Saddle Curvature Association of nsP1 Facilitates the Replication Complex Assembly of Chikungunya Virus in Cells

The replication of positive-sense RNA viruses, including the pathogenic SARS-CoV-1 and -2, DENV, and ZIKV, often takes place in curved cellular membrane compartments of tens to hundreds of nanometers in diameter within host cells. The non-structural proteins (nsPs) of viruses are found to critically control the formation and maintenance of such unique nanoscale membrane structures. However, the molecular mechanism underlying the association and assembly of nsPs around curved membranes has remained elusive, due to the technical difficulties of imaging the nanoscale interaction in live cells. In this study, we leveraged vertically aligned nanostructures to define membrane curvatures in live cells and investigated the assembly of viral nsPs on these curved membranes. Using Chikungunya virus as a model system, we found that its nsP1 was preferentially bound to and stabilized around positively curved membrane sites and the preference became more apparent as the radius of curvature decreased to 150 nm or smaller. This preferential accumulation was mainly attributed to the hydrophobic residues on recently identified membrane association loops (MA loops) of each nsP1 monomer. Molecular dynamics simulations further confirmed the improved binding kinetics and stability of nsP1 on positively curved membranes, especially when a 12-mer ring was formed by the nsP1 dodecamer. More interestingly, a saddle curvature association was enabled by the 3D coordination of nsP1 monomers along the dodecamer ring, where the MA loops of individual nsP1 monomers sensed a wide range of the positively curved membrane in the x-z plane while the rigid nsP1 dodecamer ring stabilized the negative curvature in the x-y plane. Its strong coupling to the saddle curvature fulfilled the need to constrain the neck of the membrane spherule for viral replication. Strikingly, productive CHIKV replication exhibited strong enrichment on patterned nanostructure array, proving the effectiveness of membrane curvature-guided assembly of the functional replication complex. Our findings revealed that the cell membrane facilitated the local enrichment of viral nsPs in a curvature- dependent manner. It opens up membrane curvature modulation as a new dimension to dissect the formation and regulation of membrane compartments for viral replication.