Structural and Thermodynamic Consequences of the G31U and U5C Mutations in the SARS-CoV-2 S2m

Within the 3’-untranslated region of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome, the stem-loop II motif (s2m) RNA element harbors two mutations (U5C and G31U) that alter the secondary structure and suspected roles in the viral life cycle compared to the SARS-CoV s2m sequence. From previously reported NMR and homodimerization experiments, the G31U mutation alone gives rise to a secondary structure and dimerization properties similar to SARS-CoV-2 s2m, while the U5C mutation was found to be more characteristic of SARS-CoV s2m. The individual effects of each mutation on the structure, dynamics, and thermodynamics have not been reported and could guide the discovery of potential functional differences of s2m in distinct viral genomes. Unbiased 3.5 microsecond molecular dynamics simulations were performed using previously reported three-dimensional atomistic coordinates based on NMR NOE assignments and the SARS-CoV s2m 1XJR crystallographic coordinates for each U5C and G31U mutation independently. Tertiary structural dependence on temperature was investigated by running simulations at 283 and 310 K to represent NMR and physiological temperatures, respectively. Principal component analysis was used to identify s2m conformational substates, and a quasiharmonic partition function was used to estimate the absolute entropy associated with each model to quantitate the structural and dynamical impact of each individual mutation. Starting with the SARS-CoV-2 s2m structure, the G31U simulation remains consistent base-pairing with our 1H NMR spectrum, while the U5C model loses the base pairing pattern. Our work addresses the structural and terminal loop entropy differences as interpretations for previously reported NMR and homodimerization experiments. Overall, a quantitative structural and dynamical insight with atomistic resolution for each U5C and G31U mutation contribution is provided for the experimentally observed deviations between SARS-CoV and SARS-CoV-2 s2m.