Structural, Dynamical, and Entropic Differences between SARS-CoV and SARS-CoV-2 s2m Elements Using Molecular Dynamics Simulations

Abstract

The functional role of the highly conserved stem-loop II motif (s2m) in SARS-CoV and SARS-CoV-2 in the viral lifecycle remains enigmatic and an intense area of research. Structure and dynamics of the s2m are key to establishing a structure–function connection, yet a full set of atomistic resolution coordinates is not available for SARS-CoV-2 s2m. Our work constructs three-dimensional coordinates consistent with NMR solution phase data for SARS-CoV-2 s2m and provides a comparative analysis with its counterpart SARS-CoV s2m. We employed initial coordinates based on PDB ID 1XJR for SARS-CoV s2m and two models for SARS-CoV-2 s2m: one based on 1XJR in which we introduced the mutations present in SARS-CoV-2 s2m and the second based on the available SARS-CoV-2 NMR NOE data supplemented with knowledge-based methods. For each of the three systems, 3.5 μs molecular dynamics simulations were used to sample the structure and dynamics, and principal component analysis (PCA) reduced the ensembles to hierarchal conformational substates for detailed analysis. Dilute solution simulations of SARS-CoV s2m demonstrate that the GNRA-like terminal pentaloop is rigidly defined by base stacking uniquely positioned for possible kissing dimer formation. However, the SARS-CoV-2 s2m simulation did not retain the reported crystallographic SARS-CoV motifs and the terminal loop expands to a highly dynamic “nonaloop.” Increased flexibility and structural disorganization are observed for the larger terminal loop, where an entropic penalty is computed to explain the experimentally observed reduction in kissing complex formation. Overall, both SARS-CoV and SARS-CoV-2 s2m elements have a similarly pronounced L-shape due to different motif interactions. Our study establishes the atomistic three-dimensional structure and uncovers dynamic differences that arise from s2m sequence changes, which sets the stage for the interrogation of different mechanistic pathways of suspected biological function.

Effect of the SARS-CoV-2 Delta-associated G15U mutation on the s2m element dimerization and its interactions with miR-1307-3p

“…Similarly, it is interesting to note that, despite having the same size, the monomer bands for the reference s2m and s2m G15U migrate differently in the TBM gel (Figure 5, right, arrow 1 and 1’), suggesting that they have different monomer conformations in the presence of MgCl 2 . Our MD simulations for the reference s2m revealed that it has a kinked 3D structure; however, it is possible that the additional U15-A29 base pair in the upper stem of s2m G15U results in a more linear structure that will migrate further through the TBM gel and MD simulations are in progress in our laboratories to test this hypothesis (Kensinger et al 2022). Thus, we cannot determine if the single dimer band observed for the s2m G15U in the TBM gel (Figure 5, right, arrow 3) is originating from a mixture of linear kissing dimer and duplex conformations which will migrate at the same position, or from the duplex conformation formed spontaneously from an unstable kissing dimer during incubation and migration through the gel (Shetty et al 2010).…”

“…Notably, only a two-nucleotide difference exists between the s2m of SARS-CoV-2 and SARS-CoV, the related virus responsible for the 2002-2003 outbreak of “SARS.” One of those mutations, G>U at position 31 (G31U) of the s2m (29,758 in the reference SARS-CoV-2 genome, EPI_ISL_402123), has previously been conserved as a G in all other reported s2m elements, and our earlier study revealed that G31U is the causative mutation between differences in dimerization properties in SARS-CoV-2 s2m and SARS-CoV s2m, as well as in miR-1307-3p binding (Imperatore et al 2022; Zhao et al 2020). Moreover, our molecular dynamics (MD) simulation study showed that these dimerization differences are likely caused by increases in structural flexibility of the terminal loop in SARS-CoV-2 s2m compared to the rigid GNRA-like pentaloop motif found in the SARS-CoV s2m (Kensinger et al 2022). In SARS-CoV-2 s2m, an expansion to a “nonaloop” was computed to confer an entropic penalty, potentially explaining the reduction in kissing complex formation, the intermediate step in duplex formation (Kensinger et al 2022).…”

“…The copyright holder for this preprint this version posted February 10, 2023. ; https://doi.org/10.1101/2023.02.10.528014 doi: bioRxiv preprint our molecular dynamics (MD) simulation study showed that these dimerization differences are likely caused by increases in structural flexibility of the terminal loop in SARS-CoV-2 s2m compared to the rigid GNRA-like pentaloop motif found in the SARS-CoV s2m (Kensinger et al 2022). In SARS-CoV-2 s2m, an expansion to a "nonaloop" was computed to confer an entropic penalty, potentially explaining the reduction in kissing complex formation, the intermediate step in duplex formation (Kensinger et al 2022).…”

Delta SARS-CoV-2 s2m Structure, Dynamics, and Entropy: Consequences of the G15U Mutation

“…This is facilitated by finding the eigenvectors of the covariance matrix of atomic coordinates C , where each respective eigenvalue is a proportion of the total position variance. Ultimately, this procedure enables classification of simulated structures into conformational substates (CS), organized by structural similarity and dissimilarity. , Application of the k -means clustering algorithm partitioned PCA-reduced MD data into CSs that maximize dissimilarity between substates and identify representative centroid structures. , We performed PCA on different selections of atoms, particularly, the entire s2m (nt. 1–41) and the terminal loop (nt.…”

“…We previously reported differences in the atomistic structures and dynamics of the s2m of ancestral SARS-CoV and SARS-CoV-2 . Despite only two mutations (U5C and G31U) distinguishing SARS-CoV-2 s2m from SARS-CoV s2m, experimental and computational evidence show that the secondary and tertiary structures change drastically due to a base pair register shift. ,,, Aside from U5C and G31U, it has been reported that SARS-CoV-2 s2m has obtained other mutations, including a G15U (G29742U) substitution, which was first reported in May 2020, and most recently in April 2022 within the “Deltamicron” recombinant lineage. ,− In this case, Delta 21J_AY.4 and Omicron 21K/BA.1 variant genomes recombined resulting in a hybrid displaying signature mutations from both Delta and Omicron, with G15U being characteristic of the parent Delta variant. , …”