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

Abstract

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 3’ UTR is highly conserved among coronaviruses and three other viral families. In this study, we demonstrate that the s2m element of SARS-CoV-2 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 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.

A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2

“…Typically, 2D [ 15 N, 1 H] HSQC spectra were acquired with spectral widths equivalent to 16 ppm for 1 H and 36 ppm for 15 N centered at 4.699 ppm and 117 ppm, respectively. 1D 19 F NMR experiments were performed with a spectral width of 50 ppm centered at a frequency corresponding to -70 ppm for CF3 containing molecules, while a spectral width of 150 ppm centered at -120 ppm was used for the remaining fluorine containing fragments, which were also subjected to 1 H-decoupling to prevent splitting and therefore loss of signal intensity. Ligand binding was monitored by measuring 19 F T2 relaxation with two relaxation periods (typically 5-10 ms and 200-300 ms) in the presence or absence of the macromolecules.…”

“…Here, we utilise a synergistic approach that combines NMR spectroscopy with CLIR-MS, a method that couples cross-linking and mass spectrometry (MS) 17,18 to characterise the interactions of the two structured RBDs of the N-protein with a folded RNA element, towards the final goal to identify interaction hotspots and inform a drug screening campaign (Figure 1A). As a proxy of structured viral RNA, we use s2m, a well folded and conserved RNA element that is located at the 3'UTR region and that is known to interact with the N-protein 19,20 . By combining the cross-linking sites with NMR chemical shift perturbations, we identified the interface of binding of both domains to s2m and used this information for guiding an NMR fragment screening by selecting molecules binding at these interfaces.…”

A 21L/BA.2-21K/BA.1 “MixOmicron” SARS-CoV-2 hybrid undetected by qPCR that screen for variant in routine diagnosis

“…This element is present in sarbecoviruses and in most of the SARS-CoV-2 genomes. It was proposed to be involved in RNA interference pathways, in hijacking of host protein synthesis, and in RNA recombination events (Imperatore et al, 2022; Gallaher et al, 2022), and it could have initiated viral infection and pathogenicity in various animal hosts. For instance, the s2m element was reported to interact with cellular miRNA-1307-3p in humans, being putatively able to manipulate the host immune response.…”

“…However, the acquisition by a Omicron 21L/BA.2 genome of the 3’ terminal part of a Omicron 21K/BA.1 genome is of very particular interest as this Omicron 21K/BA.1 fragment contains a short transposable element named S2m. S2m is a 41-nucleotide long stem loop motif that is present in four different families of positive-sense single-stranded RNA viruses (Robertson et al, 1998; Imperatore et al, 2022) and also shows high levels of similarity with sequences of insects (Tengs et al, 2021). This element is present in sarbecoviruses and in most of the SARS-CoV-2 genomes.…”

Targeting a conserved structural element from the SARS-CoV-2 genome using l-DNA aptamers

“…S8, ESI †). 38 Not unexpectedly, both aptamers bound the monomeric D-s2m-CoV2 hairpin but failed to bind the D-s2m-CoV1 homodimer duplex. Thus, the minor sequence differences between D-s2m-CoV2 and D-s2m-CoV1 result in dramatic conformational differences in vitro, that can be discriminated by L-aptamers.…”