RNA-Dependent RNA Polymerase (RdRp): Structure, Function, and Antiviral Targeting in RNA Viruses

RNA-dependent RNA polymerase (RdRp) is the core catalytic enzyme that mediates RNA viral replication and transcription. The polymerase facilitates template and substrate channel assembly, nucleotide selectivity, and phosphodiester bond formation, making it an essential component of the viral life cycle. RdRps differ greatly in domain organization and replication initiation mechanisms across viruses, but their catalytic cores are highly conserved. The intrinsic low-fidelity replication of RdRp accelerates viral evolution, while some viruses fine-tune replication fidelity by proofreading exonucleases or accessory proteins that balance genome stability and adaptive variation. Given its conserved structure and non-redundant function, RdRp is a key target for broad-spectrum antiviral development, and numerous nucleoside (NIs) and non-nucleoside inhibitors (NNIs) are currently undergoing clinical or preclinical evaluation. However, three major obstacles limit inhibitor development: mutation-induced drug resistance, structural RdRp heterogeneity across viral species, and unfavorable pharmacokinetic properties. This review summarizes the structural basis and catalytic mechanisms of RdRp, discusses its functions during viral replication, genetic variation and virus-host interactions, and updates NI/NNI design, mechanisms, and development. We also dissect drug resistance mechanisms and inhibitor optimization strategies using advanced high-resolution structural and functional analyses. Overall, we provide insights for the rational development of broad-spectrum, potent antiviral agents with a low risk of RdRp resistance.