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.

An alphavirus temperature-sensitive capsid mutant reveals stages of nucleocapsid assembly

“…Morphological studies indicate that the capsid pentamers in CLPs are rotated ∼10° counterclockwise with the density at the fivefold axes projecting outwards, as compared to the NC present in mature, intact virus (Mukhopadhyay et al, 2002). Cryo-EM studies of VEEV cytoplasmic NC and isolated NC from mature virus also indicate conformational differences between these NC states, as well as with that of NC in the mature intact virus (Lamb et al, 2010; Paredes et al, 2003). Variations among these results may reflect differences in the alphaviruses used in these studies and the cellular proteins present in the various NC preparations, as well the technical difficulties of the reconstructions (Mukhopadhyay et al, 2002).…”

“…Biochemical and morphological studies indicate that the structure of cytoplasmic NC differs from that of the NC in released virus particles (Coombs et al, 1984; Lamb et al, 2010), suggesting that the NC organization changes through its association with the envelope protein lattice during budding (Jose et al, 2012; Pletnev et al, 2001). However, the structure of the cytoplasmic NC is sensitive to purification (Mukhopadhyay et al, 2002), complicating its interpretation.…”

The Alphavirus E2 Membrane-Proximal Domain Impacts Capsid Interaction and Glycoprotein Lattice Formation

“…Cryo-electron microscopy (cryo-EM) reconstructions of alphavirus cytoplasmic nucleocapsids, nucleocapsids imaged within virus particles, and nucleocapsids purified from virus particles reveal structural differences (26,27). Comparative studies also suggest differences in sedimentation properties and RNase sensitivity between NCs Interactions of E1-T317 in the glycoprotein lattice.…”

The role of the IKK complex in viral infections

“…An in vitro kinase assay demonstrated that the virulence factor NSs was indeed phosphorylated by the low molecular weight IKK complex 2 alluding to the possibility that RVFV requires a kinase-active IKK complex for phosphorylation of NSs for efficient viral replication (Narayanan et al, 2012). The positive-sense ssRNA virus that belongs to the family Togaviridae, Venezuelan equine encephalitis virus (VEEV) produces a protein that interacts with IKKb (Weaver et al, 2004;Garmashova et al, 2007;Lamb et al, 2010). We have recently reported an activation of the NF-jB cascade in human astrocytes infected with the attenuated strain of VEEV, TC-83.…”

“…The positive-sense ssRNA virus that belongs to the family Togaviridae, Venezuelan equine encephalitis virus (VEEV) produces a protein that interacts with IKKb (Weaver et al, 2004;Garmashova et al, 2007;Lamb et al, 2010). We have recently reported an activation of the NF-jB cascade in human astrocytes infected with the attenuated strain of VEEV, TC-83.…”