Evolution of the 3′ Untranslated Region Enhances NS5B Binding During Cell Culture Adaptation of Classical Swine Fever Virus

Classical swine fever virus (CSFV) adapts to cell culture through progressive genetic changes that enhance viral replication and fitness. Although adaptive mutations in viral proteins have been extensively characterized, the contribution of noncoding genomic regions to this process remains poorly understood. In this study, we investigated the evolutionary dynamics of the CSFV 3′ untranslated region (3′ UTR) during serial adaptation in PK-15 cells and examined its functional interaction with the viral RNA-dependent RNA polymerase NS5B. CSFV isolated from spleen tissue was serially passaged in PK-15 cells, resulting in enhanced viral replication and the emergence of cytopathic effects at later passages. Sequencing of the 3′ UTR across multiple passages revealed the accumulation of nucleotide substitutions, insertions, and recurrent deletions, indicating strong selective pressure on this regulatory region during in vitro adaptation. Phylogenetic analysis based on alignment-derived evolutionary distances demonstrated a passage-dependent divergence of 3′ UTR sequences, with early passages clustering closely, intermediate passages forming a distinct subclade, and late passages exhibiting increased evolutionary distances, consistent with a stepwise and non-linear adaptive trajectory. Molecular docking analyses showed a progressive increase in binding affinity between evolved 3′ UTR RNA variants and NS5B across successive passages. An intermediate passage variant (P20), occupying a transitional phylogenetic position, was selected for molecular dynamics simulation and formed a stable NS5B 3′ UTR complex under explicit solvent conditions. The complex maintained structural integrity throughout the simulation, characterized by sustained hydrogen bonding, low intermolecular distances, and limited conformational fluctuations. Collectively, these findings demonstrate that evolutionary remodeling of the CSFV 3′ UTR enhances its interaction with NS5B, stabilizes the viral replication complex, and contributes to efficient cell culture adaptation, highlighting the functional importance of noncoding RNA elements in viral evolution.