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Regulation of VP30-Dependent Transcription by RNA Sequence and Structure in the Genomic Ebola Virus Promoter.

Abstract：

:Viral transcription and replication of Ebola virus (EBOV) is balanced by transcription factor VP30, an RNA binding protein. An RNA hairpin at the transcription start site (TSS) of the first gene (NP hairpin) in the 3'-leader promoter is thought to mediate the VP30 dependency of transcription. Here, we investigated the constraints of VP30 dependency using a series of monocistronic minigenomes with sequence, structure and length deviations from the native NP hairpin. Hairpin stabilizations decreased while destabilizations increased transcription in the absence of VP30, but in all cases, transcription activity was higher in the presence versus absence of VP30. This also pertains to a mutant that is unable to form any RNA secondary structure at the TSS, demonstrating that the activity of VP30 is not simply determined by the capacity to form a hairpin structure at the TSS. Introduction of continuous 3'-UN5 hexamer phasing between promoter elements PE1 and PE2 by a single point mutation in the NP hairpin boosted VP30-independent transcription. Moreover, this point mutation, but also hairpin stabilizations, impaired the relative increase of replication in the absence of VP30. Our results suggest that the native NP hairpin is optimized for tight regulation by VP30 while avoiding an extent of hairpin stability that impairs viral transcription, as well as for enabling the switch from transcription to replication when VP30 is not part of the polymerase complex.IMPORTANCE A detailed understanding is lacking how the Ebola virus (EBOV) protein VP30 regulates activity of the viral polymerase complex. Here, we studied how RNA sequence, length and structure at the transcription start site (TSS) in the 3'-leader promoter influence the impact of VP30 on viral polymerase activity. We found that hairpin stabilizations tighten the VP30 dependency of transcription but reduce transcription efficiency and attenuate the switch to replication in the absence of VP30. Upon hairpin destabilization, VP30-independent transcription - already weakly detectable at the native promoter - increases, but never reaches the same extent as in the presence of VP30. We conclude that the native hairpin structure involving the TSS (i) establishes an optimal balance between efficient transcription and tight regulation by VP30, (ii) is linked to hexamer phasing in the promoter, and (iii) favors the switch to replication when VP30 is absent.

journal_name

J Virol

journal_title

Journal of virology

authors

Bach S,Demper JC,Grünweller A,Becker S,Biedenkopf N,Hartmann RK

doi

10.1128/JVI.02215-20

subject

Has Abstract

pub_date

2020-12-02 00:00:00

eissn

0022-538X

issn

1098-5514

pii

JVI.02215-20

pub_type

杂志文章

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Regulation of VP30-Dependent Transcription by RNA Sequence and Structure in the Genomic Ebola Virus Promoter.