Turnover Rate of NS3 Proteins Modulates Bluetongue Virus Replication Kinetics in a Host-Specific Manner

Abstract

Bluetongue virus (BTV) is an arbovirus transmitted to livestock by midges of the Culicoides family and is the etiological agent of a hemorrhagic disease in sheep and other ruminants. In mammalian cells, BTV particles are released primarily by virus-induced cell lysis, while in insect cells they bud from the plasma membrane and establish a persistent infection. BTV possesses a ten-segmented double-stranded RNA genome, and NS3 proteins are encoded by segment 10 (Seg-10). The viral nonstructural protein 3 (NS3) plays a key role in mediating BTV egress as well as in impeding the in vitro synthesis of type I interferon in mammalian cells. In this study, we asked whether genetically distant NS3 proteins can alter BTV-host interactions. Using a reverse genetics approach, we showed that, depending on the NS3 considered, BTV replication kinetics varied in mammals but not in insects. In particular, one of the NS3 proteins analyzed harbored a proline at position 24 that leads to its rapid intracellular decay in ovine but not in Culicoides cells and to the attenuation of BTV virulence in a mouse model of disease. Overall, our data reveal that the genetic variability of Seg-10/NS3 differentially modulates BTV replication kinetics in a host-specific manner and highlight the role of the host-specific variation in NS3 protein turnover rate. IMPORTANCEBTV is the causative agent of a severe disease transmitted between ruminants by biting midges of Culicoides species. NS3, encoded by Seg-10 of the BTV genome, fulfills key roles in BTV infection. As Seg-10 sequences from various BTV strains display genetic variability, we assessed the impact of different Seg-10 and NS3 proteins on BTV infection and host interactions. In this study, we revealed that various Seg-10/NS3 proteins alter BTV replication kinetics in mammals but not in insects. Notably, we found that NS3 protein turnover may vary in ovine but not in Culicoides cells due to a single amino acid residue that, most likely, leads to rapid and host-dependent protein degradation. Overall, this study highlights that genetically distant BTV Seg-10/NS3 influence BTV biological properties in a host-specific manner and increases our understanding of how NS3 proteins contribute to the outcome of BTV infection. Bluetongue virus (BTV) is an arthropod-borne virus responsible for a hemorrhagic disease of domestic and wild ruminants (1, 2) and is transmitted between mammalian hosts by biting midges of the Culicoides family (3, 4). C. imicola represents the major vector species in Africa (3) and in southern Europe (5, 6), whereas C. obsoletus and C. pulicaris species have been involved in the transmission of BTV in central and northern Europe (7-9). The impact of bluetongue disease in Europe was relatively low before the 1990s (6, 10, 11). However, since 1998, several seasonal incursions of distinct BTV serotypes/strains have occurred virtually every year, causing massive economic losses to animal husbandry (7,(12)(13)(14)(15)(16). BTV is a nonenveloped virus that belongs to the Orbi...

Identification and genomic characterization of the first isolate of bluetongue virus serotype 5 detected in Australia

“…The Seg-10 of BTV-5 is also the first documented report of a Western topotype of this gene entering Australia. Segment 10 has recently been strongly implicated as a significant modulator of BTV pathogenicity (Feenstra et al 2014;Ftaich et al 2015;Pullinger et al 2016). Collectively, these data implied a potential for BTV genome segments that have been associated with highly pathogenic BTV serotypes in the Western episystem to be more rapidly transported to Southeast Asian episystems than previously thought plausible.…”

“…This comprises the inner core VP3 protein (Seg-3), a further outer core protein, VP7 (Seg-7) arranged in interconnected trimeric structures and a diffuse outercoat comprising proteins VP2 and VP5 (Seg-2 and Seg-6 respectively) (Hewat et al 1992(Hewat et al , 1994. Gene segments 5, 8 and 10 code for three non-structural proteins (NS1, NS2 and NS3 respectively) (Mertens et al 1984;Vandijk & Huismans 1988;Roy 1989Roy , 1992) and a second truncated form of NS3, lacking the first 13 aa (NS3a), is generated from a second initiation site (French et al 1989). A fourth non-structural protein (NS4) and a putative fifth non-structural protein (NS5) are expressed from overlapping ORF's (+ 1 reading frame shift) present on Seg-9 and Seg-10 respectively (Belhouchet et al 2011;Stewart et al 2015).…”

Neuroteratogenic Viruses and Lessons for Zika Virus Models

“…BTV target cells are subependymal neuronal and glial precursor cells. Susceptible mouse strains include BALB/c [81] and type I IFN receptor-deficient mice [82,83], but no BTV mouse model of TPT is reported.…”

Virus‐induced autophagic degradation of STAT 2 as a mechanism for interferon signaling blockade

“…Importantly, poly-Ub chains did not co-IP with NS4 protein (Fig EV5B), indicating that the ubiquitination is specific to NS3 protein. NS3 has two putative lysine residues at positions 13 and 15 susceptible to ubiquitination [49]. NS3 has two putative lysine residues at positions 13 and 15 susceptible to ubiquitination [49].…”

“…Ubiquitin is a globular protein that is covalently attached to target proteins through a modification known as ubiquitination (reviewed in [48]). Given NS3 banding pattern observed in Western blotting and the presence of two putative ubiquitination sites on NS3 [49], we tested by Co-IP whether NS3 bound ubiquitin. To this end, 293T cells were co-transfected with increasing amounts of HAtag Ub-expressing plasmid and a FLAG-tag NS3 expression plasmid; and Ub presence was assessed in NS3 pull downs by immune blotting (IB) ( Fig 6A).…”