Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

Abstract

Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

Structural dynamics of the SARS-CoV-2 frameshift-stimulatory pseudoknot reveal topologically distinct conformers

“…2B, black) showed a contour length change of ΔLc = 35.1 ± 0.3 nm for complete unfolding (Table S1). This result was close to the value expected for full unfolding of the pseudoknot, 34.7-36.5 nm, based on cryo-EM reconstructions 10,15 and computational modeling of likely structures 18 . Sometimes these curves contained an intermediate state, I1 (Fig.…”

“…These results confirm the suggestion from simulations and cryo-EM imaging that the SARS-CoV-2 frameshift signal can form a variety of different structures, and they reveal how these structures respond to mechanical tension similar to what the ribosome would apply during −1 PRF. The most prominent conformation, N, unfolded through the full length of the pseudoknot at moderately high force and was suppressed by occlusion of the 5′ end by the duplex handle, precisely as would be expected for a 5′-end threaded structure such as those seen in cryo-EM images on and off the ribosome 10,15 or predicted from simulations 18 . In contrast, the conformation unfolding at lower force (N′) occurred more frequently when the 5′ end was occluded, in proportion to the suppression of N, as would be expected for a conformation in which the 5′ end remains unthreaded.…”

“…1B, C). Some of these conformers involve knot-like fold topologies that have not previously been observed in frameshift-stimulatory pseudoknots, specifically conformers with the 5′ end threaded through the junction between the 3 helices to generate what we term a 'ring-knot' 10,15,18 . Such a 5′-end threaded ring-knot fold has only previously been observed in viral exoribonuclease-resistant RNAs [19][20][21] .…”

“…The pseudoknot stimulating −1 PRF in SARS-CoV-2 has a 3-stem architecture 1,10,15,16 (Fig. 1A) that is characteristic of coronaviruses, in contrast to the more common 2-stem architecture of most viral frameshift-stimulatory pseudoknots 17 .…”

“…1A) that is characteristic of coronaviruses, in contrast to the more common 2-stem architecture of most viral frameshift-stimulatory pseudoknots 17 . Cryo-EM imaging 10,15 and computational modeling 18 both suggest that the SARS-CoV-2 pseudoknot can take on several different conformers (Fig. 1B, C).…”