Alphaviral Capsid Proteins Inhibit Stress Granule Assembly via Competitive RNA Binding With G3BP1

ABSTRACT Viral infection is one of the conditions that induce stress granule (SG) formation, a cellular defense mechanism that exerts antiviral effects. To counteract this host response, viruses have evolved a broad spectrum of strategies to inhibit SG formation. However, the molecular mechanisms underlying SG inhibition remain poorly understood. The nucleocapsid proteins play a critical role in virus replication and host interaction. Here, using Semliki Forest Virus (SFV) as a model, we uncover the function of the alphavirus nucleocapsid in SG inhibition. This inhibitory function depends on oligomerization mediated by an N‐terminal α‐helix and with a positively charged intrinsically disordered region (IDR). We show that SFV capsid directly competes with G3BP1 for RNA binding, thereby disrupting G3BP1‐RNA liquid–liquid phase separation (LLPS) in vitro and SG assembly in cells. This mechanism is conserved across the alphavirus family but is not shared by the nucleocapsid of SARS‐CoV‐2 or other endemic viruses examined. Notably, expression of a peptide from SFV capsid is sufficient to inhibit SG formation induced by Amyotrophic Lateral Sclerosis (ALS)‐associated mutations, suggesting potential therapeutic applications. Our findings reveal mechanistic insight into SG modulation by the viral capsid protein and provide a possible bioengineering tool for probing SG dynamics in health and disease.