The western equine encephalitis (WEEV), an αvirus, causes fever and, in some cases, severe neurological disease in humans. We identified a 17-nucleotide stem-loop (SL1) containing a palindromic GUAC region in the 3’-UTR of the WEEV RNA genome which we showed by biophysical methods forms kissing dimers, implying a role in genome dimerization. Bioinformatics analysis revealed conservation of one or more SL1 variants in other αviruses, suggesting a shared biological function. However, a systematic investigation into structural determinants and dynamic and energetic underpinnings of kissing complex formation in αviruses has not been conducted. We address this gap by combining molecular dynamics (MD) simulations with in vitro data on WEEV SL1 and several αviral variants. Preliminary analysis showed stem-loop alterations affect dimerization; notably, SL1 loop palindrome mutations abolish kissing dimer formation whereas other mutations do not. We generated dimer structures for WEEV SL1 and two other mutant variants (C10U and U6C) using predictive Vfold3D/IsRNA pipeline and performed microsecond-scale MD simulations. Entropic analysis of the loop revealed C10U—the non-dimerizing mutant—exhibited reduced flexibility compared to WT and U6C, decreasing loop entropy by 10%. Principal-component analysis (PCA) demonstrated C10U differs significantly along the second principal component, reflecting enhanced intramolecular interactions within the stem-loop, specifically between C11 and G1 positions in a single hairpin. These interactions promote a more compact monomer, weakening intermolecular contacts required for kissing complex formation. We conclude that mutations within or adjacent to the GUAC region disrupt dimerization by suppressing intermolecular kissing interactions and promoting intramolecular folding. Our results rationalize why C10U fails to form a kissing dimer and establish a framework for probing the mutational scope of αviral kissing complexes, providing a foundation on structural determinants of RNA dimerization in viral replication.
Chartier et al. (Sun,) studied this question.