Membrane binding promotes oligomerization and functional activation of viral RNA-dependent RNA polymerase

Positive-sense, single-stranded RNA (ssRNA ⁺ ) viruses initiate infection by utilizing host ribosomes to directly translate proteins from their genomes. One of these proteins is the RNA-dependent RNA polymerase (RdRp), which is responsible for replicating the viral genome. Hepatitis C virus (HCV), a well-studied ssRNA ⁺ virus, encodes a membrane-bound RdRp, here termed NS5B, that can form oligomers. NS5B exists in two conformations; only one is believed to be capable of binding RNA. The relationship between membrane localization, oligomerization, and conformation remains unclear. We investigated whether membrane localization mediates NS5B’s oligomerization, conformation and function. In solution, NS5B exhibited temperature-dependent oligomerization that correlated with cooperative RNA binding and a conformational shift at micromolar concentrations. In contrast, total internal reflection fluorescence microscopy and atomic force microscopy revealed that membrane-bound NS5B forms stable, non-terminating oligomers at nanomolar concentrations, often localized near lipid raft domains. The presence of oligomers was correlated with membrane recruitment of RNA and nucleotides, suggesting functional activation. The lower concentration threshold for RNA binding on membranes may be explained by membrane-induced oligomerization that promotes the RNA-binding conformation of NS5B. Our findings are consistent with a model in which membrane association regulates NS5B’s function through oligomerization-driven conformational control.