Role of an Essential Triloop Hairpin and Flanking Structures in the 3' Untranslated Region of Alfalfa Mosaic Virus RNA in in Vitro Transcription

The minus-strand promoter of Alfalfa mosaic virus (AMV), a tripartite plant virus belonging to the family Bromoviridae, is located within the 3'-terminal 145 nucleotides (nt), which can adopt a tRNA-like structure (TLS). This contrasts with the subgenomic promoter for RNA4 synthesis, which requires approximately 40 nt and forms a single triloop hairpin. Detailed analysis of the minus-strand promoter now shows that a similar triloop hairpin, hairpin E (hpE), is crucial for minus-strand synthesis. The loop sequence of hpE appeared to not be essential for RNA synthesis, whereas the identity and base-pairing capability of bases below the triloop were indeed essential. Reducing the size of the bulge loop of hpE triggered transcription from an internal site similar to the process of subgenomic transcription. Similar effects were observed when deleting (part of) the TLS, suggesting that tertiary contacts between hpE and the TLS prevent internal initiation. The data indicate that the minus-strand promoter hpE and the subgenomic promoter hairpin are equivalent in binding the viral polymerase. We propose that the major role of the TLS is to enforce the initiation of transcription by polymerase at the very 3' end of the genome.