Segment-specific promoter activity for RNA synthesis in the genome of Oz virus, genus Thogotovirus

Oz virus (OZV), a tick-borne, six-segmented negative-strand RNA virus in the genus Thogotovirus, caused a fatal human infection in Japan in 2023. To study viral RNA synthesis, we developed an OZV minigenome assay using mammalian cells. This revealed variations in promoter activities among the six genome segments. The "distal duplex," a double-stranded RNA structure beginning at the 11th nucleotide on the 5' end and the 10th on the 3' end, was found in all segments. A factor affecting promoter activity was the base pairing between the 12th nucleotide at the 5' end and the 11th at the 3' end, forming either G:C or A:U pairs. Disruption of this pairing caused a significant loss of promoter activity, emphasizing the importance of the distal duplex with at least six consecutive base pairs. Comparative analysis of genome terminal sequences suggests similar structural variations in the promoters of other species in Thogotovirus. •A minigenome assay was developed to elucidate the RNA synthesis mechanism of Oz virus.•Oz virus is a six-segmented RNA virus, with variations in promoter activities observed across the six segments.•A double-stranded RNA structure starting at the 11th nucleotide on the 5' end and the 10th on the 3' end of the genome was identified in all segments.•The base pairing between the 12th nucleotide at the 5' end and the 11th at the 3' end (G:C or A:U pairs) influences promoter activity.