Oral Presentation Australasian RNA Biology and Biotechnology Association 2026 Conference

Lost in Translation: Flavivirus infection and the production of tRNA halves (144834)

Lachlan De Hayr 1 , Rhys Parry 1 , Lingjun Ye 1 , Jarvis Goh 1 , Elizaveta Reshetnikova 1 , Washington Romero Vasquez 1 , Ziyao Zhao 1 , Andrii Slonchak 1 2 3 , Alexander A Khromykh 1 2
  1. The University of Queensland, St Lucia, QLD, Australia
  2. AID, Australian Infectious Diseases Research Centre, Brisbane, QLD, Australia
  3. Systems Virology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia

Orthoflaviviruses such as West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV) remain important public health threats, yet no virus-specific therapies are available, in part because key virus–host interactions are still poorly defined. tRNA-derived stress-induced RNAs (tiRNAs) are small non-coding RNAs generated by cleavage of mature tRNAs within the anticodon loop, producing 5′ and 3′ tRNA halves. Although tiRNAs have been linked to cellular stress responses, their roles during viral infection are not well understood. Here, we investigated tiRNA biogenesis and function during orthoflavivirus infection. Small RNA sequencing of WNV-infected cells revealed a marked increase in 30–32 nt small RNAs, predominantly tiRNAs, with Glu-CTC, His-GTG and SeC-TCA 5′ fragments among the most strongly induced. Northern blotting confirmed that tiRNA production is conserved across WNV, JEV and YFV infections and in multiple WNV-infected cell types, including HEK293T, A549 and astroglial cells. Using qRT-PCR and RNA interference, we identified RNase4 as a virus-inducible nuclease required for infection-associated tiRNA production, in contrast to previous reports implicating angiogenin. Recombinant RNase4 also generated tiRNAs in vitro, confirming its direct tiRNA cleavage activity. Functional modulation using synthetic tiRNA antisense oligonucleotides supported a proviral role for tiRNAs in infected cells. Consistent with this, RNase4 knockdown substantially reduced tiRNA abundance and significantly impaired viral replication.  Additionally, the cleavage of mature tRNAs during infection depleted the cellular pool of intact tRNAs. Depletion of SeC-TCA tRNA coincides with reduced protein abundance of the selenoproteins GPX1 and GPX4 despite increased mRNA expression. Increasing GPX1 and GPX4 protein abundance prior to infection restricted viral replication. Collectively, these findings identify RNase4-driven tiRNA production as a conserved response to orthoflavivirus infection that promotes viral replication and represents a potential therapeutic target.