Assembly of lipid droplet-associated ring structures in hepatitis C virus-infected cells via liquid-liquid phase...

Hepatitis C virus (HCV) exploits lipid droplets (LDs) during the production of infectious particles. Although several host factors, including stress granule (SG) proteins, have been reported to localize to LDs in HCV-infected cells, the mechanism by which these proteins are recruited to LDs has remained unclear. In this study, we show that Ras-GAP SH3 domain-binding protein 1 (G3BP1), an essential SG component, is recruited to LDs with a time lag of several hours or more after the HCV Core protein accumulates on LDs following infection, and that G3BP1 retains its ability to assemble SGs even when localized to LDs during the late phase of infection. Treatment of HCV-infected cells with compounds that disrupt liquid-liquid phase separation (LLPS) abolished the LD localization of G3BP1, as well as another SG protein, T-cell intracellular antigen-1, whereas the LD localization of the viral proteins Core and NS5A was not affected. Finally, we demonstrate that ectopic expression of HCV Core alone is insufficient to recruit G3BP1 to LDs; however, co-expression of Core together with subgenomic HCV RNA, even a replication-defective replicon RNA, leads to the accumulation of G3BP1 around LDs. Collectively, these findings suggest that during the HCV life cycle, the initial localization of Core to LDs, followed by the recruitment of newly synthesized viral RNA to the LD surface, drives LLPS-mediated assembly of biomolecular condensates containing G3BP1 and other host SG proteins around LDs.IMPORTANCEDuring the late stages of hepatitis C virus (HCV) infection, various viral and host proteins are known to accumulate around lipid droplets (LDs), where virion assembly is thought to occur. However, it has remained unclear how the expression of a limited subset of viral factors can induce such an extensive cellular reorganization. Here, we identify that HCV infection induces the relocalization of the stress granule (SG)-associated proteins G3BP1 and TIA-1 onto LDs through liquid-liquid phase separation (LLPS). We further demonstrate that the minimal requirement for G3BP1 recruitment to LDs is the coexistence of the HCV Core and the viral RNA. Because G3BP1 plays a central role in the interaction network underlying SG formation and is capable of mobilizing additional SG components, our findings suggest that LLPS-dependent assembly of biomolecular condensates occurs around LDs during HCV infection. This work provides mechanistic insight into HCV particle formation, including viral genome packaging, and offers a conceptual basis for developing future antiviral strategies.