Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase

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.

Distinctive features of the respiratory syncytial virus priming loop compared to other non-segmented negative strand RNA viruses

“…However, at a detailed level, there are differences between them. For example, whereas rhabdoviruses, paramyxoviruses and filoviruses have a single initiation site in their promoters, the pneumoviruses, RSV and HMPV, have two initiation sites [ 9 , 15 , 31 , 40 , 41 ] and whereas most nsNSVs investigated so far initiate RNA replication at position 1 of the promoter, ebolaviruses initiate at position 2 [ 42 , 43 ]. It is of interest to understand to what extent these different mechanisms reflect differences in the polymerase protein.…”

Distinctive Features of the Respiratory Syncytial Virus Priming Loop Compared to Other Non-Segmented Negative Strand Rna Viruses

“…However, at a detailed level, there are differences between them. For example, whereas rhabdoviruses, paramyxoviruses and filoviruses have a single initiation site in their promoters, the pneumoviruses, RSV and HMPV, have two initiation sites (9, 14, 30, 39, 40) and whereas as most nsNSVs investigated so far initiate RNA replication at position 1 of the promoter, ebolaviruses initiate at position 2 (41, 42). It is of interest to understand to what extent these different mechanisms reflect differences in the polymerase protein.…”