Characterisation of the tilapia lake virus proteome and identification of an 11th protein, S9-F3

Tilapia Lake Virus (TiLV) is an emerging RNA virus threatening global tilapia aquaculture. While proteins encoded by segments 1-3 (polymerase) and 4 (nucleoprotein) are characterised, the functions of polypeptides from the remaining segments remain largely unknown. Here, we investigated the expression and subcellular localisation of all ten predicted TiLV proteins, confirming major polypeptides from each segment and identifying an additional 11th protein, S9-F3, translated from an alternative reading frame of segment 9. Microscopy of GFP-tagged constructs revealed S2 and S10 were predominantly nuclear, while S1, S3, S5, S8 and S9-F3 were mostly cytoplasmic. Bioinformatic analysis and inhibitor assays indicated a nuclear export signal in S9-F3 and confirmed its CRM1-dependent export. Evolutionary analyses showed selective pressure on S9 and S9-F3 and identified an S9-F3 homologue in a TiLV-like guppy virus. These findings uncover an alternative translation product and a regulated nuclear export mechanism in TiLV.