Klonierung der Genomsegmente des Oropouche-Virus und Charakterisierung der Interferon-antagonistischen Aktivität des S-Segment-kodierten NSs-Proteins

Abstract

Oropouche virus (OROV) is a human-pathogenic member of the genus orthobunyavirus within the family Bunyaviridae. Until now, neither an effective antiviral treatment for the dengue fever-like disease caused by OROV nor a vaccine for the prevention of OROV infections exists. Prerequisites to achieve these goals are a reverse genetics system for molecular characterization and targeted attenuation which will allow the development of a safe vaccine and a better understanding of the viral pathogenicity factors. In order to establish an OROV reverse genetics system all OROV gene segments of the OROV strain Trinidad were cloned, sequenced and all rescue and helper plasmids were generated. The activity of the cloned viral RNA-dependent RNA polymerase, which is the basis of the reverse genetics system, was investigated in minireplicon assays. The transcription and replication of an OROV-minigenome could not be achieved when the cloned OROV-polymerase was employed, however when it was replaced by a heterologous RNA polymerase of the related La Crosse virus the minireplicon system was functional. The reasons for the failure of the OROV minireplicon-system are not entirely clear. The results of the performed experiments indicate that the cloned OROV polymerase provides insufficient enzymatic activity und should be therefore re-cloned whereas the viral nucleoprotein and the non-coding ends of the minigenome which act as viral promoter are functional. Within the context of this work it was observed that OROV infections do not induce the antiviral type I interferon system. In order to identify the viral interferon-antagonistic pathogenicity factor, the S segment-encoded non-structural protein NSs was cloned and its ability to block type I interferon induction was investigated. It turned out that OROV-NSs is able to inhibit completely the activation of the IFN-β promoter. The obtained results indicate that OROV-NSs, similar to the NSs proteins of Rift Valley fever virus and Bunyamwera virus, inhibits IFN-β promoter activation by interference with cellular RNA polymerase II-mediated transcription. It could be therefore demonstrated for the first time, that OROV – like some other bunyaviruses - interfere with the type I interferon system by means of the NSs protein.