Bluetongue virus NS4 protein is an interferon antagonist and a determinant of virus virulence

Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV non-structural protein NS4 favours viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage to this virus in primary sheep endothelial cells and in IFN-competent immortalized cell lines. We determined that in cells infected with a NS4 deletion mutant (BTV8ΔNS4) there is an increased synthesis of type I interferon (IFN) compared to cells infected with wild type BTV-8. In addition, using RNAseq, we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activity of a variety of promoters such as the cytomegalovirus immediate early promoter, the IFN-β promoter and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN-antagonist and a key determinant of viral virulence. IMPORTANCE Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by Culicoides biting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study we show that a non-structural protein of BTV (NS4) is critical to counteract the innate immune response of the host. Infection of cells with a BTV mutant lacking NS4 results in increased synthesis of IFN-β and upregulation of interferon stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by favouring viral replication in sheep, the animal species most susceptible to bluetongue