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 ...