Transcription of ORFs on RNA2 and RNA4 of Rice stripe virus terminate at an AUCCGGAU sequence that is conserved in the genus Tenuivirus

•Transcription termination sites of ORFs on RSV RNA1-4 are determined.•AUCCGGAU in IR2 and IR4 is the dominant termination site for RNA2 and RNA4 transcripts.•AUCCGGAU is conserved in the genus Tenuivirus.

Rice stripe virus, the type member of the genus Tenuivirus, has four genomic RNAs. RNAs 2–4 have an ambisense coding strategy and the noncoding intergenic regions (IRs) separating the two ORFs are thought to function in termination of transcription. Sequencing the 3′-untranslated region of transcripts from RNA2 and RNA4 in virus-infected Oryza sativa (the natural host), Nicotiana benthamiana (an experimental host) and Laodelphax striatellus (the vector), showed that the sequences of p2 and pc2 transcripts on RNA2, and p4 and pc4 transcripts on RNA4 terminated with high frequency at a palindromic sequence AUCCGGAU that was located in a region predicted to form a hairpin secondary structure. The AUCCGGAU sequence is highly conserved in RNA2 and RNA4 of different RSV isolates and is also conserved among the corresponding genomic RNAs of other tenuiviruses. p3 transcripts from the three hosts all had the same dominant termination site, while pc3 transcripts from different hosts terminated at different sites. All pc1 3′-UTR sequences ended at the 3′-end of the viral complementary strand of RNA1 (data not shown), indicating that the pc1 transcript may be synthesized by runoff of viral polymerase, but had no characteristic termination sequence. This is the first experimental report determining the exact transcription termination sites of a plant ambisense virus, and has implications for understanding the transcription of RSV as well as other plant viruses with an ambisense coding strategy.