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

The s2m, a highly conserved 41-nt hairpin structure in the SARS-CoV-2 genome, serves as an attractive therapeutic target that may have important roles in the virus life cycle or interactions with the host. However, the conserved s2m in Delta SARS-CoV-2, a previously dominant variant characterized by high infectivity and disease severity, has received relatively less attention than that of the original SARS-CoV-2 virus. The focus of this work is to identify and define the s2m changes between Delta and SARS-CoV-2 and the subsequent impact of those changes upon the s2m dimerization and interactions with the host microRNA miR-1307-3p. Bioinformatics analysis of the GISAID database targeting the s2m element reveals a >99% correlation of a single nucleotide mutation at the 15th position (G15U) in Delta SARS-CoV-2. Based on 1H NMR spectroscopy assignments comparing the imino proton resonance region of s2m and the s2m G15U at 19°C, we show that the U15-A29 base pair closes, resulting in a stabilization of the upper stem without overall secondary structure deviation. Increased stability of the upper stem did not affect the chaperone activity of the viral N protein, as it was still able to convert the kissing dimers formed by s2m G15U into a stable duplex conformation, consistent with the s2m reference. However, we show that the s2m G15U mutation drastically impacts the binding of host miR-1307-3p. These findings demonstrate that the observed G15U mutation alters the secondary structure of s2m with subsequent impact on viral binding of host miR-1307-3p, with potential consequences on immune responses.

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.

Host microRNA interactions with the SARS-CoV-2 viral genome 3'-untranslated region.

The 2019 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has marked the spread of a novel human coronavirus. While the viral life cycle is well understood, most of the interactions at the virus-host interface remain elusive. Furthermore, the molecular mechanisms behind disease severity and immune evasion are still largely unknown. Conserved elements of the viral genome such as secondary structures within the 5'- and 3'-untranslated regions (UTRs) serve as attractive targets of interest and could prove crucial in furthering our understanding of virus-host interactions. It has been proposed that microRNA (miR) interactions with viral components could be used by both the virus and host for their own benefit. Analysis of the SARS-CoV-2 viral genome 3'-UTR has revealed the potential for host cellular miR binding sites, providing sites for specific interactions with the virus. In this study, we demonstrate that the SARS-CoV-2 genome 3'-UTR binds the host cellular miRNAs miR-760-3p, miR-34a-5p, and miR-34b-5p, which have been shown to influence translation of interleukin-6 (IL-6), the IL-6 receptor (IL-6R), as well as progranulin (PGRN), respectively, proteins that have roles in the host immune response and inflammatory pathways. Furthermore, recent work suggests the potential of miR-34a-5p and miR-34b-5p to target and inhibit translation of viral proteins. Native gel electrophoresis and steady-state fluorescence spectroscopy were utilized to characterize the binding of these miRs to their predicted sites within the SARS-CoV-2 genome 3'-UTR. Additionally, we investigated 2'-fluoro-D-arabinonucleic acid (FANA) analogs of these miRNAs as competitive binding inhibitors for these miR binding interactions. The mechanisms detailed in this study have the potential to drive the development of antiviral treatments for SARS-CoV-2 infection, and provide a potential molecular basis for cytokine release syndrome and immune evasion which could implicate the host-virus interface.

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.

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

The stem loop 2 motif (s2m), a highly conserved 41-nucleotide hairpin structure in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome, serves as an attractive therapeutic target that may have important roles in the virus life cycle or interactions with the host. However, the conserved s2m in Delta SARS-CoV-2, a previously dominant variant characterized by high infectivity and disease severity, has received relatively less attention than that of the original SARS-CoV-2 virus. The focus of this work is to identify and define the s2m changes between Delta and SARS-CoV-2 and subsequent impact of those changes upon the s2m dimerization and interactions with the host microRNA miR-1307-3p. Bioinformatics analysis of the GISAID database targeting the s2m element reveals a greater than 99% correlation of a single nucleotide mutation at the 15 th position (G15U) in Delta SARS-CoV-2. Based on 1 H NMR assignments comparing the imino proton resonance region of s2m and the G15U at 19°C, we find that the U15-A29 base pair closes resulting in a stabilization of the upper stem without overall secondary structure deviation. Increased stability of the upper stem did not affect the chaperone activity of the viral N protein, as it was still able to convert the kissing dimers formed by s2m G15U into a stable duplex conformation, consistent with the s2m reference. However, we find that the s2m G15U mutation drastically reduces the binding affinity of the host miR-1307-3p. These findings demonstrate that the observed G15U mutation alters the secondary structure of s2m with subsequent impact on viral binding of host miR-1307-3p, with potential consequences on the immune response.