Vida van Staden, University of Pretoria

Amy M. Mc Menamin 1 , Nhlakanipho Mtambo 1 , A. Christiaan Potgieter 2 , Vida van Staden 1 1 Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Pretoria, 0002, South Africa. 2 Deltamune (Pty) Ltd, Moraine House - The Braes, 193 Bryanston Drive, Bryanston, Gauteng, 2191, South Africa

Virus Replication: Entry, Exit and Everything in Between

African horse sickness virus (AHSV) remains a highly feared equine pathogen, causing annual disease outbreaks with severe morbidity and mortality in sub-Saharan Africa. AHSV expands its genomic coding capacity by utilising overlapping open reading frames (ORFs). Segment 10 encodes NS3 in the primary reading frame, while a second internal +1 ORF encodes the small non-structural protein NS5. The NS5 ORF is conserved across all AHSV strains, with predicted protein sizes ranging from 60 to 83 amino acids. The role of NS5 in the viral replication cycle and pathogenesis remains uncharacterised. Preliminary data indicated that AHSV NS5 localises to mitochondria, a feature seen in viral proteins that disrupt mitochondrial functions and interfere with innate immune responses via mitochondrial antiviral signalling (MAVS). This study aimed firstly to investigate the effect of NS5 knockout on virus virulence, and secondly to identify and functionally characterise mitochondrial targeting sequences (MTSs) in NS5. A recombinant NS5-knockout AHSV strain was rescued by reverse genetics and compared to wild-type virulent AHSV. Comparative growth kinetics assays and in ovo assessments allowed evaluation of virus attenuation. Following bioinformatics-based analyses, site-directed mutagenesis was employed to generate NS5-MTS mutants, which were expressed from recombinant baculoviruses and compared to wild-type NS5 by confocal laser scanning microscopy to visualise intracellular protein distribution. AHSV NS5 partially localised to the mitochondria in insect cells, and mutations in specific MTSs reduced this mitochondrial targeting. These results provide evidence to support a role for NS5 in mediating aspects of virus virulence by potentially antagonising host MAVS pathways.