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Cryo-EM structure of the mature dengue virus at 3.5-Å resolution

Nature Structural & Molecular Biology volume 20, pages 105–110 (2013)Cite this article

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Abstract

Regulated by pH, membrane-anchored proteins E and M function during dengue virus maturation and membrane fusion. Our atomic model of the whole virion from cryo–electron microscopy at 3.5-Å resolution reveals that in the mature virus at neutral extracellular pH, the N-terminal 20-amino-acid segment of M (involving three pH-sensing histidines) latches and thereby prevents spring-loaded E fusion protein from prematurely exposing its fusion peptide. This M latch is fastened at an earlier stage, during maturation at acidic pH in the trans-Golgi network. At a later stage, to initiate infection in response to acidic pH in the late endosome, M releases the latch and exposes the fusion peptide. Thus, M serves as a multistep chaperone of E to control the conformational changes accompanying maturation and infection. These pH-sensitive interactions could serve as targets for drug discovery.

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Figure 1: Overview of the cryo-EM structure of the dengue virion.
Figure 2: Atomic model of the E-M-M-E heterotetramer.
Figure 3: Hydrophobic interactions.
Figure 4: Key interactions between E and M.
Figure 5: Proposed mechanisms for maturation (stages 1–3) and exposure of the fusion peptide of E required for infection (stage 4).

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Acknowledgements

We thank V. Vordam (Centers for Disease Control Dengue Branch, San Juan, Puerto Rico) for providing the viral stock and advising about cell culture, I. Atanasov and W.H. Hui for participation in data acquisition, J. Jiang for suggestions in data processing, UCLA undergraduate students K.M. Lau, J. Chen and K. Chen and B.K. Zhou of Beverly Vista School for scanning photographic films and boxing particles, and A. Paredes and J.-Q. Zhang for preliminary efforts in viral preparation. This work is supported in part by grants from the US National Institutes of Health grant GM071940 (to Z.H.Z.), National Natural Science Foundation of China (NSFC) grant 30928003 and 30725017 (to G.B.), NSFC 30470085 and 30870480 (to Q.Z.). We acknowledge the use of instruments at the Electron Imaging Center for NanoMachines supported by National Institutes of Health (1S10RR23057) and the California NanoSystems Institute at UCLA.

Author information

Author notes

  1. Jennifer M Brannan

    Present address: Present address: US Army Medical Research Institute, Frederick, Maryland, USA.,

  2. Xiaokang Zhang and Peng Ge: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, California, USA

    Xiaokang Zhang, Peng Ge, Xuekui Yu & Z Hong Zhou

  2. California NanoSystems Institute, UCLA, Los Angeles, California, USA

    Xiaokang Zhang, Peng Ge, Xuekui Yu, Stan Schein & Z Hong Zhou

  3. Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, Texas, USA

    Xiaokang Zhang, Peng Ge, Xuekui Yu, Jennifer M Brannan & Z Hong Zhou

  4. Hefei National Laboratory for Physical Sciences at the Microscale, and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.,

    Xiaokang Zhang, Guoqiang Bi & Z Hong Zhou

  5. School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.,

    Xiaokang Zhang, Guoqiang Bi & Z Hong Zhou

  6. School of Life Sciences, State Key Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou, Guangdong, China.,

    Qinfen Zhang

  7. Department of Psychology, and the Brain Research Institute, UCLA, Los Angeles, California, USA

    Stan Schein

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Contributions

Z.H.Z., X.Z., P.G. and X.Y. designed experiments. J.M.B., X.Z. and X.Y. cultured cells and purified virus samples. X.Z., X.Y., P.G., J.M.B. and Z.H.Z. obtained cryo-EM images. Z.H.Z., J.M.B. and X.Z. participated in the image processing and three-dimensional reconstruction from the Polara data. X.Z. obtained a 7-Å structure from the Polara data. P.G. refined the structure to 3.5-Å resolution with the Titan Krios data and built the atomic models. P.G., X.Z. and Z.H.Z. interpreted the structure and drafted the manuscript. P.G., X.Z., Z.H.Z. and S.S. finalized the manuscript. G.B. and Q.Z. participated in discussion and interpretation of the results. All authors reviewed the final manuscript.

Corresponding author

Correspondence to Z Hong Zhou.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Tables 1 and 2 (PDF 4306 kb)

Supplementary Movie 1

A 3D visualization of various structures described in the figures. The animation begins with a surface rendering of the cryoEM density map, rotating around a 2-fold axis. Structural units containing membrane proteins E and M shown in the same color are equivalent by icosahedral symmetry. The differently colored structural units are quasiequivalent. Specifically, the green units fall on the icosahedral 5-fold axes, the blue on the 3-fold and the red on the 2-fold. This scene is followed by a close up view of a rhombus-shaped group of six E-M dimers, fitted with the ribbon representations of its atomic model, rotating around the horizontal axis. Next, the three quasi-equivalent E-M-M-E heterotetramers are averaged. Rotated around the horizontal axis, half of this averaged tetramer is rendered as a shaded surface representation and half is shown in semi-transparent grey, superimposed with the ribbon representations of an E monomer and an M monomer, with the same color scheme as in Figure 2. (AVI 26509 kb)

Supplementary Movie 2

Interactions between E (molecular surface) and M (sticks). First, an animated view of Figure 4b. Second, an animated view of the molecular surface of E and the ribbon and stick model of M that comprise pocket 1, as shown in Figure 4c and Supplementary Figure 8c. A second pocket 1 in a symmetry related position is also visible in the same view. Third, an animated view of the molecular surface of E and the ribbon and stick model of M that comprise pocket 2, as shown Figure 4d and Supplementary Figure 8e. The histidine in the center of this movie is His7 of M which is involved in the pH sensitive latching of E by M. The two contiguous nitrogen atoms in a nearby bulge above and to the right of this His7 belong to His209 of E. Finally, an animated view of the molecular surface of E and the ribbon and stick model of M that comprise pocket 3 as shown Figure 4e and Supplementary Figure 8g. The surrounding of the central Trp19 of M is highly hydrophobic as indicated by the atom types on the molecular surface of E. (AVI 12159 kb)

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Zhang, X., Ge, P., Yu, X. et al. Cryo-EM structure of the mature dengue virus at 3.5-Å resolution. Nat Struct Mol Biol 20, 105–110 (2013). https://doi.org/10.1038/nsmb.2463

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