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.

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 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.

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 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.

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.

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.

Projection structure of VP6, the rotavirus inner capsid protein, and comparison with bluetongue VP7.

The rotavirus nucleocapsid protein (VP6) is the major structural protein of inner capsid particles (ICP). VP6 is essential for RNA transcription and binds to a virally encoded glycoprotein receptor (NSP4) involved in the rotavirus assembly pathway. To explore the structure of VP6, two-dimensional (2D) crystals of VP6 were generated and examined by electron microscopy and image processing. Fourier transforms computed from low-dose images of negatively stained 2D VP6 crystals displayed complete data to 13 A resolution for p6 plane group symmetry. To correct for the resolution dependent fall-off of the amplitudes derived from electron microscopic images, the rotavirus VP6 amplitudes were scaled to the bluetongue VP7 amplitudes derived from the atomic model by applying a B factor of -360 A-2. The unit cell (a=b=101(+/-2)A, gamma=120(+/-1) degrees) contains two VP6 trimers, each composed of three roughly circular subunits approximately 30 A in diameter. The trimeric organization of VP6 is similar to the oligomeric structure of VP6 when assembled in T=13l icosahedral inner capsid particles at 25 to 40 A resolution. However, a channel at the center of the trimer is better resolved in our map at 15 A resolution. The projection structure of rotavirus VP6 was compared to the homologous protein (VP7) of bluetongue virus, which is also a member of the family of Reoviridae. Notably, both VP6 and bluetongue VP7 assemble as 260 capsomers on the surface of the inner capsid. To compare VP6 and VP7, a projection map of bluetongue VP7 at 15 A resolution was generated using the atomic model derived by X-ray crystallography. VP6 and VP7 both exhibit a trimeric organization with a central channel, even though the alignment identity between the 45 kDa VP6 and the 38 kDa VP7 primary sequences is only 12%. The ability of VP6 to form well-ordered 2D crystals should enable a higher resolution structure analysis by cryo-electron microscopy that will extend our understanding of the icosahedral ICP structure, clarify the mechanism by which VP6 interacts with the NSP4 receptor, and allow a more detailed comparison of VP6 and VP7.

Use of a gene-targeted phage display random epitope library to map an antigenic determinant on the bluetongue virus outer capsid protein VP5.

We describe the use of a gene-targeted random epitope library for the mapping of antigenic determinants. A DNA clone encoding the target antigen was digested randomly with DNase I to generate a population of DNA fragments of different sizes and sequences. After size fractionation, small DNA fragments (100-200 bp) were isolated and cloned into the phage expression vector fUSE2 to form an expression library displaying random polypeptide sequences as fusion proteins at the N terminus of the phage gene III protein. This library, termed a gene-targeted random epitope library to distinguish it from totally random synthetic epitope libraries, was then screened by affinity selection for recombinant phages which were specifically bound by the antibody of interest. Using this approach, we have mapped a monoclonal antibody (mAb)-defined epitope on the bluetongue virus outer capsid protein VP5. This epitope is not accessible on the intact virus surface, but is recognised by the immune system of sheep and cattle during virus infection. Although the example given here utilised a DNA fragment of known sequence and the library was screened for a mAb-defined epitope, the strategy described should be equally applicable to genes of unknown sequence and for screening of epitopes using polyclonal antibodies. The approach can also be extended to identify immunodominant epitope from much more complex genome-targeted random epitope library for virus, bacteria and eukaryotic organisms. Other applications of recombinant phages expressing defined immunodominant epitopes include serodiagnosis and vaccine development.

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.

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.

Bluetongue virus: surface exposure of VP7.

The exposed proteins of bluetongue virus serotype 17 were determined using surface labeling and reactivity with monoclonal antibodies. Iodination of amino groups predominantly labeled VP2; however, iodination of tyrosine residues labeled both VP2 and VP5, with VP7 labeled to a significantly lesser degree. To investigate the exposure of VP7 on the intact virion further, monoclonal antibodies that reacted with this protein were used. At least two antibodies, reacting with different epitopes on VP7, bound to intact virions, as determined by adsorption of infectious particles, electron microscopic observation of antibody-bound virus, and co-sedimentation of antibody and virus. Surface iodination of viral cores was used to show that VP7 and VP3 are major exposed proteins on these particles. We conclude that a major core protein, VP7, has at least two epitopes exposed on the virus surface.

New generation of African horse sickness virus vaccines based on structural and molecular studies of the virus particles.

African horse sickness virus (AHSV) is a member of the genus Orbivirus, which also includes bluetongue virus (BTV) and epizootic haemorrhagic disease (EHDV) virus. These orbiviruses have similar morphological and biochemical properties, with distinctive pathobiological properties and host ranges. Sequencing studies of the capsid proteins have revealed evolutionary relationships between these viruses. Biochemical studies of the viruses together with the expression of individual proteins and protein complexes have resulted in the development of new generation vaccines. Baculovirus expressed AHSV VP2 provides protection against death caused by AHSV challenge. Similarly, BTV VP2 alone elicits protective neutralising antibodies against BTV in sheep, which is enhanced in the presence of VP5. Recent developments in biotechnology (multiple gene expression baculovirus systems) have made it possible to synthesise orbivirus particles that biochemically and immunologically mimic authentic virions but lack the genetic material. Particle doses as low as 10 micrograms elicit responses that are sufficient to protect sheep 15 months post vaccination, against virulent virus challenge. Moreover, knowledge of the three dimensional structure of these particles enables us to engineer them to deliver multiple foreign peptide components representing other viral epitopes (e.g. foot and mouth disease virus and influenza virus) in order to elicit protective immunity.

The use of immuno-gold silver staining in bluetongue virus adsorption and neutralisation studies.

The immuno-gold-silver staining (IGSS) technique was used in scanning electron microscopy for the detection and semi-quantitation of low copy antigens on the surface of cells. The methodology was exploited in experiments designed to examine the interaction of small numbers of virus particles with the surface of susceptible host cells. Using bluetongue virus (BTV) as an example, IGSS procedures confirmed that maximum adsorption occurred within 60 min and that adsorbed virus particles were distributed randomly on the surface of the cell. Neutralising antibody did not prevent binding of BTV to the plasma membrane, but abrogated virus uptake. The use of IGSS in the study of virus-cell interactions was validated by transmission electron microscopy and classical biochemical experiments utilising radioactively-labelled virus.

Improved method for counting virus and virus like particles.

An improved method for counting virus and virus like particles by electron microscopy (EM) was developed. The procedure involves the determination of the absolute concentration of pure or semi-pure particles once deposited evenly on EM grids using either centrifugation or antibody capture techniques. The counting of particles was done with a Microfiche unit which enlarged approximately 50 x the image of particles on a developed negative film which had been taken at a relatively low magnification (2500 x) by EM. Initially, latex particles of a known concentration were counted using this approach, to prove the accuracy of the technique. The latex particles were deposited evenly on an EM grid using centrifugation (Modified Beckmen EM-90 Airfuge technique). Subsequently, recombinant Bluetongue virus (BTV) core-like particles (CLPs) captured by a Monoclonal antibody using a novel sample loading method were counted by the Microfiche unit method and by a direct EM method. Comparison of the simplified counting method developed with a conventional method, showed good agreement. The method is simple, accurate, rapid, and reproducible when used with either pure particles or with particles from crude cell culture extracts.

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.

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).

From genes to complex structures of bluetongue virus and their efficacy as vaccines.

Bluetongue virus-like and core-like structures consisting of multiproteins in different molar ratios, have been synthesized using baculovirus multiple expression vectors. These particles lacking genetic materials, mimic the single- and double-shelled authentic virus particles and have been shown to be highly immunogenic and protective for sheep challenged with infectious virus. The formation of virus-like particles, using this new technology, offers a novel approach to vaccine development.