Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Sep 19;15(9):e1007996.
doi: 10.1371/journal.ppat.1007996. eCollection 2019 Sep.

Molecular Basis of Dengue Virus Serotype 2 Morphological Switch From 29°C to 37°C

Affiliations
Free PMC article

Molecular Basis of Dengue Virus Serotype 2 Morphological Switch From 29°C to 37°C

Xin-Ni Lim et al. PLoS Pathog. .
Free PMC article

Abstract

The ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to "bumpy" surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become "bumpy". These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. DENV2 NGC strains have different morphologies at 37°C which may be attributed to differences in their E protein sequences.
(A) CryoEM micrographs of the lab-adapted DENV2 NGC strains with different passage histories (NGC-1 and NGC-2) at 29°C and 37°C. (B) (Top panel) Sequence alignment of NGC-1 with NGC-2, and (bottom panel) their residue differences indicated on an E protein raft on the virus surface. (C) List of mutations in M1-4 viruses, and (D) the cryoEM micrographs showing their morphologies at 29°C and 37°C. Scale bar is 50nm.
Fig 2
Fig 2. Characterization of the thermal stability and growth curve of the DENV2 strains and mutants.
Thermal stability of (A) DENV2 NGC strains and mutants, and (B) clinical isolates (Table 1) at various temperatures. The y-axis shows the percentage of PFU at different temperatures divided by that of the same virus at 4°C. *P<0.01 (paired t-test for A, B). (C) Growth curve of DENV2 NGC-1, M1, M3 and M4 in both mammalian (HuH-7, BHK-21) and mosquito cells (C6/36) detected by plaque assays (HuH-7, BHK-21), focus-forming assay (C6/36) and RT-PCR (HuH-7, BHK-21 and C6/36). Dashed line represents the limit of detection of the assay. All assays have been repeated at least 3 times.
Fig 3
Fig 3. Morphology of DENV2 clinical strains at different temperatures.
(A) CryoEM micrographs of DENV2 clinical isolates (PVP94/07, PVP103/07, 05K4155 and SL56) at pH7.4 at 4°C, 37°C and 40°C. Scale bar is 50nm. (B) (Right) Sequence alignment of PVP94/07 and PVP103/07 showing only one residue difference at position 262 of E protein, and (left) its location on an E protein raft on the virus surface.
Fig 4
Fig 4. Characterization of the smooth PVP94/07 and bumpy surfaced PVP103/07 DENV2 clinical isolates.
(A-E) Neutralization profiles of MAbs (A) 4G2, (B) 4.8A, (C) 1A1D-2 (D) 2D22 and (E) C10 against PVP94/07 and PVP103/07. Data points represent average of n = 3 experiments with SD error bar. (F) Comparison of the attachment ability of PVP103/07 and PVP94/07 to C6/36 and HuH-7 cells. Y-axis shows genome copies level relative to PVP94/07, details of the formula is in the methods section. More bumpy surface particles (PVP103/07) are observed to bind to HuH-7 than the smooth surfaced PVP94/07 while no significant difference is detected when these virus are exposed to C6/36. Each data point represents one independent experiment with error bar representing SD. (* p<0.05) (G) Fusion assay of PVP94/07 and PVP103/07 with liposomes showed that bumpy surfaced PVP103/07, has a better fusion efficiency to liposomes. The average with standard error calculated from averaging the replicates of three individual experiments each with triplicates is presented. A two-way ANOVA followed with Fisher’s LSD test was carried out to compare the extent of fusion (%) of DENV2 PVP94/07 with DENV2 PVP103/07 at pH 5.0. ***p = 0.0010 shows the fusion efficiency is significantly different between PVP94/07 and PVP103/07.
Fig 5
Fig 5. MD simulations showing the stability of the N-terminal loop of the E protein protomers within a dimer in the EI6 and EM6 systems.
The initial (t = 0 ns, top) and final (t = 300 ns, bottom) conformations of the N-terminal loop (residues 1–10) are shown for both protomers (chains A and B) of the (A) EI6 and (B) EM6 systems. The protein backbone is shown as ribbons and colored according to the secondary structure. Residue number 6 is shown as sphere (carbon in cyan, nitrogen in blue, oxygen in red and hydrogen in grey). The per-residue secondary structure propensity for the N-terminal loop over simulation time is shown for each chain in the (C) EI6 and (D) EM6 systems.
Fig 6
Fig 6. MD simulations showing stability of the central helical interface of the E proteins within a dimer in both ET262 and EM262 systems.
The initial (t = 0 ns, top) and final (t = 300 ns, bottom) states, visualized along the dimer axis, are shown for the (A) ET262 and (B) EM262 systems. Key residues side chains are shown as stick representation (carbon in cyan, nitrogen in blue, oxygen in red and hydrogen in grey) and are accordingly labeled. The remainder of the protein is shown as ribbon and colored according to the secondary structure (310-helix in blue, α-helix in purple, extended configuration in yellow, loop in cyan, coil in white). A side-on view of this region (residues 250–265 of both chains) is shown for the final states of (C) ET262 and (D) EM262, with either chain colored in blue or red. In (E), the minimum distance over simulation time between residue number 262 from opposing chains is shown for the two systems. In (F), existence maps for the intra-helical hydrogen bond between the T262 side chain hydroxyl and the G258 backbone carbonyl oxygen are shown for each E protein chain in the ET262 system.
Fig 7
Fig 7. MD simulations showing conformational changes in the E protein dimer induced by EI6 to EM6 mutation.
The two extreme conformations generated by principal component analysis (PCA) from a filtered trajectory representing the dominant motion of the E protein dimer in EM6 are shown. The protein is shown as ribbons, with red, yellow, and blue corresponding to domains I, II, and III, respectively. The helical interface (residues 250–265) between the two chains of the dimer is shown in cyan. M6 backbone atoms are shown as green spheres. Curly arrows represent the directions of motion of domains I, II, and III.
Fig 8
Fig 8. MD simulations showing conformational changes in the E protein dimer induced by mutation of ET262 to EM262.
(A) The total buried surface area between helical residues 250–265 from opposing chains is shown with respect to simulation time for both systems. (B) The two extreme conformations generated by principal component analysis (PCA) from a filtered trajectory representing the dominant motion of the E protein dimer in EM262 The protein is shown as ribbons, with red, yellow and blue corresponding to domains I, II, and III respectively. M262 backbone atoms are shown as green spheres. Curly arrows represent the directions of motion of domains I, II, and III.

Similar articles

See all similar articles

Cited by 1 article

References

    1. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al. The global distribution and burden of dengue. Nature. 2013;496(7446):504–7. 10.1038/nature12060 - DOI - PMC - PubMed
    1. The live attenuated dengue vaccine TV003 elicits complete protection against dengue in a human challenge model.pdf>. - PubMed
    1. Rico-Hesse R. Molecular evolution and distribution of dengue viruses type 1 and 2 in nature. Virology. 1990;174(2):479–93. 10.1016/0042-6822(90)90102-w - DOI - PubMed
    1. Holmes E, Twiddy S. The origin, emergence and evolutionary genetics of dengue virus. Infection, Genetics and Evolution. 2003;3(1):19–28. - PubMed
    1. Halstead SB, O'Rourke EJ. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J Exp Med. 1977;146(1):201–17. 10.1084/jem.146.1.201 - DOI - PMC - PubMed

Publication types

MeSH terms

Feedback