Bluetongue virus capsid protein VP5 perforates membranes at low endosomal pH during viral entry.

Bluetongue virus (BTV) is a non-enveloped virus and causes substantial morbidity and mortality in ruminants such as sheep. Fashioning a receptor-binding protein (VP2) and a membrane penetration protein (VP5) on the surface, BTV releases its genome-containing core (VP3 and VP7) into the host cell cytosol after perforation of the endosomal membrane. Unlike enveloped ones, the entry mechanisms of non-enveloped viruses into host cells remain poorly understood. Here we applied single-particle cryo-electron microscopy, cryo-electron tomography and structure-guided functional assays to characterize intermediate states of BTV cell entry in endosomes. Four structures of BTV at the resolution range of 3.4-3.9 Å show the different stages of structural rearrangement of capsid proteins on exposure to low pH, including conformational changes of VP5, stepwise detachment of VP2 and a small shift of VP7. In detail, sensing of the low-pH condition by the VP5 anchor domain triggers three major VP5 actions: projecting the hidden dagger domain, converting a surface loop to a protonated ß-hairpin that anchors VP5 to the core and stepwise refolding of the unfurling domains into a six-helix stalk. Cryo-electron tomography structures of BTV interacting with liposomes show a length decrease of the VP5 stalk from 19.5 to 15.5 nm after its insertion into the membrane. Our structures, functional assays and structure-guided mutagenesis experiments combined indicate that this stalk, along with dagger domain and the WHXL motif, creates a single pore through the endosomal membrane that enables the viral core to enter the cytosol. Our study unveils the detailed mechanisms of BTV membrane penetration and showcases general methods to study cell entry of other non-enveloped viruses.

An Early Block in the Replication of the Atypical Bluetongue Virus Serotype 26 in Culicoides Cells Is Determined by Its Capsid Proteins.

Arboviruses such as bluetongue virus (BTV) replicate in arthropod vectors involved in their transmission between susceptible vertebrate-hosts. The "classical" BTV strains infect and replicate effectively in cells of their insect-vectors (Culicoides biting-midges), as well as in those of their mammalian-hosts (ruminants). However, in the last decade, some "atypical" BTV strains, belonging to additional serotypes (e.g., BTV-26), have been found to replicate efficiently only in mammalian cells, while their replication is severely restricted in Culicoides cells. Importantly, there is evidence that these atypical BTV are transmitted by direct-contact between their mammalian hosts. Here, the viral determinants and mechanisms restricting viral replication in Culicoides were investigated using a classical BTV-1, an "atypical" BTV-26 and a BTV-1/BTV-26 reassortant virus, derived by reverse genetics. Viruses containing the capsid of BTV-26 showed a reduced ability to attach to Culicoides cells, blocking early steps of the replication cycle, while attachment and replication in mammalian cells was not restricted. The replication of BTV-26 was also severely reduced in other arthropod cells, derived from mosquitoes or ticks. The data presented identifies mechanisms and potential barriers to infection and transmission by the newly emerged "atypical" BTV strains in Culicoides.

Bluetongue virus assembly and exit pathways.

Bluetongue virus (BTV) is an insect-vectored emerging pathogen of wild ruminants and livestock in many parts of the world. The virion particle is a complex structure of consecutive layers of protein surrounding a genome of 10 double-stranded (ds) RNA segments. BTV has been studied extensively as a model system for large, nonenveloped dsRNA viruses. A combination of recombinant proteins and particles together with reverse genetics, high-resolution structural analysis by X-ray crystallography and cryo-electron microscopy techniques have been utilized to provide an order for the assembly of the capsid shell and the protein sequestration required for it. Further, a reconstituted in vitro assembly system and RNA-RNA interaction assay, have defined the individual steps required for the assembly and packaging of the 10-segmented RNA genome. In addition, various microscopic techniques have been utilized to illuminate the stages of virus maturation and its egress via multiple pathways. These findings have not only given an overall understanding of BTV assembly and morphogenesis but also indicated that similar assembly and egress pathways are likely to be used by related viruses and provided an informed starting point for intervention or prevention.

Bluetongue Virus Nonstructural Protein 3 Orchestrates Virus Maturation and Drives Non-Lytic Egress via Two Polybasic Motifs.

Bluetongue virus (BTV) is an arthropod-borne virus that infects domestic and wild ruminants. The virion is a non-enveloped double-layered particle with an outer capsid that encloses a core containing the segmented double-stranded RNA genome. Although BTV is canonically released by cell lysis, it also exits non-lytically. In infected cells, the BTV nonstructural glycoprotein 3 (NS3) is found to be associated with host membranes and traffics from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane. This suggests a role for NS3 in BTV particle maturation and non-lytic egress. However, the mechanism by which NS3 coordinates these events has not yet been elucidated. Here, we identified two polybasic motifs (PMB1/PMB2), consistent with the membrane binding. Using site-directed mutagenesis, confocal and electron microscopy, and flow cytometry, we demonstrated that PBM1 and PBM2 mutant viruses retained NS3 either in the Golgi apparatus or in the endoplasmic reticulum, suggesting a distinct role for each motif. Mutation of PBM2 motif decreased NS3 export to the cell surface and virus production. However, both mutant viruses produced predominantly inner core particles that remained close to their site of assembly. Together, our data demonstrates that correct trafficking of the NS3 protein is required for virus maturation and release.

In situ structures of RNA-dependent RNA polymerase inside bluetongue virus before and after uncoating.

Bluetongue virus (BTV), a major threat to livestock, is a multilayered, nonturreted member of the Reoviridae, a family of segmented dsRNA viruses characterized by endogenous RNA transcription through an RNA-dependent RNA polymerase (RdRp). To date, the structure of BTV RdRp has been unknown, limiting our mechanistic understanding of BTV transcription and hindering rational drug design effort targeting this essential enzyme. Here, we report the in situ structures of BTV RdRp VP1 in both the triple-layered virion and double-layered core, as determined by cryo-electron microscopy (cryoEM) and subparticle reconstruction. BTV RdRp has 2 unique motifs not found in other viral RdRps: a fingernail, attached to the conserved fingers subdomain, and a bundle of 3 helices: 1 from the palm subdomain and 2 from the N-terminal domain. BTV RdRp VP1 is anchored to the inner surface of the capsid shell via 5 asymmetrically arranged N termini of the inner capsid shell protein VP3A around the 5-fold axis. The structural changes of RdRp VP1 and associated capsid shell proteins between BTV virions and cores suggest that the detachment of the outer capsid proteins VP2 and VP5 during viral entry induces both global movements of the inner capsid shell and local conformational changes of the N-terminal latch helix (residues 34 to 51) of 1 inner capsid shell protein VP3A, priming RdRp VP1 within the capsid for transcription. Understanding this mechanism in BTV also provides general insights into RdRp activation and regulation during viral entry of other multilayered, nonturreted dsRNA viruses.

A blind deconvolution approach for improving the resolution of cryo-EM density maps.

Cryo-electron microscopy (cryo-EM) plays an increasingly prominent role in structure elucidation of macromolecular assemblies. Advances in experimental instrumentation and computational power have spawned numerous cryo-EM studies of large biomolecular complexes resulting in the reconstruction of three-dimensional density maps at intermediate and low resolution. In this resolution range, identification and interpretation of structural elements and modeling of biomolecular structure with atomic detail becomes problematic. In this article, we present a novel algorithm that enhances the resolution of intermediate- and low-resolution density maps. Our underlying assumption is to model the low-resolution density map as a blurred and possibly noise-corrupted version of an unknown high-resolution map that we seek to recover by deconvolution. By exploiting the nonnegativity of both the high-resolution map and blur kernel, we derive multiplicative updates reminiscent of those used in nonnegative matrix factorization. Our framework allows for easy incorporation of additional prior knowledge such as smoothness and sparseness, on both the sharpened density map and the blur kernel. A probabilistic formulation enables us to derive updates for the hyperparameters; therefore, our approach has no parameter that needs adjustment. We apply the algorithm to simulated three-dimensional electron microscopic data. We show that our method provides better resolved density maps when compared with B-factor sharpening, especially in the presence of noise. Moreover, our method can use additional information provided by homologous structures, which helps to improve the resolution even further.

A novel method for purifying bluetongue virus with high purity by co-immunoprecipitation with agarose protein A.

BACKGROUND: Bluetongue virus (BTV) is an icosahedral non-enveloped virus within the genus Orbivirus of Reoviridae and exists as 24 distinct serotypes. BTV can infect all ruminant species and causes severe sickness in sheep. Recently, it was reported that BTV can infect some human cancer cells selectively. Because of the important oncolysis of this virus, we developed a novel purifying method for large-scale production. The purifying logic is simple, which is picking out all the components unwanted and the left is what we want. The process can be summarized in 4 steps: centrifugation, pulling down cell debrises and soluble proteins by co-immunoprecipitation with agarose Protein A, dialysis and filtration sterilization after concentration. RESULTS: The result of transmission electron microscope (TEM) observation showed that the sample of purified virus has a very clear background and the virions still kept intact. The result of 50% tissue culture infective dose (TCID(50)) assay showed that the bioactivity of purified virus is relatively high. CONCLUSIONS: This method can purify BTV-10 with high quality and high biological activity on large-scale production. It also can be used for purifying other BTV serotypes.

Bluetongue virus: dissection of the polymerase complex.

Bluetongue is a vector-borne viral disease of ruminants that is endemic in tropical and subtropical countries. Since 1998 the virus has also appeared in Europe. Partly due to the seriousness of the disease, bluetongue virus (BTV), a member of genus Orbivirus within the family Reoviridae, has been a subject of intense molecular study for the last three decades and is now one of the best understood viruses at the molecular and structural levels. BTV is a complex non-enveloped virus with seven structural proteins arranged in two capsids and a genome of ten double-stranded (ds) RNA segments. Shortly after cell entry, the outer capsid is lost to release an inner capsid (the core) which synthesizes capped mRNAs from each genomic segment, extruding them into the cytoplasm. This requires the efficient co-ordination of a number of enzymes, including helicase, polymerase and RNA capping activities. This review will focus on our current understanding of these catalytic proteins as derived from the use of recombinant proteins, combined with functional assays and the in vitro reconstitution of the transcription/replication complex. In some cases, 3D structures have complemented this analysis to reveal the fine structural detail of these proteins. The combined activities of the core enzymes produce infectious transcripts necessary and sufficient to initiate BTV infection. Such infectious transcripts can now be synthesized wholly in vitro and, when introduced into cells by transfection, lead to the recovery of infectious virus. Future studies thus hold the possibility of analysing the consequence of mutation in a replicating virus system.

Mapping the assembly pathway of Bluetongue virus scaffolding protein VP3.

The structure of the Bluetongue virus (BTV) core and its outer layer VP7 has been solved by X-ray crystallography, but the assembly intermediates that lead to the inner scaffolding VP3 layer have not been defined. In this report, we addressed two key questions: (a) the role of VP3 amino terminus in core assembly and its interaction with the transcription complex (TC) components; and (b) the assembly intermediates involved in the construction of the VP3 shell. To do this, deletion mutants in the amino terminal and decamer-decamer interacting region of VP3 (DeltaDD) were generated, expressed in insect cells using baculovirus expression systems, and their ability to assemble into core-like particles (CLPs) and to incorporate the components of TC were investigated. Deletion of the N-terminal 5 (Delta5N) or 10 (Delta10N) amino acids did not affect the ability to assemble into CLPs in the presence of VP7 although the cores assembled using the 10 residue mutant (Delta10N) deletion were very unstable. Removal of five residues also did not effect incorporation of the internal VP1 RNA polymerase and VP4 mRNA capping enzyme proteins of the TC. Removal of the VP3-VP3 interacting domain (DeltaDD) led to failure to assemble into CLPs yet retained interaction with VP1 and VP4. In solution, purified DeltaDD mutant protein readily multimerized into dimers, pentamers, and decamers, suggesting that these oligomers are the authentic assembly intermediates of the subcore. However, unlike wild-type VP3 protein, the dimerization domain-deleted assembly intermediates were found to have lost RNA binding ability. Our study emphasizes the requirement of the N-terminus of VP3 for binding and encapsidation of the TC components, and defines the role of the dimerization domain in subcore assembly and RNA binding.

The bluetongue virus core: a nano-scale transcription machine.

The replication phase of the bluetongue virus (BTV) infection cycle is initiated when the virus core is delivered into the cytoplasm of a susceptible host cell. The 10 segments of the viral genome remain packaged within the core throughout the replication cycle, helping to prevent the activation of host defence mechanisms that would be caused by direct contact between the dsRNA and the host cell cytoplasm. However, the BTV core is a biochemically active 'nano-scale' machine, which can simultaneously and repeatedly transcribe mRNA from each of the 10 genome segments, which are packaged as a liquid crystal array within a central cavity. These mRNAs, which are also capped and methylated within the core, are extruded into the cytoplasm through pores at the vertices of the icosahedral structure, where they are translated into viral proteins. One copy of each of the viral mRNAs is also assembled with these newly synthesised proteins to form nascent virus particles, which mature by a process that involves -ve RNA strand synthesis on the +ve stand template, thereby reforming dsRNA genome segments within progeny virus cores. The structure of the BTV core particle has been determined to atomic resolution by X-ray crystallography, revealing the organisation and interactions of its major protein components (VP3(T2)-subcore shell and VP7(T13) outer core layer) and important features of the packaged dsRNA. By soaking crystals of BTV cores with metal ions and substrates/products of the transcription reactions prior to analysis by X-ray crystallography, then constructing difference maps, it has been possible to identify binding sites and entry/exit routes for these ions, substrates and products. This has revealed how BTV solves the many logistical problems of multiple and simultaneous transcription from the 10 genome segments within the confined space of the core particle. The crystal structure of the BTV core has also revealed an outer surface festooned with dsRNA. This may represent a further protective strategy adopted by the virus to prevent host cell shut-off, by sequestering any dsRNA that may be released from damaged particles.

Nature and duration of protective immunity to bluetongue virus infection.

Genetic engineering offers a variety of approaches to producing viral vaccines. An exciting advance in this field is the ability to construct virus-like particles (VLPs) that resemble their natural counterparts but lack genetic information. To develop a rationally designed vaccine for bluetongue disease of sheep that is caused by virus (BTV), we have synthesised individual BTV proteins and BTV-like particles (VLPs and CLPs) using baculovirus expression systems and insect cell cultures. A series of clinical trials were undertaken using these proteins and particles in BTV-susceptible sheep. The accumulated data obtained from these studies are: (i) the two surface proteins when used in high doses (approximately 100 microg/dose) could afford complete protection in sheep against virulent virus challenge; (ii) in contrast, only 5-10 microg of VP2 of a related virus, African horse sickness virus (AHSV) afforded protection in horses against virulent virus challenges when vaccinated in the presence of appropriate adjuvant; (iii) vaccination with as little as 10 microg VLPs (consisting of all four major proteins) gave long lasting protection (at least for 14 months) against homologous BTV challenge; (iv) cross-protection was also achieved depending on the challenge virus and amounts of antigen used for vaccination and (v) limited vaccination trials with CLPs (containing only two highly conserved internal proteins) afforded partial (with slight fever) protection against homologous and heterologous virus challenges. Since CLPs are conserved across the twenty four BTV serotypes, CLPs could have potential for a candidate vaccine that may at least mitigate the disease and inhibit virus spread. In summary, VLPs and CLPs offer completely safe and efficacious vaccines as their particles are devoid of any detectable amount of insect, baculovirus proteins or nucleic acids and thus pose no potential adverse effects.

Virus-derived tubular structure displaying foreign sequences on the surface elicit CD4+ Th cell and protective humoral responses.

Particulate vector systems for the presentation of immunogenic epitopes provide an alternate and powerful approach for the delivery of immunogens of interest. In this article, we have exploited a viral protein of unknown function, bluetongue virus (BTV) nonstructural protein NS1, which forms distinct tubular aggregates in infected cells, as an immunogen delivery system. Tubules are helical assemblies of NS1 protein that present the C-terminus of the protein to the outer edge effectively displaying appended residues in a regular and repeating array akin to the coat of a filamentous phage. To assess the breadth of response induced following tubule-based immunization, two different immunodominant foreign peptides were inserted at the C-terminus of NS1 and chimeric tubules generated following expression in the baculovirus expression system. Both constructs, one carrying a peptide of foot and mouth disease virus (FMDV) (aa 135-144 of VP1) and the other, a peptide of influenza A virus (aa 186-205 of HA), effectively assembled into tubules and were easily purified. Subsequently, using in vitro assay systems, we demonstrated that each purified chimeric particle was capable of eliciting strong immune responses. Further, NS1-FMDV chimeric tubules could induce a potent immune response that could protect against disease.

IMIRS: a high-resolution 3D reconstruction package integrated with a relational image database.

Recent advances in electron cryomicroscopy instrumentation and single particle reconstruction have created opportunities for high-throughput and high-resolution three-dimensional (3D) structure determination of macromolecular complexes. However, it has become impractical and inefficient to rely on conventional text file data management and command-line programs to organize and process the increasing numbers of image data required in high-resolution studies. Here, we present a distributed relational database for managing complex datasets and its integration into our high-resolution software package IMIRS (Image Management and Icosahedral Reconstruction System). IMIRS consists of a complete set of modular programs for icosahedral reconstruction organized under a graphical user interface and provides options for user-friendly, step-by-step data processing as well as automatic reconstruction. We show that the integration of data management with processing in IMIRS automates the tedious tasks of data management, enables data coherence, and facilitates information sharing in a distributed computer and user environment without significantly increasing the time of program execution. We demonstrate the applicability of IMIRS in icosahedral reconstruction toward high resolution by using it to obtain an 8-A 3D structure of an intermediate-sized dsRNA virus.

High resolution structural studies of complex icosahedral viruses: a brief overview.

Structural descriptions of viral particles are key to our understanding of their assembly mechanisms and properties. We will describe the application of X-ray crystallography and electron cryomicroscopy to the structural determination of the bluetongue virus core and the herpesvirus capsid. These represent the highest resolution structural studies carried out by these techniques on such complex and large icosahedral virus particles. The bluetongue virus core consists of two layers of distinct proteins with different protein packing symmetries, while the herpes virus capsid is made up of four types of proteins with 3.3 MDa per asymmetric unit. The structural results reveal that each of these proteins has distinct folds and they are packed uniquely to form stable particles.

RGD tripeptide of bluetongue virus VP7 protein is responsible for core attachment to Culicoides cells.

Bluetongue virus (BTV) is an arthropod-borne virus transmitted by Culicoides species to vertebrate hosts. The double-capsid virion is infectious for Culicoides vector and mammalian cells, while the inner core is infectious for only Culicoides-derived cells. The recently determined crystal structure of the BTV core has revealed an accessible RGD motif between amino acids 168 to 170 of the outer core protein VP7, whose structure and position would be consistent with a role in cell entry. To delineate the biological role of the RGD sequence within VP7, we have introduced point mutations in the RGD tripeptide and generated three recombinant baculoviruses, each expressing a mutant derivative of VP7 (VP7-AGD, VP7-ADL, and VP7-AGQ). Each expressed mutant protein was purified, and the oligomeric nature and secondary structure of each was compared with those of the wild-type (wt) VP7 molecule. Each mutant VP7 protein was used to generate empty core-like particles (CLPs) and were shown to be biochemically and morphologically identical to those of wt CLPs. However, when mutant CLPs were used in an in vitro cell binding assay, each showed reduced binding to Culicoides cells compared to wt CLPs. Twelve monoclonal antibodies (MAbs) was generated using purified VP7 or CLPs as a source of antigen and were utilized for epitope mapping with available chimeric VP7 molecules and the RGD mutants. Several MAbs bound to the RGD motif on the core, as shown by immunogold labeling and cryoelectron microscopy. RGD-specific MAb H1.5, but not those directed to other regions of the core, inhibited the binding activity of CLPs to the Culicoides cell surface. Together, these data indicate that the RGD motif present on BTV VP7 is responsible for Culicoides cell binding activity.

Functional dissection of the major structural protein of bluetongue virus: identification of key residues within VP7 essential for capsid assembly.

A lattice of VP7 trimers forms the surface of the icosahedral bluetongue virus (BTV) core. To investigate the role of VP7 oligomerization in core assembly, a series of residues for substitution were predicted based on crystal structures of BTV type 10 VP7 molecule targeting the monomer-monomer contacts within the trimer. Seven site-specific substitution mutations of VP7 have been created using cDNA clones and were employed to produce seven recombinant baculoviruses. The effects of these mutations on VP7 solubility, ability to trimerize and formation of core-like particles (CLPs) in the presence of the scaffolding VP3 protein, were investigated. Of the seven VP7 mutants examined, three severely affected the stability of CLP, while two other mutants had lesser effect on CLP stability. Only one mutant had no apparent effect on the formation of the stable capsid. One mutant in which the conserved tyrosine at residue 271 (lower domain helix 6) was replaced by arginine formed insoluble aggregates, implying an effect in the folding of the molecule despite the prediction that such a change would be accommodated. All six soluble VP7 mutants were purified, and their ability to trimerize was examined. All mutants, including those that did not form stable CLPs, assembled into stable trimers, implying that single substitution may not be sufficient to perturb the complex monomer-monomer contacts, although subtle changes within the VP7 trimer could destabilize the core. The study highlights some of the key residues that are crucial for BTV core assembly and illustrates how the structure of VP7 in isolation underrepresents the dynamic nature of the assembly process at the biological level.

Structures of virus and virus-like particles.

Virus structures continue to be the basis for mechanistic virology and serve as a paradigm for solutions to problems concerning macromolecular assembly and function in general. The use of X-ray crystallography, electron cryomicroscopy and computational and biochemical methods has provided not only details of the structural folds of individual viral components, but also insights into the structural basis of assembly, nucleic acid packaging, particle dynamics and interactions with cellular molecules.

Virus crystallography.

Virus crystallography can provide atomic resolution structures for intact isometric virus particles and components thereof. The methodology is illustrated by reference to a particularly complex example, the core of the bluetongue virus (700 A).

Quantification of recombinant core-like particles of bluetongue virus using immunosorbent electron microscopy.

Immunosorbent electron microscopy was used to quantify recombinant baculovirus-generated bluetongue virus (BTV) core-like particles (CLP) in either purified preparations or lysates of recombinant baculovirus-infected cells. The capture antibody was an anti-BTV VP7 monoclonal antibody. The CLP concentration in purified preparations was determined to be 6.6 x 10(15) particles/l. CLP concentration in lysates of recombinant baculovirus-infected cells was determined at various times post-infection and shown to reach a value of 3 x 10(15) particles/l of culture medium at 96 h post-infection. The results indicated that immunosorbent electron microscopy, aided by an improved particle counting method, is a simple, rapid and accurate technique for the quantification of virus and virus-like particles produced in large scale in vitro systems.

Courageous science: structural studies of bluetongue virus core.

The structure of the bluetongue virus core was recently reported and represents the largest structure determined to atomic resolution. As a biological machine capable of RNA transcription, the structure has immense biological significance.