G3BP/Rin-Binding Motifs Inserted into Flexible Regions of nsP2 Support RNA Replication of Chikungunya Virus

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus. In infected cells, its positive-sense RNA genome is translated into polyproteins that are subsequently processed into four nonstructural proteins (nsP1 to 4), the virus-encoded subunits of the RNA replicase. However, for RNA replication, interactions between nsPs and host proteins are also needed. These interactions are mostly mediated through the intrinsically disordered C-terminal hypervariable domain (HVD) in nsP3. Duplicate FGDF motifs in the HVD are required for interaction with mammalian RasGAP SH3-binding proteins (G3BPs) and their mosquito homolog Rin; these interactions are crucial for CHIKV RNA replication. In this study, we inactivated G3BP/Rin-binding motifs in the HVD and inserted peptides containing either native or inactivated G3BP/Rin-binding motifs into flexible regions of nsP1, nsP2, or nsP4. Insertion of native motifs into nsP1 or nsP2 but not into the C terminus of nsP4 activated CHIKV RNA replication in human cells in a G3BP-dependent manner. In mosquito cells, activation also resulted from the insertion of inactive motifs after residue 8 or 466 in nsP2; however, the effect was significantly larger when the inserted sequence contained native motifs. Nonetheless, CHIKV mutants harboring mutations in the HVD and containing insertions of native motifs in nsP2 were not viable in mosquito cells. In contrast, mutant genomes containing native motifs after residue 466 or 618 in nsP2 replicated in BHK-21 cells, with the latter mutant forming infectious progeny. Thus, the binding of G3BPs to nsP2 can support CHIKV RNA replication and restore the infectivity of viruses lacking G3BP-binding motifs in the HVD of nsP3. IMPORTANCE CHIKV is a reemerging alphavirus that has spread throughout more than 60 countries and is the causative agent of chikungunya fever. No approved drugs or vaccines are available for the treatment or prevention of CHIKV infection. CHIKV replication depends on the ability of its replicase proteins to interact with host cell factors, and a better understanding of host cell factor roles in viral infection will increase our understanding of CHIKV RNA replication and provide new strategies for viral infection attenuation. Here, we demonstrate that the motifs required for the binding of host G3BP/Rin proteins remain functional when transferred from their natural location in nsP3 to different replicase proteins and may enable mutant viruses to complete a full replication cycle. To our knowledge, this is the first demonstration of interaction motifs for crucial host factors being successfully transferred from one replicase protein to another subunit of alphavirus replicase. CHIKV is a reemerging alphavirus that has spread throughout more than 60 countries and is the causative agent of chikungunya fever. No approved drugs or vaccines are available for the treatment or prevention of CHIKV infection.