The role of African horse sickness virus non-structural protein NS4 in virulence and host immunity

Abstract

African horse sickness virus (AHSV) causes African horse sickness (AHS), a highly infectious vector-borne disease that impacts significantly on animal health. A live-attenuated vaccine is currently used to control AHS but has many associated risks and problems. Little is known about the function of the recently identified fourth AHSV non-structural protein, NS4. AHSV NS4 exists as one of three variants: NS4-I, NS4-II or NLS-NS4-II and whilst orbiviruses replicate exclusively in the cytoplasm, the NS4 proteins of AHSV and the related bluetongue virus (BTV) are nucleocytoplasmic and bind dsDNA. BTV NS4 is an interferon antagonist and virulence factor, and its early expression and localisation to the plasma membrane suggest possible roles in virus entry and/or exit. AHSV NS4 may have an analogous role to BTV NS4. This study aimed to investigate the role of AHSV NS4 in virus virulence and host immunity, to increase our understanding of the function of NS4.

The interaction (if any) of NS4 with mitochondria and other AHSV non-structural proteins associated with virus entry and exit was investigated. Overall, limited colocalisation with NS4 was observed at the perimeter of NS1 tubule bundles, NS2 viral inclusion bodies (VIBs) and perinuclear NS3. No NS4 was observed in the matrix of VIBs, and NS4 did not colocalise with NS3/A at the plasma membrane. Therefore, AHSV NS4 is unlikely to be involved in virus assembly or exit. Some limited colocalisation of NS4 was observed at the perimeter of mitochondria clusters. Hence it is possible that NS4 associates with the outer mitochondrial membrane, perhaps interfering with RIG-I-like receptor signalling in innate immunity.

To further understand the role of NS4 in virus replication, virulence and pathogenesis, reverse genetics was used to generate AHSV NS4 knockout and reassortant viruses based on virulent AHSV5 (expressing NS4-II) and attenuated AHSV4LP (expressing NS4-I). In vitro assays showed the expression and intracellular localisation of NS4 is dependent on Seg-9, not the backbone into which it is incorporated. This segment encodes both VP6 and NS4, therefore this could be due to one, or both, of the proteins. It was also shown that NS4-II, and/or VP6 encoded by AHSV5 Seg-9 (S95), may give AHSV a replication advantage in BSR cells.

All reverse genetics-derived viruses were injected into embryonated chicken eggs (ECEs), and virulence and pathogenesis were compared to wild-type viruses. Viruses containing S95 (NS4-II) remained virulent whereas those lacking NS4 expression were attenuated, suggesting that NS4-II is a virulence factor in ECEs. Vaccine trials undertaken at Deltamune (Pty) Ltd established that rAHSV5 (NS4-II) was virulent in horses, and that the same virus with NS4 knocked out (rAHSV5minNS4) was attenuated. Comparing the transcriptional response in a subset of these horses suggested that the absence of NS4 allows the host to launch an earlier innate immune response. Further investigations indicated that the NS4 protein interfered with the nuclear accumulation of STAT1 during JAK-STAT signalling.

This study highlights the importance of AHSV NS4 in virulence, and suggests a mechanism of immune system evasion by AHSV.