RNA secondary structure at the transcription start site influences EBOV transcription initiation and replication in a length- and stability-dependent manner

Bach, Simona; Demper, Jana-Christin; Biedenkopf, Nadine; Becker, Stephan; Hartmann, Roland K.

doi:10.1080/15476286.2020.1818459

Cited by 7 publications

(9 citation statements)

References 42 publications

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“…RNA-Seq, as introduced in Fig 2 , was performed with RNA from cells transfected with the wt NP MG, with a MG construct harboring a destabilized hairpin (NheI NP) and with the MG variant Δ5’ spacer lacking the capacity to form a hairpin structure owing to a 12-nt deletion in the spacer between PE1 and PE2 ( Fig 8A ) [ 40 , 44 ]. In line with the qRT-PCR results, we observed an increase in leader RNA amounts in the absence of VP30 for the wt (NP) MG and the two mutant MGs ( Fig 8B , red columns), while mRNA levels largely decreased in the absence of VP30 ( Fig 8C ).…”

Section: Resultsmentioning

confidence: 99%

“… ( A ) Illustration of the MG encoding the wt NP HP and mutant derivatives either encoding the NheI HP [ 44 ] destabilized particularly on the mRNA level or the Δ5’ spacer variant [ 40 ] devoid of any hairpin structure on the genomic and mRNA level. Minimum free energies (MFE; ΔG) of the secondary structures were predicted by RNAfold using the default parameters [ 80 ].…”

Section: Resultsmentioning

confidence: 99%

“…MG variants were constructed as described [ 18 , 40 , 41 ], using Dpn I-based site-directed mutagenesis techniques (see S1 Fig ). Cloning primers are specified in S1 Table .…”

Section: Methodsmentioning

confidence: 99%

“…The TSS (in cyan) and and a spacer sequence (orange residues) form the NP hairpin. Hexamer phasing in the leader promoter, manifesting as the need for a multiple of 6 nt between position 51 and 80, is crucial for EBOV replication and transcription initiation at the 3’-leader promoter [ 13 , 18 , 40 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…MG variants were constructed as described [ 18 , 40 , 41 ], using Dpn I-based site-directed mutagenesis techniques (see S1 Fig ). Cloning primers are specified in S1 Table .…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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“…These viruses share a common architecture of their genomes. The RNA genome length varies between 12 (RSV, VSV) and 19 (filoviruses) kb in length and contains essential untranslated regions (UTRs) at their 3 -(leader) and 5 -(trailer) terminal ends important for viral transcription, replication, and encapsidation [61][62][63][64][65]. While the number of genes encoded by nsNSV varies among its families (from 5 to 10), the organization and relative position of the structural genes is highly conserved: 3'-leader-Nucleoprotein N-Phosphoprotein P-Matrix protein (M)-Glycoprotein (G)-RNA-dependent RNA polymerase (L for large protein)-trailer-5 (Figure 2A,B).…”

Section: Viral Replication Cycle Of Negative Strand Rna Viruses (Nsv)mentioning

confidence: 99%

New Perspectives on the Biogenesis of Viral Inclusion Bodies in Negative-Sense RNA Virus Infections

Dolnik

Gerresheim

Biedenkopf

2021

Cells

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Infections by negative strand RNA viruses (NSVs) induce the formation of viral inclusion bodies (IBs) in the host cell that segregate viral as well as cellular proteins to enable efficient viral replication. The induction of those membrane-less viral compartments leads inevitably to structural remodeling of the cellular architecture. Recent studies suggested that viral IBs have properties of biomolecular condensates (or liquid organelles), as have previously been shown for other membrane-less cellular compartments like stress granules or P-bodies. Biomolecular condensates are highly dynamic structures formed by liquid-liquid phase separation (LLPS). Key drivers for LLPS in cells are multivalent protein:protein and protein:RNA interactions leading to specialized areas in the cell that recruit molecules with similar properties, while other non-similar molecules are excluded. These typical features of cellular biomolecular condensates are also a common characteristic in the biogenesis of viral inclusion bodies. Viral IBs are predominantly induced by the expression of the viral nucleoprotein (N, NP) and phosphoprotein (P); both are characterized by a special protein architecture containing multiple disordered regions and RNA-binding domains that contribute to different protein functions. P keeps N soluble after expression to allow a concerted binding of N to the viral RNA. This results in the encapsidation of the viral genome by N, while P acts additionally as a cofactor for the viral polymerase, enabling viral transcription and replication. Here, we will review the formation and function of those viral inclusion bodies upon infection with NSVs with respect to their nature as biomolecular condensates.

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Molecular mechanism of de novo replication by the Ebola virus polymerase

Peng,

Yuan,

Cheng

et al. 2023

Nature

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RNA secondary structure at the transcription start site influences EBOV transcription initiation and replication in a length- and stability-dependent manner

Cited by 7 publications

References 42 publications

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

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

New Perspectives on the Biogenesis of Viral Inclusion Bodies in Negative-Sense RNA Virus Infections

Molecular mechanism of de novo replication by the Ebola virus polymerase

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