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. 2005 Mar;79(6):3822-30.
doi: 10.1128/JVI.79.6.3822-3830.2005.

Complementarity in the supramolecular design of arenaviruses and retroviruses revealed by electron cryomicroscopy and image analysis

Benjamin W Neuman  1 , Brian D Adair, John W Burns, Ronald A Milligan, Michael J Buchmeier, Mark Yeager
Affiliations

Complementarity in the supramolecular design of arenaviruses and retroviruses revealed by electron cryomicroscopy and image analysis

Benjamin W Neuman et al. J Virol. 2005 Mar.

Abstract

Arenaviruses are rodent-borne agents of diseases, including potentially lethal human hemorrhagic fevers. These enveloped viruses encapsidate a bisegmented ambisense single-stranded RNA genome that can be packaged in variable copy number. Electron cryomicroscopy and image analysis of New World Pichinde and Tacaribe arenaviruses and Old World lymphocytic choriomeningitis virus revealed pleomorphic enveloped particles ranging in diameter from approximately 400 to approximately 2,000 A. The surface spikes were spaced approximately 100 A apart and extended approximately 90 A from the maximum phospholipid headgroup density of the outer bilayer leaflet. Distinctive stalk and head regions extended radially approximately 30 and approximately 60 A from the outer bilayer leaflet, respectively. Two interior layers of density apposed to the inner leaflet of the viral lipid bilayer were assigned as protein Z and nucleoprotein (NP) molecules on the basis of their appearance, spacing, and projected volume. Analysis of en face views of virions lacking the GP-C spikes showed reflections consistent with paracrystalline packing of the NP molecules in a lattice with edges of approximately 57 and approximately 74 A. The structural proteins of retroviruses and arenaviruses assemble with similar radial density distributions, using common cellular components.

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Figures

FIG. 1.
FIG. 1.
Electron cryomicroscopy of arenaviruses. Images of native frozen-hydrated Pic (A), Tac (B), and LCM (C) virions are shown. Bar, 500 Å.
FIG. 2.
FIG. 2.
Comparison of arenavirus morphology and size distribution. Electron micrographs of frozen-hydrated virions of Pic (A and F), Tac (B), LCM (C), 1 M LiCl-treated LCM (D), and 1 M NaCl- and pH 5.0-treated LCM (E) are shown. The images in panels A to E were recorded at −1.5 to −1.9 μm under focus to emphasize the two leaflets of the lipid bilayer. The image in panel F was recorded at −3.0 μm under focus to emphasize the surface spikes. The distribution of native Pic, Tac, and LCM virion diameters is shown in panel G. Histograms depict the average number of virions per 4-Å-diameter increment, averaged within a 48-Å sliding window. Bar, 200 Å.
FIG. 3.
FIG. 3.
Structural features of Pic, Tac, and LCM arenaviruses revealed by rotationally averaged radial density profiles. Twelve well-centered, circular virion projections displaying bilayer leaflets of approximately even intensity were aligned and averaged to produce each curve. The schematic interpretation identifies the peaks in the radial density plots with the viral glycoprotein oligomers (GP-C), the lipid bilayer (LB), inner track 1 (IT-1), and inner track 2 (IT-2).
FIG. 4.
FIG. 4.
Surface features as well as inner densities (IT-1, IT-2) revealed by analysis of side views. Class averages of side views reveal the lipid bilayer (LB) and GP-C ectodomain as well as inner densities. A single globular density (D, bottom) or double-lobed projection (D, top) was visible outside the membrane of Pic (A), Tac (B), and LCM (C) viruses. Class averages had double-lobed (D, top) or single-lobed (D, bottom) LCM GP-C projections. Class averages constructed from larger fields of view revealed two concentric layers of density, designated IT-1 and IT-2, for Pic, Tac, and LCM (E, F, and G, respectively) that were closely apposed to the inner bilayer leaflet. Beneath the GP-C density were thin threads of density that connected the IT-1 and IT-2 layers. Bars, 50 Å. The images were masked so as to concentrate on two different regions: the GP-C ectodomain (A, B, and C) and the inner track densities (IT-1 and IT-2) at lower radii (E, F, and G). Boxes A to D have been resized relative to E to G to highlight structural features.
FIG. 5.
FIG. 5.
Paracrystalline lattice of NP revealed by analysis of en face images. Computed diffraction patterns of class averages (large images) were constructed for en face views from Pic (A and E), Tac (B and F), LCM (C and G), and LiCl-treated LCM (D and H). Images were analyzed individually (large images A to D), or raw images were aligned to class averages, Fourier transformed, and averaged in real space to produce composite images (large images E to H). The dimensions of the paracrystalline lattice are shown in panel I.
FIG. 6.
FIG. 6.
(A) Schematic representation of the arenavirus and murine leukemia virus organization (based on reference used with permission of the National Academy of Sciences). The murine leukemia virus Gag domains matrix (MA), P12, capsid (CA), and nucleocapsid (NC) are shown assembled at the viral lipid bilayer (LB) on the left, while the arenavirus GP, Z, and NP proteins are depicted on the right. (B) Schematic representation of clathrin-coated vesicle organization, showing the position of the “cargo” protein, adaptor protein complex (AP), and clathrin coat (CLA). Bar, ∼100 Å.
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