Alphavirus-host interactions : how to unravel the proviral activity of G3BP

Abstract: The genus Alphavirus belongs to the virus family Togaviridiae, a group of arthropod-borne enveloped RNA viruses with single-stranded, positive-sense genome. Members of this genus can be found worldwide and are geographically distinguished into Old World and New World alphaviruses. Old World alphaviruses include the re-emerging human pathogen chikungunya virus (CHIKV) and the model virus Semliki Forest virus (SFV). Upon cellular infection, the released viral genome is directly translated to produce the non-structural polyprotein, which is subsequently processed into four non-structural proteins. The non-structural protein 3 (nsP3) of Old World alphaviruses contains two FGDF motifs, which facilitate binding to the NTF2-like domain of the host protein G3BP and its recruitment to viral replication complexes. G3BP1 and G3BP2 (hereafter jointly referred to as G3BP) are homologous proteins, best characterized for their ability to assemble stress granules in response to a variety of cellular stressors, such as viral infection. In paper I we investigated the structural and functional significance of the two FGDF motifs for the Old World alphaviruses SFV and CHIKV. A 3-dimensional structure of the NTF2-like domain of G3BP1 in complex with a SFV nsP3-derived peptide showed that the two FGDF motifs crosslink dimers of G3BP1 into a nsP3:G3BP1 oligomer. Mutational analysis of the FGDF motifs furthermore revealed that both motifs are required for efficient growth of SFV and suggest a critical role for the formation of nsP3:G3BP oligomeric structures for SFV. CHIKV is non-viable if the nsP3:G3BP1 interaction is abrogated through mutation of both motifs. The presence of a single functional FGDF motif is sufficient to rescue CHIKV replication, albeit to a lesser extent than in the presence of both motifs. Together, the results of this paper highlight similarities, but also discrepancies between SFV and CHIKV for the two G3BP-binding motifs. In paper II we studied potential proviral roles of G3BP for SFV and CHIKV. To this end, we used a panel of human osteosarcoma (U2OS) cell lines lacking endogenous G3BP proteins and stably expressing G3BP1 mutants and truncation variants. SFV replication is attenuated in the absence of G3BP and efficiently rescued by the presence of only the NTF2-like domain of G3BP1, which is accompanied by clustering of replication complexes. On the contrary, CHIKV strictly depends on the presence of the NTF2-like and the RGG domains of G3BP1. By immunoprecipitation we show that the RGG domain of G3BP1 facilitates binding of nsP3:G3BP1 complexes to 40S ribosomal, which correlates with enhanced localized translational activity in close proximity to viral replication complexes. The results suggest that G3BP exerts several proviral activities by mediating clustering of viral replication complexes and the recruitment of the translation initiation machinery. The results of paper III demonstrate that Old Wold alphaviruses differ remarkably in their dependence on G3BP. We describe a role for the P4 residue of the cleavage site between nsP1 and nsP2 (1/2 site) of the alphavirus non-structural polyprotein in conferring the extent of G3BP sensitivity. An Arg residue at the P4 position of the 1/2 site, as for CHIKV, is associated with fast a cleavage rate at this site and a high sensitivity towards G3BP deletion. An His residue at this position, as for SFV4, confers a slower cleavage rate at this site, accompanied with partial resistance towards G3BP deletion. Arg-to-His substitution of the P4 residue of CHIKV allows partially rescues replication even in the absence of G3BP. However, our data suggest that G3BP proteins do not influence the processing of the non-structural polyprotein. Instead we propose a critical role for G3BP proteins during the initiation of viral RNA replication. In summary, the work presented in this thesis improves our understanding of alphavirus-host interactions and highlights previously unanticipated proviral roles of G3BP for Old World alphavirus infections. The results mark G3BP as a potential target for the development of antivirals and provide a platform for future investigations.