In part 1 of the thesis quantitative proteomics was used to determine changes in abundance and phosphorylation status of host proteins during infection with the human pathogen chikungunya virus ... Show moreIn part 1 of the thesis quantitative proteomics was used to determine changes in abundance and phosphorylation status of host proteins during infection with the human pathogen chikungunya virus (CHIKV). Several proteins were identified that may be specifically downregulated during CHIKV infection to create a suitable environment for viral replication. eEF2 was identified as a factor that becomes strongly phosphorylated during infection with several viruses which may be a mechanism to stall translation in response to viral infection. In part 2 of the thesis the discovery of a novel and unusual -2/-1 programmed ribosomal frameshifting (PRF) mechanism is described that is used during translation of the nonstructural open reading frame of the economically important porcine reproductive and respiratory syndrome virus (PRRSV). This mechanism relies on a slippery site and stimulatory RNA signal in the PRRSV genome and is stimulated by the viral protein nsp1beta and host poly (C) binding proteins 1 and 2. Frameshifting results in the synthesis of two previously unidentified proteins, nsp2 TF (-2 PRF) and nsp2N (-1 PRF). Virus mutants that can no longer make the frameshift products are attenuated and may be used for vaccine development. Show less
The order Nidovirales comprises a monophyletic group of viruses with positive-stranded RNA genomes that are classified in the families Arteriviridae, Coronaviridae, Mesoniviridae, and Roniviridae.... Show moreThe order Nidovirales comprises a monophyletic group of viruses with positive-stranded RNA genomes that are classified in the families Arteriviridae, Coronaviridae, Mesoniviridae, and Roniviridae. They share a conserved genome organization and a characteristic set of key replicative proteins. Although, in principle, this suggests a conserved replication mechanism, it is currently unclear how far exactly the resemblance extends on a more detailed level. This is foremost due to our poor understanding of the role of most viral proteins in the replication cycle. In addition, most of the knowledge that was obtained predominantly derives from studies of only few coronaviruses, the nidovirus subgroup with the largest known genome and therefore presumably employing the most complex replication strategy. In contrast, thus far only limited attention was given to the RNA rep_licating and processing enzymes of arteriviruses, and none at all to those of mesoni- and roniviruses, whose genome sizes are (much) smaller than those of coronaviruses. The work described in this thesis addresses some poorly or uncharacterized (domains of ) nonstructural proteins (nsps) that are likely involved in one or multiple steps of RNA replication and/or transcription of the prototypic arterivirus equine arteritis virus (EAV). Show less
