Carmen Butan

Cryo-electron microscopy reconstruction shows poliovirus 135S particles poised for membrane interaction and RNA release

ABSTRACT During infection, binding of mature poliovirus to cell surface receptors induces an irreversible expansion of the capsid, to form an infectious cell-entry intermediate particle that sediments at 135S. In these expanded virions, the major capsid proteins (VP1 to VP3) adopt an altered icosahedral arrangement to open holes in the capsid at 2-fold and quasi-3-fold axes, and internal polypeptides VP4 and the N terminus of VP1, which can bind membranes, become externalized. Cryo-electron microscopy images for 117,330 particles were collected using Leginon and reconstructed using FREALIGN. Improved rigid-body positioning of major capsid proteins established reliably which polypeptide segments become disordered or rearranged. The virus-to-135S transition includes expansion of 4%, rearrangements of the GH loops of VP3 and VP1, and disordering of C-terminal extensions of VP1 and VP2. The N terminus of VP1 rearranges to become externalized near its quasi-3-fold exit, binds to rearranged GH loops of VP3 and VP1, and attaches to the top surface of VP2. These details improve our understanding of subsequent stages of infection, including endocytosis and RNA transfer into the cytoplasm.

Spiral Architecture of the Nucleoid in Bdellovibrio bacteriovorus

We present a cryo-electron tomographic analysis of the three-dimensional architecture of a strain of the Gram-negative bacterium Bdellovibrio bacteriovorus in which endogenous MreB2 was replaced with monomeric teal fluorescent protein (mTFP)-labeled MreB2. In contrast to wild-type Bdellovibrio cells that predominantly displayed a compact nucleoid region, cells expressing mTFP-labeled MreB2 displayed a twisted spiral organization of the nucleoid. The more open structure of the MreB2-mTFP nucleoids enabled clear in situ visualization of ribosomes decorating the periphery of the nucleoid. Ribosomes also bordered the edges of more compact nucleoids from both wild-type cells and mutant cells. Surprisingly, MreB2-mTFP localized to the interface between the spiral nucleoid and the cytoplasm, suggesting an intimate connection between nucleoid architecture and MreB arrangement. Further, in contrast to wild-type cells, where a single tight chemoreceptor cluster localizes close to the single polar flagellum, MreB2-mTFP cells often displayed extended chemoreceptor arrays present at one or both poles and displayed multiple or inaccurately positioned flagella. Our findings provide direct structural evidence for spiral organization of the bacterial nucleoid and suggest a possible role for MreB in regulation of nucleoid architecture and localization of the chemotaxis apparatus.

Structure and Assembly of the RNA Binding Domain of Bluetongue Virus Non-structural Protein 2*

Bluetongue virus non-structural protein 2 belongs to a class of highly conserved proteins found in orbiviruses of the Reoviridae family. Non-structural protein 2 forms large multimeric complexes and localizes to cytoplasmic inclusions in infected cells. It is able to bind single-stranded RNA non-specifically, and it has been suggested that the protein is involved in the selection and condensation of the Bluetongue virus RNA segments prior to genome encapsidation. We have determined the x-ray structure of the N-terminal domain (sufficient for the RNA binding ability of non-structural protein 2) to 2.4 Å resolution using anomalous scattering methods. Crystals of this apparently insoluble domain were obtained by in situ proteolysis of a soluble construct. The asymmetric unit shows two monomers related by non-crystallographic symmetry, with each monomer folded as a β sandwich with a unique topology. The crystal structure reveals extensive monomer-monomer interactions, which explain the ability of the protein to self-assemble into large homomultimeric complexes. Of the entire surface area of the monomer, one-third is used to create the interfaces of the curved multimeric assembly observed in the x-ray structure. The structure reported here shows how the N-terminal domain would be able to bind single-stranded RNA non-specifically protecting the bound regions in a heterogeneous multimeric but not polymeric complex.

Visualization of the Two-Step Fusion Process of the Retrovirus Avian Sarcoma/Leukosis Virus by Cryo-Electron Tomography

ABSTRACT Retrovirus infection starts with the binding of envelope glycoproteins to host cell receptors. Subsequently, conformational changes in the glycoproteins trigger fusion of the viral and cellular membranes. Some retroviruses, such as avian sarcoma/leukosis virus (ASLV), employ a two-step mechanism in which receptor binding precedes low-pH activation and fusion. We used cryo-electron tomography to study virion/receptor/liposome complexes that simulate the interactions of ASLV virions with cells. Binding the soluble receptor at neutral pH resulted in virions capable of binding liposomes tightly enough to alter their curvature. At virion-liposome interfaces, the glycoproteins are ∼3-fold more concentrated than elsewhere in the viral envelope, indicating specific recruitment to these sites. Subtomogram averaging showed that the oblate globular domain in the prehairpin intermediate (presumably the receptor-binding domain) is connected to both the target and the viral membrane by 2.5-nm-long stalks and is partially disordered, compared with its native conformation. Upon lowering the pH, fusion took place. Fusion is a stochastic process that, once initiated, must be rapid, as only final (postfusion) products were observed. These fusion products showed glycoprotein spikes on their surface, with their interiors occupied by patches of dense material but without capsids, implying their disassembly. In addition, some of the products presented a density layer underlying and resolved from the viral membrane, which may represent detachment of the matrix protein to facilitate the fusion process.

Insights into the role of the non-structural protein 2 (NS2) in Bluetongue virus morphogenesis.

Bluetongue virus (BTV) non-structural protein 2 (NS2) belongs to a class of highly conserved proteins found in Orbiviruses of the Reoviridae family. NS2 forms large multimeric complexes and localizes to cytoplasmic inclusions in infected cells. Due to its ability to bind single-stranded RNA (ssRNA), it has been suggested that the protein participates in the selection and sequestration of the 10 different BTV-ssRNA segments, prior to their encapsidation and conversion into the BTV double-stranded RNA (dsRNA) genome. Recent advances in understanding how BTV-NS2 is organized and functions are largely inferred from structural studies. The X-ray crystal structure of the N-terminal domain of NS2 suggests that the full-length protein could assemble as homomultimers of maximally 10-11 subunits. The crystallographic structural information combined with small-angle X-ray scattering experiments on the C-terminal domain as well as negative-stain electron microscopy on the full-length protein give us a first glimpse of how the two protein domains associate and function. Herein, we survey biochemical and recent structural investigations on NS2 important to the understanding of the molecular events underlying the process of BTV morphogenesis. We also present a phylogenetic analysis of the NS2 sequences.

Suppression of a Morphogenic Mutant in Rous Sarcoma Virus Capsid Protein by a Second-Site Mutation: a Cryoelectron Tomography Study

ABSTRACT Retrovirus assembly is driven by polymerization of the Gag polyprotein as nascent virions bud from host cells. Gag is then processed proteolytically, releasing the capsid protein (CA) to assemble de novo inside maturing virions. CA has N-terminal and C-terminal domains (NTDs and CTDs, respectively) whose folds are conserved, although their sequences are divergent except in the 20-residue major homology region (MHR) in the CTD. The MHR is thought to play an important role in assembly, and some mutations affecting it, including the F167Y substitution, are lethal. A temperature-sensitive second-site suppressor mutation in the NTD, A38V, restores infectivity. We have used cryoelectron tomography to investigate the morphotypes of this double mutant. Virions produced at the nonpermissive temperature do not assemble capsids, although Gag is processed normally; moreover, they are more variable in size than the wild type and have fewer glycoprotein spikes. At the permissive temperature, virions are similar in size and spike content as in the wild type and capsid assembly is restored, albeit with altered polymorphisms. The mutation F167Y-A38V (referred to as FY/AV in this paper) produces fewer tubular capsids than wild type and more irregular polyhedra, which tend to be larger than in the wild type, containing ∼30% more CA subunits. It follows that FY/AV CA assembles more efficiently in situ than in the wild type and has a lower critical concentration, reflecting altered nucleation properties. However, its infectivity is lower than that of the wild type, due to a 4-fold-lower budding efficiency. We conclude that the wild-type CA protein sequence represents an evolutionary compromise between competing requirements for optimization of Gag assembly (of the immature virion) and CA assembly (in the maturing virion).

Chapter 4:Three-dimensional Structures of Pleiomorphic Viruses from Cryo-Electron Tomography

The past two decades have seen remarkable progress in structural studies of viruses that possess icosahedral capsids, stemming from technical advances in cryo-EM1,2 and X-ray crystallography.3 Among other insights, it has emerged that protein subunits with some half-dozen different folds assemble in...