Identification of the Bunyamwera bunyavirus transcription termination signal

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Abstract
Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae, which comprises segmented RNA viruses. Each of the BUNV negative-strand segments, small (S), medium (M) and large (L), serves as template for two distinct RNA-synthesis activities: (i) replication to generate antigenomes that are in turn replicated to yield further genomes; and (ii) transcription to generate a single species of mRNA. BUNV mRNAs are truncated at their 3′ ends relative to the genome template, presumably because the BUNV transcriptase terminates transcription before reaching the 5′ terminus of the genomic template. Here, identification of the transcription termination signal responsible for 3′-end truncation of BUNV S-segment mRNA was carried out. It was shown that efficient transcription termination was signalled by a 33 nt sequence within the 5′ non-translated region (NTR) of the S segment. A 6 nt region (3′-GUCGAC-5′) within this sequence was found to play a major role in termination signalling, with other nucleotides possessing individually minor, but collectively significant, signalling ability. By abrogating the signalling ability of these 33 nt, we identified a second, functionally independent termination signal located 32 nt downstream. This downstream signal was 9 nt in length and contained a pentanucleotide sequence, 3′-UGUCG-5′, that overlapped the 6 nt major signalling component of the upstream signal. The pentanucleotide sequence was also found within the 5′ NTR of the BUNV L segment and in several other members of the genus Orthobunyavirus, suggesting that the mechanism responsible for BUNV transcription termination may be common to other orthobunyaviruses.