Biomolecular condensation by liquid-liquid phase separation (LLPS) is increasingly recognized as a mechanism underlying SARS-CoV-2 replication. We investigate how the domain architecture of nonstructural protein 8 (Nsp8) contributes to condensate formation, material properties, and mesoscale organization. Using polyethylene glycol (PEG-8000) as a macromolecular crowder, full-length Nsp8 forms confocally visible droplets with liquid-like recovery by FRAP. Domain truncations reveal complementary contributions: N-terminal fragments (1-76, 1-85) and a C-terminal core (67-198) undergo LLPS, whereas 85-198 primarily aggregates. Fluorescent mixing shows the disordered 1-76 fragment partitions into 67-198 condensates, suggesting a client-like role for the N terminus in tuning condensate composition. Condensate material state depends on concentration. At high protein and PEG levels, droplets adopt gel-like properties with slow FRAP recovery, while lower concentrations yield faster exchange. Far-UV circular dichroism indicates a molten-globule-like conformation of the C terminus, consistent with recent studies, while predictions for the N terminus support high disorder that increases multivalency and lowers the LLPS threshold. These observations support a model in which a sticker-rich C-terminal core scaffolds condensates, while the disordered N terminus modulates their architecture. Nsp7, a known binding partner, co-localizes within Nsp8 condensates, suggesting that such assemblies can enrich replication cofactors. RNA (poly-U22) further promotes condensation and alters material properties, consistent with multivalent protein-RNA interactions. Small-angle neutron scattering (SANS) measurements performed at 16% D 2 O buffer conditions—where PEG is contrast matched—reveal low-Q structural correlations in condensates. The scattering profiles are analyzed using Teubner-Strey correlation-length models, probing mesoscale organization, and testing the possibility of PEG partitioning within dense phases. Together, these findings show how Nsp8’s modular domain composition and molecular partners regulate condensate formation and viscoelasticity, providing a structural framework for understanding viral replication assemblies.
Khan et al. (Sun,) studied this question.