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

Abstract

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 15th position (G15U) in Delta SARS-CoV-2. Based on 1H 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.

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

“…Yet, as discussed in greater detail below, the latter half of the 310 K simulation allowed for additional sampling of terminal loop conformations. In contrast, the system simulated at 283 K adopted and maintained a secondary structure consistent with our NMR assignments …”

“…In contrast to SARS-CoV-2 s2m, no long-range interaction that could serve to stabilize the backbone for a hinge point for an L-shaped kink was found. This result rationalizes observed differences in migration patterns in PAGE experiments, where the Delta s2m monomer band is slightly more mobile than the SARS-CoV-2 s2m monomer, suggestive of a difference in shape.…”

“…In contrast, the system simulated at 283 K adopted and maintained a secondary structure consistent with our NMR assignments. 23 s2m Dynamics. The total structure had an average backbone heavy atom RMSD of 8.4 ± 1.5 Å at 310 K (Figure 2A).…”

“…17,23 We have reported the ability of SARS-CoV and SARS-CoV-2 s2m to form an extended duplex conformation mediated by a kissing complex intermediate, and our homodimerization experiments show the additional G15U mutation in Delta SARS-CoV-2 s2m results in further kinetic differences in the formation of the kissing complex and extended duplex relative to both SARS-CoV and SARS-CoV-2 s2m. 13,23 Given our previous success in providing physical context and validation to our homodimerization experiments, we presently aim to expand our knowledge to include the Delta s2m.…”

“…Figure1. H 1 ,H 1 -NOESY base pair assignments reported for the s2m in (A) SARS-CoV-217 and (B) Delta SARS-CoV-2 23. Relative to SARS-CoV s2m, we have the U5C mutation in green, G31U in red, and G15U in blue.…”