Comparative ultrastructural characterization of African horse sickness virus-infected mammalian and insect cells reveals a novel potential virus release mechanism from insect cells.

African horse sickness virus (AHSV) is an arbovirus capable of successfully replicating in both its mammalian host and insect vector. Where mammalian cells show a severe cytopathic effect (CPE) following AHSV infection, insect cells display no CPE. These differences in cell death could be linked to the method of viral release, i.e. lytic or non-lytic, that predominates in a specific cell type. Active release of AHSV, or any related orbivirus, has, however, not yet been documented from insect cells. We applied an integrated microscopy approach to compare the nanomechanical and morphological response of mammalian and insect cells to AHSV infection. Atomic force microscopy revealed plasma membrane destabilization, integrity loss and structural deformation of the entire surface of infected mammalian cells. Infected insect cells, in contrast, showed no morphological differences from mock-infected cells other than an increased incidence of circular cavities present on the cell surface. Transmission electron microscopy imaging identified a novel large vesicle-like compartment within infected insect cells, not present in mammalian cells, containing viral proteins and virus particles. Extracellular clusters of aggregated virus particles were visualized adjacent to infected insect cells with intact plasma membranes. We propose that foreign material is accumulated within these vesicles and that their subsequent fusion with the cell membrane releases entrapped viruses, thereby facilitating a non-lytic virus release mechanism different from the budding previously observed in mammalian cells. This insect cell-specific defence mechanism contributes to the lack of cell damage observed in AHSV-infected insect cells.

Characterization of the nucleic acid binding activity of inner core protein VP6 of African horse sickness virus.

Minor structural protein VP6 is the putative helicase of African horse sickness virus (AHSV), of the genus Orbivirus in the Reoviridae family. We investigated how the protein interacts with double-stranded (ds) RNA and other nucleic acids. Binding was assayed using an electrophoretic migration retardation assay and a nucleic acid overlay protein blot assay. VP6 bound double and single stranded RNA and DNA in a NaCl concentration sensitive reaction. Of six truncated VP6 peptides investigated, two partially overlapping peptides were found to bind dsRNA at pH 7.0, while other peptides with the same overlap did not. The distinction between the peptides appeared to be the pI which ranged from more than 8.0 to just above 6.0. Changing the pH of the binding buffer modified the binding activity. Regardless of assay conditions, only peptides with a specific region of amino acids in common, showed evidence of binding activity. No sequence homology was identified with other binding domains, however, the presence of charged amino acids are assumed to be important for binding activity. The results suggested dsRNA binding in the blot assay was strongly affected by the net charge on the peptide.

A comparison of different orbivirus proteins that could affect virulence and pathogenesis.

The factors that determine the virulence and pathogenesis characteristics of bluetongue virus (BTV), African horse sickness virus (AHSV) and other orbiviruses are not well known. With respect to the viral proteins that are expected to play a role it may be assumed that proteins, such as the outer capsid proteins VP2 and VP5, that are involved in the attachment of virus particles to target cells and influence replication efficiency are particularly important. Equally important are viral proteins such as non-structural protein NS3 that influence the release of virus particles from a target host or vector cell. The authors compare the amino acid sequence variation, structural motifs and some phenotypic characteristics of proteins VP2, VP5 and NS3 of different orbiviruses, such as AHSV, BTV and equine encephalosis virus (EEV). The most variable protein is VP2 and a pair-wise alignment of VP2 sequences of different serotypes of both BTV and AHSV indicated variation of between 48% to 64% and 46% to 52% for most isolates, respectively. Several regions of high variability can be identified. VP5 of BTV is much less variable than VP2 but still more so than the cognate AHSV VP5. In contrast, the NS3 protein of AHSV is much more variable than its BTV or EEV counterpart with maximum levels of NS3 variation up to 36% as compared to 10% for BTV. The AHSV NS3 variation is clustered into three discreet phylogenetic groups. All orbivirus NS3/NS3A proteins share a number of highly conserved structural features that include two hydrophobic domains (HD1 and HD2) that are involved in the interaction with the membrane. Most of the NS3 variation is located in HD1 and the adjacent variable region between HD1 and HD2. In the case of AHSV this region only has 13% identity compared to 64% in the case of BTV. NS3 of AHSV is also a highly toxic protein and mutation analysis has indicated that the toxicity is associated with the two hydrophobic domains. Expression of NS3 deletion mutants in bacterial cells has shown that both HD1 and HD2 are necessary for cytotoxicity and that removal of the adjacent N-terminal domains increases cytotoxicity. Preliminary results with different AHSV strains and the corresponding NS3 equivalent have indicated that the membrane permeabilisation effect of the individual NS3 proteins correlate with the permeabilisation effect of the corresponding viruses. These results would suggest that characterisation of the NS3 protein by itself might predict some phenotypic characteristics and potential membrane destabilisation effect of the corresponding virus.

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.

Membrane association of African horsesickness virus nonstructural protein NS3 determines its cytotoxicity.

The smallest RNA genome segment of African horsesickness virus (AHSV) encodes the nonstructural protein NS3 (24K). NS3 localizes in areas of plasma membrane disruption and is associated with events of viral release. Conserved features in all AHSV NS3 proteins include the synthesis of a truncated NS3A protein from the same open reading frame as that of NS3, a proline-rich region, a region of strict sequence conservation and two hydrophobic domains. To investigate whether these features are associated with the cytotoxicity of NS3 or altered membrane permeability, a series of mutants were constructed and expressed in the BAC-TO-BAC baculovirus-expression system. Our results indicate that mutations in either of the two hydrophobic domains do not prevent membrane targeting of the mutant proteins but abolish their membrane anchoring. This prevents their localization to the cell surface and obviates their cytotoxic effect. The cytotoxicity of NS3 is therefore dependent on its membrane topography and thus involves both hydrophobic domains. NS3 has many of the characteristics of lytic viral proteins that play a central role in viral pathogenesis through modifying membrane permeability.

Variation of African horsesickness virus nonstructural protein NS3 in southern Africa.

NS3 protein sequences of recent African horsesickness virus (AHSV) field isolates, reference strains and current vaccine strains in southern Africa were determined and compared. The variation of AHSV NS3 was found to be as much as 36.3% across serotypes and 27.6% within serotypes. NS3 proteins of vaccine and field isolates of a specific serotype were found to differ between 2.3% and 9.7%. NS3 of field isolates within a serotype differed up to 11.1%. Our data indicate that AHSV NS3 is the second most variable AHSV protein, the most variable being the major outer capsid protein, VP2. The inferred phylogeny of AHSV NS3 corresponded well with the described NS3 phylogenetic clusters. The only exception was AHSV-8 NS3, which clustered into different groups than previously described. No obvious sequence markers could be correlated with virulence. Our results suggest that NS3 sequence variation data could be used to distinguish between field isolates and live attenuated vaccine strains of the same serotype.

Cloning, sequencing and expression of the gene that encodes the major neutralisation-specific antigen of African horsesickness virus serotype 9.

A marked improvement in the efficiency of cloning the large double stranded RNA (dsRNA) genome segments of African horsesickness virus (AHSV) was achieved when the dsRNA polyadenylation step was carried out with undenatured rather than strand-separated dsRNA. It is a prerequisite to use dsRNA of very high purity because in the presence of even trace amounts of single stranded RNA, the dsRNA appears to be poorly polyadenylated as judged by its effectiveness as a template for oligo-dT-primed cDNA synthesis. The full-length VP2 gene of AHSV-9, cloned by this approach, was sequenced and it was found to show the highest percentage identity (60%) to VP2 of AHSV-6, providing an explanation of why these two serotypes show some cross protection. The VP2 protein was also expressed in Spodoptera frugiperda (Sf9) cells by means of a baculovirus recombinant. The yield of the expressed VP2 was high, but the protein was found to be largely insoluble. Nine smaller, truncated VP2 peptides were subsequently expressed in insect cells, but no significant improvement in solubility of the peptides, as compared to that of the full-sized protein, was observed. A western immunoblot analysis of the overlapping peptides indicated the presence of a strong linear epitope located within a large hydrophilic domain between amino acids 369 and 403.

Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries.

VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.

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.

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.

Sequence analysis of the RNA polymerase gene of African horse sickness virus.

The gene encoding the inner core protein VP1 of African horse sickness virus (AHSV) serotype 9 has been cloned, expressed in vitro and entirely sequenced, completing molecular characterization of the AHSV genome. An analysis of the sequence supporting the identity of AHSV VP1 as the putative viral RNA polymerase is presented.

Intracellular production of African horsesickness virus core-like particles by expression of the two major core proteins, VP3 and VP7, in insect cells.

To gain more insight into the structure of the African horsesickness virus (AHSV) core particle, we have cloned, partially characterized and expressed the two major core proteins, VP3 and VP7, of AHSV-9. VP7 was found to be highly conserved amongst different serotypes. The VP3 and VP7 genes were subsequently expressed in insect cells by means of recombinant baculoviruses. VP7 was synthesized to very high levels and aggregated into distinctive crystals. Co-expression of VP3 and VP7 resulted in the intracellular formation of core-like particles which structurally resembled empty AHSV cores.

Characterization of tubular structures composed of nonstructural protein NS1 of African horsesickness virus expressed in insect cells.

The characteristic tubules that are produced during the orbivirus infection cycle are composed of a major viral nonstructural protein, NS1. To characterize the NS1 gene and gene product of African horsesickness virus (AHSV), a full-length cDNA copy of the NS1 gene of AHSV-6 was cloned and the nucleotide sequence determined. NS1 was highly conserved within the AHSV serogroup with between 95-98% conservation of amino acids among NS1 of AHSV-6, AHSV-4 and AHSV-9. The structure of AHSV NS1 tubules was investigated by in vitro translation of the AHSV-6 NS1 gene followed by expression of the gene in insect cells. The NS1 protein assembled in tubular structures with a diameter of approximately 23 nm and lengths of up to 4 microns. The absence of a ladder-like structure and lower sedimentation value of AHSV NS1 tubules clearly distinguished them from those of bluetongue virus.

Characterization of the gene encoding core protein VP6 of two African horsesickness virus serotypes.

The genes encoding the inner core protein VP6 of African horsesickness virus (AHSV) serotypes 3 and 6 have been cloned and sequenced. The genes are 1169 nucleotides in length and both encode a largely hydrophilic protein of 369 amino acids. The VP6 amino acid sequence is highly conserved between the two serotypes with an overall similarity of 95 percent. Comparison of the AHSV VP6 amino acid sequences with those of bluetongue virus serotype 10 VP6 revealed that it is 41 amino acids longer with an overall amino acid identity of 29 percent. The similarity is mainly confined to a short but highly conserved 13 amino acid region at the N terminus, a short seven amino acid region at the C terminus and a 22 amino acid region close to the C terminus. Within this last region is a smaller 11 amino acid region from 318 to 328 with a 91 percent similarity to the Rep helicase of Escherichia coli.

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.

Identification of a short domain within the non-structural protein NS2 of epizootic haemorrhagic disease virus that is important for single strand RNA-binding activity.

The role that a conserved amino acid motif, found in the non-structural protein NS2 of orbiviruses, plays in the interaction of this protein with single stranded (ss) RNA was investigated by mutation analysis of the NS2 of epizootic haemorrhagic disease virus. An NS2 mutant in which this motif (amino acids 75 to 83) was deleted was expressed in Spodoptera frugiperda cells by a recombinant baculovirus and found to be unable to bind to poly(U)-Sepharose. The deletion mutant also differed from wild-type NS2 in that it did not appear to be complexed with ssRNA in cells infected with the baculovirus recombinant. Furthermore, the deletion exerted an adverse effect on the ability of NS2 to form inclusion bodies in the cytoplasm of baculovirus-infected insect cells. To further characterize the role of this motif in RNA-binding, specific residues within the region were substituted by site-directed mutagenesis and the mutants were expressed in Escherichia coli as fusion proteins. Analysis of the different mutant proteins indicated that in each case ssRNA-binding was impaired relative to that of the wild-type NS2 control. The degree of impairment corresponded to the number of amino acid substitutions and the largest effects were associated with non-conserved substitutions. It is suggested that the conserved motif is an important structural determinant in the interaction of NS2 with ssRNA.

Site-specific mutations in the NS2 protein of epizootic haemorrhagic disease virus markedly affect the formation of cytoplasmic inclusion bodies.

The importance of a conserved amino acid motif in the nonstructural protein NS2 of different orbiviruses was investigated with regard to virus inclusion body (VIB) formation. A number of epizootic haemorrhagic disease virus NS2 deletion and substitution mutants were prepared and expressed as baculovirus recombinants. Deletion of the motif or substitution of at least three residues within the region, had a detrimental effect on VIB formation in insect cells. Furthermore, these NS2 mutants were not complexed with single-stranded RNA in infected cells and appeared to be cytotoxic. Cumulatively, the results suggest that this motif is an essential structural determinant for NS2 function.

Detection of bluetongue virus and African horsesickness virus in co-infected cell cultures with NS1 gene probes.

The serogroup specificity of the bluetongue virus (BTV) NS1 and VP3 gene probes was confirmed by means of northern blot hybridization. Under high-stringency conditions both probes hybridized to 22 BTV serotypes (18 South African serotypes, BTV3 from Cyprus and BTV16 from Pakistan) but not to serotypes that originate from Australia and India. Furthermore, NS1 gene probes of BTV and African horsesickness virus (AHSV) were used in a dot-spot in situ hybridization procedure to differentiate between BTV and AHSV in co-infected cell cultures. The method detects viral RNA directly i glutaraldehyde-fixed infected cell cultures without prior nucleic-acid extraction or purification. AHSV could be detected in cells infected with AHSV at a multiplicity of infection of 10(-4) PFU/cell in the presence of a hundred excess of co-infecting BTV. The method may have an application in epidemiological surveys to detect different orbiviruses in the same Culicoides population.

The multimeric nonstructural NS2 proteins of bluetongue virus, African horsesickness virus, and epizootic hemorrhagic disease virus differ in their single-stranded RNA-binding ability.

The structure and single-stranded (ss) RNA-binding by the nonstructural protein NS2 of three different orbiviruses were studied and compared. African horsesickness virus (AHSV), bluetongue virus (BTV), and epizootic hemorrhagic disease virus (EHDV) were analyzed in recombinant baculovirus-infected cells and in cells infected with BTV and AHSV. Sedimentation analysis and nonreducing SDS-PAGE revealed that NS2 of all three orbiviruses is a 7S multimer with both inter- and intramolecular disulfide bonds, probably consisting of six or more NS2 molecules. The 7S NS2 multimer of all three viruses binds ssRNA but there is a marked disparity in the ssRNA-binding ability between the three proteins. At physiological salt concentration, BTV NS2 binds ssRNA very efficiently, whereas AHSV NS2 shows only a low efficiency for binding ssRNA. EHDV NS2 binds with intermediate efficiency. The result was the same irrespective of whether poly(U)-Sepharose or viral mRNA was used, indicating that ssRNA-binding by NS2 is nonspecific. The difference in RNA-binding ability may be related to the alpha-helix content of the respective proteins. NS2 of BTV has the highest predicted alpha-helix content followed by EHDV and AHSV. The ability of the NS2 proteins to form virus inclusion body-like structures in baculovirus-infected cells is not affected by the ssRNA-binding disparity.

Expression of nonstructural protein NS3 of African horsesickness virus (AHSV): evidence for a cytotoxic effect of NS3 in insect cells, and characterization of the gene products in AHSV infected Vero cells.

The smallest genome segment of African horsesickness virus (AHSV), segment 10 (S10), encodes two minor nonstructural proteins, NS3 and NS3A. While the cognate bluetongue virus (BTV) proteins have been suggested to play a role in the release of virus particles from infected cells, no function has yet been ascribed to AHSV NS3/NS3A. When the AHSV-3 S10 gene was expressed in a baculovirus system only a single NS3 protein (24 K) was synthesized, at lower levels than expected. It was shown that this could be due to a membrane association of NS3, leading to an alteration in host cell membrane permeability and eventual cell death. Based on computer predictions a general model for the membrane-associated topology of NS3 of five different orbiviruses was proposed. Studies on AHSV-3 infected Vero cells showed that equimolar amounts of NS3 and NS3A were synthesized. No evidence was found for the glycosylation of NS3. The S10 genes and NS3/3A proteins of AHSV-3 and AHSV-7 were shown to be closely related, and clearly distinct from the cognate proteins of the other 7 AHSV serotypes. This distinguishes the AHSV S10 gene product from that of BTV NS3, which appears to be much more conserved.