Highly conserved s2m element of SARS-CoV-2 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 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.

The highly conserved stem-loop II motif is important for the lifecycle of astroviruses but dispensable for SARS-CoV-2

“…Any host pathway affected by the s2m is likely to be highly conserved since the s2m has been identified in viruses that are predicted to infect mammals, birds, and insects 6 . It is also possible that the s2m is a key element for viral RNA-RNA interactions during the viral lifecycle, as suggested by others 33,34 .…”

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

“…7,8 Specifically, conserved elements within coronavirus genomes are enticing targets for antiviral intervention due to an implied biological function and phylogenetic stability unlike other targets that vary due to constant mutation and contribute to immune escape. [9][10][11] However, complete atomistic structures of conserved sequences from experimental data have yet to be reported to guide the design and development of SARS-CoV-2 antiviral therapies.…”

“…[12][13][14][15][16][17][18][19][20][21] Although there is abundant speculation on the function of s2m in the field, 10,13,[18][19][20][21][22][23] we have reported that s2m contains a palindromic sequence in its terminal loop (GUAC, nt 20-23, Figure 1), a striking similarity to the conserved dimerization initiation site (DIS) in the human immunodeficiency virus (HIV) and X55 region in the hepatitis C virus (HCV). 11,24,25 During kissing complex formation between monomer RNA hairpin loops, unpaired terminal loop nucleotides form intermolecular base pairs facilitated through palindromic sequences. 26 Functionally, in these viral systems, these hairpins form kissing complexes that are converted to extended duplexes by the viral capsid protein, being involved in the genome dimerization.…”

“…26 Functionally, in these viral systems, these hairpins form kissing complexes that are converted to extended duplexes by the viral capsid protein, being involved in the genome dimerization. 11 However, mutations in SARS-CoV-2 s2m reorganize the hairpin secondary structure and impact kissing complex and extended duplex formation compared to SARS-CoV s2m. 24,25 While prior to SARS-CoV-2 the s2m element showed high conservancy, relative to the SARS-CoV s2m (Tor2 isolate), the SARS-CoV-2 s2m (Wuhan-Hu-1 isolate) differs in sequence by two nucleotides: U29732C and G29758U, named U5C and G31U in this study for simplicity (Figure 1).…”

“…24,25 While prior to SARS-CoV-2 the s2m element showed high conservancy, relative to the SARS-CoV s2m (Tor2 isolate), the SARS-CoV-2 s2m (Wuhan-Hu-1 isolate) differs in sequence by two nucleotides: U29732C and G29758U, named U5C and G31U in this study for simplicity (Figure 1). 11,[27][28][29] Our experimental results showed that the SARS-CoV s2m readily forms kissing dimers, whereas SARS-CoV-2 s2m exists in an equilibrium between monomers and kissing dimers. Moreover, the viral N protein can only convert the SARS-CoV-2 s2m kissing dimers into the extended duplex conformation, but not the SARS-CoV s2m.…”