The SARS-CoV-2 Programmed −1 Ribosomal Frameshifting Element Crystal Structure Solved to 2.09 Å Using Chaperone-Assisted RNA Crystallography

Abstract

The programmed −1 ribosomal frameshifting element (PFSE) of SARS-CoV-2 is a well conserved structured RNA found in all coronaviruses’ genomes. By adopting a pseudoknot structure in the presence of the ribosome, the PFSE promotes a ribosomal frameshifting event near the stop codon of the first open reading frame Orf1a during translation of the polyprotein pp1a. Frameshifting results in continuation of pp1a via a new open reading frame, Orf1b, that produces the longer pp1ab polyprotein. Polyproteins pp1a and pp1ab produce nonstructural proteins NSPs 1–10 and NSPs 1–16, respectively, which contribute vital functions during the viral life cycle and must be present in the proper stoichiometry. Both drugs and sequence alterations that affect the stability of the −1 programmed ribosomal frameshifting element disrupt the stoichiometry of the NSPs produced, which compromise viral replication. For this reason, the −1 programmed frameshifting element is considered a promising drug target. Using chaperone assisted RNA crystallography, we successfully crystallized and solved the three-dimensional structure of the PFSE. We observe a three-stem H-type pseudoknot structure with the three stems stacked in a vertical orientation stabilized by two triple base pairs at the stem 1/stem 2 and stem 1/stem 3 junctions. This structure provides a new conformation of PFSE distinct from the bent conformations inferred from midresolution cryo-EM models and provides a high-resolution framework for mechanistic investigations and structure-based drug design.

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

“…It was demonstrated that the PFS element sequence alone could recapitulate the PFS activity without a protein cofactor in SARS-CoV ( Baranov et al, 2005 ). The pseudoknotted structure was observed in NMR ( Liphardt et al, 1999 ), chemical probing ( Huston et al, 2021 ), cryo-EM (complexed with an elongating ribosome) ( Bhatt et al, 2021 ), and X-ray crystallography ( Roman et al, 2021 ).…”

Geneticin shows selective antiviral activity against SARS-CoV-2 by interfering with programmed -1 ribosomal frameshifting

“…The high flexibility and plasticity of the FSE is an essential requirement for its biological activity (23,(46)(47)(48)(49). This unique characteristic is also supported by cryo-EM and x-ray structures recently published (34,44,51). In particular, the pseudoknot structure seems to be highly dynamic before encountering the ribosome.…”

Viral and cellular translation during SARS‐CoV‐2 infection

“…The presence of a central ring is shown in yellow. (D) Crystallographic structures of the free PFSE (PDB:7mlx) [ 26 ] (left) and (PDB:7mky) [ 27 ] (right). The stems S1 (green), S2 (blue) and S3 (orange) are shown.…”

“…Then, X‐ray crystallography studies showed that the structure of the pseudoknot is formed by three H‐type stems stacked in a vertical orientation (Fig. 1D ): These structures bring interaction details at atomic resolution that will be useful for the identification of binding sites of specific ligands and for the drug design of antiviral compounds that will target specifically the PFSE [ 26 , 27 ]. In addition, a short isoform of the host zinc‐finger antiviral protein ZAP‐S directly interacts with the PFSE and thereby modifies its folding, leading to downregulation of −1 frameshifting [ 28 ].…”