Untersuchungen zur Replikation und Transkription von Marburg- und Ebolavirus

Filoviren, zu denen das Marburg- (MARV) und das Ebolavirus Zaire (EBOV-Z) gehören, zählen zu den tödlichsten Humanpathogenen, gegen die es weder ein Therapeutikum noch einen Impfstoff gibt. Innerhalb der Mononegavirales besitzen sie das längste Genom (etwa 19 kb) und als einzige Mitglieder neben den...

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Bibliographic Details
Main Author: Enterlein, Sven
Contributors: Klenk, Hans-Dieter (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2005
Online Access:PDF Full Text
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The two filoviruses Marburg (MARV) and Ebolavirus Zaire (EBOV-Z) are two of the most lethal human pathogens. There are currently no cures or vaccines available. Filoviruses have the longest genome within the order Mononegavirales, and along with the Pneumoviruses, possess a fourth nucleocapsid protein (VP30). In the case of EBOV VP30 acts as an activator for transcription whereas its role for MARV is unknown. In this work, the cis-acting signals for replication and transcription of MARV, EBOV-Z and Ebolavirus Reston (EBOV-R) were examined, and common motifs compared. Furthermore, a full-length rescue system for MARV was established, that was used for studies on VP30. In collaboration with the USAMRIID synthetic DNA analogues were analysed for their ability to interfere with replication of EBOV-Z in cell culture. The secondary structure of the transcription start signal (tss) of MARV was determined by chemical modification and was shown to differ significantly from the EBOV-Z tss. Disruption of the secondary structure of the EBOV-Z tss resulted in a transcription independent of VP30. Chimeras of the two secondary structures were hardly replicated and showed no transcriptional activity. Additional experiments supported the notion that rather the primary sequence than the secondary structure is important for replication and transcription. The EBOV-Z-specific minigenome system was used to intensely study the replication promoter. It was already known, that two promoter elements (PE) existed separated by exchangeable sequence. However, the sequence between the two PEs could only be extended or shortened by a multiple of 6 nts without effect on replication. In this work I could show that only 3 out of 8 adjacent Hexamers UN5 were necessary to support minimal replication. This motif is also found in MARV and EBOV-R. The current data, however, suggests that MARV possesses a single promoter in which signals for replication and transcription overlap. As a useful tool to examine MARV in detail, a full-length rescue system was established. Recombinant MARV was generated by transfection of a full-length antigenome along with plasmids encoding the nucleocapsid proteins L, VP30, VP35, and NP. This allowed the introduction of specific mutations into the genome and their effects on the viral life cycle. This project was done in close collaboration with Prof. Volchkov in Lyon. Using this system the role of VP30 was found to be essential to rescue recombinant MARV, although rather a structural than a catalytic function is proposed. Additionally, it was possible to rescue a full-length MARV that contained 18 nucleotides of the EBOV-Z replication promoter. Finally, the inhibition of EBOV-Z replication in cell culture by peptide-conjugated phosphorodiamidate-morpholino oligomers (P-PMOs) was examined. One P-PMO targeted against the translation start sequence of the VP35 gene exhibited good prophylactic properties and even some therapeutic effect when applied postinfection. The results could be verified in the mouse model by collaborators at the USAMRIID. The use of P-PMOs as a prophylactic or even therapeutic approach looks promising.