Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome

Abstract

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5′ end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration.

The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

“…6 , Supplementary Table 1 ), which agreed with the expected value for the full-length PK reported previously Fig. 5B ) 51 , 52 . Moreover, the majority (80%) of RNA molecules unfolded at forces (F U ) of 15–20 pN (Supplementary Fig.…”

“…6 , Supplementary Table 1 ). Such a hysteresis during refolding is commonly reported with PKs and other highly structured RNAs 51 , 52 . When we performed the measurements in the presence of ZAP-S RNA unfolding trajectories remained almost/mostly unaffected, suggesting that the interaction neither stabilizes nor destabilizes the RNA structure (Fig.…”

“…To this end, an RNA containing the 68 nucleotides long wild type SARS-CoV-2 PK (nucleotides 13475–13542 of SARS-CoV-2 genome) was hybridized to DNA handles and immobilized on polystyrene beads. We employed exclusively the sequence corresponding to the putative PK to preclude the formation of alternative conformers 49 , 51 – 53 . We used the force-ramp method to probe the forces required for (un)folding of the RNA in the presence and absence of ZAP-S.…”

“…The force was gradually increased until the RNA was fully unfolded (bottom). B 3D structure of SARS-CoV-2 pseudoknot RNA (PK) derived from 51 and colored according to the scheme used in Fig. 4 .…”

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

“…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.…”