Characterization of the Citrus Leaf Blotch Virus AlkB Domain and Its Involvement in Viral Accumulation

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 harbor 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 open reading frame 1 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 characterization of a viral AlkB domain and provide a critical reference for future mechanistic interrogation of AlkB-domain function across viral systems.