Functional role of non-structural protein NS4 in the African horse sickness virus infection cycle

Abstract

African horse sickness (AHS) is a non-contagious disease causing severe illness and mortality in infected horses, and is listed as a notifiable disease by the World Organisation for Animal Health. The orbivirus African horse sickness virus (AHSV), which is transmitted via Culicoides biting midges, causes AHS. The only current viable AHS vaccine is the live attenuated vaccine approved in South Africa, however it has a number of limitations and new vaccines that meet international approval criteria are required. AHSV encodes seven structural proteins (VP1-VP7) and four non-structural proteins (NS1-NS4), but limited information is available on the function of the smallest non-structural protein NS4. Whilst the full replication cycle of AHSV occurs in the cytoplasm, NS4 localises to both the cytoplasm and nucleus of infected cells. This study aimed to investigate the functional role of the AHSV non-structural protein NS4 in the nucleus, and to gain insight into its role in AHSV replication, virulence and pathogenesis. All AHSV strains were shown to encode one of three variants of NS4, termed NS4-I, NS4-II or NLS-NS4-II, which differed in amino acid sequence and protein size. Whilst all NS4 types were present in the nucleus and the cytoplasm of infected cells, differences were apparent in the nuclear to cytoplasmic ratios. NS4-I had a statistically significant higher nuclear content than NS4-II, which had a higher cytoplasmic content. NLS-NS4-II displayed a homogeneous distribution in the cytoplasm with punctate foci in the nucleus. The presence of NS4 within specific nuclear compartments was investigated. All types of NS4 colocalised with promyelocytic leukaemia nuclear bodies (PML-NBs), while the shape of these PML-NBs was altered when cells were infected with AHSV lacking the expression of NS4. PML-NBs often play a role in host antiviral defense. In addition, novel intracellular distributions or interactions were described for two other non-structural proteins. NS1 localised in close proximity to PML-NBs in the nucleus, and NS2 in viral inclusion bodies colocalised with nuclear speckles in the cytoplasm. Bioinformatic analyses of NS4 predicted a nuclear export signal, a leucine zipper-like domain and phosphorylation sites, which were all present on the highly conserved C-terminal region. These functional domains may contribute to the unique intracellular localisation pattern of each NS4 type, as some functional domains were specific to an NS4 type. Truncations of NS4 revealed that the entire NS4 protein may be involved in DNA binding, and evidence was obtained indicating the phosphorylation of NS4-II. Reverse genetics was utilised to generate AHSV mutants, where either the leucine zipper domain or the phosphorylation sites in NS4 were abolished. Although these mutations did not impact AHSV replication and cell death in tissue culture, the mutant NS4 proteins showed a significantly lower nuclear presence than the wild type NS4. These AHSV mutants were also attenuated compared to the virulent wild type strain when inoculated into embryonated chicken eggs, resulting in milder or no clinical signs. These findings highlight the importance of NS4 in virulence and pathogenicity, and its potential to contribute to vaccine development for AHSV.