# Distinct 5′ UTR Requirements for Translation of the Bicistronic X/P mRNA Among Avian Orthobornaviruses

## TL;DR

The study shows that different avian bornaviruses have distinct requirements for the 5′ UTR in their X/P mRNA to control protein translation and replication.

## Contribution

The study reveals clade-specific differences in 5′ UTR requirements for X/P mRNA translation in avian orthobornaviruses.

## Key findings

- PaBV-5 requires a long 5′ UTR with a stem–loop and upstream ORF to maintain X/P translational balance.
- PaBV-4 can tolerate a PaBV-5 5′ UTR without affecting translation or replication.
- ABBV-1 replication is efficient with PaBV-5 5′ UTR but is strongly impaired with PaBV-4 5′ UTR.

## Abstract

Orthobornaviruses express X and the phosphoprotein (P) from a bicistronic X/P mRNA, and these proteins regulate polymerase activity. In mammalian orthobornaviruses, the 5′ untranslated region (5′ UTR) of the X/P mRNA controls the translational balance between X and P and thereby promotes efficient replication. Avian bornaviruses (ABVs) belong to two clades, clade‐2 and ‐3, that differ in the structure and length of the 5′ UTR of the X/P mRNA. However, the functional consequences of these differences remain unclear. Using reverse genetics, we generated chimeric viruses by reciprocally exchanging the 5′ UTR of the X/P mRNA among clade 2 parrot bornavirus 5 (PaBV‐5) and aquatic bird bornavirus 1 (ABBV‐1) and clade 3 PaBV‐4. In PaBV‐5, a long 5′ UTR with a stem–loop and an upstream ORF was required to maintain the X‐to‐P translational balance. On the other hand, replacing the 5′ UTR of the X/P mRNA from PaBV‐5 with that from PaBV‐4 reduced X expression and markedly impaired viral growth. However, PaBV‐4 tolerated the 5′ UTR of the X/P mRNA from PaBV‐5 without detectable effects on translation or replication, which suggests that translation of PaBV‐4 X/P mRNA does not depend on the origin of the 5′ UTR. Furthermore, ABBV‐1 replicated efficiently with the 5′ UTR of the X/P mRNA from PaBV‐5 but was strongly attenuated with that from PaBV‐4. Taken together, these results demonstrate a clade‐dependent requirement for the 5′ UTR for translation of the X/P mRNA and provide novel insights into the evolution of translational control in orthobornaviruses.