Functional characterisation of the Tilapia Lake virus (TiLV) proteome

Abstract

Tilapia lake virus (TiLV) is a novel RNA virus posing a significant threat to global tilapia aquaculture and the food security of millions of people. The viral genome comprises 10 segments of linear negative-sense, single-stranded RNA. While the protein coded by segment 1 shares minimal sequence similarity to the PB1 protein of influenza C virus, the remaining proteins show no sequence homology to any other known sequences. Several features, including the presence of similar complementary sequences at the noncoding ends of all TiLV segments, a short uninterrupted uridine stretch at the 5'-terminus of TiLV genomic RNAs, and the nuclear transcription site of TiLV messenger RNAs, indicate similarities of TiLV to orthomyxoviruses. Accordingly, recent classification has designated TiLV as a new species (Tilapia tilapinevirus), within a novel family Amnoonviridae, under the same order as orthomyxoviruses (Articulavirales). Although the viral nucleoprotein has recently been proposed to derive from segment 4, there is still limited knowledge regarding the molecular characterisation of the remaining viral proteins, with currently no specific treatments or commercial vaccines available to combat the infection. In this study, the expression and cellular localisation of all ten TiLV proteins were investigated both in vitro and in eukaryotic cells. The in vitro results revealed major polypeptides translated from all segments, as well as suggesting the possibility of alternative translation initiation in certain segments. When GFP-fusion proteins were expressed in transfected cells, a combined cytoplasmic and nuclear localisation was observed for most polypeptides. Exceptions were the S2 and S10 polypeptides that accumulated predominantly in the nucleus, S1 that was exclusively cytoplasmic, and S5 that partially localised to the endoplasmic reticulum. Further sequence analysis demonstrated the presence of a signal peptide motif and N-linked glycosylation sites in segments 5 and 6, implying their involvement in membrane protein-associated secretory pathways. To characterise the viral membrane proteins, further biochemical and mutagenic analyses were conducted on segment 5. The results revealed possible glycosylation and the usage of alternative AUG codons in 5' end of the messenger RNA for translation initiation. A single-step infection was conducted to examine a single infectious cycle of TiLV in tilapia cells. This replication curve indicated a potential time frame of ~2.5 days required by the virus to complete its life cycle. A multi-step replication curve demonstrated the kinetics of virus spread within a population of cells over 10 days. Mass spectrometry analysis of the virus-infected cells confirmed the production of all primary segment polypeptides and revealed an additional protein from an overlapping open reading frame of segment 9, referred to as S9.3 protein. Further investigation of the sequence of S9.3 indicated the presence of a nuclear export signal, which was supported by localisation studies. In normal cells, S9.3 showed a purely cytoplasmic localisation, which upon treatment with the nuclear export inhibitor leptomycin B was reversed to being largely nuclear, suggesting CRM1-dependent nuclear export of S9.3. Moreover, an in-depth proteomic analysis of partially-purified virus samples unveiled potential viral structural proteins encoded by segments 1-4, 7, 8, 10 and perhaps 9, although further validation is necessary to elucidate the structural features of the S5 and S6 proteins. In summary, this study provides preliminary steps towards the functional characterisation of the polypeptides encoded by TiLV. These findings contribute to a deeper comprehension of TiLV virology and its evolutionary parallels with other viruses, paving the way for the development of vaccines and therapeutics.