Identification and characterization of a novel non-structural protein of bluetongue virus.

Maxime Ratinier,Massimo Palmarini,Sandro Filipe Nunes,Frazer Rixon,Amr Bayoumy,Alessia Armezzani,Kathryn Allan,Andrew Shaw,Marco Caporale,Giulia Franzoni,Matthew Golder,Mark Heise

doi:10.1371/journal.ppat.1002477

Maxime Ratinier, Massimo Palmarini + Show 10 more

Open Access

https://doi.org/10.1371/journal.ppat.1002477

Copy DOI

Abstract

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77–79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell.

Highlights

Bluetongue is a major infectious disease of ruminants caused by an arbovirus (Bluetongue virus, BTV) transmitted by biting midges (Culicoides spp.) [1,2,3]
In this study we have shown that BTV expresses a previously uncharacterised non- structural protein that favours viral replication in cells in an antiviral state
By constructing deletion mutants by reverse genetics, we showed that NS4 is dispensable for viral replication in vitro, both in mammalian and insect cells, and in vivo in murine experimental models

Summary

Introduction

Bluetongue is a major infectious disease of ruminants caused by an arbovirus (Bluetongue virus, BTV) transmitted by biting midges (Culicoides spp.) [1,2,3]. In the last decade BTV has spread extensively in several geographical areas including Southern Europe and unexpectedly, in Northern Europe causing a serious burden to both animal health and the economy [5,6]. BTV is a member of the Orbivirus genus, within the Reoviridae family, and possesses a doublestranded RNA genome formed by 10 segments (Seg-1 to Seg-10) of approximately 19200 base pairs in total [1,3]. The BTV genome has been shown to encode for 7 structural and 3 non-structural proteins. The BTV genome is packaged within a triple layered icosahedral protein capsid of approximately 90 nm in diameter [1,7,8,9,10]. VP1 (RNA dependent RNA polymerase), VP4 (capping enzyme and transmethylase) and VP6 (RNA dependent ATPase and helicase) are contained within the core that is transcriptionally active in infected cells [15,16,17,18,19,20,21]

Methods

Results

Conclusion