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 bound to an FGDF-containing peptide, which we
validated by site-directed mutagenesis.
Our findings present a common FGDF motif to bind G3BP, which has a negative regulatory
effect on the SG-nucleating function of G3BP. This molecular mechanism and the presented 3Dmodel
demonstrate the therapeutic potential of targeting this interaction
