The Inhibition of RNA-Polymerase II-Mediated Expression by the Non-Structural Protein NSs of the Oropouche Virus and Establishing an Oropouche Virus Minireplicon System

by Thomas Essien
Doctoral thesis
Date of Examination:2015-06-02
Date of issue:2015-06-02
Advisor:Prof. Dr. Frank Torsten Hufert
Referee:Prof. Dr. Sabine Mihm
Referee:Prof. Dr. Martin Oppermann
Persistent Address: http://dx.doi.org/10.53846/goediss-5110

 

 

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Abstract

English

The OROV is a RNA virus that has a segmented, single stranded genome of negative polarity. It belongs to the Bunyaviridae family. Infection by the OROV causes an acute very debilitating febrile episode. This RNA virus is responsible for multiple and massive epidemic outbreaks in South America involving hundreds of thousands of infected people. The viruses of this family express a NSs protein as their major virulence factor, which inhibits the expression of IFN-β. IFN-β is a critical antiviral protein, whose expression, if inhibited, allows for the replication of virus particles. The experiments carried out in this thesis aim at characterizing the pathogenic mechanism of the NSs protein of the OROV. There are three transcriptional factors that are necessary for IFN-β gene expression and they are activated after viral recognition: IRF-3, NF-κB and AP-1. The OROV-NSs protein blocks the activation of the IFN-β promoter as well as the activation of the artificial IRF-3-, NF-κB- and AP-1-responsive promoters, and the constitutively active SV40 promoter. The inhibitory effect of the OROV-NSs does not lie in translation inhibition since mRNA can still be translated to protein in the presence of the NSs protein, irrespective of the cap-/IRES-status of the mRNA. Instead, the OROV-NSs inhibits IFN-β gene transcription probably by inhibiton of the RNAP II. A T7 polymerase based expression system can bypass the NSs inhibitory effect, showing certain specificity for inhibition of transcription mediated by mammalian RNAP II. In contrast to the NSs of its close relatives, BUNV and RVFV, the OROV-NSs does not influence the phosphorylation of the serine 2 (elongation and 3’-end processing) of the heptapeptide repeat at the CTD of the RNAP II. The OROV-NSs also does not prevent the phosphorylation of serine 5. Its transcription inhibitory effect is not due to the induction of degradation of the RNAP II. The OROV-NSs induces a reorganization of active RNAP II in the nucleus of some cells and is probably associated with the RNAP II-containing transcription complex. The second part of this thesis involved setting up a functional minireplicon system for the OROV, as this has never been done before. A minireplicon system is critical in understanding a viruses transcription and replication processes. Experiments carried out, showed that the functional OROV minireplicon system could not be established probably due to sequence errors either in the OROV-L ORF or in the NTRs of the OROV minigenome, which were present in the expression constructs due to the faulty OROV reference sequences in the international gene bank (NCBI).
Keywords: oropouche; orov nss; non structural protein; oropouche virus; oropouche minireplicon; oropouche rnap ii; oropouche rna polymerase ii
 
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