Translation of SARS-CoV-2 gRNA Is Extremely Efficient and Competitive despite a High Degree of Secondary Structures and the Presence of an uORF

Figure 1. Scheme of the SARS-CoV-2 gRNA and sgRNAs. The cap and poly(A) tail structures are indicated. The 5′UTR of each viral mRNA is underlined and is composed of the common leader sequence (black square) and the variable region (orange square). The other different colors correspond to the different ORFs as indicated on the top.

Figure 2. Modelization of the structure of the SARS-CoV-2 gRNA 5′UTR. This model, adapted from Miao et al. (2021) [73], shows the different stem-loop (SL) RNA structures. Position of the AUG codons of uORF and main ORF are highlighted in red, the coding region in orange and the TRS-L in green.

Figure 3. Translation efficiency of the SARS-CoV-2 5′UTRs. (A) Scheme of the gRNA and the different sgRNAs, with the leader sequence in black and the variable region in orange, which represent each 5′UTR (this panel was obtained from Figure 1). The sequences corresponding to each 5′UTR have been cloned upstream of the Renilla ORF. (B,C) RRL and (D) untreated RRL have been programmed with capped and polyadenylated mRNAs harboring specifically the 5′UTR of the ORF1ab, ORF-S, ORF-3a, ORF-E, ORF-M, ORF-6, ORF-7a, ORF-7b, ORF-8 or ORF-N. The resulting protein products have been resolved on (B) an SDS-PAGE and quantified with a phosphoImager, or (C,D) analyzed by luciferase assay. (E) Jurkat cells were electroporated with capped and polyadenylated mRNAs as indicated. After 90 min of incubation, the cellular extracts were used for RT-qPCR and luciferase assay. All the results are normalized to the globin 5′UTR expression (set at 100%). Values are the mean (+/− S.D.) for three independent experiments (panels (B,C)) or (+/− S.E.M.) for four independent experiments (panels (D,E)).

Figure 4. Divergence in translation efficiency between the 5′UTRs of MERS-CoV and SARS-CoV gRNA. (A) Percentage of identity between the 5′UTRs of MERS-CoV, SARS-CoV-1 and SARS-CoV-2 gRNAs resulting from a MUSCLE alignment (Figure S2). (B) Scheme of capped and polyadenylated mRNAs coding for the Renilla reporter gene with the leader or the 5′UTRORF1ab from SARS-CoV-1/2 and MERS-CoV. (C) RRL was programmed with the corresponding in vitro transcribed reporter mRNAs. Expression products were quantified by luciferase assays and normalized to globin 5′UTR expression (set at 100%). (D) Jurkat cells were electroporated with capped and polyadenylated mRNAs as indicated. After 90 min of incubation, the cellular extracts were used for luciferase assay and RT-qPCR. All the results are normalized to the globin 5′UTR expression (set at 100%). Values shown are the mean (+/− S.E.M.) for 6 (C) and 5 (D) independent experiments.

Figure 5. The effects of the cap and poly(A) tail on viral translation. Capped/polyadenylated (+/+), capped/non-polyadenylated (+/−), uncapped/polyadenylated (−/+) or uncapped/non-polyadenylated (−/−) Globin Renilla, 5′UTRORF1ab Renilla and leader Renilla RNAs were translated in (A) the RRL or in (B) the untreated RRL. Translational products were analyzed by luciferase assays and normalized to the (−/−) condition (arbitrary set at 1). Values shown are the mean +/− S.D. for three independent experiments.

Figure 6. Requirement for the eIF4F complex. (A) Schematic cartoon of the eIF4F complex, which is composed of eIF4E, eIF4G and eIF4A. The interactions with the PIC, cap analog, L protease and hippuristanol are depicted on the figure. (B) RRL was treated with cap analog (1 µM) or with water (ctrl) and then programmed with capped and polyadenylated globin-Renilla and SARS-CoV-2 leader or 5′UTRORF1ab-Renilla mRNAs. Products of expression were quantified by luciferase assays and normalized to the control condition (set at 100%). (C–F) Untreated RRL was pre-incubated alone (ctrl) or in presence of different amounts of L protease (µL) as indicated, and then programmed with (D) globin-Renilla, (E) EMCV IRES-Renilla, (F) SARS-CoV-2 leader or 5′UTRORF1ab-Renilla mRNAs. Translation products were (C) resolved by a 7.5% SDS-PAGE and then transferred to a PVDF membrane for Western blotting analysis against eIF4G, or (D–F) analyzed by luciferase assay and normalized to the control condition (set at 100%). (G) RRL pre-treated with DMSO or increasing amounts (µM) of hippuristanol (hipp.) was programmed for 30 min with globin-Renilla, HCV-IRES Renilla, SARS-CoV-2 leader or ORF-1ab 5′UTR-Renilla mRNAs.…

Figure 7. The 5′UTR uORF differentially regulates translation from SARS-CoV-2 and MERS-CoV. (A) Scheme of the reporter mRNA with the 5′UTRORF1ab of SARS-CoV-2 or MERS-CoV gRNA with their respective uORF and positions. (B) Capped and polyadenylated mRNA harboring the SARS-CoV-2 5′UTR gRNA WT or the Mut-AUG107 or uORF-phase mutations were translated in the RRL. Translation products were resolved by 12% SDS-PAGE and quantified with a phosphoImager. (C) Capped and polyadenylated mRNA harboring the MERS-CoV 5′UTR gRNA WT or the Mut-AUG158 mutation, were translated in the RRL. Translation products were resolved by 12% SDS-PAGE and quantified with a phosphoImager. (D) Jurkat cells were electroporated with capped and polyadenylated mRNAs as indicated. After 90 min of incubation, the cellular extracts were used for luciferase assay and RT-qPCR. Values shown are the mean (+/− S.D.) for three independent experiments.