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

Bioinformatic analysis of the Delta SARS-CoV-2 genome reveals a single nucleotide mutation (G15U) in the stem-loop II motif (s2m) relative to ancestral SARS-CoV-2. Despite sequence similarity, unexpected differences between SARS-CoV-2 and Delta SARS-CoV-2 s2m homodimerization experiments require the discovery of unknown structural and thermodynamic changes necessary to rationalize the data. Using our reported SARS-CoV-2 s2m model, we induced the G15U substitution and performed 3.5 microseconds of unbiased molecular dynamics simulation at 283 and 310 K. The resultant Delta s2m adopted a secondary structure consistent with our reported NMR data, resulting in significant deviations in the tertiary structure and dynamics from our SARS-CoV-2 s2m model. First, we find differences in the overall three-dimensional structure, where the characteristic 90° L-shaped kink of the SARS-CoV-2 s2m did not form in the Delta s2m resulting in a "linear” hairpin with limited bending dynamics. Delta s2m helical parameters are calculated to align closely with A-form RNA, effectively eliminating a hinge point to form the L-shape kink by correcting an upper stem defect in SARS-CoV-2 induced by a noncanonical and dynamic G:A base pair. Ultimately, the shape difference rationalizes the migration differences in reported electrophoresis experiments. Second, increased fluctuation of the Delta s2m palindromic sequence, within the terminal loop, compared to SARS-CoV-2 s2m results in an estimated increase of entropy of 6.8 kcal/mol at 310 K relative to the SARS-CoV-2 s2m. The entropic difference offers a unique perspective on why the Delta s2m homodimerizes less spontaneously, forming fewer kissing dimers and extended duplexes compared to SARS-CoV-2. In this work, both the L-shape reduction and palindromic entropic penalty provides an explanation of our reported in vitro electrophoresis homodimerization results. Ultimately, the structural, dynamical, and entropic differences between the SARS-CoV-2 s2m and Delta s2m serve to establish a foundation for future studies of the s2m function in the viral lifecycle.

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

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.

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

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.

Highly conserved s2m element of SARS-CoV-2 dimerizes via a kissing complex and interacts with host miRNA-1307-3p.

The ongoing COVID-19 pandemic highlights the necessity for a more fundamental understanding of the coronavirus life cycle. The causative agent of the disease, SARS-CoV-2, is being studied extensively from a structural standpoint in order to gain insight into key molecular mechanisms required for its survival. Contained within the untranslated regions of the SARS-CoV-2 genome are various conserved stem-loop elements that are believed to function in RNA replication, viral protein translation, and discontinuous transcription. While the majority of these regions are variable in sequence, a 41-nucleotide s2m element within the genome 3' untranslated region is highly conserved among coronaviruses and three other viral families. In this study, we demonstrate that the SARS-CoV-2 s2m element dimerizes by forming an intermediate homodimeric kissing complex structure that is subsequently converted to a thermodynamically stable duplex conformation. This process is aided by the viral nucleocapsid protein, potentially indicating a role in mediating genome dimerization. Furthermore, we demonstrate that the s2m element interacts with multiple copies of host cellular microRNA (miRNA) 1307-3p. Taken together, our results highlight the potential significance of the dimer structures formed by the s2m element in key biological processes and implicate the motif as a possible therapeutic drug target for COVID-19 and other coronavirus-related diseases.

Molecular Dynamic Simulations of Folding and Dimerization in Mutations Found in the s2m Motif of SARS-Cov-2

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, contains a highly conserved stem-loop within the 3? untranslated region called the s2m motif. Evidence suggests that the s2m motif plays a key role in replication and recombination of the virus. In SARS-CoV-2 we see slight differences in the s2m sequence from SARS-CoV-1, and the functional result of this mutation is unknown. We have also observed other mutations in the s2m motif commonly reported in COVID-19 patients in positions previously thought to be invariant. To better understand dimerization in the s2m region we used molecular dynamics simulations to analyze the folding of the mutant as monomers. Using AmberTools and NAMD, systems for the sequences were created, minimized, equilibrated, and then submitted for production. The results were aligned to the SARS-CoV-2 reference sequence using VMD and analyzed to find changes in thermodynamics, positions of the bases, and RSMD between the reference sequence and the mutants.