Base-paired structure in the 5′ untranslated region is required for the efficient amplification of negative-strand RNA3 in the bromovirus melandrium yellow fleck virus

• We analyzed the role of the 5′ UTR of RNA3 during MYFV replication.
• A base-paired structure in the 5′ UTR is required for efficient RNA3 amplification.
• In vitro assays showed the structure's importance in negative-strand amplification.
• A similar structure functions in BMV RNA3 with MYFV replicase but not that of BMV.
• Specific interactions may occur between base-paired structures and MYFV replicase.

Melandrium yellow fleck virus belongs to the genus Bromovirus, which is a group of tripartite plant RNA viruses. This virus has an approximately 200-nucleotide direct repeat sequence in the 5′ untranslated region (UTR) of RNA3 that encodes the 3a movement protein. In the present study, protoplast assays suggested that the duplicated region contains amplification-enhancing elements. Deletion analyses of the 5′ UTR of RNA3 showed that mutations in the short base-paired region, which is located dozens of bases upstream of the initiation codon of the 3a gene, greatly reduced the accumulation of RNA3. Disruption and restoration of the base-paired structure caused the accumulation of RNA3 to be decreased and restored, respectively. In vitro translation/replication assays demonstrated that the base-paired structure is important for the efficient amplification of negative-stand RNA3. A similar base-paired structure in RNA3 of another bromovirus, brome mosaic virus (BMV), also facilitated the efficient amplification of BMV RNA3, but only in combination with melandrium yellow fleck virus (MYFV) replicase and not with BMV replicase, thereby suggesting specific interactions between base-paired structures and MYFV replicase.