plot represents the sum of such single sigma s.d.'s of each of the two conditions being compared. Orthogonal views of the differences in deuterium exchange in E-protein peptides with temperature (37 °C in DENV2 (c,d) and 40 °C in DENV1 (g,h)) mapped onto the cryo-EM structure of E-M heterotetramer with one of the heterodimer rendered transparent for clarity. Regions with no peptide coverage are in grey.

Figure 4. Expansion of DENV2 and DENV1 captured by TR-FRET.

(a) Representative fluorescence lifetime decay curve of AF488-TFP for dual labelled DENV2 at 25 (black) and 37 °C (grey), including the impulse response function (IRF) of the instrument. The lifetime traces are fitted with a two-component model. The average fluorescence lifetimes (<τ_f>) of AF488-TFP for donor-only labeled, <τ_f,SL> (grey triangles), and dual labelled, <τ_f,SL> (black circles), for DENV2 (NGC) (b) and DENV1 (PVP 159) (c). Error bars represent s.d.'s of six different experimental replicates in both DENV2 and DENV1 viruses. The temperature in these two cases was increased from 25 to 40 °C. The midpoint temperature of the transition was at ∼33 °C and greater than 37 °C for DENV2 and DENV1, respectively.

Figure 5. Temperature expansion loci differ between DENV2 and DENV1.

Isotopic mass spectral envelopes corresponding to representative homologous peptides of equivalent lengths of DENV2 (a) and DENV1 (b) E-proteins spanning the two, three and five-fold interaction interfaces after deuterium exchange of t=0 and 1 min at 28, 37 and 40 °C. Dashed lines represent centroid of mass envelope. Differences in sequence between DENV2 and DENV1 are indicated in black. (c) Temperature-specific changes at 37 °C mapped onto unexpanded DENV2 structure (PDB ID: 3J27). (d) Temperature-specific changes at 40 °C mapped onto unexpanded DENV1 structure (PDB ID: 4CCT). Regions with no peptide coverage are grey.

Figure 6. Quaternary contacts alter deuterium exchange: Comparison of HDXMS of unassembled E-protein with E-protein from DENV particles.

RFU of unassembled DENV2 E-protein (a) at 28 °C for time points (1, 2, 5, 10 and 60 min) and DENV1 E-protein (b) at 28 °C for deuterium labelling time point of 1 min are shown as a modified mirror plot. E-protein peptides are listed from N- to C-terminus (X-axis). Error bars for each peptide are shown as shaded regions along the X-axis and are colour coded according to the RFU plots. The s.e. for a given peptide represents the sum of the single sigma s.d.'s over all the time-points from three independent HDXMS measurements. RFU after 1 min of deuterium exchange from unassembled DENV2 (c) and DENV1 (d) E-protein is overlaid onto the respective E-protein structures (PDB ID:1OAN, 4CCT). RFU is colour coded according to key. The range has been maintained the same as in for better visual comparison between virion and unassembled E-protein. Comparison of deuterium uptake of identical peptides between viral E-protein and unassembled E-protein from DENV2 (e) and DENV1 (g). SD represents the single s.d. of deuterium uptake for each listed peptide and differences in deuterium uptake were calculated by subtracting deuterium uptake of unassembled E-protein peptides with viral E-protein peptides at 28 °C. Peptides showing large differences in deuterium exchange (indicated in blue) between unassembled and viral E-proteins in DENV2 and DENV1 are mapped onto a raft of three parallel E-dimers (f). Black arrows indicate the regions where the largest magnitude differences in deuterium exchange between unassembled E-protein and viral E-protein were observed.