Detection of a Fourth Orbivirus Non-Structural Protein

Abstract

The genus Orbivirus includes both insect and tick-borne viruses. The orbivirus genome, composed of 10 segments of dsRNA, encodes 7 structural proteins (VP1–VP7) and 3 non-structural proteins (NS1–NS3). An open reading frame (ORF) that spans almost the entire length of genome segment-9 (Seg-9) encodes VP6 (the viral helicase). However, bioinformatic analysis recently identified an overlapping ORF (ORFX) in Seg-9. We show that ORFX encodes a new non-structural protein, identified here as NS4. Western blotting and confocal fluorescence microscopy, using antibodies raised against recombinant NS4 from Bluetongue virus (BTV, which is insect-borne), or Great Island virus (GIV, which is tick-borne), demonstrate that these proteins are synthesised in BTV or GIV infected mammalian cells, respectively. BTV NS4 is also expressed in Culicoides insect cells. NS4 forms aggregates throughout the cytoplasm as well as in the nucleus, consistent with identification of nuclear localisation signals within the NS4 sequence. Bioinformatic analyses indicate that NS4 contains coiled-coils, is related to proteins that bind nucleic acids, or are associated with membranes and shows similarities to nucleolar protein UTP20 (a processome subunit). Recombinant NS4 of GIV protects dsRNA from degradation by endoribonucleases of the RNAse III family, indicating that it interacts with dsRNA. However, BTV NS4, which is only half the putative size of the GIV NS4, did not protect dsRNA from RNAse III cleavage. NS4 of both GIV and BTV protect DNA from degradation by DNAse. NS4 was found to associate with lipid droplets in cells infected with BTV or GIV or transfected with a plasmid expressing NS4.

Sensing and Control of Bluetongue Virus Infection in Epithelial Cells via RIG-I and MDA5 Helicases

“…It is caused by the BT virus (BTV), a viral agent of the Orbivirus genus of the Reoviridae family (40,55). The viral genome is composed of 10 segments of double-stranded RNA (dsRNA) that encode seven structural proteins (VP1 to VP7) and five nonstructural proteins (NS1 to NS4, NS3A) (3,48,49). There are currently 26 recognized serotypes (BTV-1 to BTV-26) worldwide that induce serotype-specific immunity (34).…”

Bluetongue virus genetic and phenotypic diversity: Towards identifying the molecular determinants that influence virulence and transmission potential

“…Non-structural protein NS4 appears to localize in the nucleolus of infected cells early during the viral replication cycle and contains "coiled-coil" domains that are similar to nucleic acid binding proteins (Belhouchet et al, 2011;Ratinier et al, 2011). Nucleic acid protection assays have demonstrated that NS4 is able to bind to double stranded DNA (Belhouchet et al, 2011) and it has been suggested that the protein may be involved in subverting host cell antiviral defences, possibly by modulating the transcription of genes in the nucleolus (Ratinier et al, 2011). Indeed it has been demonstrated that NS4 is able to confer a replication advantage to wild type BTV-8 in infected mammalian cells that are in an interferon induced anti-viral state in contrast to a BTV-8 mutant lacking the ability to express the protein (Ratinier et al, 2011).…”

Balance of RNA sequence requirement and NS3/NS3a expression of segment 10 of orbiviruses

“…Orbiviruses are non-enveloped, architecturally complex viruses *80 nm in diameter, which are composed of structural proteins VP1-7 organized in three capsid shells (Hewat et al, 1992). In addition, at least four non-structural (NS) viral proteins are expressed after infection (Belhouchet et al, 2011;Ratinier et al, 2011;Roy, 2005;Stewart et al, 2015;Zwart et al, 2015). NS3 (encoded by Seg-10, *26 kDa) and its N-terminaltruncated form NS3a (*24 kDa), expressed from a downstream in-frame start codon, function in virus release from infected cells (Celma & Roy, 2009;Wu et al, 1992).…”