Investigating the Role of African Horse Sickness Virus VP7 Protein Crystalline Particles on Virus Replication and Release.

African horse sickness is a deadly and highly infectious disease of equids, caused by African horse sickness virus (AHSV). AHSV is one of the most economically important members of the Orbivirus genus. AHSV is transmitted by the biting midge, Culicoides, and therefore replicates in both insect and mammalian cell types. Structural protein VP7 is a highly conserved major core protein of orbiviruses. Unlike any other orbivirus VP7, AHSV VP7 is highly insoluble and forms flat hexagonal crystalline particles of unknown function in AHSV-infected cells and when expressed in mammalian or insect cells. To examine the role of AHSV VP7 in virus replication, a plasmid-based reverse genetics system was used to generate a recombinant AHSV that does not form crystalline particles. We characterised the role of VP7 crystalline particle formation in AHSV replication in vitro and found that soluble VP7 interacted with viral proteins VP2 and NS2 similarly to wild-type VP7 during infection. Interestingly, soluble VP7 was found to form uncharacteristic tubule-like structures in infected cells which were confirmed to be as a result of unique VP7-NS1 colocalisation. Furthermore, it was found that VP7 crystalline particles play a role in AHSV release and yield. This work provides insight into the role of VP7 aggregation in AHSV cellular pathogenesis and contributes toward the understanding of the possible effects of viral protein aggregation in other human virus-borne diseases.

Generation of a Soluble African Horse Sickness Virus VP7 Protein Capable of Forming Core-like Particles.

A unique characteristic of the African horse sickness virus (AHSV) major core protein VP7 is that it is highly insoluble, and spontaneously forms crystalline particles in AHSV-infected cells and when expressed in vitro. The aggregation of AHSV VP7 into these crystals presents many problems in AHSV vaccine development, and it is unclear whether VP7 aggregation affects AHSV assembly or contributes to AHSV pathogenesis. Here, we set out to abolish VP7 self-assembly by targeting candidate amino acid regions on the surface of the VP7 trimer via site-directed mutagenesis. It was found that the substitution of seven amino acids resulted in the complete disruption of AHSV VP7 self-assembly, which abolished the formation of VP7 crystalline particles and converted VP7 to a fully soluble protein still capable of interacting with VP3 to form core-like particles. This work provides further insight into the formation of AHSV VP7 crystalline particles and the successful development of AHSV vaccines. It also paves the way for future research by drawing comparisons with similar viral phenomena observed in human virology.

African horse sickness virus NS4 protein is an important virulence factor and interferes with JAK-STAT signaling during viral infection.

African horse sickness virus (AHSV) non-structural protein NS4 is a nucleocytoplasmic protein that is expressed in the heart, lung, and spleen of infected horses, binds dsDNA, and colocalizes with promyelocytic leukemia nuclear bodies (PML-NBs). The aim of this study was to investigate the role of AHSV NS4 in viral replication, virulence and the host immune response. Using a reverse genetics-derived virulent strain of AHSV-5 and NS4 deletion mutants, we showed that knockdown of NS4 expression has no impact in cell culture, but results in virus attenuation in infected horses. RNA sequencing (RNA-seq) was used to investigate the transcriptional response in these horses, to see how the lack of NS4 mediates the transition of the virus from virulent to attenuated. The presence of NS4 was shown to result in a 24 hour (h) delay in the transcriptional activation of several immune system processes compared to when the protein was absent. Included in these processes were the RIG-I-like, Toll-like receptor, and JAK-STAT signaling pathways, which are key pathways involved in innate immunity and the antiviral response. Thus, it was shown that AHSV NS4 suppresses the host innate immune transcriptional response in the early stages of the infection cycle. We investigated whether AHSV NS4 affects the innate immune response by impacting the JAK-STAT signaling pathway specifically. Using confocal laser scanning microscopy (CLSM) we showed that AHSV NS4 disrupts JAK-STAT signaling by interfering with the phosphorylation and/or translocation of STAT1 and pSTAT1 into the nucleus. Overall, these results showed that AHSV NS4 is a key virulence factor in horses and allows AHSV to overcome host antiviral responses in order to promote viral replication and spread.

African horse sickness virus NS4 is a nucleocytoplasmic protein that localizes to PML nuclear bodies.

African horse sickness virus (AHSV) is the causative agent of the often fatal disease African horse sickness in equids. The non-structural protein NS4 is the only AHSV protein that localizes to the nucleus. Here we report that all AHSV reference and representative field strains express one of the two forms of NS4, i.e. NS4-I or NS4-II. Both forms of NS4 are nucleocytoplasmic proteins, but NS4-I has a stronger nuclear presence whilst NS4-II has a proportionally higher cytoplasmic distribution. A subtype of NS4-II containing a nuclear localization signal (NLS), named NLS-NS4-II, displays distinct punctate foci in the nucleus. We showed that NS4 likely enters the nucleus via passive diffusion as a result of its small size. Colocalization analysis with nuclear compartments revealed that NS4 colocalizes with promyelocytic leukaemia nuclear bodies (PML-NBs), suggesting a role in the antiviral response or interferon signalling. Interestingly, we showed that two other AHSV proteins also interact with nuclear components. A small fraction of the NS1 tubules were present in the nucleus and associated with PML-NBs; this was more pronounced for a virus strain lacking NS4. A component of nuclear speckles, serine and arginine rich splicing factor 2 (SRSF2) was recruited to viral inclusion bodies (VIBs) in the cytoplasm of AHSV-infected cells and colocalized with NS2. Nuclear speckles are important sites for cellular mRNA transcript processing and maturation. Collectively, these results provide data on three AHSV non-structural proteins interacting with host cell nuclear components that could contribute to overcoming antiviral responses and creating conditions that will favour viral replication.

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.

A Dual Laser Scanning Confocal and Transmission Electron Microscopy Analysis of the Intracellular Localization, Aggregation and Particle Formation of African Horse Sickness Virus Major Core Protein VP7.

The bulk of the major core protein VP7 in African horse sickness virus (AHSV) self-assembles into flat, hexagonal crystalline particles in a process appearing unrelated to viral replication. Why this unique characteristic of AHSV VP7 is genetically conserved, and whether VP7 aggregation and particle formation have an effect on cellular biology or the viral life cycle, is unknown. Here we investigated how different small peptide and enhanced green fluorescent protein (eGFP) insertions into the VP7 top domain affected VP7 localization, aggregation, and particle formation. This was done using a dual laser scanning confocal and transmission electron microscopy approach in conjunction with analyses of the solubility, aggregation, and fluorescence profiles of the proteins. VP7 top domain modifications did not prevent trimerization, or intracellular trafficking, to one or two discrete sites in the cell. However, modifications that resulted in a misfolded and insoluble VP7-eGFP component blocked trafficking, and precluded protein accumulation at a single cellular site, perhaps by interfering with normal trimer-trimer interactions. Furthermore, the modifications disrupted the stable layering of the trimers into characteristic AHSV VP7 crystalline particles. It was concluded that VP7 trafficking is driven by a balance between VP7 solubility, trimer forming ability, and trimer-trimer interactions.

Characterising Non-Structural Protein NS4 of African Horse Sickness Virus.

African horse sickness is a serious equid disease caused by the orbivirus African horse sickness virus (AHSV). The virus has ten double-stranded RNA genome segments encoding seven structural and three non-structural proteins. Recently, an additional protein was predicted to be encoded by genome segment 9 (Seg-9), which also encodes VP6, of most orbiviruses. This has since been confirmed in bluetongue virus and Great Island virus, and the non-structural protein was named NS4. In this study, in silico analysis of AHSV Seg-9 sequences revealed the existence of two main types of AHSV NS4, designated NS4-I and NS4-II, with different lengths and amino acid sequences. The AHSV NS4 coding sequences were in the +1 reading frame relative to that of VP6. Both types of AHSV NS4 were expressed in cultured mammalian cells, with sizes close to the predicted 17-20 kDa. Fluorescence microscopy of these cells revealed a dual cytoplasmic and nuclear, but not nucleolar, distribution that was very similar for NS4-I and NS4-II. Immunohistochemistry on heart, spleen, and lung tissues from AHSV-infected horses showed that NS4 occurs in microvascular endothelial cells and mononuclear phagocytes in all of these tissues, localising to the both the cytoplasm and the nucleus. Interestingly, NS4 was also detected in stellate-shaped dendritic macrophage-like cells with long cytoplasmic processes in the red pulp of the spleen. Finally, nucleic acid protection assays using bacterially expressed recombinant AHSV NS4 showed that both types of AHSV NS4 bind dsDNA, but not dsRNA. Further studies will be required to determine the exact function of AHSV NS4 during viral replication.

Factors that affect the intracellular localization and trafficking of African horse sickness virus core protein, VP7.

African horse sickness virus (AHSV) VP7 is the major core protein of the virion. Apart from its role in virus assembly, VP7 forms crystalline-like particles during infection and when expressed in insect cells. The aim of this study was to investigate the process of VP7 crystalline-like particle formation. The intracellular distribution of VP7 was characterized in different systems and the association of VP7 with virus factories during AHSV infection was investigated. It was shown that the majority of VP7 is sequestered into these particles, and is therefore not available for new virion assembly. This is likely to have a negative impact on virus assembly and yield. By using specific markers and inhibitors of host trafficking pathways, VP7 localization was shown to be independent of host trafficking mechanisms and evaded host defenses against aggregation. Studying the process of VP7 crystalline-like particle formation will help us further understand AHSV replication and assembly.

Utilization of cellulose microcapillary tubes as a model system for culturing and viral infection of mammalian cells.

Cryofixation by high-pressure freezing (HPF) and freeze substitution (FS) gives excellent preservation of intracellular membranous structures, ideal for ultrastructural investigations of virus infected cells. Conventional sample preparation methods of tissue cultured cells can however disrupt the association between neighboring cells or of viruses with the plasma membrane, which impacts upon the effectiveness whereby virus release from cells can be studied. We established a system for virus infection and transmission electron microscopy preparation of mammalian cells that allowed optimal visualization of membrane release events. African horse sickness virus (AHSV) is a nonenveloped virus that employs two different release mechanisms from mammalian cells, i.e., lytic release through a disrupted plasma membrane and a nonlytic budding-type release. Cellulose microcapillary tubes were used as support layer for culturing Vero cells. The cells grew to a confluent monolayer along the inside of the tubes and could readily be infected with AHSV. Sections of the microcapillary tubes proved easy to manipulate during the HPF procedure, showed no distortion or compression, and yielded well preserved cells in their native state. There was ample cell surface area available for visualization, which allowed detection of both types of virus release at the plasma membrane at a significantly higher frequency than when utilizing other methods. The consecutive culturing, virus infection and processing of cells within microcapillary tubes therefore represent a novel model system for monitoring intracellular virus life cycle and membrane release events, specifically suited to viruses that do not grow to high titers in tissue culture.

Genome segment reassortment identifies non-structural protein NS3 as a key protein in African horsesickness virus release and alteration of membrane permeability.

The role of African horsesickness virus (AHSV) nonstructural membrane protein NS3 in determining the effects of AHSV infection on Vero cells was examined. NS3 protein sequences are highly variable and cluster into three phylogenetic groups, alpha, beta, and gamma. Three AHSV strains, with NS3 from alpha, beta, or gamma, were shown to have quantitatively different phenotypes in Vero cells. Reassortants between these strains, in which the S10 genome segment encoding NS3 was exchanged alone or with other segments, were generated and compared to parental strains. Exchange of the NS3 gene resulted in changes in virus release, membrane permeability and total virus yield, indicating an important role for NS3 in these viral properties. Differences in the cytopathicity and the effect on cell viability between the parental strains could not be associated with NS3 alone, and it is likely that a number of viral and host factors play a role.