Characterisation of the subcellular localisation of African horse sickness virus non-structural protein NS5

African horse sickness virus (AHSV) is an economically important orbivirus affecting equids throughout sub-Saharan Africa. Genome segment 10 encodes NS3 and an alternative +1 ORF predicted to encode non-structural protein NS5. In silico analysis of 105 unique NS5 ORFs predicted conserved N-terminal mitochondrial targeting features and a putative nuclear localisation signal. Mutagenesis suggested arginine residues at positions 21 and 22 contribute to mitochondrial targeting scores. Recombinant baculovirus constructs of wild-type and mutant NS5 fused to Strep-tag, Twin-Strep-tag, or eGFP were expressed in Sf9 cells. Confocal microscopy showed Strep-tagged NS5 predominantly nuclear, while eGFP fusions showed mixed nuclear and partial mitochondrial localisation, supporting dual organelle localisation and informing future functional studies.