Unbranched rod-like RNA is required for RNA editing of hepatitis delta virus genotype 2 and genotype 4

•RNA editing of hepatitis delta virus genotype 2 and 4 involves the unbranched rod RNA•Editing rates are influenced by the base pairing surrounding of the editing site•Editing rates are controlled by the base pairing upstream of the editing core•The size of the upstream bulge of the editing site controls editing rates•Studying RNA editing across different genotypes offers insights into viral evolution RNA editing of the hepatitis delta virus (HDV) is essential for generating the large delta antigen, which is crucial for virion assembly. In HDV genotype 1 (HDV-1), editing occurs within the context of the unbranched rod-like structure characteristic of HDV RNA, while RNA editing in HDV-3 requires a branched double-hairpin structure. The regulation of RNA editing in HDV-2 and HDV-4 remains uncertain. Based on predictions of the unbranched rod-like RNA structures of HDV-2 and HDV-4, the editing site occurs as an A.C mismatch pair, surrounded by four base pairs upstream and two base pairs downstream of the editing site, respectively. To investigate HDV-2 and HDV-4 RNA editing, cultured cells were transfected with non-replicating editing reporters carrying wild-type sequences or specific mutations. The results revealed that the editing rates observed for wild-type HDV-2 and HDV-4 were fairly similar, albeit lower than that of HDV-1. Like HDV-1, both HDV-2 and HDV-4 showed a reduction in editing rate when the A.C mismatch pair and the immediately upstream base-paired region were disturbed. Notably, extending the downstream base-paired region from two to three or four (forming a structure identical to that of HDV-1) base pairs increased editing rate. Furthermore, we presented novel evidence that indicates the importance of the first bulge's size, located upstream of the editing site, and the base-pairing length within 7–13 and 28-39 nucleotides downstream of the editing site in influencing the HDV-4 editing rate. To summarize, our analyses suggest that the unbranched rod-like structures surrounding the editing site of HDV-2 and HDV-4 play a crucial role in regulating their RNA editing rates.