LLPS of FXR proteins drives replication organelle clustering for β-coronaviral proliferation

Abstract

β-Coronaviruses remodel host endomembranes to form double-membrane vesicles (DMVs) as replication organelles (ROs) that provide a shielded microenvironment for viral RNA synthesis in infected cells. DMVs are clustered, but the molecular underpinnings and pathophysiological functions remain unknown. Here, we reveal that host fragile X–related (FXR) family proteins (FXR1/FXR2/FMR1) are required for DMV clustering induced by expression of viral non-structural proteins (Nsps) Nsp3 and Nsp4. Depleting FXRs results in DMV dispersion in the cytoplasm. FXR1/2 and FMR1 are recruited to DMV sites via specific interaction with Nsp3. FXRs form condensates driven by liquid–liquid phase separation, which is required for DMV clustering. FXR1 liquid droplets concentrate Nsp3 and Nsp3-decorated liposomes in vitro. FXR droplets facilitate recruitment of translation machinery for efficient translation surrounding DMVs. In cells depleted of FXRs, SARS-CoV-2 replication is significantly attenuated. Thus, SARS-CoV-2 exploits host FXR proteins to cluster viral DMVs via phase separation for efficient viral replication.

Molecular Mechanisms and Potential Antiviral Strategies of Liquid–Liquid Phase Separation during Coronavirus Infection

“…The mechanism of coronavirus-induced LLPS involves the interaction between viral N proteins and RNA. LLPS is a shared intrinsic property among seven homologous N proteins from different HCoVs, undergoing phase separation under distinct in vitro conditions [37]. The initiation of LLPS by a major CoV N protein depends on its RNA binding and post-translational modification.…”

“…The multimerization interaction of CoV Nsps with RNA may be a key factor in the regulation of LLPS [52]. Host fragile X-related (FXR) family proteins (FXR1/FXR2/FMR1) are recruited by SARS-CoV-2 Nsp3 to cluster viral DMVs via LLPS for efficient viral replication [37].…”