Mapping the impact of 1'-, 2'- and 4'-nucleotide modifications on the Respiratory Syncytial Virus RNA-dependent RNA...

Human respiratory syncytial virus (RSV) causes pediatric bronchiolitis and severe respiratory illness in the elderly. Despite recent advancements in vaccines and antibody therapies, the search for antiviral agents remains a significant public health challenge. We designed nucleotide analogs (NAs) with ribose modifications to assess their incorporation by the RSV polymerase. Biochemical assays and structural modelling revealed that these NAs effectively disrupt RNA synthesis elongation. They act as chain-terminators via a unique mechanism mediated by the 4'-modification, whereas 2'-F alone has no effect and 1'-modification slows-down RNA synthesis. We evaluated the ability of the polymerase to discriminate between natural nucleotides and NAs through incorporation efficiency/competitive assays, correlating these findings with RSV replication inhibition in infected cell cultures. Our ranking of compounds indicates that cytidine analogs demonstrate the strongest antiviral activity, due to their phosphorylation efficiency and intracellular concentration relative to natural nucleotides as well as their ready incorporation into the growing RNA chain. 4'-modifications are accepted by the RSV polymerase due to structural differences between the active sites of (+) and (-) RNA virus polymerases.