Decoding the genome of SARS-CoV-2: a pathway to drug development through translation inhibition

Figures

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  Figure 1.. SARS-CoV-2 genome organization and the canonical sg-mRNAs. The secondary structures of the 5 ′UTR, 3 ′UTR and PFSE are shown in insets. Orange lines indicate pseudoknot interaction. Dotted blue line in the PFSE indicates region that forms stem 3 or long-range interactions outside the PFSE. gRNA, genomic RNA; sg-mRNAs, sub-genomic messenger RNAs; ORF, open reading frame; S, spike protein; E, envelope protein; M, membrane protein; N, nucleocapsid protein; UTR, untranslated region; SL (sl), stem-loop; TRS, transcription-regulating sequence; TRS-L, TRS-leader; PFSE, programmed − 1 frameshift stimulation element; PRF, programmed ribosomal frameshift; uORF, upstream ORF; NSPs, non-structural proteins; BSL, bulged stem-loop; HVR, hypervariable region; SPEAR, sarbecoviral pan-end activating RNA; s2m, stem–loop II-like motif; ONM, octa-nucleotide motif; AH, attenuator hairpin; SS, slippery sequence.

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  Figure 2.. SARS-CoV-2 life cycle and targeted viral RNA cleavage. (A) SARS-CoV-2 life cycle and proposed strategies to induce global viral RNA cleavage. SARS-CoV-2 attaches to ACE2 and enters host cell by surface fusion or endosomal entry. After uncoated, ORF1a/b on the gRNA is translated to generate pp1a/b which is then cleaved into NSPs. The NSPs assemble into RTCs for RNA synthesis in DMVs. Resultant sg-mRNAs encode viral structural and accessory proteins. Structural proteins S, E and M are inserted into ER and follow a secretory path. New gRNAs can be translated or used for RNA synthesis or virion packaging. New virion assembly starts with gRNA encapsulation by N, budding into ERGIC and acquiring a lipid bilayer. Mature virions are transported to plasma membrane and released by exocytosis. Potential strategies like siRNAs, gapmers, or CRISPR/Cas13 crRNAs targeting the shared 5’ leader sequence, including SL1 and TRS, can induce targeted cleavage of both sgRNA and sg-mRNAs, hindering global viral protein translation. Red X-marks denote the targeted cleavage of RNA. (B) Experimentally verified siRNAs, gapmers, CRISPR/Cas13 crRNAs, XNAzymes and their targets spanning the SARS-CoV-2 genome.…

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  Figure 3.. Models proposed for SARS-CoV-2 protein translation and targeted inhibition of viral RNA element function. (A) Cap-dependent scanning. eIF4E, 4 G and PABP interact for gRNA and sg-mRNAs to pseudo-circularize; 5’ UTR SLs need to be unfolded by eIF4A. (B) Cap-independent internal ribosome entry. The 5 ′-3 ′ interaction via end-to-end base pairing or potential macromolecular interaction with both ends promotes mRNA circularization and translation. IGF2BP1 interacts with both ends and may circularize mRNA. SL5 and s2m both may interact with RP, rRNA and eIFs. Dependent on UTR structures and end-to-end crosstalk, gRNA can start translation cap-independently. For sg-mRNA, without SL4 and SL5, its internal ribosome recruitment may be weakened, and its end-to-end base pairing needs further study. (A and B) SL1 redirects host translation to virus via interaction with NSP1; PFSE mediates PRF on the gRNA to translate ORF1b. ASOs and small molecule drugs like MTDB, merafloxacin, geneticin, aminoquinazoline derivatives, and DMA are designed to block translation-related functions to combat SARS-CoV-2. Question marks indicate the need for further investigation. gRNA, genomic RNA; sg-mRNA,…

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  Figure 4.. Schematic of the s2m secondary structures of SARS-CoV (A) and SARS-CoV-2 (B-G). Blue arrows show the base substitutions: U5C and G31U. Red star denotes GUAC palindrome. Blue star denotes GAGUA sequence that forms a GNRA-like motif in SARS-CoV (A) and SARS-CoV-2 (E).

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  Figure 5.. Model proposed for switch-like mechanism of SARS-CoV-2 s2m. SARS-CoV-2 s2m forms alternative secondary structures. Its GUAC palindrome mediates the formation of homo-dimeric kissing complex, which can turn into a stable duplex with the help of the viral N protein. The monomer binds host miRNA but the dimer doesn’t. The GNRA-like terminal loop is related to 43S complex recruitment via interaction with RP, rRNA or eIFs, and dimerization hinders this as it makes the GNRA-like loop a paired stem. eIFs, eukaryotic initiation factors; N, nucleocapsid; RP, ribosomal protein; rRNA, ribosomal RNA.

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  Figure 6.. Schematic of SARS-CoV-2 PFSE secondary structures. (A) Canonical H-type PK. (B-E) alternative secondary structures. Asterisk denotes ORF1a stop codon. Orange lines indicate pseudoknot interaction. Dotted blue line indicates the region that forms stem 3 or long-range interactions outside the PFSE. PK, pseudoknot; S1, stem 1; S2, stem 2; S3, stem 3; AS1, alternative stem 1; AH, attenuator hairpin; SS, slippery sequence.