Characterising Non-Structural Protein NS4 of African Horse Sickness Virus

Abstract

African horse sickness is a serious equid disease caused by the orbivirus African horse sickness virus (AHSV). The virus has ten double-stranded RNA genome segments encoding seven structural and three non-structural proteins. Recently, an additional protein was predicted to be encoded by genome segment 9 (Seg-9), which also encodes VP6, of most orbiviruses. This has since been confirmed in bluetongue virus and Great Island virus, and the non-structural protein was named NS4. In this study, in silico analysis of AHSV Seg-9 sequences revealed the existence of two main types of AHSV NS4, designated NS4-I and NS4-II, with different lengths and amino acid sequences. The AHSV NS4 coding sequences were in the +1 reading frame relative to that of VP6. Both types of AHSV NS4 were expressed in cultured mammalian cells, with sizes close to the predicted 17–20 kDa. Fluorescence microscopy of these cells revealed a dual cytoplasmic and nuclear, but not nucleolar, distribution that was very similar for NS4-I and NS4-II. Immunohistochemistry on heart, spleen, and lung tissues from AHSV-infected horses showed that NS4 occurs in microvascular endothelial cells and mononuclear phagocytes in all of these tissues, localising to the both the cytoplasm and the nucleus. Interestingly, NS4 was also detected in stellate-shaped dendritic macrophage-like cells with long cytoplasmic processes in the red pulp of the spleen. Finally, nucleic acid protection assays using bacterially expressed recombinant AHSV NS4 showed that both types of AHSV NS4 bind dsDNA, but not dsRNA. Further studies will be required to determine the exact function of AHSV NS4 during viral replication.

Synthesis of empty african horse sickness virus particles

“…The AHSV genome is composed of 10 segments of linear double-stranded (ds) RNA, which encode four nonstructural proteins (NS1 to NS4) and seven structural proteins (Bremer, 1976;Grubman and Lewis, 1992;Zwart et al, 2015). The virion is a nonenveloped icosahedral particle (Oellermann et al, 1970;Burroughs et al, 1994) composed of three concentric protein layers (Manole et al, 2012).…”

Immunoinformatics Prediction of Peptide-Based Vaccine Against African Horse Sickness Virus

“…There were 5 epitopes that was addressed as potentially promising epitopes as they bound the highest number of alleles. 'YTFGNKFLL' epitope was found to bind 8 showed affinity for 5 MHC I alleles. As a matter of confirmation, YTFGNKFLL, YAYCLILAL and WFFDYYATL peptides were docked with the equine MHC molecule using the patchdock server.…”

“…It has nine antigenically different identified serotypes that belongs to the Orbivirus genus of the Reoviridae family [3,8,9,15,16]. (AHSV) is a double-stranded RNA (dsRNA) virus with ten genome segments encoding seven structural proteins (VP1-VP7) and four non-structural proteins (NS1, NS2, NS3, NS3A) [1,6].…”

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).…”