Programmed -1 Ribosomal Frameshifting in coronaviruses: A therapeutic target

Abstract

Human population growth, climate change, and globalization are accelerating the emergence of novel pathogenic viruses. In the past two decades alone, three such members of the coronavirus family have posed serious threats, spurring intense efforts to understand their biology as a way to identify targetable vulnerabilities. Coronaviruses use a programmed -1 ribosomal frameshift (-1 PRF) mechanism to direct synthesis of their replicase proteins. This is a critical switch in their replication program that can be therapeutically targeted. Here, we discuss how nearly half a century of research into -1 PRF have provided insight into the virological importance of -1 PRF, the molecular mechanisms that drive it, and approaches that can be used to manipulate it towards therapeutic outcomes with particular emphasis on SARS-CoV-2.

Keywords: Antiviral; Coronavirus; Covid-19; Frameshifting; Ribosome; SARS-CoV; SARS-CoV-2; Therapeutics; Vaccine; Virus.

Graphical abstract

Fig. 1

Betacoronavirus gene organization and expression flowchart. A. Map of the betacoronavirus genomic RNA (gRNA). Open reading frames are color-coded and the −1 PRF signal is indicated inside of the yellow diamond. B. Flowchart of the intracellular coronavirus (CoV) replication program. Upon infection and release of viral genomic RNA into the cytoplasm, ORF1a-encoded proteins are synthesized first, initiating Stage 1 of the program. Their function is to “hijack” the cell by securing the ribosomes and disrupting the host cellular innate immune response. Approximately one quarter of translating ribosomes are induced to shift reading frame at the −1 PRF signal. This −1 PRF signal represents a decision point: to continue with Stage 1 or to move into Stage 2, wherein proteins expressed from ORF1b are synthesized in order to transcribe new viral RNAs, including new genomic and subgenomic RNAs. New gRNAs also provide feedback to reinforce cellular takeover by the virus. The transition to Stage 2 may either be rapid, requiring the accumulation of a critical mass of ORF1b products to generate a rapid burst of RNA synthesis (e.g. the rate of viral factory assembly may be determined by −1 PRF rates), or it may be a gradual process instead. In Stage 3, structural proteins encoded in the subgenomic RNAs package the genomic RNAs to produce new viral particles, which exit to repeat the infectious cycle. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

−1 PRF in SARS-CoV and SARS-CoV-2. A-B. Cartoon showing elements involved in −1 PRF and example of shift in reading frame. Elongating ribosomes pause at the 3-stemmed pseudoknot with A- and P-site tRNAs base-paired respectively to AAG and UUA codons in slippery site; upon slippage, non-wobble bases of tRNAs can re-pair to −1 frame codons AAA and UUU. C-E. Comparison of the two-dimensional representations of the SARS-CoV-2 −1 PRF signals. Data from (Bhatt et al., 2020; Kelly et al., 2020; Zhang et al., 2020) are labeled and color-coded as indicated. The nucleotides that differ between SARS-CoV-2 and SARS-CoV are boxed in grey. The dimerization domain identified in (Ishimaru et al., 2013) is circled in cyan. F–H. Space-filled models of the SARS-CoV-2 three stemmed pseudoknot. From left to right, an example of a 5′-end threaded conformation generated by molecular dynamics simulations (Omar et al., 2020), the cryo-EM structure of an isolated pseudoknot (Zhang et al., 2020), and the cryo-EM image of the pseudoknot in the context of a paused ribosome (Bhatt et al., 2020). I. A model of the dimerized SARS-CoV-2 −1 PRF signal from molecular dynamics simulations (Omar et al., 2020). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Small-molecule inhibitors of −1 PRF in SARS-CoV-2. A. Three examples of small molecules that have been found to inhibit −1 PRF: MTDB (Kelly et al., 2020), merafloxacin (Sun et al., 2020), and ivacaftor (Chen et al., 2020). B. Binding site of MTDB on SARS-CoV-2 pseudoknot. Model of the binding site of MTDB (red) found from docking calculations and molecular dynamics simulations of the bound complex shows the ligand binds in a cleft formed because of 5′-end threading in the pseudoknot. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)