Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome

Abstract

RNA structural elements occur in numerous single stranded (+)-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3′ UTR in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including SARS-CoV and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts implicates a key role of s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodelled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as anti-viral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The anti-viral potential of this conserved element represents a promising start for further research into targeting conserved elements in RNA viruses.

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

“…In this study, we contemplate the implications of a fine-tuned molecular switch from s2m element dimerization to miRNA-1307-3p binding, with the latter mechanism potentially aiding in severe COVID-19 symptom development and/or viral propagation. Altogether, the results of this study implicate dimerization of the highly conserved s2m element of SARS-CoV-2 as a potentially significant molecular target for antiviral therapeutics, such as antisense oligonucleotides, for COVID-19 as well as future coronavirus-related disease outbreaks ( 101 ).…”

Inhibition of SARS-CoV-2 by Targeting Conserved Viral RNA Structures and Sequences

“…For example, replacing the natural phosphodiester bridge with a phosphorothioate group in the ASO would significantly increase its half-life in vivo due to high serum protein binding and nuclease resistance ( Temsamani et al, 1993 ). Phosphorothioate linkage in ASOs retains the RNase H1 recognition and is usually used throughout gapmers ( Lulla et al, 2021 ). Alkylation of the 2’-OH in the ribose with a methoxyethyl group (MOE) in the ASO would enhance the hybridization stability and lessen the nonspecific binding ( Dhuri et al, 2020 ).…”

“…Two 2’-MOE/phosphorothioate-modified ASOs targeting this region, SE_ORF1ab_6449 and SE_ORF1ab_9456, were reported to effectively inhibit SARS-CoV infection in Vero E6 cells ( Figure 5 ) ( Sun et al, 2021a ). Gapmers 2 and 5 targeting the conserved S2M sequences in the 3’ UTR were demonstrated to have efficacy in degrading the viral RNA genome ( Figure 5 ) ( Lulla et al, 2021 ). The current development of ASO-based anti-SARS-CoV-2 agents is summarized in Table 1 .…”

“…RNA-targeting antisense oligonucleotides (ASO) or small molecules will degrade the viral RNA genome or hinder RNA translation ( Li et al, 2021b , Li et al, 2021a. ; Lulla et al, 2021 ; Rosenke et al, 2021 ; Sun et al, 2021a ; Zhang et al, 2021 ; Zhu et al, 2021 ). The SARS-CoV-2 main protease (M pro ) is also an attractive drug target.…”

No species-level losses of s2m suggests critical role in replication of SARS-related coronaviruses

“…The effect of swapping the 3′ UTR regions between s2m-containg and s2m-deficient virus strains has also been investigated, but the effect on viral fitness seems subtle 27 . Due to s2m’s highly conserved nature, it has also been suggested as a potential drug target 28 , 29 and as a polymerase chain reaction (PCR) primer site for exploring virus diversity 30 . Further studies are needed in order to elucidate the role of s2m in virus replication, not just within the coronaviruses, but in all virus families where this horizontally transferred element has been detected.…”