Abstract:

Structures of prM-containing dengue and yellow fever virus particles were determined to 16 and 25 A resolution, respectively, by cryoelectron microscopy and image reconstruction techniques. The closely similar structures show 60 icosahedrally organized trimeric spikes on the particle surface. Each spike consists of three prM:E heterodimers, where E is an envelope glycoprotein and prM is the precursor to the membrane protein M. The pre-peptide components of the prM proteins in each spike cover the fusion peptides at the distal ends of the E glycoproteins in a manner similar to the organization of the glycoproteins in the alphavirus spikes. Each heterodimer is associated with an E and a prM transmembrane density. These transmembrane densities represent either an EE or prMprM antiparallel coiled coil by which each protein spans the membrane twice, leaving the C-terminus of each protein on the exterior of the viral membrane, consistent with the predicted membrane-spanning domains of the unprocessed polyprotein.

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Structures of immature flavivirus particles

Department of Biological Sciences, Lilly Hall, 915 West State Street, Purdue University, West Lafayette, IN 47907-2054 and ^{Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA}

^{Present address: Department of Medical Microbiology, Leiden University Medical Center (LUMC), NL-2300 RC Leiden, The Netherlands}

^{Present address: NCI-Frederick MCL, Building 539, Frederick, MD 21702, USA}

^{Corresponding authors e-mail:}ude.eudrup.oib.ggarb@nhukjr or ude.eudrup.oib.anaidni@rgm

Received 2003 Jan 29; Revised 2003 Apr 4; Accepted 2003 Apr 9.

Abstract

Structures of prM-containing dengue and yellow fever virus particles were determined to 16 and 25 Å resolution, respectively, by cryoelectron microscopy and image reconstruction techniques. The closely similar structures show 60 icosahedrally organized trimeric spikes on the particle surface. Each spike consists of three prM:E heterodimers, where E is an envelope glycoprotein and prM is the precursor to the membrane protein M. The pre-peptide components of the prM proteins in each spike cover the fusion peptides at the distal ends of the E glycoproteins in a manner similar to the organization of the glycoproteins in the alphavirus spikes. Each heterodimer is associated with an E and a prM transmembrane density. These transmembrane densities represent either an EE or prMprM antiparallel coiled coil by which each protein spans the membrane twice, leaving the C-terminus of each protein on the exterior of the viral membrane, consistent with the predicted membrane-spanning domains of the unprocessed polyprotein.

Keywords: conformational changes/dengue virus/EM reconstruction/immature particles

Abstract

For more details of the EMfit program parameters, see Rossmann et al., 2001.

^{Mol, red, blue and green correspond to the E monomers shown in Figure}2B.

^sumf₁, sumf₂, and sumf₃ are the average densities of C_α atoms for domains I, II and III, respectively, expressed as a percentage of the highest density in the map.

^-den is the percentage of atoms in negative density.

^centx, centy and centz are the refined coordinates of the center of mass of the E monomer in the map. Note the consistency of the results, independent of the order of fitting.

^θ₁, θ₂ and θ₃ are the refined Eulerian angles required to rotate the atomic coordinates of E into position. Note the consistency of the results.

^{Possible fitting procedures: (1) blue molecule (first), followed by green molecule (second), followed by red molecule (third); (2) red molecule (first), followed by blue molecule (second), followed by green molecule (third); (3) green molecule (first), followed by red molecule (second), followed by blue molecule (third).}

^{Differentiation between E and prM was established by fitting the structure of three Es into the map. The pre-peptide volume was assumed to be all the globular volume covering the top of E. The volumes are the average of three copies of each protein.}

^{The microscope magnification was a nominal 33 000 for all images.}

^{PFT CC is the polar Fourier transform correlation coefficient (}Baker and Cheng, 1996).

Acknowledgements

We thank Suchetana (Tuli) Mukhopadhyay and Ellen Strauss for many helpful discussions, Rob Ashmore, Chuan (River) Xiao, Yongchang Ji and Dan Marinescu for the use of their various computer programs that were essential for calculating the image reconstruction of the prM particles, and Cheryl Towell and Sharon Wilder for help in preparing the manuscript. We are grateful for an equipment grant from the Keck Foundation that provided the CM300 electron microscope. The work was supported by an NIH Project Program Grant (AI 45976) to M.G.R., R.J.K. and T.S.B., NIH grants to T.S.B. (GM33050) and J.H.S. (AI 10793) and an NSF grant to T.S.B. (DBI 9986316).

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