Targeted mutational analysis to unravel the complexity of African horse sickness virus NS3 function in mammalian cells.

The African horse sickness virus non-structural protein 3 (NS3) is involved in the final stages of infection. To gain insight into the function of different NS3 domains, we generated reverse genetics-derived mutants, each expressing a modified version of the protein. A functional comparison of these mutants to the wild-type virus in mammalian cells indicated the variable contribution of the different domains to the cytopathic effect and in ensuring effective virus trafficking and release. The transmembrane domains were determined as essential mediators of NS3 localisation, as the abnormal processing of these mutant proteins resulted in their nuclear localisation and interaction with NS1. NS3 cytoplasmic domain disruptions resulted in increased cytosolic virus particle accumulation and abnormal virion tethering to plasma membranes. Other aspects of infection were also affected, such as VIB formation and distribution of the outer capsid proteins. Overall, these results illustrate the intricate role of NS3 in the infection cycle.

Analysis of the three-dimensional structure of the African horse sickness virus VP7 trimer by homology modelling.

VP7 is the major core protein of orbiviruses and is essential for virion assembly. African horse sickness virus (AHSV) VP7 self-assembles into highly insoluble crystalline particles - an attribute that may be related to the role of AHSV VP7 in virus assembly but also prevents crystallization. Given that this inherent insolubility is unique to AHSV VP7, we use amino acid sequence conservation analysis between AHSV VP7 and other orbiviruses to identify putative key residues that drive AHSV VP7 self-assembly. A homology model of the AHSV VP7 trimer was generated to analyze surface properties of the trimer and to identify surface residues as candidates for the AHSV VP7 trimer-trimer interactions that drive AHSV VP7 self-assembly. Nine regions were identified as candidate residues for future site-directed mutagenesis experiments that will likely result in a soluble AHSV VP7 protein. Additionally, we identified putative residues that function in the intermolecular interactions within the AHSV VP7 trimer as well as several epitopes. Given the many previous efforts of solubilizing AHSV VP7, we propose a useful strategy that will yield a soluble AHSV VP7 that can be used to study AHSV assembly and increase yield of recombinant vaccine preparations.

Variation in the NS3 gene and protein in South African isolates of bluetongue and equine encephalosis viruses.

Bluetongue virus (BTV) and equine encephalosis virus (EEV) are agriculturally important orbiviruses transmitted by biting midges of the genus Culicoides. The smallest viral genome segment, S10, encodes two small nonstructural proteins, NS3 and NS3A, which mediate the release of virus particles from infected cells and may subsequently influence the natural dispersion of these viruses. The NS3 gene and protein sequences of South African isolates of these viruses were determined, analysed and compared with cognate orbivirus genes from around the world. The South African BTV NS3 genes were found to have the highest level of sequence variation for BTV (20 %), while the highest level of protein variation of BTV NS3 (10 %) was found between South African and Asian BTV isolates. The inferred NS3 gene phylogeny of the South African BTV isolates grouped them with BTV isolates from the United States, while the Asian BTV isolates grouped into a separate lineage. The level of variation found in the NS3 gene and protein of EEV was higher than that found for BTV and reached 25 and 17 % on the nucleotide and amino acid levels, respectively. The EEV isolates formed a lineage independent from that of the other orbiviruses. This lineage segregated further into two clusters that corresponded to the northern and southern regions of South Africa. The geographical distribution of these isolates may be related to the distribution of the Culicoides subspecies that transmit them.

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.

Expression of the major core structural proteins VP3 and VP7 of African horse sickness virus, and production of core-like particles.

The genome segments encoding the seven structural proteins of African horse sickness virus (AHSV), including the largest coding for VP1, were cloned and sequenced. Analysis of the VP1 sequence supports the putative identity of this protein as an RNA polymerase. The genes encoding the two major core proteins, VP3 and VP7, were also cloned and expressed by both in vitro translation and by means of recombinant baculoviruses. Co-infection of insect cells with VP3 and VP7 recombinant baculoviruses resulted in the intracellular formation of multimeric particles with a diameter of 72 nm, which structurally resembled authentic AHSV cores (core like particles: CLP). The complete genome of AHSV has now been cloned and sequenced.

Structural studies of orbivirus particles.

We are using crystallographic methods to investigate the structure of AHSV and BTV. Our initial approach was to investigate the structure of the major protein component of the viral core, VP7(T13). This trimeric protein has been studied in several crystal forms from both orbiviruses and reveals a structure made up of conserved domains, capable of conformational changes and possessing a cleavage site. Further crystallographic analyses of native particles have provided a picture of the VP7(T13) and VP3(T2) layers of the BTV core. The VP7(T13) layer consists of 260 trimers arranged rather symmetrically and possessing very similar structures, thereby following the rules of quasi equivalence. The VP3(T2) layer is thin and contains 120 copies of 100 kDa protein arranged as 60 approximate dimers. This type of icosahedral construction has not been observed before and appears to contain a genome which is highly ordered. We anticipate that all of these features will be common to AHSV.

Characterization of two African horse sickness virus nonstructural proteins, NS1 and NS3.

Each of the ten segments of the African horse sickness virus (AHSV) genome encodes at least one viral polypeptide. This report focuses on the nonstructural proteins NS1 and NS3, which are encoded by genome segments 5 and 10 respectively. The NS1 protein assembles into tubular structures, which are characteristically produced during orbivirus replication in infected cells. NS1 expressed by a recombinant baculovirus in Sf9 cells also forms tubules, which were analysed electron microscopically. These tubules had an average diameter of 23 +/- 2 nm, which is less than half the width of the corresponding bluetongue virus (BTV) tubules. They were also more fragile at high salt concentrations or pH. The cytotoxic effects produced by NS3 were examined by constructing of mutated versions and expressing them in insect cells. Substitution of amino acids 76-81 in a conserved region (highly conserved amongst all AHSV NS3 proteins, as well as other orbiviruses) with similar amino acids, did not influence the cytotoxicity of the mutant protein. However, mutation of four amino acids, from hydrophobic to charged amino residues, (aa 165-168) in a predicted transmembrane region of NS3, largely abolished its cytotoxic effect. It is considered likely that the mutant protein is unable to interact with cellular membrane components, thereby reducing its toxicity.

Phylogenetic analysis of African horse sickness virus segment 10: sequence variation, virulence characteristics and cell exit.

African horse sickness virus (AHSV) genome segment 10 encodes the non-structural proteins NS3/NS3a, which is involved in release of virus from cells. Full length segment 10 cDNAs were amplified by reverse transcription-polymerase chain reaction, from isolates of AHSV serotypes 2, 3, 4, 5, 7, 8 and 9. These cDNAs were cloned, sequenced and their phylogenetic relationships analysed. High levels of sequence homology were detected in segment 10 from some isolates of different serotypes, confirming that they could be grouped on this basis (serotypes 4, 5, 6 and 9 (group alpha); serotypes 3 and 7 (group beta); serotypes 1, 2, and 8 (group gamma). However, data from bluetongue virus (the prototype orbivirus) indicate that the AHSV serotype is determined exclusively by the structural outer coat proteins VP2 and VP5, encoded by genome segments 2 and 5 respectively. Therefore, as a direct consequence of genome segment reassortment between AHSV strains from different serotypes, the differences observed in segment 10 do not give a reliable indication of virus serotype. Segment 10 of AHSV 3 (virulent) and AHSV 3att (attenuated) were also analysed. These strains, together with AHSV 8, have been used to study of the genetic basis of virulence using reassortment (O'Hara et al., this publication). Virus release studies, using Culicoides cell cultures, indicate that differences in segment 10 of AHSV 3att and 8 can influence the timing of virus release from the infected cell.

The complete sequence of four major structural proteins of African horse sickness virus serotype 6: evolutionary relationships within and between the orbiviruses.

The amino acid sequences of four major capsid proteins of African horse sickness virus (serotype 6, AHSV-6) have been determined from analyses of cDNA clones representing the L2, L3, M6 and S7 RNA segments. The AHSV-6 L3 RNA segment has an open reading frame of 2715 base pairs and encodes the inner capsid protein VP3 which comprises 905 amino acids. The VP3 layer forms the subcore of the virion and is surrounded by the VP7 protein which is encoded by the S7 gene. The AHSV-6 S7 gene was found to be 1047 nucleotides in length with a coding capacity for the VP7 protein of 349 amino acids. These core proteins are encapsulated by the outer capsid proteins VP5 and VP2 which are encoded by the M6 and L2 genes respectively. The M6 gene of AHSV-6 was determined to be 1564 nucleotides in length and encoded a protein product of 504 amino acids while the L2 gene comprised 3203 nucleotides which encoded a predicted protein product of 1051 amino acids. Comparison of these four sequences with the core protein sequences of other serotypes of African horse sickness virus, Bluetongue virus which infects sheep, and Epizootic haemorrhagic disease virus of deer, demonstrated that despite the pathobiological properties and host range of these distinct orbiviruses, extreme conservation is evident within the capsid genes. Sequence analyses also suggested that the similarity levels between serogroups depict the structure and function of the individual capsid proteins. The data indicated that the evolution of the capsid genes of gnat transmitted orbiviruses is strongly influenced by functional and structural constraints.

Crystal structure of the top domain of African horse sickness virus VP7: comparisons with bluetongue virus VP7.

The baculovirus-expressed core protein VP7 of African horse sickness virus serotype 4 (AHSV-4) has been purified to homogeneity and crystallized in the presence of 2.8 M urea. The X-ray structure has been solved to a 2.3-Angstroms (1 Angstrom = 0.1 nm) resolution with an Rfactor of 19.8%. The structure of AHSV VP7 reveals that during crystallization, the two-domain protein is cleaved and only the top domain remains. A similar problem was encountered previously with bluetongue virus (BTV) VP7 (whose structure has been reported), showing that the connections between the top and the bottom domains are rather weak for these two distinct orbiviruses. The top domains of both BTV and AHSV VP7 are trimeric and structurally very similar. The electron density maps show that they both possess an extra electron density feature along their molecular threefold axes, which is most likely due to an unidentified ion. The characteristics of the molecular surface of BTV and AHSV VP7 suggest why AHSV VP7 is much less soluble than BTV VP7 and indicate the possibility of attachment to the cell via attachment of an Arg-Gly-Asp (RGD) motif in the top domain of VP7 to a cellular integrin for both of these orbiviruses.

Subcellular localization of the nonstructural protein NS3 of African horsesickness virus.

The subcellular localization of the minor nonstructural protein NS3 of African horsesickness virus (AHSV) has been investigated by means of immunogold electron-microscopical analysis. NS3 was observed in perturbed regions of the plasma membrane of AHSV-infected VERO cells, and its presence appears to be associated with events of viral release. These events are budding, whereby released viruses acquire fragments from the host-cell membrane, as well as by the extrusion of nonenveloped particles through the cell membrane. The membrane association of NS3 was confirmed by its detection in the disrupted plasma membranes of cells infected with an NS3 baculovirus recombinant. The absence of NS3 on intact cell membranes suggests that the protein is not exposed extracellularly.

African horse sickness virus structure.

African horse sickness virus (AHSV), of which there are nine serotypes (AHSV-1, -2, etc.), is a member of Orbivirus genus within the Reoviridae family. Both in morphology and molecular constituents AHSV particles are comparable to those of bluetongue virus (BTV), the prototype virus of the genus. The two viruses have seven structural proteins (VP1-7) organized in two layered capsid. The outer capsid is composed of VP2 and VP5. The inner capsid, or core, is composed of two major proteins, VP3 and VP7, and three minor proteins, VP1, VP4 and VP6. Within the core is the virus genome. This genome consists of 10 double-stranded (ds)RNA segments of different sizes, three large, designated L1-L3, three medium, M4-M6, and four small, S7-S10. In addition to the seven structural proteins that are coded by seven of the RNA species, four non-structural proteins, NS1, NS2, NS3 and NS3A, are coded by three RNA segments, M5, S8 and S10. The two smallest proteins (NS3 and NS3A) are synthesized by the S10 RNA segment, probably from different in-frame translation initiation codons. Nucleotide sequences of eight RNA segments (L2, L3, M4, M5, M6, S7, S8 and S10) and the predicted amino acid sequences of the encoded gene products are also available, mainly representing one serotype, AHSV-4. In this review the properties of the AHSV genes and gene products are discussed. The sequence and hybridization analyses of the different AHSV dsRNA segments indicate that the segments that code for the core proteins, as well as those that code for NS1 and NS2 proteins, are highly conserved between the different virus serotypes. However, the RNA encoding NS3 and NS3A, and the two segments encoding the outer capsid proteins, are more variable between the AHSV serotypes. A close phylogenetic relationship between AHSV, BTV and epizootic haemorrhagic disease virus (EHDV), three Culicoides-transmitted orbiviruses, has been revealed when the equivalent sequences of genes and gene products are compared. Recently, the four major AHSV capsid proteins have been expressed using recombinant baculoviruses. Biochemically and antigenically these proteins are similar to the authentic proteins. Since the AHSV VP7 protein is highly conserved among the different serotypes, it has been utilized as a diagnostic reagent. The expressed VP7 protein has also been purified to homogeneity and crystallized for three-dimensional X-ray analysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of African horsesickness virus serotype 4-induced polypeptides in Vero cells and their reactivity in Western immunoblotting.

The structural and non-structural proteins induced by African horsesickness virus serotype 4 (AHSV-4) in infected Vero cells were analysed by SDS-PAGE. Twenty-two virus-induced polypeptides were detected in infected cells by comparison with the polypeptides of mock-infected cells, of which four major (VP2, VP3, VP5 and VP7) and three minor (VP1, VP4 and VP6) structural proteins and four non-structural proteins (P58, P48, P21 and P20) were shown to be virus-coded, as deduced from electrophoretic and antigenic studies of purified virions and infected cells. The proteins that elicit the major antibody responses both in vaccinated and naturally or experimentally infected horses were shown to be three structural proteins, VP2, VP5 and VP7, and the four major non-structural proteins, P58, P48, P21 and P20, as deduced by radioimmunoprecipitation and immunoblotting assays. The cross-reactivity between AHSV-4 and sera obtained from horses experimentally infected with seven other serotypes was also determined. The results showed that VP5, VP7, P48, P21 and P20 are conserved and can be used to diagnose the infection of any of these eight serotypes.

Identification and characterization of the structural and nonstructural proteins of African horsesickness virus and determination of the genome coding assignments.

Proteins present in purified African horsesickness virus (AHSV) and in infected cells were analyzed by SDS-polyacrylamide gel electrophoresis. Twelve viral proteins were identified, one minor and four major structural proteins, three major and two minor nonstructural proteins, as well as variable amounts of two additional structural proteins. Cell-free translation of total AHS virion RNA in a rabbit reticulocyte system resulted in the synthesis of proteins which were qualitatively and quantitatively similar to those found in infected cells. The in vivo and in vitro synthesized proteins were viral specific as demonstrated by immunoprecipitation. The coding assignments of all the purified genome segments were determined by in vitro translation and confirmed by immunoprecipitation.