Mosquito-borne alphaviruses like Chikungunya virus (CHIKV) and Mayaro virus (MAYV), which caused endemic outbreaks involving millions of patients over the past decades, pose an emerging threat due to climate-driven expansion of the virus vectors. Recently, the first vaccine against CHIKV was approved by the FDA, however, the lack of antiviral treatments underscores the need for a deeper understanding of virus-host interactions, potentially leading to the development of novel antiviral strategies. In this thesis, we investigate the role of ubiquitin-specific protease 10 (USP10) in the context of CHIKV and MAYV. Our findings demonstrate that USP10 overexpression significantly reduces infection rates for both alphaviruses. This antiviral effect is independent of USP10's deubiquitinase activity and instead relies on its interaction with the stress granule protein G3BP, mediated by a specific motif (FGDF) within USP10. Notably, USP10 overexpression disrupts the formation of stress granules during CHIKV infection, likely by sequestering G3BP and preventing its aggregation. Exploring the impact of USP10 on different stages of the viral replication cycle, our findings suggest that USP10 primarily targets the later stages, inhibiting the formation and release of new virus particles. In contrast, CHIKV glycoprotein-mediated entry and viral RNA replication are not significantly affected by USP10. Analogously to USP10, CHIKV nonstructural protein 3 (nsP3) binds G3BP via two FGDF motifs. Cells infected with G3BP binding-deficient mutants displayed no reduction in viral infection upon USP10 overexpression, underlining a critical role of the G3BP-nsP3 interaction in the antiviral effect of USP10. Finally, we explored the emergence of potential escape variants after serial passaging of CHIKV under selection pressure by USP10 overexpression. Notably, mutations were observed in the nsP2 and E2 proteins, suggesting potential rescue mutations in these proteins counteracting the antiviral activity of USP10. This highlights the selective pressure exerted by USP10, highlighting its importance as an antiviral factor. In conclusion, this thesis establishes USP10 as a potent cellular inhibitor of alphavirus infection. By targeting the G3BP-nsP3 interaction, USP10 offers a promising path for the development of novel antiviral strategies.
