AbstractsBiology & Animal Science

Stress granules (SG) are dynamic RNA/protein assemblies in the cytoplasm of the cell, formed under conditions of oxidative stress, heat shock or viral infections. These stress conditions trigger a sudden translational arrest, leading to a rapid switch of translation from housekeeping genes to stress-related factors. SGs fulfil multiple roles in the cell one of which is acting as triage centres for mRNA, where the mRNA is stored pending either degradation or reinitiation of translation. Many proteins are sequestered to SGs, among them signalling molecules, which make SGs signal centres to communicate a “state of emergency”. The importance of SGs is also underlined by the fact that they restrict viral propagation. The assembly of SGs is dependent on many RNA-binding proteins, one of which is G3BP (Ras-GAP SH3 domain binding protein). Semliki Forest virus (SFV) belongs to the alphaviruses, a large group of arthropod-borne animal viruses including several relevant human pathogens such as the re-emerging Chikungunya virus (CHIKV). Alphavirus infections lead to fever, rashes, arthralgia and can be lethal. Recent CHIKV outbreaks in the Caribbean area and the US, brings alphavirus research back on the agenda. Therefore there is a need to understand the molecular mechanisms how alphaviruses interact with their host. The aim of this thesis was to dissect virus-host cell interactions in the early response to alphavirus infection. Alphavirus infection leads to the formation of SGs at very early time points. Interestingly, they dissolve in the vicinity of viral replication complexes at later time points. In paper I, we showed that the non-structural protein nsP3 of SFV is responsible for sequestration of G3BP to replication complexes, by doing so, actively disassembling SGs and blocking their reformation. We mapped the binding site for G3BP to two C-terminal repeat domains of nsP3. A recombinant virus mutant lacking these repeats showed a longer and more persistent stress response and was attenuated in growth. In paper II, we extended this finding to the closely related CHIKV. Our results show that nsP3 of both SFV and CHIKV interact with G3BP via two C-terminal repeat domains and that the proline-rich region of nsP3 is dispensable for this interaction. In paper III we investigated the interaction between nsP3 and G3BP in molecular detail and determined that the residues FGDF in the C-terminal repeats of nsP3 are the G3BP binding motif. We further asked whether other proteins use the same mechanism as nsP3 to bind G3BP and whether this interaction inhibits the formation of SGs. We revealed that the phenylalanines and the glycine in the FGDF are essential for binding G3BP. We further demonstrated that the cellular ubiquitin-specific protease 10 (USP10) and the herpes simplex virus (HSV) protein ICP8 (infected cell protein 8) also bind G3BP via an FGDF motif. In addition we show that the FGDFmediated binding to G3BP leads to a negative regulation of G3BP’s SG-nucleating function. Lastly we present a 3D-model of G3BP…