Analyse des Ebolavirus 3'-Leader-Promotors – mechanistische Beiträge von VP30, Promotorarchitektur, RNA-Sequenzelementen und RNA-Sekundärstrukturen zur viralen Transkriptions- und Replikationsinitiation

In recent years the human pathogen Ebola virus (EBOV) has been the causative agent of large epidemics in Central and Western Africa with fatality rates of up to 90 %. Up to date efficacious antiviral treatments are still missing. The Democratic Republic of the Congo is currently threatened by another severe EBOV epidemic. The EBOV genome is a non-segmented negative sense (NNS) RNA. Regulatory elements for viral replication and transcription are located at its 3’- and 5’-terminal regions (3’-leader, 5’-trailer). EBOV replication is conducted by a protein complex comprising three viral proteins, the RNA-dependent RNA polymerase L, its cofactor viral protein 35 (VP35) and the nucleoprotein NP. Viral transcription requires an additional transcription activator, viral protein 30 (VP30). It is thought that the viral polymerase uses a single entry site at the genome's very 3’ end to initiate transcription and replication. In case of some other NNS viruses evidence was provided that this mechanism entails the synthesis of an abortive transcript antisense to the 3‘-leader prior to initiation of mRNA synthesis at the first transcription start sequence (TSS) which is located at some distance to the genome 3' end. The genomic replication promoter is bipartite. Promoter elements 1 and 2 (PE1, PE2) are separated by the first TSS and additional nucleotides (spacer). To assure efficient replication, the spacer has to follow the „rule of 6“. Thus, it can only be extended or shortened by a multiple of 6 nucleotides. Furthermore, PE2 comprises 8 consecutive 3'-UN5 hexamers. Three of them are essential for residual replication activity. Furthermore, UN5 hexamer phasing was identified in this work to extend to the TSS-spacer region and the 5'-terminal part of PE1. However, here hexamer continuity in the TSS-spacer region is interrupted by a guanosine residue (instead of a uridine) at nt -75. TSS and spacer are predicted to form RNA secondary structures both on the genomic as well as on the mRNA level (NP hairpin). This NP hairpin was previously reported to be involved in VP30-dependent transcription regulation. The present study aimed to gain insight into initiation of polymerization at the genome 3‘-leader promoter. Apart from analyses on the replication and transcription promoter architecture, we focused on the analysis of regulatory RNA sequences, constraints of the TSS hairpin structure as well as on regulation by VP30. By use of selective replication-competent and replication-deficient minigenome variants combined with reporter gene assays and qRT-PCR, we demonstrated that PE1 also encodes the EBOV transcription promoter. Furthermore, our data shows that the “rule of 6” is not only key to productive replication initiation but to transcription initiation as well. Substantial replication and transcription activity was still detected when the spacer region was expanded by up to 66 nt, depending on sequence and structure context. The function of spacer and PE2 UN5 hexamers remains elusive. Presumably, they are involved in positioning NP on the RNA template in a certain phase that ensures productive promoter recognition by the viral polymerase, or they coordinate NP dissociation/reassociation from the RNA template while threading the RNA through the active site of the polymerase. Making UN5 hexamer phasing continuous between PE1 and PE2 by introduction of a G-75 to U point mutation led to an overall increase in viral transcription and replication. Though, this was at the expense of regulatory fine-tuning of both processes by VP30 (transcription activation and replication repression). A spacer deletion of 12 nt which simultaneously abolished NP hairpin structure formation still allowed replication and transcription activities > 50 % and was basically maintaining the regulation by VP30. This stresses the notion that RNA secondary structures are neither essential to VP30-dependent transcription nor to replication. However, we observed a trend toward relaxed VP30 dependency with destabilized hairpin structures, while stabilizations tightened the VP30 dependency of transcription. An incremental extension of helical double strand regions in the TSS hairpin by strategic insertion of G:C base pairs resulted in severe activity losses or elimination of viral polymerization, depending on the extent of stabilization on antigenomic/mRNA level. Thus, we provide first indirect evidence that TSS RNA secondary structures indeed form during viral RNA synthesis. In general, any deviations from the native NP hairpin with regard to sequence, structure, stability and length led to a relaxed VP30 dependency of transcription. Hence, the NP hairpin appears to be optimized for a balanced combination of tight regulation by VP30 and efficient transcription and replication. Moreover, RNA sequencing and Northern Blot experiments confirmed the synthesis of abortive leaderRNAs that are initiated opposite to genome position -2, exactly like replicative RNA (cRNA). In contrast, preservation of the first genome nucleotide increased transcription ~ 4-fold. Both initiation at nt -2 or termination in the range of nt -60 to -80 occurred irrespective of deviations in RNA sequence/structure or the absence of VP30. In the presence of VP30, overall leaderRNA synthesis was reduced. Steady-state leaderRNA levels were 9-fold or 68-fold lower than mRNA levels 24 h post EBOV infection or 48 h post minigenome transfection. In conclusion, our data suggest that leaderRNAs are products of abortive antigenome synthesis rather than obligatory pre-products of viral transcription.