Abstract
Transcription of non-segmented negative sense (NNS) RNA viruses follows a stop-start mechanism and is thought to be initiated at the genome’s very 3’-end. The synthesis of short abortive leader transcripts (leaderRNAs) has been linked to transcription initiation for some NNS viruses. Here, we identified the synthesis of abortive leaderRNAs (as well as trailer RNAs) that are specifically initiated opposite to (anti)genome nt 2; leaderRNAs are predominantly terminated in the region of nt ~ 60–80. LeaderRNA synthesis requires hexamer phasing in the 3’-leader promoter. We determined a steady-state NP mRNA:leaderRNA ratio of ~10 to 30-fold at 48 h after Ebola virus (EBOV) infection, and this ratio was higher (70 to 190-fold) for minigenome-transfected cells. LeaderRNA initiation at nt 2 and the range of termination sites were not affected by structure and length variation between promoter elements 1 and 2, nor the presence or absence of VP30. Synthesis of leaderRNA is suppressed in the presence of VP30 and termination of leaderRNA is not mediated by cryptic gene end (GE) signals in the 3’-leader promoter. We further found different genomic 3’-end nucleotide requirements for transcription versus replication, suggesting that promoter recognition is different in the replication and transcription mode of the EBOV polymerase. We further provide evidence arguing against a potential role of EBOV leaderRNAs as effector molecules in innate immunity. Taken together, our findings are consistent with a model according to which leaderRNAs are abortive replicative RNAs whose synthesis is not linked to transcription initiation. Rather, replication and transcription complexes are proposed to independently initiate RNA synthesis at separate sites in the 3’-leader promoter, i.e., at the second nucleotide of the genome 3’-end and at the more internally positioned transcription start site preceding the first gene, respectively, as reported for Vesicular stomatitis virus.
Highlights
Ebola virus (EBOV), a member of the Filoviridae family in the order Mononegavirales, causes a severe febrile illness with high fatality rates [1]
We identified the synthesis of abortive replicative RNAs that are initiated opposite to genome nt 2 and predominantly terminated in the region of nt ~ 60–80 near the transcription start site (TSS)
Our findings further argue against the model that leaderRNA synthesis is a prerequisite for each transcription initiation event at the TSS
Summary
Ebola virus (EBOV), a member of the Filoviridae family in the order Mononegavirales, causes a severe febrile illness with high fatality rates [1]. The terminal regions of the EBOV genome encode promoters for initiation of RNA synthesis by the viral polymerase complex as well as signals important for encapsidation. The genomic 3’-leader sequence harbors the EBOV replication and transcription promoter PE2 harbors eight consecutive 3’-UN5 hexamers that may encode encapsidation signals as assumed for similar sequence elements in the promoter of the paramyxovirus Nipah virus [12]. Deletion of the terminal 55 or 56 nt of the trailer promoter prevented rescue of full-length EBOV, suggesting that this region is important for replication and/or encapsidation [14,15]. 6 nt of the RNA are bound per NP molecule [19,20] This makes it likely that hexamer phasing in the 3’-leader promoter is functionally linked to NP coverage
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