Inferring Protein Function in an Emerging Virus: Detection of the Nucleoprotein in Tilapia Lake Virus

Reem Abu Rass, Talia Kustin, Rachel Zamostiano, Nechama Smorodinsky, Daniella Ben Meir, Daniel Feder, Nischay Mishra, W. Ian Lipkin, Avi Eldar, Marcelo Ehrlich, Adi Stern, Eran Bacharach*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Emerging viruses impose global threats to animal and human populations and may bear novel genes with limited homology to known sequences, necessitating the development of novel approaches to infer and test protein functions. This challenge is dramatically evident in tilapia lake virus (TiLV), an emerging "orthomyxo-like" virus that threatens the global tilapia aquaculture and food security of millions of people. The majority of TiLV proteins have no homology to known sequences, impeding functionality assessments. Using a novel bioinformatics approach, we predicted that TiLV's Protein 4 encodes the nucleoprotein, a factor essential for viral RNA replication. Multiple methodologies revealed the expected properties of orthomyxoviral nucleoproteins. A modified yeast three-hybrid assay detected Protein 4-RNA interactions, which were independent of the RNA sequence, and identified specific positively charged residues involved. Protein 4-RNA interactions were uncovered by R-DeeP and XRNAX methodologies. Immunoelectron microscopy found that multiple Protein 4 copies localized along enriched ribonucleoproteins. TiLV RNA from cells and virions coimmunoprecipitated with Protein 4. Immunofluorescence microscopy detected Protein 4 in the cytoplasm and nuclei, and nuclear Protein 4 increased upon CRM1 inhibition, suggesting CRM1-dependent nuclear export of TiLV RNA. Together, these data reveal TiLV's nucleoprotein and highlight the ability to infer protein functionality, including novel RNA-binding proteins, in emerging pathogens.

Original languageEnglish
Article numbere01757-21
JournalJournal of Virology
Issue number6
StatePublished - Mar 2022


  • RNA-binding protein
  • emerging virus
  • nucleoprotein
  • tilapia lake virus


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