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

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,...

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Autores principales:	Simone Bach, Jana-Christin Demper, Paul Klemm, Julia Schlereth, Marcus Lechner, Andreas Schoen, Lennart Kämper, Friedemann Weber, Stephan Becker, Nadine Biedenkopf, Roland K Hartmann
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/c6ad5159461e4d67be7bcc56880b63d8
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spelling	oai:doaj.org-article:c6ad5159461e4d67be7bcc56880b63d82021-12-02T19:59:58ZIdentification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase.1553-73661553-737410.1371/journal.ppat.1010002https://doaj.org/article/c6ad5159461e4d67be7bcc56880b63d82021-10-01T00:00:00Zhttps://doi.org/10.1371/journal.ppat.1010002https://doaj.org/toc/1553-7366https://doaj.org/toc/1553-7374Transcription 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.Simone BachJana-Christin DemperPaul KlemmJulia SchlerethMarcus LechnerAndreas SchoenLennart KämperFriedemann WeberStephan BeckerNadine BiedenkopfRoland K HartmannPublic Library of Science (PLoS)articleImmunologic diseases. AllergyRC581-607Biology (General)QH301-705.5ENPLoS Pathogens, Vol 17, Iss 10, p e1010002 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
spellingShingle	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 Simone Bach Jana-Christin Demper Paul Klemm Julia Schlereth Marcus Lechner Andreas Schoen Lennart Kämper Friedemann Weber Stephan Becker Nadine Biedenkopf Roland K Hartmann Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase.
description	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.
format	article
author	Simone Bach Jana-Christin Demper Paul Klemm Julia Schlereth Marcus Lechner Andreas Schoen Lennart Kämper Friedemann Weber Stephan Becker Nadine Biedenkopf Roland K Hartmann
author_facet	Simone Bach Jana-Christin Demper Paul Klemm Julia Schlereth Marcus Lechner Andreas Schoen Lennart Kämper Friedemann Weber Stephan Becker Nadine Biedenkopf Roland K Hartmann
author_sort	Simone Bach
title	Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase.
title_short	Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase.
title_full	Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase.
title_fullStr	Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase.
title_full_unstemmed	Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase.
title_sort	identification and characterization of short leader and trailer rnas synthesized by the ebola virus rna polymerase.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/c6ad5159461e4d67be7bcc56880b63d8
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Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase.

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