Abstract
Respiratory syncytial virus (RSV) remains a leading cause of lower respiratory tract infections (LRTIs) and infant mortality worldwide. Despite recent advances in prophylactic interventions, effective therapeutics for active RSV infection are still lacking. Small molecule non-nucleoside inhibitors (NNIs) targeting the RSV L protein, particularly its polyribonucleotidyltransferase (PRNTase) domain, represent a promising antiviral strategy. Here, we evaluate the genetic variability of the PRNTase domain and the binding pocket of two NNIs, MRK-1 and MRK-2, to assess the potential for preexisting resistance. A comprehensive analysis of 28,140 RSV L protein sequences from NCBI Virus and GISAID EpiRSV databases revealed low overall variability within the PRNTase domain and near-complete conservation of the MRK-1/2 binding pocket. Resistance-associated mutations identified through in vitro dose-escalation studies localized to this pocket but were absent in global sequence datasets. These findings support the PRNTase domain as a genetically stable and viable target for NNI-based RSV therapeutics and suggest a low likelihood of preexisting resistance among circulating strains.