ミカン葉斑紋ウイルスAlk Bドメインとそのウイルス蓄積への関与の特性評価

Chemical modifications are prevalent on the genomic and messenger RNAs of RNA viruses. The AlkB family proteins are a class of demethylases that have the capacity to modulate the abundance and distribution of chemical modifications on nucleic acids in a dynamic manner, thereby dictating the functional competence of viral RNAs and, ultimately, affecting viral infectivity and replication. A subset of positive-sense RNA viruses harbour an AlkB domain within their polyproteins; however, whether this domain displays enzymatic activity equivalent to that of canonical AlkB enzymes, and thus participates in virus-host interactions, remains unresolved. Here, we characterized the biological role of the AlkB domain embedded in ORF1 of citrus leaf blotch virus (CLBV). Site-directed mutagenesis of catalytic core residues within the domain did not abolish infectivity in Nicotiana benthamiana, but markedly delayed cell-to-cell movement, reduced viral accumulation in emerging leaves, and attenuated plant stunting. Moreover, m⁶A peaks in total RNA were significantly more abundant, and their distribution patterns on both host mRNAs and the viral genomic RNA were altered, in plants inoculated with the AlkB-mutated infectious clone. Collectively, these data suggest that the CLBV-encoded AlkB domain likely possesses m⁶A demethylase activity that is analogous to that of cellular AlkB proteins. The results of the present study provide the first functional characterisation of a viral AlkB domain and provide a critical reference for future mechanistic interrogation of AlkB-domain function across viral systems.