Regulation der Transkriptions-(Re)initiation durch RNA-Sekundärstrukturen an den Genstartstellen des Ebolavirus-Genoms
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Philipps-Universität Marburg
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Abstract
Ebola virus (EBOV) is a human-pathogenic virus with lethality rates of ~35 to 80 %. To date, there is no effective causal therapy. For developing such curative treatments, it is important to understand the functions of the virus at the molecular level. Within this work, the molecular basis of viral replication and transcription was investigated.
EBOV belongs to the non-segmented negative strand (NNS) RNA viruses. It´s genome encodes seven structural proteins. The genome end regions, 3'-leader and 5'-trailer, contain important regulatory sequences for viral replication and transcription. Replication of the viral genome is accomplished by the RNA-dependent RNA polymerase L, its cofactor VP35 (viral protein 35), and the nucleoprotein (NP). In addition to these three viral proteins, the specific transcription factor VP30 is required for viral transcription. The activity of VP30 is regulated by phosphorylation. The transcription start sequences of the individual genes are involved in RNA secondary structures that differ in length, structure and stability. One aim of this work was to gain a better understanding of the initiation of RNA synthesis at the transcription start site (TSS) in the 3'-leader promoter in comparison with structural constraints for reinitiation at internal gene start (GS) sites. Using mutant monocistronic minigenomes, the structural requirements for efficient transcription initiation at the 3'-leader promoter were investigated at the viral RNA level. Furthermore, bicistronic minigenomes were used to evaluate the conditions of reinitiation, in particular with regard to the differing secondary structures at the individual internal gene start sites. The regulation of viral transcription by VP30 was also analyzed in much detail.
By means of minigenome-based reporter gene assays and qRT-PCR quantifications using mutated monocistronic minigenome variants, it was shown that transcription and replication of EBOV are influenced by the length and stability of the secondary structure at the TSS. An extension of the spacer sequence between promoter elements 1 and 2 of up to 54 nucleotides is tolerated, while incremental stabilizations of the secondary structures rapidly decrease polymerase activity. In this way, the formation of the secondary structures could be indirectly demonstrated for the first time. Furthermore, small changes in length, structure or stability at the NP hairpin changed the VP30 dependence of viral transcription. Hairpin stabilizations reduce transcription activity in the absence of VP30, while hairpin destabilizations enhance VP30-independent transcription activity. However, in all instances transcriptional activity was higher with VP30 than without VP30, which also pertains to mutants that are unable to form any RNA secondary structure at the TSS. This finding demonstrates that VP30 function is not simply determined by the RNA's capacity to form a hairpin structure. Altogether, a picture emerges according to which the native NP hairpin has evolved to enable tight regulation by VP30 while avoiding hairpin stabilities that substantially impair viral transcription. In addition, the native NP hairpin is optimized for efficient switching from transcription to replication when VP30 is expelled from the polymerase complex owing to phosphorylation.
Another result is the identification of abortive leader RNAs (~60 to 80 nt) by RNA sequencing. Standard curves for different viral RNA species allowed us to quantify the ratio between viral RNA and leader RNA upon EBOV infection or minigenome transfection. It could be shown that leader RNA levels, upon EBOV infection or minigenome transfection, are lower than those of the first (NP) mRNA. Also, the presence of VP30 suppresses leader RNA synthesis and the structural requirements at the very 3'-end of the viral genome are different for transcription and replication. The leader RNAs thus appear to be termination products of antigenome synthesis. These findings support a mechanistic model according to which the viral polymerase complex recognizes the 3'-leader promoter differently and independently in the replicative versus transcription mode to initiate RNA synthesis at different positions.
Furthermore, it could be shown that the transcription factor VP30 is not only required for initiation at the transcription start site (TSS), but also plays an essential role in reinitiation at internal genes. The VP30 dependency at internal GS sites seems to be even more stringent than at the TSS. It was also demonstrated that the polymerase complex encounters different structural constraints during reinitiation at internal genes than during initiation at the TSS. Specifically, higher stabilizations of hairpin structures are tolerated at internal GS sites without loss of function, and hexamer phasing does not play a role at internal genes. Moreover, the function of the transcription promoter was investigated in more detail. Previous deletion analyses (Bach et al., 2020) had provided evidence that nucleotides -54 and -55 of promoter element 1 (PE1) adjacent to the TSS are an essential part of the transcription promoter. This assumption could not be confirmed in its generality. Rather, the role of these two nucleotides is context-dependent, i.e., in the context of the native NP hairpin, their deletion only leads to a partial loss of transcriptional activation, whereas deletion in the context of other hairpins derived from internal genes results in complete transcriptional inactivation. It was also shown that the base identities at positions -54/-55 as well as at position -58 of the TSS play a substantial role in transcription activation.
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Created: 2023Issued: 2025-05-15Updated: 2025-05-15
Faculty
Fachbereich Pharmazie
Publisher
Philipps-Universität Marburg
Language
ger
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DoctoralThesis
DFG-subjects
ReinitiationbicistronischBiowissenschaftenEbolavirusBiologieReplikationVirologieMinigenomTranskriptionInitiationmonocistronisch
DDC-Numbers
570
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Demper, Jana-Christin (M.Sc.): Regulation der Transkriptions-(Re)initiation durch RNA-Sekundärstrukturen an den Genstartstellen des Ebolavirus-Genoms. : Philipps-Universität Marburg 2025-05-15. DOI: https://doi.org/10.17192/z2024.0081.