Structure of a Venezuelan equine encephalitis virus assembly intermediate isolated from infected cells

Abstract

Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to the mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.

Figure 1

Electron micrographs of negatively stained thin-sections of BHK cells. (A) Uninfected BHK cells. (B) Sections of BHK cells 19 hr post-infection with VEEV TC-83. (C) Immuno-gold labeled thin-section of uninfected BHK cells. (D) Immuno-gold labeled thin-section of BHK cells infected with VEEV TC-83. The sections of BHK cells were labeled with primary antibody against the C-terminus of VEEV capsid protein and 5 nm gold-conjugated secondary antibody. In panels (B) and (D), the arrows point to pre-viral VEEV nucleocapsids. Scale bars = 100 nm.

Figure 2

Electron micrographs of purified pre-viral nucleocapsid particles. (A) Electron micrograph of negatively stained pre-viral nucleocapsid particles recovered from the cushion stage of particle purification. Scale bar = 100 nm. (B) Electron micrograph of selected immuno-gold labeled (anti-VEEV capsid antibody) pre-viral nucleocapsid particles recovered from the cushion stage of particle purification. Scale bar = 50 nm. (C) Electron micrograph of vitrified pre-viral nucleocapsid particles recovered from the cushion stage of particle purification. Inserts in panels A and C show representative particles enlarged for clarity. Scale bar = 100 nm.

Figure 3

Protein composition of purified pre-viral nucleocapsids. (A) Coomassie blue staining of proteins from pre-viral nucleocapsid particles recovered from the cushion stage of particle purification and separated with SDS-PAGE. Molecular weight markers are shown along the left-hand side. (B) Coomassie blue staining of proteins from pre-viral nucleocapsid particles recovered from iodixanol gradient fractions and separated with SDS-PAGE. The iodixanol concentration of each fraction is listed above each lane. (C) Western blot (anti-VEEV capsid antibody) of proteins from pre-viral nucleocapsid particles recovered from iodixanol gradient fractions and separated with SDS-PAGE.

Figure 4

Pre-viral nucleocapsid diameters. (A) Frequency distribution plot of diameters of pre-viral nucleocapsids (n=200) recovered from the cushion stage of purification. The dashed line shows a Gaussian curve fit to the data points. Particle diameters were measured from micrographs of vitrified particles. Measurement errors were ±1.8 nm, based on multiple diameter measurements for each particle. (B) Average diameter of pre-viral nucleocapsids (n=100) recovered from distinct iodixanol gradient fractions. Particle diameters were measured from electron micrographs of negatively stained particles. Error bars were ±2 nm, based on multiple diameter measurements for each particle.

Figure 5

Three-dimensional reconstruction of an icosahedral VEEV pre-viral nucleocapsid assembly state. (A) Map of the pre-viral nucleocapsid viewed along an icosahedral 3-fold axis. Starting model for this reconstruction was an Auto3dem-generated map. (B) Orientations of particles used for the pre-viral nucleocapsid reconstruction plotted relative to the asymmetric unit.

Figure 6

Radial analysis of icosahedral pre-viral nucleocapsid particles. (A) Central section through the pre-viral nucleocapsid particle, viewed along an icosahedral 2-fold axis. (B) Radial density plot of the pre-viral nucleocapsid map.

Figure 7

Capsid proteins modeled within the pre-viral nucleocapsid. (A) Twelve C-terminal domains of the VEEV capsid protein positioned within an icosahedral face of the pre-viral nucleocapsid particle and viewed along an icosahedral 3-fold axis. (B) Side view of the pre-viral nucleocapsid highlighting the arrangement of twelve C-terminal domains of the VEEV capsid protein within an icosahedral face of the particle.