The small genome segment of Bunyamwera orthobunyavirus harbours a single transcription-termination signal

Transcription termination of the mRNA produced from the small (S) genome segment of Bunyamwera orthobunyavirus (BUNV) has previously been mapped to two cis-acting sequences located within the 5′ UTR using a virus-free replication assay. The ability of these sequences to terminate transcription was attributed to the shared pentanucleotide motif 3′-UGUCG-5′. Taking advantage of our plasmid-based rescue system to generate recombinant viruses, we re-evaluated the importance of both pentanucleotide motifs as well as that of two other conserved sequences in transcription termination in vivo. Analysis of the 3′ ends of positive-stranded viral RNAs derived from the S segment revealed that only the region around the upstream pentanucleotide motif mediated transcription termination in cells infected with wild-type BUNV, leading to mRNAs that were about 100 nt shorter than antigenome RNA. Furthermore, the downstream motif was not recognized in recombinant viruses in which the upstream signal has been disrupted. Our results suggest that in the context of virus infection transcription termination of the BUNV S genome segment mRNA is exclusively directed by the upstream-termination signal. Interestingly, within this region we identified a motif similar to a transcription-termination sequence used by Rift Valley fever phlebovirus.