Determinants of Chikungunya virus replication and pathogenesis in the mammalian host and the insect vector

" Chikungunya virus (CHIKV) is a mosquito-borne alphavirus posing a global health threat. Since its explosive reemergence in the Indian Ocean in 2004, it has spread worldwide causing large-scale outbreaks, most recently on La Réunion Island. CHIKV causes an acute febrile illness marked by severe arthralgia and myalgia, with symptoms that can persist for months to years, greatly impairing patients' quality of life. Despite the recurring CHIKV epidemics, no effective interventions- vector targeted, antiviral, or prophylactic- exist or are broadly used. This lack of countermeasures highlights critical gaps in our understanding of CHIKV biology, its replication strategies and the molecular mechanisms of pathogenesis. Closing these gaps is essential for developing novel therapeutic and preventive strategies. CHIKV replication is driven by a viral replication complex (vRC) composed of four CHIKV non-structural proteins (nsP1-nsP4) which remodel the host plasma membrane into spherules, the specialized sites for viral RNA synthesis. The mechanisms governing vRC assembly and the molecular coupling between RNA synthesis and nucleocapsid formation remain largely elusive. Among the CHIKV-encoded proteins, nsP3 stands out as the most enigmatic. It is essential for both viral replication and host adaptation, yet its precise molecular functions are unclear. Early in the infection process, nsP3 localizes to spherules, where it recruits host factors crucial for replication. We demonstrated that nsP3 hijacks the muscle-specific factor FHL1 to mediate replication and tissue-specific pathogenesis; however, the underlying mechanisms remain unknown. As infection progresses, nsP3 forms cytoplasmic organelles termed alphagranules that are a hallmark of alphavirus infection. We discovered that nsP3 assembles into a honeycomb-like tubular scaffold underpinning the architecture of alphagranules in infected cells. Alphagranules concentrate viral RNA, capsid protein and host factors important for infection such as FHL1 or G3BP proteins. nsP3 also contributes to mosquito vector specificity, which is critical for CHIKV global spread. Despite its central role in multiple processes, the molecular mechanisms governing nsP3's diverse functions remain elusive. Elucidating them will uncover previously unrecognized targets for antiviral therapy. The CHICAGO project aims to elucidate the molecular and cellular mechanisms governing CHIKV replication, host interactions, and pathogenesis, with a central focus on the multifunctional protein nsP3. The proposal builds on a foundation of novel concepts and seminal discoveries made by consortium members and is driven by the conviction that fundamental research into CHIKV biology is essential for advancing future antiviral and vaccine development. Spanning 36 months, the project is organized into five tightly integrated work packages (WPs): WP1- Define the architecture and dynamics of the CHIKV vRC using cryo-electron microscopy. WP2- Elucidate the composition, formation and function of alphagranules through biochemical reconstitution, super-resolution imaging, RNA sequencing, and proteomic analyses. WP3- Apply cryo-electron tomography to resolve the in situ organization of alphagranules and study their role in nucleocapsid assembly WP4- Uncover the molecular mechanisms by which FHL1 regulates CHIKV infection and disease, using in vivo models of muscle regeneration. WP5- Characterize how nsP3 interacts with mosquito-specific host factors to influence vector competence and alphavirus emergence potential. CHICAGO will deliver transformative insights into alphavirus biology by establishing a structural and functional basis of the CHIKV replication machinery and elucidating the central role of nsP3 in viral replication, pathogenesis and vector adaptation. The project will lay the conceptual and molecular foundation for novel antiviral therapies and vector-targeted interventions. Aligned with the ANRS-MIE scientific roadmap, CHICAGO is a consortium of recognized experts with complementary expertise and a strong track record of successful collaboration."