CRISPR screening reveals a dependency on ribosome recycling for efficient SARS-CoV-2 programmed ribosomal frameshifting and viral replication

Frederick Rehfeld; Jennifer L Eitson; Maikke B Ohlson; Tsung-Cheng Chang; John W Schoggins; Joshua T Mendell

doi:10.1016/j.celrep.2023.112076

. 2023 Jan 30;42(2):112076. doi: 10.1016/j.celrep.2023.112076

CRISPR screening reveals a dependency on ribosome recycling for efficient SARS-CoV-2 programmed ribosomal frameshifting and viral replication

Frederick Rehfeld ¹, Jennifer L Eitson ², Maikke B Ohlson ², Tsung-Cheng Chang ¹, John W Schoggins ², Joshua T Mendell ^1,^3,^4,^5,^6,^∗

PMCID: PMC9884621 PMID: 36753415

Abstract

During translation of the genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus in the COVID-19 pandemic, host ribosomes undergo programmed ribosomal frameshifting (PRF) at a conserved structural element. Although PRF is essential for coronavirus replication, host factors that regulate this process have not yet been identified. Here we perform genome-wide CRISPR-Cas9 knockout screens to identify regulators of SARS-CoV-2 PRF. These screens reveal that loss of ribosome recycling factors markedly decreases frameshifting efficiency and impairs SARS-CoV-2 viral replication. Mutational studies support a model wherein efficient removal of ribosomal subunits at the ORF1a stop codon is required for frameshifting of trailing ribosomes. This dependency upon ribosome recycling is not observed with other non-pathogenic human betacoronaviruses and is likely due to the unique position of the ORF1a stop codon in the SARS clade of coronaviruses. These findings therefore uncover host factors that support efficient SARS-CoV-2 translation and replication.

Keywords: SARS-CoV-2, coronavirus, host factors, programmed ribosomal frameshifting, ribosome recycling, ABCE1, DENR, DOHH, EIF5A, diphthamide

Graphical abstract

Programmed ribosomal frameshifting is essential for SARS-CoV-2 replication. Through genome-wide CRISPR screening, Rehfeld et al. find a requirement for ribosome recycling factors in efficient frameshifting and viral replication. The unique position of the ORF1a stop codon in SARS-CoV-2 necessitates the efficient removal of post-termination ribosomes to enable frameshifting of trailing ribosomes.

Introduction

During protein synthesis, ribosomes decode the genetic information carried by messenger RNAs (mRNAs) into polypeptide chains. During the elongation phase of translation, ribosomes move in steps of three nucleotides, codon by codon, as they translate the message from the initiation to the termination codon. Ribosomes that deviate from the selected open reading frame (ORF) will produce truncated, misfolded, and potentially toxic proteins. The rate of spontaneous frameshifting is reported to be very low, with a rate of <10⁻⁵ events per codon.¹ ^, ² ^, ³ However, on certain transcripts, ribosomal frameshifting occurs at much higher rates and has evolved as a regulatory mechanism to expand the coding capacity of a given mRNA sequence. This “intentional” abandoning of co-linearity of the protein coding sequence is termed programmed ribosomal frameshifting (PRF) and is triggered by specific cis-elements encoded in the mRNA.⁴ ^, ⁵ PRF is most prevalent in viral transcriptomes but is utilized as a mechanism of gene regulation in all domains of life. As an example, in eukaryotic cells, polyamine biosynthesis is subject to homeostatic regulation through +1 frameshifting of the ornithine decarboxylase antizyme mRNA.⁶ ^, ⁷

Several pathogenic human viruses utilize PRF. These include the West Nile and Japanese encephalitis viruses,⁸ both members of the Flaviviridae family; the human immunodeficiency virus (HIV),⁹ a retrovirus; and severe acute respiratory syndrome coronavirus (SARS-CoV)-1 and SARS-CoV-2, belonging to the Coronaviridae (CoV) family.¹⁰ ^, ¹¹ ^, ¹² ^, ¹³ Efficient PRF is required for replication of all of these viruses.⁸ ^, ¹⁴ ^, ¹⁵ Controlled frameshifting not only increases the coding capacity of the viral genome, but also allows differential protein expression from overlapping ORFs at the expense of minimal additional sequence. For example, HIV frameshifting from the canonical gag-encoding ORF into the pol-encoding −1-shifted ORF occurs with a relatively low efficiency, so the stoichiometry of the two translated proteins is approximately 20:1. This ratio is necessary for proper assembly of the structural (gag) and non-structural (pol) HIV subunits into virions, illustrating the importance of PRF as a regulatory mechanism.⁹ ^, ¹⁵

PRF plays a similarly important role in the life cycle of SARS-CoV-2. Like all coronaviruses, SARS-CoV-2 contains a capped and polyadenylated positive-stranded RNA genome (Figure 1A). Upon release of the viral RNA into the host cytosol, ORF1a and the −1-frameshifted ORF1b are translated by the host. These two overlapping ORFs encode large non-structural polyproteins that are cleaved by viral proteases, forming the replication transcription complex (RTC) that subsequently transcribes all subgenomic RNAs and replicates the viral genome.¹⁶ Between ORF1a and ORF1b lies the coronavirus frameshifting element (FSE), consisting of a heptanucleotide slippery sequence (U UUA AAC; the underlined codons represent the 0 frame) located 7 nucleotides upstream of a three-stemmed pseudoknot secondary structure (Figure 1B).¹⁰ ^, ¹¹ ^, ¹³ The majority of ribosomes read through the slippery sequence and terminate translation at a stop codon present in the first stem of the pseudoknot. Some ribosomes, however, stall at the pseudoknot and undergo a −1 frameshift at the slippery sequence, leading to translation of an ORF1ab fusion polypeptide. Recent cryogenic electron microscopy (cryo-EM) structures of the ribosome engaged in a frameshifting-primed state showed that the pseudoknot of the SARS-CoV-2 FSE adopts a corkscrew-like conformation that contacts the ribosomal mRNA entry channel at multiple positions.¹³ These interactions contribute to ribosome stalling and might explain the strict spacing constraints between the slippery sequence and the pseudoknot that are required for frameshifting.

Genome-wide CRISPR-Cas9 screen identifies host-encoded regulators of SARS-CoV-2 frameshifting

(A) Schematic of the SARS-CoV-2 genome. Dotted box indicates close up of region shown in (B) harboring the coronavirus frameshifting element (FSE).

(B) Secondary structure of the SARS-CoV-2 FSE containing the slippery sequence and three-stemmed pseudoknot. Based on structural data from Bhatt et al.¹³

(C) Schematic representation of the SARS-CoV-2 frameshifting reporter (PRF−1) and the in-frame control reporter (PRF0). The UAA stop codon in the PRF−1 reporter is indicated with a red box. The additional nucleotide in PRF0 that creates the in-frame fusion of mCherry and eGFP ORFs is indicated with a yellow box.

(D) Flow cytometry analysis of eGFP and mCherry fluorescence in representative PRF0 and PRF−1 reporter clones.

(E) Frameshifting rate of SARS-CoV-2 and human immunodeficiency virus (HIV) PRF−1 reporter cell lines, each normalized to the respective PRF0-expressing cells. Means ± SD shown, with individual replicates plotted. Biological replicates: n = 18 for SARS-CoV-2 PRF0, n = 16 for SARS-CoV-2 PRF−1, n = 3 for HIV PRF0 and PRF−1.

(F) Overview of SARS-CoV-2 PRF CRISPR screen. (1) Transduction of HCT116 reporter cell lines with the Brunello human CRISPR knockout pooled library at low multiplicity of infection (MOI). (2) Selection in puromycin and collection of the top 1% eGFP/mCherry and bottom 0.75% eGFP/mCherry cell populations by fluorescence-activated cell sorting (FACS). (3) Isolation of genomic DNA (gDNA) of sorted and unsorted populations and PCR amplification of sgRNA sequences with barcoded adapters. (4) Deep sequencing and subsequent sgRNA enrichment analysis of genes in sorted pools compared with unsorted cells by model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK).

(G and H) Genes plotted by their MAGeCK p values from the PRF0 control (x axis) and PRF−1 CRISPR screens (y axis). Genes enriched in the top eGFP/mCherry population, representing candidate inhibitors of frameshifting, are shown in (G), while hits from the bottom eGFP/mCherry population, representing putative positive regulators of frameshifting, are shown in (H). High-ranking hits in the PRF−1 screens that clustered within functional categories are labeled in color and listed on the right of each plot (gene names ordered by rank in screen). See also Figure S1 and Table S1.

Ribosomal frameshifting of SARS-CoV-2 is essential for viral replication and therefore represents a potentially druggable step in the viral life cycle. Despite our knowledge of the RNA cis-elements and the structural conformation of frameshifting ribosomes, essential host factors for this process have not yet been identified. Intriguingly, reporter assays that measured coronavirus PRF efficiency exhibited significant variability across different cell types (15%–40%) and were generally less efficient than natural frameshifting on coronavirus genomic RNA, as measured by ribosome profiling of infected cells.¹⁷ ^, ¹⁸ These observations raise the question of whether host or viral factors are required for, or regulate the efficiency of, PRF. The identification of such factors could uncover fundamental mechanisms underlying frameshifting and might enable new therapeutic strategies that target proteins that regulate this essential step in the replication cycle of SARS-CoV-2.

To identify human host factors that regulate SARS-CoV-2 frameshifting, we performed genome-wide CRISPR-Cas9 knockout screens using dual-fluorescence frameshifting reporter constructs. This led to the identification of factors that positively or negatively regulate SARS-CoV-2 PRF. Impaired biosynthesis of the special amino acid diphthamide, a known regulator of translational fidelity, and loss of RNA polymerase III (Pol III) components led to an increase in frameshifting. Conversely, loss of the 80S ribosome recycling factor ABCE1 or the 40S ribosome recycling factor DENR impaired PRF. Moreover, loss of ribosome recycling reduced frameshifting during SARS-CoV-2 infection and inhibited viral replication. Mutational studies of the SARS-CoV-2 FSE suggested a model in which efficient removal of a post-termination ribosome on the ORF1a stop codon by the ribosome recycling machinery is necessary for a trailing ribosome to stall at the slippery sequence and undergo frameshifting. These findings therefore reveal host-viral interactions that are essential for the SARS-CoV-2 life cycle.

Results

A genome-wide CRISPR-Cas9 screen for regulators of SARS-CoV-2 frameshifting

To perform a genome-wide CRISPR-Cas9 knockout screen for regulators of SARS-CoV-2 PRF, we first established a reporter system that provided a fluorescent readout of frameshifting activity. Dual-luciferase reporter assays have been used in the past to study the SARS-CoV-1 and highly similar SARS-CoV-2 FSEs.¹⁰ ^, ¹¹ ^, ¹² Based on these studies, we designed a fluorescent lentiviral reporter containing an mCherry ORF followed by the SARS-CoV-2 FSE and a −1-frameshifted P2A-eGFP ORF (PRF−1; Figure 1C). The ratio of eGFP to mCherry produced from this reporter thereby served as a readout of the efficiency of frameshifting (Figure S1A). We also generated a control construct with a single nucleotide insertion prior to the slippery sequence in the FSE, thus placing mCherry and eGFP in the same reading frame (PRF0). This reporter enabled the identification of factors that regulate mCherry or eGFP expression independent of frameshifting. Clonal reporter lines expressing PRF−1 and PRF0 were established using HCT116 cells, a stably diploid human cell line well suited to large-scale genetic screens.¹⁹ ^, ²⁰

As expected, mCherry expression was comparable between PRF−1 and PRF0 reporter cells, while eGFP produced from PRF−1 was markedly reduced (Figure 1D). Quantification of frameshifting efficiency, calculated by determining the mean fluorescence intensity of eGFP relative to mCherry produced from the PRF−1 reporter normalized to the same ratio produced from the PRF0 reporter, revealed a highly reproducible rate of frameshifting of approximately 20% among multiple PRF−1 SARS-CoV-2 reporter clones (Figure 1E). Immunoblotting for mCherry and eGFP proteins further validated these results and showed a similarly reduced eGFP-to-mCherry ratio of PRF−1 compared with PRF0 (Figures S1B and S1C). These data are concordant with previous reports of SARS coronavirus frameshifting efficiency.¹⁰ ^, ¹¹ ^, ¹² ^, ²¹ By comparison, reporter cell lines with an identical reporter design carrying the HIV FSE exhibited a frameshifting rate of approximately 5%, matching the reported rate for this virus⁹ (Figure 1E).

We next utilized these fluorescent reporters of SARS-CoV-2 frameshifting in genome-wide CRISPR-Cas9 knockout screens to identify regulators of this process (Figure 1F). Two independent PRF0 and PRF−1 reporter clones were transduced with the Brunello pooled lentiviral CRISPR library targeting ∼19,000 human genes.²² After 10 days of puromycin selection, fluorescence-activated cell sorting (FACS) was used to collect the population with the highest eGFP/mCherry expression ratio, representing cells with an increase in frameshifting, and the population with the lowest eGFP/mCherry ratio, representing cells with reduced frameshifting. Single-guide RNA (sgRNA) abundance in sorted and unsorted populations was quantified by high-throughput sequencing, and genes targeted by sgRNAs enriched in the sorted pools were identified and ranked using model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK)²³ (Table S1).

Far more significant hits were recovered in PRF−1 cells compared with PRF0 cells, suggesting that few genes regulate the reporter constructs independent of frameshifting (Figures 1G and 1H). Notable exceptions were NAA10, NAA15, and HYPK, which scored as significant hits in both PRF−1 and PRF0 reporter cells in the top eGFP/mCherry population (Figure S1D). All three genes encode essential components of the N-acetyltransferase complex (NAT) that co-translationally acetylates the N termini of proteins. These hits were most likely retrieved due to the self-cleavable P2A peptide sequence inserted between the mCherry and the eGFP ORFs. Loss of N-terminal acetylation will result in destabilization of mCherry but not eGFP, whose N terminus is non-acetylated and generated through P2A cleavage, thereby elevating the eGFP/mCherry ratio.

Host factors that inhibit SARS-CoV-2 frameshifting

Analysis of genes whose loss of function increased the efficiency of frameshifting (uniquely recovered in the top eGFP/mCherry population of the PRF−1 reporter screen) revealed two notable sets of enriched genes: those that encode components of the diphthamide biosynthesis pathway (DPH1, DPH2, DPH3) and subunits of RNA Pol III (CRCP, POLR3B, POLR3C, POLR3E, POLR3F, POLR3H, POLR3K; Figure 1G). As described below, regulation of frameshifting efficiency by these genes is consistent with the known functions of these factors and thus these hits provided validation that this screening strategy could identify bona fide PRF regulators.

Diphthamide is a special amino acid that is generated through the post-translational modification of a histidine residue in archaeal and eukaryotic translation elongation factor 2 (eEF2; Figure 2A). Consistent with the results of the screen, we observed an increase in the ratio of eGFP to mCherry fluorescence in the PRF−1 reporter, but not in the PRF0 reporter, after CRISPR-mediated knockout of genes required for diphthamide biosynthesis (Figures 2B, 2C, S2A, and S2B). Loss of this modification was previously shown to result in reduced translational fidelity,²⁴ providing a mechanistic explanation for an increase in frameshifting in diphthamide-deficient cells.

Loss of diphthamide biosynthesis and RNA polymerase III components increases SARS-CoV-2 PRF

(A) Chemical structure of diphthamide, a special amino acid in eEF2 that is required for translational fidelity.

(B and C) Flow cytometry of SARS-CoV-2 PRF−1 (B) and PRF0 (C) reporter cell lines transduced with lentiviral constructs encoding Cas9 and either non-target control sgRNA (gray plots) or sgRNA targeting *DPH1* (colored plots). Left and center plots show fluorescence histograms. Right plots show contour plots for mCherry (x axis) and eGFP (y axis) fluorescence.

(D) Human Pol III subunit organization, based on structure from Girbig et al.²⁵ Subunits shared among more than one DNA-dependent RNA polymerase are italicized. Genes retrieved as significant hits in the CRISPR screen are labeled in orange. BRF1 and GTF3C1 are tRNA transcription factors.

(E) Flow cytometry of SARS-CoV-2 PRF−1 reporter cell lines transduced with lentiviral constructs encoding Cas9 and either non-target control sgRNA (gray plots) or sgRNA targeting *POLR3K* (colored plots). See also Figure S2.

Pol III is a large complex comprising 17 subunits that transcribes various classes of small non-coding RNAs, including tRNAs.²⁵ ^, ²⁶ Intriguingly, only genes encoding subunits exclusive to Pol III were retrieved as putative frameshifting repressors, not genes that encode subunits that are also required for other DNA-dependent RNA polymerases (Figure 2D). In addition, BRF1 and GTF3C1, two genes encoding transcription factors required for tRNA transcription, were highly ranked in the screen.²⁷ It is therefore probable that loss of these Pol III subunits or transcription factors resulted in globally reduced tRNA abundance. Previous in vitro studies of HIV frameshifting established that reduced levels of tRNAs necessary for decoding the slippery sequence resulted in an increase in −1 frameshifting.²⁸ This effect is likely a consequence of enhanced stalling of inefficiently translating ribosomes at the FSE. Consistent with these prior data, we observed an increase in SARS-CoV-2 reporter frameshifting when genes encoding Pol III subunits were knocked out (Figures 2E and S2C) or when Pol III was inhibited pharmacologically (Figure S2D).

Ribosome recycling is required for efficient SARS-CoV-2 frameshifting

Whereas the hits that functioned as negative regulators of frameshifting largely affected this process through established mechanisms, the most significant hits whose loss of function impaired frameshifting (uniquely recovered in the bottom eGFP/mCherry population of the PRF−1 reporter screen) were not previously linked to PRF. Among these putative host factors for SARS-CoV-2 frameshifting, the most highly represented pathway was post-termination ribosome recycling (Figure 1H). Ribosome recycling refers to the process whereby ribosomes are removed from template mRNAs after translation termination (Figure 3A). Following stop codon recognition and peptidyl-tRNA hydrolysis by release factors eRF1 and eRF3, ABCE1 binds the post-termination 80S ribosome and splits the ribosomal subunits, thereby releasing the large subunit from the transcript. Next, DENR, in complex with MCTS1, binds the 40S ribosomal P site and ejects the remaining deacylated tRNA, resulting in release of the small subunit from the mRNA.²⁹ ^, ³⁰ ^, ³¹ ABCE1 (rank 6) and DENR (rank 10) were among the most highly significant hits in the screen. Other significant hits included ORAOV1 (rank 1) and YAE1D1 (rank 3), which are required for cytosolic loading of an essential iron-sulfur cluster into ABCE1.³²

Loss of ribosome recycling factors impairs SARS-CoV-2 PRF

(A) Overview of ribosome recycling following stop codon recognition. Following translation termination and peptide release by eRF1 and eRF3, ABCE1 splits the two ribosomal subunits. After 60S release, DENR in complex with MCTS1 binds the ribosomal P site and ejects the deacylated tRNA, resulting in disassociation of the 40S subunit.

(B) Western blotting of the indicated proteins after transduction of PRF−1 reporter cells with sgRNA-encoding lentiviruses.

(C–E) Flow cytometry of SARS-CoV-2 PRF−1 reporter cell lines transduced with lentiviral constructs encoding Cas9 and either non-target control sgRNA (gray plots) or sgRNA targeting *ABCE1* (C), *DENR* (D), or *EIF2D* (E) (colored plots). Fluorescence histograms shown. See also Figure S3.

Knockout of ABCE1, DENR, ORAOV1, or YAE1D1 led to a marked decrease in SARS-CoV-2 PRF, validating the screening results (Figures 3B–3D and S3A–S3F). We were unable to identify sgRNAs that resulted in robust depletion of MCTS1 (data not shown), leaving the role of this DENR co-factor in SARS-CoV-2 PRF undetermined. We also knocked out EIF2D, which encodes a single polypeptide paralog of the DENR-MCTS1 dimer, to investigate whether this alternative 40S recycling factor is required for SARS-CoV-2 frameshifting (Figure 3B). EIF2D loss did not result in reduced frameshifting (Figure 3E), which might be explained by the recent finding that EIF2D plays a minor role in bulk 40S recycling.³³

SARS-CoV-2 stop codon position confers dependency of frameshifting on ribosome recycling

We next investigated the mechanistic role of 80S and 40S recycling factors in frameshifting at the SARS-CoV-2 PRF element. We hypothesized that a non-recycled post-termination ribosome located at the ORF1a stop codon might influence the frameshifting outcome of a trailing ribosome. In fact, the distance between the ORF1a termination codon and the slippery sequence is 18 nucleotides, much less than a single ribosomal footprint (∼30 nucleotides) (Figure 4A). Thus, obstruction of the slippery sequence by a post-termination ribosome dwelling at the ORF1a stop codon would be expected to impede frameshifting of a trailing ribosome. In addition, a post-termination ribosome at the ORF1a stop codon might keep the pseudoknot in an unfolded state, reducing stalling and frameshifting of a trailing ribosome.

The dependency of PRF on ribosome recycling is determined by the distance between the frame 0 stop codon and the slippery sequence

(A) Secondary structure of the SARS-CoV-2 FSE showing the distance between the PRF0 stop codon and the slippery sequence.

(B) Schematic of the dual-luciferase frameshifting reporter with a nano-luciferase (nLuc) ORF followed by the FSE followed by a firefly luciferase (ffLuc) ORF. Red box indicates the position of the frame 0 stop codon. PRF−1 and PRF0 reporter constructs were generated for all tested FSE variants.

(C) Basal frameshifting rate of reporter constructs harboring the indicated FSEs. Frameshifting rate was determined as the ratio of ffLuc/nLuc for the PRF−1 construct normalized to the ffLuc/nLuc ratio of the PRF0 control construct. Means ± SD are shown, with individual replicates plotted. The p values were calculated by one-way ANOVA with Dunnett’s multiple comparisons test; n.s., not significant. Biological replicates: n = 12 for SARS-CoV-2, n = 6 for SARS-CoV-2-UUA, n = 6 for SARS-CoV-1, n = 12 for OC43, and n = 8 for HKU1.

(D–H) Top: sequence and secondary structure of tested FSEs. Bottom: effect of loss of ABCE1 or DENR on frameshifting rate of each construct. Data are represented as the mean ± SD. The p values were calculated by unpaired, two-tailed Student’s t test; ^∗p ≤ 0.05, ^∗∗p ≤ 0.01. Biological replicates: n = 6 for SARS-CoV-2, n = 3 for SARS-CoV-2-UUA, n = 3 for SARS-CoV-1, n = 6 for OC43, and n = 4 for HKU1. See also Figures S4 and S5.

To investigate these possible mechanisms, we employed a dual-luciferase-based reporter system consisting of lentiviral vectors expressing a nano-luciferase ORF followed by an FSE and a frameshifted firefly luciferase ORF (Figure 4B). The frameshifting activity of the SARS-CoV-2 FSE in this reporter was comparable to that observed using fluorescent PRF reporters (Figure 4C). Loss of either ABCE1 or DENR resulted in an approximately 2-fold reduction in frameshifting of the SARS-CoV-2 FSE luciferase reporter (Figure 4D), confirming our previous results.

In addition to ribosome recycling factors, we also noticed that multiple components of the hypusine biosynthesis pathway (DOHH, DHPS) scored as highly significant hits in the screen (Figure 1H). Hypusine is a modified lysine residue found in translation elongation factor EIF5A that is required for its activity. Hypusine-modified EIF5A binds to the ribosomal E site to promote peptide bond formation of sterically unfavorable amino acid combinations, such as polyproline, and plays a critical role in peptidyl-tRNA hydrolysis after stop codon recognition (Figure S4A).³⁴ Notably, EIF5A loss of function results in the accumulation of ribosomes at termination codons. In keeping with the screening results, knockout of DOHH or EIF5A impaired frameshifting of the PRF−1 luciferase reporter (Figure S4B), providing further support for the model that efficient clearance of ribosomes from the ORF1a stop codon promotes frameshifting of trailing ribosomes.

To test whether the proximity of the ORF1a stop codon to the slippery sequence conferred a requirement for efficient ribosome removal from the FSE for frameshifting, we generated a mutant version of the SARS-CoV-2 FSE in which the ORF1a stop codon was moved farther downstream (SARS-CoV-2-UUA; Figure 4E). Two complementary nucleotide substitutions left the secondary structure and free energy of the first stem of the pseudoknot unaltered but increased the distance between the slippery sequence and the termination codon to 33 nucleotides, a distance greater than a ribosomal footprint. This mutation did not significantly alter the baseline rate of frameshifting compared with the wild-type FSE (Figure 4C). Nevertheless, the dependency of frameshifting on ABCE1 or DENR was completely abolished by the SARS-CoV-2-UUA mutation (Figure 4E). Likewise, frameshifting at the mutant FSE was insensitive to depletion of DOHH or EIF5A (Figure S4C). Importantly, dwelling of a terminating or post-termination ribosome at the repositioned stop codon in the SARS-CoV-2-UUA construct would still be expected to unfold the FSE secondary structure. Therefore, these results strongly implicate the proximity of the stop codon to the slippery sequence as the key feature that necessitates efficient termination and ribosome recycling for frameshifting at the SARS-CoV-2 FSE.

In some cases, cryptic promoter activity of cloned sequences or cryptic splicing of polycistronic reporter transcripts has been associated with artifactual results arising from the use of dual-luciferase reporter constructs.³⁵ ^, ³⁶ ^, ³⁷ To rule out a contribution of these effects to our data supporting the importance of ribosome recycling and stop codon position in SARS-CoV-2 PRF, we further validated our findings using full-length in vitro-transcribed dual-luciferase reporter mRNA carrying the SARS-CoV-2 FSE (Figures S5A and S5B). Direct transfection of reporter mRNA confirmed that fully efficient frameshifting on the wild-type FSE required ABCE1 and DENR, while the UUA stop codon-mutant FSE was insensitive to depletion of these factors (Figures S5C and S5D).

Human betacoronaviruses exhibit variability with respect to the position of the ORF1a stop codon within the FSE and its proximity to the slippery sequence. If obstruction of the slippery sequence by a non-recycled post-termination ribosome underlies the requirement for ribosome recycling factors in efficient frameshifting, the FSEs of different coronaviruses may differ in this requirement. To investigate this possibility, we cloned the FSEs of SARS-CoV-1 and two related non-pathogenic human betacoronaviruses, OC43 and HKU1, into the dual-luciferase reporter. SARS-CoV-1, which is highly similar in sequence to SARS-CoV-2, exhibited the same basal frameshifting activity as SARS-CoV-2 and was similarly sensitive to depletion of either ABCE1 or DENR (Figure 4F). In contrast, the FSEs of OC43 and HKU1, whose ORF1a stop codons are located farther downstream, did not require ABCE1 or DENR for fully efficient frameshifting (Figures 4G and 4H). Thus, these experiments revealed a dependency of the SARS clade of betacoronaviruses on the ribosome recycling machinery for efficient PRF that is absent in other human betacoronaviruses.

Reduced ribosomal load abolishes the dependency of frameshifting on ribosome recycling

To further test the model that efficient ribosome recycling is needed to remove a post-termination ribosome from the ORF1a stop codon in order to allow a trailing ribosome to access the slippery sequence, we generated a series of reporter constructs with reduced rates of translation. We reasoned that a decrease in ribosome load would provide an opportunity for a post-termination ribosome to be cleared from the FSE, even in the setting of inefficient ribosome recycling. To accomplish this, the AUG initiation codon was replaced with one of the cognate start codons CUG, GUG, or ACG (Figure 5A), all of which initiate translation much less efficiently compared with a canonical AUG initiation codon.³⁸ Accordingly, these mutations resulted in an approximately 20-fold reduction in firefly luciferase expression from the in-frame PRF0 reporter compared with the AUG-initiated reporter (Figure 5B). Basal rates of frameshifting were modestly reduced on the CUG-initiated reporter transcript, while the GUG and ACG constructs showed an approximately 2-fold impairment in PRF (Figure 5C). We then tested whether these mutant constructs depend upon ribosome recycling factors for PRF. While depletion of ABCE1 or DENR impaired frameshifting on the AUG-initiated construct as expected, frameshifting on all three cognate start codon reporters was insensitive to loss of either ABCE1 or DENR (Figures 5D and 5E). These data further support a model in which a non-recycled post-termination ribosome at the ORF1a stop codon negatively influences the frameshifting outcome of a trailing ribosome.

Reduced ribosomal load abolishes dependency of SARS-CoV-2 PRF on ribosome recycling

(A) Schematic of SARS-CoV-2 dual-luciferase FSE reporters with canonical AUG initiation codon or non-canonical, near-cognate initiation codon (CUG, GUG, or ACG) that reduces ribosomal load.

(B) Relative firefly luciferase activity of the near-cognate start codon SARS-CoV-2 PRF0 reporters normalized to the canonical AUG-initiated reporter. Data are represented as the mean ± SD with individual replicates plotted; n = 6 biological replicates.

(C) Basal frameshifting rate of reporter constructs with different initiation codons. Data are represented as the mean ± SD with individual replicates plotted; n = 6 biological replicates. The p values were calculated by one-way ANOVA with Dunnett’s multiple comparisons test; ^∗∗∗p ≤ 0.001.

(D and E) Frameshifting rate of AUG-initiated or near-cognate start codon-initiated reporters upon loss of ABCE1 (D) or DENR (E). Frameshifting rate was normalized to a non-target control sgRNA for each construct. Data are represented as the mean ± SD with individual replicates plotted. The p values were calculated by two-way ANOVA with Šidák’s multiple comparisons test; ^∗∗∗p ≤ 0.001; n.s., not significant; n = 3 biological replicates.

Ribosome recycling is required for SARS-CoV-2 viral replication and frameshifting

Given the robust requirement for 80S and 40S ribosome recycling factors for frameshifting of reporter transcripts carrying the SARS-CoV-2 FSE, we next tested whether natural frameshifting on the SARS-CoV-2 genomic RNA similarly requires these factors and whether impaired ribosome recycling has an impact on viral replication. To this end, we generated multiple clonal HCT116 cell lines expressing ACE2 (ACE2-1 and ACE2-2), the cell-surface receptor for SARS-CoV-2, and then depleted ABCE1 and DENR using CRISPR (Figures 6A and 6B). Cells were then infected with SARS-CoV-2 and collected 7 h later in order to assess the efficiency of the initial round of infection and viral replication. The expression of nucleocapsid mRNA, a transcript produced only after successful translation of ORF1ab and assembly of the RTC, served as a readout of viral replication. Consistent with our finding that ribosome recycling factors are required for PRF, depletion of ABCE1 or DENR led to a marked reduction in nucleocapsid expression (Figures 6C and 6D).

Loss of ribosome recycling factors inhibits SARS-CoV-2 replication and reduces ribosomal frameshifting during infection

(A) Experimental workflow for testing the effect of ribosome recycling on SARS-CoV-2 replication. (1) Lentiviral expression of ACE2 in HCT116 cells. (2) CRISPR-Cas9-mediated knockout of *ABCE1* or *DENR*. (3) Infection with SARS-CoV-2. (4) Sample collection 7 h post-infection and qRT-PCR analysis of nucleocapsid (N) expression.

(B) Immunoblotting of ABCE1 and DENR in HCT116-ACE2 CRISPR knockout pools.

(C and D) qRT-PCR measurement of nucleocapsid mRNA expression 7 h after SARS-CoV-2 infection in cells transduced with non-target control sgRNA (sgNeg) or sgRNAs targeting *ABCE1* (C) or *DENR* (D). Two distinct sgRNAs were used per gene in two independent ACE2-expressing HCT116 cell lines (ACE2-1 and ACE2-2). Nucleocapsid expression was normalized to host GAPDH expression.

(E) Schematic of SARS-CoV-2 ORF1a and ORF1b non-structural proteins (NSPs). Antibody symbols indicate upstream (NSP1) and downstream (NSP16) NSPs that were detected by immunoblotting to assess relative frameshifting rate.

(F) Representative western blot for NSP1, NSP16, and nucleocapsid from uninfected cells, infected control cells (sgNeg) and infected *ABCE1* knockout pools generated with two independent sgRNAs (sgABCE1-1 and sgABCE1-2).

(G) Quantification of NSP1, NSP16, and nucleocapsid protein levels, normalized to host GAPDH expression, from three independent experiments. Data are represented as the mean ± SD with individual replicates plotted. The p values for qRT-PCR experiments were calculated by two-way ANOVA with Dunnett’s multiple comparisons test. The p values for the immunoblotting results were calculated by two-way ANOVA with Tukey’s multiple comparisons test; ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001; n = 3 biological replicates for all experiments.

To assess whether frameshifting on the SARS-CoV-2 genomic RNA was affected by impaired ribosome recycling, we examined the abundance of NSP16, which is encoded in ORF1b and translated only after a productive frameshifting event, relative to NSP1, encoded upstream of the FSE in ORF1a (Figure 6E). We detected a strong reduction in NSP16 protein expression in infected cells depleted of ABCE1 and, consistent with our previous measurements of nucleocapsid transcript levels, a similar reduction in nucleocapsid protein expression (Figures 6F and 6G). NSP1 protein expression was also measurably reduced in cells with impaired ribosome recycling, but to a lesser extent compared with NSP16 or nucleocapsid. Since any reduction in the rate of frameshifting will lower the expression of RTC components encoded in ORF1b and lead to a concomitant reduction in SARS-CoV-2 genomic RNA copies per cell, a decrease in overall NSP1 translation is expected. In addition, translated upstream ORFs (uORFs) have been detected in the 5′ leader sequences of SARS-CoV-2 and other coronaviruses.¹⁷ ^, ¹⁸ ^, ³⁹ Given the known role of ribosome recycling factors in mediating translation reinitiation after uORFs,⁴⁰ ^, ⁴¹ a direct contribution of ABCE1 to ORF1a translation initiation is also possible. Nevertheless, the stronger dependency of NSP16 and nucleocapsid protein expression on ABCE1 compared with NSP1 provides additional evidence for the importance of ribosome recycling for efficient frameshifting on the SARS-CoV-2 genomic RNA.

Discussion

Due to its essential role in coronavirus replication, PRF represents a potentially druggable step in the viral life cycle. Thus far, most efforts to leverage this potential vulnerability have focused on targeting the FSE in the viral genomic RNA. Multiple studies recently identified small-molecule compounds as well as antisense oligonucleotides that inhibit SARS-CoV-2 frameshifting and exhibit antiviral activity in cell culture infection models.¹² ^, ¹³ ^, ²¹ ^, ⁴² ^, ⁴³ ^, ⁴⁴ ^, ⁴⁵ Although existing compounds lack the potency required for viable drug candidates, these studies nevertheless established that viral frameshifting can be targeted pharmacologically and provided a foundation for structure-guided optimization of frameshifting inhibitors.

Identification of host factors that are essential for SARS-CoV-2 PRF could provide a complementary approach for targeting this step in the viral life cycle. Although PRF was discovered over four decades ago as a gene regulatory mechanism⁴⁶ ^, ⁴⁷ and has subsequently been observed in all domains of life, the role of host-encoded factors other than core translation components in this process has remained poorly understood. Here we describe a flexible genetic screening approach, based on dual-fluorescence reporters, that enables the facile detection of host factors that are required for the activity of any discrete FSE. Application of this strategy to identify host factors required for SARS-CoV-2 PRF led to the discovery that the SARS clade of betacoronaviruses depends upon efficient ribosome recycling for frameshifting and ultimately viral replication.

ABCE1 and DENR function to remove post-termination ribosomes from stop codons. Our discovery that these factors are essential for efficient SARS-CoV-2 PRF strongly suggests that rapid clearance of ribosomal subunits that have reached the frame 0 termination codon in the FSE is a prerequisite for frameshifting of a trailing ribosome. This conclusion is also supported by our finding that hypusine-modified EIF5A, which is important for efficient translation termination, also promotes frameshifting at the SARS-CoV-2 FSE. This dependency is likely due to the proximity of this stop codon to the slippery sequence, located less than one ribosomal footprint upstream. Thus, a trailing ribosome would be sterically inhibited from reaching the slippery sequence if a terminating or post-termination ribosome in the FSE is not rapidly removed. In support of this model, we demonstrated that relocation of the frame 0 termination codon farther downstream eliminates the requirement for ribosome recycling, as does reducing the ribosome load on the transcript. Moreover, other betacoronaviruses whose frame 0 stop codons are naturally located farther downstream within the FSE do not require ribosome recycling factors for efficient frameshifting.

These findings complement a recent structural study of ribosomes engaged with the SARS-CoV-2 FSE.¹³ Interestingly, this study reported that complete removal of the stop codon from the FSE led to a strong downregulation of frameshifting. The authors attributed this effect to the stalling of ribosomes on the FSE, which, like a failure of ribosome recycling or inefficient termination, would negatively affect the ability of a trailing ribosome to undergo a productive frameshifting event. Our findings, together with those of Bhatt et al.,¹³ therefore point toward a mechanism in which the stop codon in the first stem of the SARS-CoV-2 FSE, in concert with the activity of the ribosome recycling machinery, plays a key role in the efficient removal of non-frameshifted ribosomes from the secondary structure and subsequent frameshifting by incoming ribosomes.

These findings raise the question of whether there is an evolutionary advantage for SARS coronaviruses to uniquely position their frame 0 termination codon in close proximity to the slippery sequence. It has been proposed that this configuration increases the overall efficiency of PRF by preventing a trailing ribosome from encountering the slippery sequence while the FSE is engaged by, and therefore unwound by, a leading ribosome.¹³ While this arrangement increases the probability that the FSE will be refolded and competent to induce the requisite stalling of an incoming ribosome, it also creates a dependency upon the ribosome recycling machinery to rapidly remove post-termination ribosomes from the FSE.

As demonstrated by studies of SARS-CoV-1, even small perturbations in PRF efficiency can strongly impair coronavirus replication.¹⁴ Although ABCE1 and DENR are essential genes,⁴⁸ a therapeutic window might exist in which partial inhibition of ribosome recycling is tolerated by the host cell but still cripples SARS-CoV-2 replication. Moreover, ABCE1 contains an essential iron-sulfur cluster co-factor that is highly sensitive to iron depletion or oxidative stress,⁴⁹ ^, ⁵⁰ ^, ⁵¹ ^, ⁵² providing additional avenues for therapeutic targeting of this protein. Further investigation of the role of ribosome recycling factors in SARS-CoV-2 frameshifting, as well as the role of other putative regulators of PRF identified in this study, promises to improve our understanding of this critical step in the viral life cycle and may reveal new therapeutic strategies for COVID-19 and other viral illnesses.

Limitations of the study

Several additional putative regulators of frameshifting that remain to be validated and mechanistically dissected were identified in the CRISPR knockout screen described here. Furthermore, the CRISPR screen was unlikely to identify redundant factors or pathways that regulate PRF. A complementary gain-of-function screen might identify additional regulators of this process.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

anti-ABCE1	Invitrogen	Cat# MA5-35761; RRID: AB_2849661
anti-DENR	Proteintech	Cat# 10656-1-AP; RRID: AB_2092124
anti-GAPDH	Cell Signaling	Cat# 2118; RRID: AB_561053
anti-GFP	Cell Signaling	Cat# 2956S; RRID: AB_1196615
anti-mCherry	Invitrogen	Cat# M11217; RRID: AB_2536611
anti-SARS-CoV-2-Nucleocapsid	Cell Signaling	Cat# 33717
anti-SARS-CoV2-NSP1	MRC-PPU	Cat# DA103
anti-SARS-CoV2-NSP16	MRC-PPU	Cat# DA113
anti-VINCULIN	Sigma	Cat# V9131; RRID: AB_477629
anti-Mouse secondary IR Dye 800CW for western blotting	LI-COR	Cat# 926-32212; RRID: AB_621847
anti-Mouse secondary IR Dye 680RD for western blotting	LI-COR	Cat# 926-68072; RRID: AB_10953628
anti-Rabbit secondary IR Dye 800CW for western blotting	LI-COR	Cat# 926-32213; RRID: AB_621848
anti-Rabbit secondary IR Dye 680RD for western blotting	LI-COR	Cat# 926-68073; RRID: AB_10954442
anti-Rat secondary IR Dye 800CW for western blotting	LI-COR	Cat# 926-32219; RRID: AB_1850025
anti-Sheep Alexa Fluor 790	Jackson	Cat# 713-655-147; RRID: AB_2340754

Bacterial and virus strains

E.coli Stbl3	Invitrogen	Cat# C7373-03
Isolate hCoV-19/Japan/TY7-503/2021 (Brazil P.1)	BEI Resources	N/A

Chemicals, peptides, and recombinant proteins

Blasticidin	Invivogen	Cat# ant-bl-1
Dulbecco′s Modified Eagle′s Medium (DMEM)	ThermoFisher Scientific	Cat# 11995-073
Fetal Bovine Serum (FBS)	Sigma	Cat# F2442
FuGENE HD transfection reagent	Promega	Cat# E2312
MEM Non-Essential Amino Acids Solution	ThermoFisher Scientific	Cat# 11140050
Passive Lysis 5X Buffer	Promega	Cat# E1941
Penicillin-Streptomycin	ThermoFisher Scientific	Cat# 15140122
Polybrene	Millipore	Cat# TR-1003-G
Protease Inhibitor Cocktail Set III, EDTA-Free	Millipore	Cat# 539134
Puromycin	ThermoFisher Scientific	Cat# A11138-03
TRIzol	Invitrogen	Cat# 15596026

Critical commercial assays

Agencourt AMPure XP beads	Beckman Coulter Life Sciences	Cat# A63882
Direct-zol RNA Miniprep Kit	Zymo Research	Cat# R2052
Herculase II Fusion DNA Polymerase	Agilent	Cat# 600679
MasterPure Complete DNA and RNA Purification Kit	Lucigen	Cat# MC85200
Nano-Glo Dual-Luciferase Reporter Assay System	Promega	Cat# N1630
NEBuilder HiFi DNA Assembly Master Mix	New England Biolabs	Cat# E2621S
Phusion High-Fidelity DNA Polymerase	New England Biolabs	Cat# M0530S
Qubit dsDNA HS Assay Kit	ThermoFisher Scientific	Cat# Q32851
TaqMan Fast Virus 1-Step Master Mix	ThermoFisher Scientific	Cat# 4444432
HiScribe T7 ARCA mRNA Kit (with tailing)	New England Biolabs	Cat# E2060S
MEGAclear Transcription Clean-Up Kit	ThermoFisher Scientific	Cat# AM1908

Deposited data

CRISPR screening data	This paper	GEO: GSE206101

Experimental models: Cell lines

HCT116	ATCC	CCL-247; RRID: CVCL_0291
HEK293T	ATCC	CRL-3216; RRID: CVCL_0063
VeroE6	ATCC	CRL-1586; RRID: CVCL_0574
HCT116-SARS-CoV-2-PRF-1 reporter cell line 1	This paper	N/A
HCT116-SARS-CoV-2-PRF-1 reporter cell line 2	This paper	N/A
HCT116-SARS-CoV-2-PRF-0 reporter cell line 1	This paper	N/A
HCT116-SARS-CoV-2-PRF-0 reporter cell line 2	This paper	N/A
HCT116-ACE2-Blast cell line 1	This paper	N/A
HCT116-ACE2-Blast cell line 2	This paper	N/A

Oligonucleotides

Sequences of oligonucleotides used in this study are provided in Table S2	This paper	N/A

Recombinant DNA

Human CRISPR Knockout Pooled Library (Brunello)	Addgene (David Root, John Doench)	Cat# 73179; RRID: Addgene_73179
lentiCas9-Blast	Addgene (Feng Zhang)	Cat# 52962; RRID: Addgene_52962
lentiCRISPR v2	Addgene (Feng Zhang)	Cat# 52961; RRID: Addgene_52961
pMD2.G	Addgene (Didier Trono)	Cat# 12259; RRID: Addgene_12259
psPAX2	Addgene (Didier Trono)	Cat# 12260; RRID: Addgene_12260
pSCRBBL-ACE2-Blasticidin	John Schoggins lab	N/A
lenti-mCh-HIV-PRF-1-P2A-eGFP	This paper	N/A
lenti-mCh-HIV-PRF-0-P2A-eGFP	This paper	N/A
lenti-mCh-SARS-CoV2-PRF-1-P2A-eGFP	This paper	N/A
lenti-mCh-SARS-CoV2-PRF-0-P2A-eGFP	This paper	N/A
lenti-nLuc-HKU1-PRF-1-ffLuc	This paper	N/A
lenti-nLuc-HKU1-PRF-0-ffLuc	This paper	N/A
lenti-nLuc-OC43-PRF-1-ffLuc	This paper	N/A
lenti-nLuc-OC43-PRF-0-ffLuc	This paper	N/A
lenti-nLuc-SARS-CoV1-PRF-1-ffLuc	This paper	N/A
lenti-nLuc-SARS-CoV1-PRF-0-ffLuc	This paper	N/A
lenti-nLuc-SARS-CoV2-PRF-1-ffLuc	This paper	N/A
lenti-nLuc-SARS-CoV2-PRF-0-ffLuc	This paper	N/A
lenti-nLuc-SARS-CoV2-UUA-PRF-1-ffLuc	This paper	N/A
lenti-nLuc-SARS-CoV2-UUA-PRF-0-ffLuc	This paper	N/A
lentiCRISPR-v2-sgRNA-hsa-ABCE1-1	This paper	N/A
lentiCRISPR-v2-sgRNA-hsa-ABCE1-2	This paper	N/A
lentiCRISPR-v2-sgRNA-hsa-DENR-1	This paper	N/A
lentiCRISPR-v2-sgRNA-hsa-DENR-2	This paper	N/A
lentiCRISPR-v2-gRNA-hsa-DOHH	This paper	N/A
lentiCRISPR-v2-gRNA-hsa-DPH1	This paper	N/A
lentiCRISPR-v2-gRNA-hsa-DPH3	This paper	N/A
lentiCRISPR-v2-gRNA-hsa-EIF2D	This paper	N/A
lentiCRISPR-v2-gRNA-hsa-EIF5A	Manjunath et al.⁵³	N/A
lentiCRISPR-v2-gRNA-hsa-POLR3K	This paper	N/A
lentiCRISPR-v2-sgRNA-hsa-ORAOV1	This paper	N/A
lentiCRISPR-v2-sgRNA-hsa-YAE1D1	This paper	N/A

Software and algorithms

MAGeCK	Li et al.²³	N/A
FlowJo 10.7.1	www.flowjo.com	N/A
GraphPad Prism 9.5.0	www.graphpad.com	N/A

Open in a new tab

Resource availability

Lead contact

Requests for further information or reagents should be directed to the lead contact, Joshua T. Mendell (joshua.mendell@utsouthwestern.edu).

Materials availability

All reagents generated in this study are available upon request from the lead contact with a completed Materials Transfer Agreement.

Experimental model and subject details

Cell lines

HCT116 (male), HEK293T (female), and VeroE6-C1008 cells (female) were obtained from ATCC. HCT116, HEK293T, and all derived reporter cell lines were cultured in DMEM (high glucose, pyruvate) supplemented with 10% FBS (Sigma) and 1X penicillin-streptomycin antibiotic (Thermo Fisher Scientific). VeroE6-C1008 cells were maintained in MEM that contained 10% FBS, 1X non-essential amino acids, and 1X sodium pyruvate (Gibco). Cell lines were confirmed to be free of mycoplasma contamination.

Method details

Generation of SARS-CoV-2 PRF reporter cell lines

mCherry and P2A-eGFP were PCR amplified with primers containing a BstBI restriction site and adequate overhangs (primer sequences provided in Table S2). Both PCR fragments were inserted into XbaI and EcoRI digested lentiviral backbone (Addgene #52962) in a single NEBuilder HiFi DNA Assembly (NEB) reaction. The resulting vector was digested with BstBI. Annealed SARS-CoV-2 or HIV FSE (PRF-1 and PRF0) oligonucleotides were inserted by NEBuilder HiFi DNA Assembly (NEB). The resulting vector (Lenti-mCh-PRF-P2A-eGFP) was then used to generate lentivirus. 1x10⁶ HEK293T cells were seeded per well in a 6-well plate. 24 hours after seeding, cells were transfected with 1 μg Lenti-mCh-PRF-P2A-eGFP, 0.6 μg psPAX2 (Addgene #12260), and 0.4 μg pMD2.G (Addgene #12259) using 5 μl FugeneHD (Promega) transfection reagent. The medium was changed 24 hours post transfection and 48 hours after transfection, viral supernatant was collected and passed through a 0.45 μm filter. HCT116 cells were transduced with viral supernatant in the presence of 5 μg/ml polybrene at a low multiplicity of infection. 48 hours after transduction, single mCherry and eGFP positive cells were sorted into 96-well plates and clonal cell lines were established.

CRISPR-Cas9 screens

Genome-wide CRISPR-Cas9 knockout screens were performed as previously described in detail.¹⁹ In brief, two independent clones from each reporter were used for the screen and 1.4x10⁸ cells were infected with the Brunello pooled lentiviral CRISPR library (Addgene #73179) in the presence of 5 μg/ml polybrene at a multiplicity of infection between 0.3 to 0.5. Two days after transduction, the cells were selected in medium containing 1 μg/ml puromycin. Cells were passaged every 48-72 hours and a minimum of 5x10⁷ cells were reseeded in medium containing 1 μg/ml puromycin. After 8 days of selection, puromycin concentration was reduced to 0.5 μg/ml. After 10 days of puromycin selection, the top 1% eGFP/mCherry population and bottom 0.75% eGFP/mCherry population were sorted on an FACS Aria 2 cell sorter (BD Biosciences). gDNA from sorted pools was isolated by phenol-chloroform extraction. gDNA from 6x10⁷ unsorted cells was isolated with the Masterpure DNA isolation kit (Lucigen). Sequencing libraries were generated from the isolated gDNA through two sequential PCR reactions using Herculase II DNA polymerase (Agilent) (primer sequences provided in Table S2). After 18 rounds of initial PCR amplification, 5% of the reaction product was used for a second round of PCR (11-13 cycles) with primers that introduced Illumina sequencing adapters and barcodes. The final PCR products were purified using AMPure XP beads (Beckman Coulter). Libraries were sequenced on a NextSeq500 (Illumina) with 75 bp single-end reads at an average sequencing depth of ∼2x10⁷ reads per sample. sgRNAs sequences were extracted from fastq files through an in-house Galaxy script and normalized read counts were calculated. Genes with enriched sgRNA abundance in sorted versus unsorted cells were identified using MAGeCK.²³

CRISPR-Cas9 mediated gene knockout

sgRNA sequences for genes of interest were cloned into the lentiCRISPRv2 vector (Addgene #52961) (oligonucleotide sequences provided in Table S2). Lentivirus production and infection was performed as described for the PRF reporter constructs. Two days after transduction, cells were selected with 1 μg/mL puromycin. ABCE1 knockout pools were selected for 4-5 days while knockout pools for all other genes were selected for 6-7 days before analysis.

Flow cytometry analysis

Reporter cell lines were washed with PBS and incubated with 0.05% Trypsin-EDTA (Thermo Fisher Scientific) at 37°C for 3 minutes. Cells were collected with DMEM medium supplemented with 10% FBS and spun at 300 g for 3 minutes. Cell pellets were resuspended in flow buffer (3% FBS, 2 mM EDTA in PBS) and analyzed on an Accuri C6 flow cytometer (BD Biosciences).

Protein isolation and immunoblotting

Cells were washed with PBS and lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mM NaCl₂, 25 mM Tris-HCl pH 7.4, 2 mM EDTA, 1X proteinase inhibitor cocktail). After lysis, samples were briefly vortexed and kept on ice for 10 minutes. Lysates were spun for 15 minutes at 14,000 g and supernatant was stored at -80°C until use. Proteins were separated on 4-12% or 10% NuPAGE Bis-Tris polyacrylamide gels and wet-blotted to 0.45 μm nitrocellulose membranes. Blots were blocked in PBS with 0.1% Tween-20 (PBST) containing 5% non-fat milk and incubated with primary antibodies in PBST containing either 3% BSA or 5% non-fat milk (for SARS-CoV-2 NSP antibodies) overnight at 4°C. The blots were incubated with secondary antibodies in PBST with 5% non-fat milk for one hour at room temperature and images were acquired on an Odyssey fluorescent imaging system (LI-COR Biosystems).

Dual-luciferase frameshifting assays

The luciferase-based reporter was designed analogously to the fluorescent reporter. Different coronavirus FSEs were cloned between a NanoLuc luciferase (nLuc) ORF at the 5′ end and firefly luciferase (ffLuc) ORF at the 3′ end. No P2A sequence was included in this reporter. nLuc and ffLuc were amplified by PCR with primers containing appropriate overhangs and both PCR products were inserted together with a gBlock double-stranded gene fragment (IDT) encoding the FSE into XbaI and EcoRI digested lentiviral backbone (Addgene #52962) through NEBuilder HiFi DNA Assembly (NEB) (oligonucleotide sequences provided in Table S2). Lentivirus was produced from the resulting constructs as described above. For luciferase assays, knockout pools undergoing puromycin selection were transduced with dual-luciferase reporter encoding lentivirus two days before analysis. For infection, 500 μl of knockout-pool cell suspension containing 6 μg/ml polybrene was seeded per well in a 24-well plate and immediately mixed with 100 μl of lentiviral supernatant. For cell harvest, the medium was removed and 150 μl of passive lysis buffer (Promega) was added to each well. Samples were incubated on an orbital shaker (150 rpm) for 8 minutes at room temperature. 50 μl of cell lysate were transferred to an opaque 96-well plate and luciferase activity was measured on a Glomax Discover plate reader system (Promega) with the Nano-Glo Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. For analysis, PRF-1 ffLuc activity was divided by nLuc activity and this ratio was then normalized to the ffLuc/nLuc ratio of the PRF0 reporter to determine the frameshifting rate.

In vitro transcription and transfection of dual-luciferase mRNAs

Dual-luciferase frameshifting reporter constructs were amplified by PCR with a forward primer harboring the minimal T7 promoter sequence to generate T7 RNA-polymerase in vitro transcription templates. 5′ anti-reverse cap analog (ARCA) modified and 3′ poly-A tailed PRF reporter mRNA was generated with the HiScribe T7 ARCA mRNA Kit with tailing (NEB). Following column purification with the MEGAclear Transcription Clean-Up Kit (ThermoScientific), 0.5 μg of reporter mRNA was transfected with 1 μl of Lipofectamine MessengerMAX into HCT116 cells plated in a 24-well plate the day prior. 24 hours post transfection, cells were lysed, and luciferase activity was measured as described above.

SARS-CoV-2 virus production and infection

Isolate hCoV-19/Japan/TY7-503/2021 (P.1, Gamma variant) was obtained from BEI Resources. To generate a working stock of P.1 virus, 50 μl of virus was diluted into 10 ml MEM-2% FCS media containing 1X NEAA. 5 ml of diluted virus was added to a T175 flask containing 7x10⁶ VeroE6-C1008 cells. The flask was incubated at 37°C and rocked every 5-10 minutes for a total of 45 minutes. Inoculum was removed and 22 ml MEM-2%FCS media containing 1X NEAA was added back to flask. Supernatant was collected 3 days later, cleared by centrifugation, and aliquoted. Viral titer was determined by plaque assay on Vero E6-C1008 cells.

Cells were infected with SARS-CoV-2 virus at an approximate MOI of 0.3. Cells were incubated with virus for 30 minutes followed by addition of complete media. Cells were harvested at 7 hours for analysis by qRT-PCR or western blot. Protein samples were lysed in SARS-CoV-2 lysis buffer (20 mM Tris-HCl pH 7.4, 0.5% NP40, 100 mM NaCl, 5 mM MgCl₂).

RNA isolation and qRT-PCR

RNA was isolated using the Direct-zol RNA mini prep kit following manufacturer’s instructions (ZymoResearch). A 20 μl reaction contained 5 μl RNA, 5 μl TaqMan Fast Virus 1-Step Master Mix, and 1.8 μl of SARS-CoV-2 or GAPDH primer/probe set containing 6.7 μM each primer/1.7 μM probe (final concentration 600 nM primer/150 nM probe). SARS-CoV-2 primers and probe were designed as recommended by the Centers for Disease Control (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html). RT was performed at 50°C for 5 minutes, followed by inactivation at 95°C for 2 minutes, and 40 cycles of PCR (95°C for 3 seconds, 60°C for 30 seconds) on a QuantStudio 3 (Applied Biosystems).

Quantification and statistical analysis

Statistical analysis

All experiments were repeated with a minimum of three biological replicates. To determine statistical significance, Studentʹs t test, and ANOVA were calculated using GraphPad Prism 9.5.0. Values are reported as mean ± SD in all figures.

Acknowledgments

We thank John Doench, David Root, Didier Trono, and Feng Zhang for plasmids; Vanessa Schmid and Jo Wagner in the McDermott Center Next Generation Sequencing Core for assistance with high-throughput sequencing; Angie Mobley and the UT Southwestern Flow Cytometry Core facility for assistance with FACS; and Kathryn O’Donnell and members of the Mendell laboratory for helpful feedback on the manuscript. This work was supported by grants from NIH (DP1AI158124 to J.W.S), CPRIT (RP220309 to J.T.M.), the Welch Foundation (I-1961-20210327 to J.T.M.), and the Hamon Center for Regenerative Science and Medicine at UT Southwestern (to J.T.M.). J.W.S. holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. J.T.M. is an Investigator of the Howard Hughes Medical Institute. This article is subject to HHMI’s Open Access to Publications policy. HHMI lab heads have previously granted a non-exclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication.

Author contributions

F.R., J.W.S., and J.T.M. designed the experiments and interpreted the results. F.R., J.L.E., M.B.O., and T.-C.C. performed experiments. F.R. and J.T.M wrote the manuscript.

Declaration of interests

J.T.M. is a scientific advisor for Ribometrix, Inc., and owns equity in Orbital Therapeutics, Inc.

Inclusion and diversity

We support inclusive, diverse, and equitable conduct of research.

Published: January 30, 2023

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2023.112076.

Supplemental information

Document S1. Figures S1–S5

mmc1.pdf^{(910.5KB, pdf)}

Table S1. MAGeCK analysis of SARS-CoV-2 PRF screens, related to Figure 1

mmc2.xlsx^{(3.8MB, xlsx)}

Table S2. Oligonucleotides, related to STAR Methods

mmc3.xlsx^{(13.3KB, xlsx)}

Document S2. Article plus supplemental information

mmc4.pdf^{(3.5MB, pdf)}

Data and code availability

•
High-throughput sequencing data from the SARS-CoV-2 PRF CRISPR screen have been deposited in GEO and are publicly available as of the date of publication. Accession number provided in the key resources table.
•
No custom code was generated in this study.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

References

1.Garofalo R., Wohlgemuth I., Pearson M., Lenz C., Urlaub H., Rodnina M.V. Broad range of missense error frequencies in cellular proteins. Nucleic Acids Res. 2019;47:2932–2945. doi: 10.1093/nar/gky1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Manickam N., Nag N., Abbasi A., Patel K., Farabaugh P.J. Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors. RNA. 2014;20:9–15. doi: 10.1261/rna.039792.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Kramer E.B., Vallabhaneni H., Mayer L.M., Farabaugh P.J. A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae. RNA. 2010;16:1797–1808. doi: 10.1261/rna.2201210. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Rodnina M.V., Korniy N., Klimova M., Karki P., Peng B.Z., Senyushkina T., Belardinelli R., Maracci C., Wohlgemuth I., Samatova E., Peske F. Translational recoding: canonical translation mechanisms reinterpreted. Nucleic Acids Res. 2020;48:1056–1067. doi: 10.1093/nar/gkz783. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Atkins J.F., Loughran G., Bhatt P.R., Firth A.E., Baranov P.V. Ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use. Nucleic Acids Res. 2016;44:7007–7078. doi: 10.1093/nar/gkw530. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Matsufuji S., Matsufuji T., Miyazaki Y., Murakami Y., Atkins J.F., Gesteland R.F., Hayashi S. Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell. 1995;80:51–60. doi: 10.1016/0092-8674(95)90450-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Ivanov I.P., Matsufuji S., Murakami Y., Gesteland R.F., Atkins J.F. Conservation of polyamine regulation by translational frameshifting from yeast to mammals. EMBO J. 2000;19:1907–1917. doi: 10.1093/emboj/19.8.1907. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Melian E.B., Hinzman E., Nagasaki T., Firth A.E., Wills N.M., Nouwens A.S., Blitvich B.J., Leung J., Funk A., Atkins J.F., et al. NS1' of Flaviviruses in the Japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness. J. Virol. 2010;84:1641–1647. doi: 10.1128/Jvi.01979-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Jacks T., Power M.D., Masiarz F.R., Luciw P.A., Barr P.J., Varmus H.E. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature. 1988;331:280–283. doi: 10.1038/331280a0. [DOI] [PubMed] [Google Scholar]
10.Plant E.P., Pérez-Alvarado G.C., Jacobs J.L., Mukhopadhyay B., Hennig M., Dinman J.D. A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal. PLoS Biol. 2005;3:e172. doi: 10.1371/journal.pbio.0030172. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Baranov P.V., Henderson C.M., Anderson C.B., Gesteland R.F., Atkins J.F., Howard M.T. Programmed ribosomal frameshifting in decoding the SARS-CoV genome. Virology. 2005;332:498–510. doi: 10.1016/j.virol.2004.11.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Kelly J.A., Olson A.N., Neupane K., Munshi S., San Emeterio J., Pollack L., Woodside M.T., Dinman J.D. Structural and functional conservation of the programmed -1 ribosomal frameshift signal of SARS coronavirus 2 (SARS-CoV-2) J. Biol. Chem. 2020;295:10741–10748. doi: 10.1074/jbc.AC120.013449. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Bhatt P.R., Scaiola A., Loughran G., Leibundgut M., Kratzel A., Meurs R., Dreos R., O'Connor K.M., McMillan A., Bode J.W., et al. Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome. Science. 2021;372:1306–1313. doi: 10.1126/science.abf3546. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Plant E.P., Sims A.C., Baric R.S., Dinman J.D., Taylor D.R. Altering SARS coronavirus frameshift efficiency affects genomic and subgenomic RNA production. Viruses. 2013;5:279–294. doi: 10.3390/v5010279. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Biswas P., Jiang X., Pacchia A.L., Dougherty J.P., Peltz S.W. The human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy. J. Virol. 2004;78:2082–2087. doi: 10.1128/Jvi.78.4.2082-2087.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.V'kovski P., Kratzel A., Steiner S., Stalder H., Thiel V. Coronavirus biology and replication: implications for SARS-CoV-2. Nat. Rev. Microbiol. 2021;19:155–170. doi: 10.1038/s41579-020-00468-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Irigoyen N., Firth A.E., Jones J.D., Chung B.Y.W., Siddell S.G., Brierley I. High-resolution analysis of coronavirus gene expression by RNA sequencing and ribosome profiling. PLoS Pathog. 2016;12:e1005473. doi: 10.1371/journal.ppat.1005473. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Finkel Y., Mizrahi O., Nachshon A., Weingarten-Gabbay S., Morgenstern D., Yahalom-Ronen Y., Tamir H., Achdout H., Stein D., Israeli O., et al. The coding capacity of SARS-CoV-2. Nature. 2021;589:125–130. doi: 10.1038/s41586-020-2739-1. [DOI] [PubMed] [Google Scholar]
19.Zhu X., Zhang H., Mendell J.T. Ribosome recycling by ABCE1 links lysosomal function and iron homeostasis to 3’ UTR-directed regulation and nonsense-mediated decay. Cell Rep. 2020;32:107895. doi: 10.1016/j.celrep.2020.107895. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Golden R.J., Chen B., Li T., Braun J., Manjunath H., Chen X., Wu J., Schmid V., Chang T.C., Kopp F., et al. An Argonaute phosphorylation cycle promotes microRNA-mediated silencing. Nature. 2017;542:197–202. doi: 10.1038/nature21025. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Sun Y., Abriola L., Niederer R.O., Pedersen S.F., Alfajaro M.M., Silva Monteiro V., Wilen C.B., Ho Y.C., Gilbert W.V., Surovtseva Y.V., et al. Restriction of SARS-CoV-2 replication by targeting programmed -1 ribosomal frameshifting. Proc. Natl. Acad. Sci. USA. 2021;118 doi: 10.1073/pnas.2023051118. e2023051118. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Doench J.G., Fusi N., Sullender M., Hegde M., Vaimberg E.W., Donovan K.F., Smith I., Tothova Z., Wilen C., Orchard R., et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 2016;34:184–191. doi: 10.1038/nbt.3437. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Li W., Xu H., Xiao T., Cong L., Love M.I., Zhang F., Irizarry R.A., Liu J.S., Brown M., Liu X.S. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 2014;15:554. doi: 10.1186/s13059-014-0554-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Liu S., Bachran C., Gupta P., Miller-Randolph S., Wang H., Crown D., Zhang Y., Wein A.N., Singh R., Fattah R., Leppla S.H. Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development. Proc. Natl. Acad. Sci. USA. 2012;109:13817–13822. doi: 10.1073/pnas.1206933109. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Girbig M., Misiaszek A.D., Vorländer M.K., Lafita A., Grötsch H., Baudin F., Bateman A., Müller C.W. Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states. Nat. Struct. Mol. Biol. 2021;28:210–219. doi: 10.1038/s41594-020-00555-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Ramsay E.P., Abascal-Palacios G., Daiß J.L., King H., Gouge J., Pilsl M., Beuron F., Morris E., Gunkel P., Engel C., Vannini A. Structure of human RNA polymerase III. Nat. Commun. 2020;11:6409. doi: 10.1038/s41467-020-20262-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.White R.J. Transcription by RNA polymerase III: more complex than we thought. Nat. Rev. Genet. 2011;12:459–463. doi: 10.1038/nrg3001. [DOI] [PubMed] [Google Scholar]
28.Korniy N., Goyal A., Hoffmann M., Samatova E., Peske F., Pöhlmann S., Rodnina M.V. Modulation of HIV-1 Gag/Gag-Pol frameshifting by tRNA abundance. Nucleic Acids Res. 2019;47:5210–5222. doi: 10.1093/nar/gkz202. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Lomakin I.B., Stolboushkina E.A., Vaidya A.T., Zhao C., Garber M.B., Dmitriev S.E., Steitz T.A. Crystal structure of the human ribosome in complex with DENR-MCT-1. Cell Rep. 2017;20:521–528. doi: 10.1016/j.celrep.2017.06.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Skabkin M.A., Skabkina O.V., Hellen C.U.T., Pestova T.V. Reinitiation and other unconventional posttermination events during eukaryotic translation. Mol. Cell. 2013;51:249–264. doi: 10.1016/j.molcel.2013.05.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Skabkin M.A., Skabkina O.V., Dhote V., Komar A.A., Hellen C.U.T., Pestova T.V. Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling. Genes Dev. 2010;24:1787–1801. doi: 10.1101/gad.1957510. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Paul V.D., Mühlenhoff U., Stümpfig M., Seebacher J., Kugler K.G., Renicke C., Taxis C., Gavin A.C., Pierik A.J., Lill R. The deca-GX3 proteins Yae1-Lto1 function as adaptors recruiting the ABC protein Rli1 for iron-sulfur cluster insertion. Elife. 2015;4:e08231. doi: 10.7554/eLife.08231. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Young D.J., Meydan S., Guydosh N.R. 40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling. Nat. Commun. 2021;12:2976. doi: 10.1038/s41467-021-23223-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Schuller A.P., Wu C.C.C., Dever T.E., Buskirk A.R., Green R. eIF5A functions globally in translation elongation and termination. Mol. Cell. 2017;66:194–205.e5. doi: 10.1016/j.molcel.2017.03.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Loughran G., Howard M.T., Firth A.E., Atkins J.F. Avoidance of reporter assay distortions from fused dual reporters. RNA. 2017;23:1285–1289. doi: 10.1261/rna.061051.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Khan Y.A., Loughran G., Steckelberg A.L., Brown K., Kiniry S.J., Stewart H., Baranov P.V., Kieft J.S., Firth A.E., Atkins J.F. Evaluating ribosomal frameshifting in CCR5 mRNA decoding. Nature. 2022;604:E16–E23. doi: 10.1038/s41586-022-04627-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Akirtava C., May G.E., McManus C.J. False-positive IRESes from Hoxa9 and other genes resulting from errors in mammalian 5’ UTR annotations. Proc. Natl. Acad. Sci. USA. 2022;119 doi: 10.1073/pnas.2122170119. e2122170119. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Kearse M.G., Wilusz J.E. Non-AUG translation: a new start for protein synthesis in eukaryotes. Genes Dev. 2017;31:1717–1731. doi: 10.1101/gad.305250.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Wu H.Y., Guan B.J., Su Y.P., Fan Y.H., Brian D.A. Reselection of a genomic upstream open reading frame in mouse hepatitis coronavirus 5’-untranslated-region mutants. J. Virol. 2014;88:846–858. doi: 10.1128/JVI.02831-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Bohlen J., Harbrecht L., Blanco S., Clemm von Hohenberg K., Fenzl K., Kramer G., Bukau B., Teleman A.A. DENR promotes translation reinitiation via ribosome recycling to drive expression of oncogenes including ATF4. Nat. Commun. 2020;11:4676. doi: 10.1038/s41467-020-18452-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Schleich S., Strassburger K., Janiesch P.C., Koledachkina T., Miller K.K., Haneke K., Cheng Y.S., Kuechler K., Stoecklin G., Duncan K.E., Teleman A.A. DENR-MCT-1 promotes translation re-initiation downstream of uORFs to control tissue growth. Nature. 2014;512:208–212. doi: 10.1038/nature13401. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Haniff H.S., Tong Y., Liu X., Chen J.L., Suresh B.M., Andrews R.J., Peterson J.M., O'Leary C.A., Benhamou R.I., Moss W.N., Disney M.D. Targeting the SARS-CoV-2 RNA genome with small molecule binders and ribonuclease targeting chimera (RIBOTAC) degraders. ACS Cent. Sci. 2020;6:1713–1721. doi: 10.1021/acscentsci.0c00984. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Zhang K., Zheludev I.N., Hagey R.J., Haslecker R., Hou Y.J., Kretsch R., Pintilie G.D., Rangan R., Kladwang W., Li S., et al. Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome. Nat. Struct. Mol. Biol. 2021;28:747–754. doi: 10.1038/s41594-021-00653-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Munshi S., Neupane K., Ileperuma S.M., Halma M.T.J., Kelly J.A., Halpern C.F., Dinman J.D., Loerch S., Woodside M.T. Identifying inhibitors of -1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses. Viruses. 2022;14:177. doi: 10.3390/v14020177. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Park S.J., Kim Y.G., Park H.J. Identification of RNA pseudoknot-binding ligand that inhibits the -1 ribosomal frameshifting of SARS-coronavirus by structure-based virtual screening. J. Am. Chem. Soc. 2011;133:10094–10100. doi: 10.1021/ja1098325. [DOI] [PubMed] [Google Scholar]
46.Atkins J.F., Steitz J.A., Anderson C.W., Model P. Binding of mammalian ribosomes to MS2 phage RNA reveals an overlapping gene encoding a lysis function. Cell. 1979;18:247–256. doi: 10.1016/0092-8674(79)90044-8. [DOI] [PubMed] [Google Scholar]
47.Dunn J.J., Studier F.W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 1983;166:477–535. doi: 10.1016/s0022-2836(83)80282-4. [DOI] [PubMed] [Google Scholar]
48.Tsherniak A., Vazquez F., Montgomery P.G., Weir B.A., Kryukov G., Cowley G.S., Gill S., Harrington W.F., Pantel S., Krill-Burger J.M., et al. Defining a cancer dependency map. Cell. 2017;170:564–576.e16. doi: 10.1016/j.cell.2017.06.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Sudmant P.H., Lee H., Dominguez D., Heiman M., Burge C.B. Widespread accumulation of ribosome-associated isolated 3’ UTRs in neuronal cell populations of the aging brain. Cell Rep. 2018;25:2447–2456.e4. doi: 10.1016/j.celrep.2018.10.094. [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Hughes C.E., Coody T.K., Jeong M.Y., Berg J.A., Winge D.R., Hughes A.L. Cysteine toxicity drives age-related mitochondrial decline by altering iron homeostasis. Cell. 2020;180:296–310.e18. doi: 10.1016/j.cell.2019.12.035. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Weber R.A., Yen F.S., Nicholson S.P.V., Alwaseem H., Bayraktar E.C., Alam M., Timson R.C., La K., Abu-Remaileh M., Molina H., Birsoy K. Maintaining iron homeostasis is the key role of lysosomal acidity for cell proliferation. Mol. Cell. 2020;77:645–655.e7. doi: 10.1016/j.molcel.2020.01.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Alhebshi A., Sideri T.C., Holland S.L., Avery S.V. The essential iron-sulfur protein Rli1 is an important target accounting for inhibition of cell growth by reactive oxygen species. Mol. Biol. Cell. 2012;23:3582–3590. doi: 10.1091/mbc.E12-05-0413. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Manjunath H., Zhang H., Rehfeld F., Han J., Chang T.C., Mendell J.T. Suppression of ribosomal pausing by eIF5A is necessary to maintain the fidelity of start codon selection. Cell Rep. 2019;29:3134–3146.e6. doi: 10.1016/j.celrep.2019.10.129. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S5

mmc1.pdf^{(910.5KB, pdf)}

Table S1. MAGeCK analysis of SARS-CoV-2 PRF screens, related to Figure 1

mmc2.xlsx^{(3.8MB, xlsx)}

Table S2. Oligonucleotides, related to STAR Methods

mmc3.xlsx^{(13.3KB, xlsx)}

Document S2. Article plus supplemental information

mmc4.pdf^{(3.5MB, pdf)}

Data Availability Statement

•
High-throughput sequencing data from the SARS-CoV-2 PRF CRISPR screen have been deposited in GEO and are publicly available as of the date of publication. Accession number provided in the key resources table.
•
No custom code was generated in this study.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

[bib1] 1.Garofalo R., Wohlgemuth I., Pearson M., Lenz C., Urlaub H., Rodnina M.V. Broad range of missense error frequencies in cellular proteins. Nucleic Acids Res. 2019;47:2932–2945. doi: 10.1093/nar/gky1319. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib2] 2.Manickam N., Nag N., Abbasi A., Patel K., Farabaugh P.J. Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors. RNA. 2014;20:9–15. doi: 10.1261/rna.039792.113. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib3] 3.Kramer E.B., Vallabhaneni H., Mayer L.M., Farabaugh P.J. A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae. RNA. 2010;16:1797–1808. doi: 10.1261/rna.2201210. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib4] 4.Rodnina M.V., Korniy N., Klimova M., Karki P., Peng B.Z., Senyushkina T., Belardinelli R., Maracci C., Wohlgemuth I., Samatova E., Peske F. Translational recoding: canonical translation mechanisms reinterpreted. Nucleic Acids Res. 2020;48:1056–1067. doi: 10.1093/nar/gkz783. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5.Atkins J.F., Loughran G., Bhatt P.R., Firth A.E., Baranov P.V. Ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use. Nucleic Acids Res. 2016;44:7007–7078. doi: 10.1093/nar/gkw530. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib6] 6.Matsufuji S., Matsufuji T., Miyazaki Y., Murakami Y., Atkins J.F., Gesteland R.F., Hayashi S. Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell. 1995;80:51–60. doi: 10.1016/0092-8674(95)90450-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib7] 7.Ivanov I.P., Matsufuji S., Murakami Y., Gesteland R.F., Atkins J.F. Conservation of polyamine regulation by translational frameshifting from yeast to mammals. EMBO J. 2000;19:1907–1917. doi: 10.1093/emboj/19.8.1907. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Melian E.B., Hinzman E., Nagasaki T., Firth A.E., Wills N.M., Nouwens A.S., Blitvich B.J., Leung J., Funk A., Atkins J.F., et al. NS1' of Flaviviruses in the Japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness. J. Virol. 2010;84:1641–1647. doi: 10.1128/Jvi.01979-09. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib9] 9.Jacks T., Power M.D., Masiarz F.R., Luciw P.A., Barr P.J., Varmus H.E. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature. 1988;331:280–283. doi: 10.1038/331280a0. [DOI] [PubMed] [Google Scholar]

[bib10] 10.Plant E.P., Pérez-Alvarado G.C., Jacobs J.L., Mukhopadhyay B., Hennig M., Dinman J.D. A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal. PLoS Biol. 2005;3:e172. doi: 10.1371/journal.pbio.0030172. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib11] 11.Baranov P.V., Henderson C.M., Anderson C.B., Gesteland R.F., Atkins J.F., Howard M.T. Programmed ribosomal frameshifting in decoding the SARS-CoV genome. Virology. 2005;332:498–510. doi: 10.1016/j.virol.2004.11.038. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib12] 12.Kelly J.A., Olson A.N., Neupane K., Munshi S., San Emeterio J., Pollack L., Woodside M.T., Dinman J.D. Structural and functional conservation of the programmed -1 ribosomal frameshift signal of SARS coronavirus 2 (SARS-CoV-2) J. Biol. Chem. 2020;295:10741–10748. doi: 10.1074/jbc.AC120.013449. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib13] 13.Bhatt P.R., Scaiola A., Loughran G., Leibundgut M., Kratzel A., Meurs R., Dreos R., O'Connor K.M., McMillan A., Bode J.W., et al. Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome. Science. 2021;372:1306–1313. doi: 10.1126/science.abf3546. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib14] 14.Plant E.P., Sims A.C., Baric R.S., Dinman J.D., Taylor D.R. Altering SARS coronavirus frameshift efficiency affects genomic and subgenomic RNA production. Viruses. 2013;5:279–294. doi: 10.3390/v5010279. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib15] 15.Biswas P., Jiang X., Pacchia A.L., Dougherty J.P., Peltz S.W. The human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy. J. Virol. 2004;78:2082–2087. doi: 10.1128/Jvi.78.4.2082-2087.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib16] 16.V'kovski P., Kratzel A., Steiner S., Stalder H., Thiel V. Coronavirus biology and replication: implications for SARS-CoV-2. Nat. Rev. Microbiol. 2021;19:155–170. doi: 10.1038/s41579-020-00468-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib17] 17.Irigoyen N., Firth A.E., Jones J.D., Chung B.Y.W., Siddell S.G., Brierley I. High-resolution analysis of coronavirus gene expression by RNA sequencing and ribosome profiling. PLoS Pathog. 2016;12:e1005473. doi: 10.1371/journal.ppat.1005473. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib18] 18.Finkel Y., Mizrahi O., Nachshon A., Weingarten-Gabbay S., Morgenstern D., Yahalom-Ronen Y., Tamir H., Achdout H., Stein D., Israeli O., et al. The coding capacity of SARS-CoV-2. Nature. 2021;589:125–130. doi: 10.1038/s41586-020-2739-1. [DOI] [PubMed] [Google Scholar]

[bib19] 19.Zhu X., Zhang H., Mendell J.T. Ribosome recycling by ABCE1 links lysosomal function and iron homeostasis to 3’ UTR-directed regulation and nonsense-mediated decay. Cell Rep. 2020;32:107895. doi: 10.1016/j.celrep.2020.107895. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib20] 20.Golden R.J., Chen B., Li T., Braun J., Manjunath H., Chen X., Wu J., Schmid V., Chang T.C., Kopp F., et al. An Argonaute phosphorylation cycle promotes microRNA-mediated silencing. Nature. 2017;542:197–202. doi: 10.1038/nature21025. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib21] 21.Sun Y., Abriola L., Niederer R.O., Pedersen S.F., Alfajaro M.M., Silva Monteiro V., Wilen C.B., Ho Y.C., Gilbert W.V., Surovtseva Y.V., et al. Restriction of SARS-CoV-2 replication by targeting programmed -1 ribosomal frameshifting. Proc. Natl. Acad. Sci. USA. 2021;118 doi: 10.1073/pnas.2023051118. e2023051118. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib22] 22.Doench J.G., Fusi N., Sullender M., Hegde M., Vaimberg E.W., Donovan K.F., Smith I., Tothova Z., Wilen C., Orchard R., et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 2016;34:184–191. doi: 10.1038/nbt.3437. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib23] 23.Li W., Xu H., Xiao T., Cong L., Love M.I., Zhang F., Irizarry R.A., Liu J.S., Brown M., Liu X.S. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 2014;15:554. doi: 10.1186/s13059-014-0554-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib25] 24.Liu S., Bachran C., Gupta P., Miller-Randolph S., Wang H., Crown D., Zhang Y., Wein A.N., Singh R., Fattah R., Leppla S.H. Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development. Proc. Natl. Acad. Sci. USA. 2012;109:13817–13822. doi: 10.1073/pnas.1206933109. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib24] 25.Girbig M., Misiaszek A.D., Vorländer M.K., Lafita A., Grötsch H., Baudin F., Bateman A., Müller C.W. Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states. Nat. Struct. Mol. Biol. 2021;28:210–219. doi: 10.1038/s41594-020-00555-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib26] 26.Ramsay E.P., Abascal-Palacios G., Daiß J.L., King H., Gouge J., Pilsl M., Beuron F., Morris E., Gunkel P., Engel C., Vannini A. Structure of human RNA polymerase III. Nat. Commun. 2020;11:6409. doi: 10.1038/s41467-020-20262-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib27] 27.White R.J. Transcription by RNA polymerase III: more complex than we thought. Nat. Rev. Genet. 2011;12:459–463. doi: 10.1038/nrg3001. [DOI] [PubMed] [Google Scholar]

[bib28] 28.Korniy N., Goyal A., Hoffmann M., Samatova E., Peske F., Pöhlmann S., Rodnina M.V. Modulation of HIV-1 Gag/Gag-Pol frameshifting by tRNA abundance. Nucleic Acids Res. 2019;47:5210–5222. doi: 10.1093/nar/gkz202. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib29] 29.Lomakin I.B., Stolboushkina E.A., Vaidya A.T., Zhao C., Garber M.B., Dmitriev S.E., Steitz T.A. Crystal structure of the human ribosome in complex with DENR-MCT-1. Cell Rep. 2017;20:521–528. doi: 10.1016/j.celrep.2017.06.025. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib30] 30.Skabkin M.A., Skabkina O.V., Hellen C.U.T., Pestova T.V. Reinitiation and other unconventional posttermination events during eukaryotic translation. Mol. Cell. 2013;51:249–264. doi: 10.1016/j.molcel.2013.05.026. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib31] 31.Skabkin M.A., Skabkina O.V., Dhote V., Komar A.A., Hellen C.U.T., Pestova T.V. Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling. Genes Dev. 2010;24:1787–1801. doi: 10.1101/gad.1957510. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib32] 32.Paul V.D., Mühlenhoff U., Stümpfig M., Seebacher J., Kugler K.G., Renicke C., Taxis C., Gavin A.C., Pierik A.J., Lill R. The deca-GX3 proteins Yae1-Lto1 function as adaptors recruiting the ABC protein Rli1 for iron-sulfur cluster insertion. Elife. 2015;4:e08231. doi: 10.7554/eLife.08231. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib33] 33.Young D.J., Meydan S., Guydosh N.R. 40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling. Nat. Commun. 2021;12:2976. doi: 10.1038/s41467-021-23223-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib34] 34.Schuller A.P., Wu C.C.C., Dever T.E., Buskirk A.R., Green R. eIF5A functions globally in translation elongation and termination. Mol. Cell. 2017;66:194–205.e5. doi: 10.1016/j.molcel.2017.03.003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib35] 35.Loughran G., Howard M.T., Firth A.E., Atkins J.F. Avoidance of reporter assay distortions from fused dual reporters. RNA. 2017;23:1285–1289. doi: 10.1261/rna.061051.117. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib36] 36.Khan Y.A., Loughran G., Steckelberg A.L., Brown K., Kiniry S.J., Stewart H., Baranov P.V., Kieft J.S., Firth A.E., Atkins J.F. Evaluating ribosomal frameshifting in CCR5 mRNA decoding. Nature. 2022;604:E16–E23. doi: 10.1038/s41586-022-04627-y. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib37] 37.Akirtava C., May G.E., McManus C.J. False-positive IRESes from Hoxa9 and other genes resulting from errors in mammalian 5’ UTR annotations. Proc. Natl. Acad. Sci. USA. 2022;119 doi: 10.1073/pnas.2122170119. e2122170119. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib38] 38.Kearse M.G., Wilusz J.E. Non-AUG translation: a new start for protein synthesis in eukaryotes. Genes Dev. 2017;31:1717–1731. doi: 10.1101/gad.305250.117. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib39] 39.Wu H.Y., Guan B.J., Su Y.P., Fan Y.H., Brian D.A. Reselection of a genomic upstream open reading frame in mouse hepatitis coronavirus 5’-untranslated-region mutants. J. Virol. 2014;88:846–858. doi: 10.1128/JVI.02831-13. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib40] 40.Bohlen J., Harbrecht L., Blanco S., Clemm von Hohenberg K., Fenzl K., Kramer G., Bukau B., Teleman A.A. DENR promotes translation reinitiation via ribosome recycling to drive expression of oncogenes including ATF4. Nat. Commun. 2020;11:4676. doi: 10.1038/s41467-020-18452-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib41] 41.Schleich S., Strassburger K., Janiesch P.C., Koledachkina T., Miller K.K., Haneke K., Cheng Y.S., Kuechler K., Stoecklin G., Duncan K.E., Teleman A.A. DENR-MCT-1 promotes translation re-initiation downstream of uORFs to control tissue growth. Nature. 2014;512:208–212. doi: 10.1038/nature13401. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib42] 42.Haniff H.S., Tong Y., Liu X., Chen J.L., Suresh B.M., Andrews R.J., Peterson J.M., O'Leary C.A., Benhamou R.I., Moss W.N., Disney M.D. Targeting the SARS-CoV-2 RNA genome with small molecule binders and ribonuclease targeting chimera (RIBOTAC) degraders. ACS Cent. Sci. 2020;6:1713–1721. doi: 10.1021/acscentsci.0c00984. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib43] 43.Zhang K., Zheludev I.N., Hagey R.J., Haslecker R., Hou Y.J., Kretsch R., Pintilie G.D., Rangan R., Kladwang W., Li S., et al. Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome. Nat. Struct. Mol. Biol. 2021;28:747–754. doi: 10.1038/s41594-021-00653-y. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib44] 44.Munshi S., Neupane K., Ileperuma S.M., Halma M.T.J., Kelly J.A., Halpern C.F., Dinman J.D., Loerch S., Woodside M.T. Identifying inhibitors of -1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses. Viruses. 2022;14:177. doi: 10.3390/v14020177. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib45] 45.Park S.J., Kim Y.G., Park H.J. Identification of RNA pseudoknot-binding ligand that inhibits the -1 ribosomal frameshifting of SARS-coronavirus by structure-based virtual screening. J. Am. Chem. Soc. 2011;133:10094–10100. doi: 10.1021/ja1098325. [DOI] [PubMed] [Google Scholar]

[bib46] 46.Atkins J.F., Steitz J.A., Anderson C.W., Model P. Binding of mammalian ribosomes to MS2 phage RNA reveals an overlapping gene encoding a lysis function. Cell. 1979;18:247–256. doi: 10.1016/0092-8674(79)90044-8. [DOI] [PubMed] [Google Scholar]

[bib47] 47.Dunn J.J., Studier F.W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 1983;166:477–535. doi: 10.1016/s0022-2836(83)80282-4. [DOI] [PubMed] [Google Scholar]

[bib48] 48.Tsherniak A., Vazquez F., Montgomery P.G., Weir B.A., Kryukov G., Cowley G.S., Gill S., Harrington W.F., Pantel S., Krill-Burger J.M., et al. Defining a cancer dependency map. Cell. 2017;170:564–576.e16. doi: 10.1016/j.cell.2017.06.010. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib49] 49.Sudmant P.H., Lee H., Dominguez D., Heiman M., Burge C.B. Widespread accumulation of ribosome-associated isolated 3’ UTRs in neuronal cell populations of the aging brain. Cell Rep. 2018;25:2447–2456.e4. doi: 10.1016/j.celrep.2018.10.094. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib50] 50.Hughes C.E., Coody T.K., Jeong M.Y., Berg J.A., Winge D.R., Hughes A.L. Cysteine toxicity drives age-related mitochondrial decline by altering iron homeostasis. Cell. 2020;180:296–310.e18. doi: 10.1016/j.cell.2019.12.035. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib51] 51.Weber R.A., Yen F.S., Nicholson S.P.V., Alwaseem H., Bayraktar E.C., Alam M., Timson R.C., La K., Abu-Remaileh M., Molina H., Birsoy K. Maintaining iron homeostasis is the key role of lysosomal acidity for cell proliferation. Mol. Cell. 2020;77:645–655.e7. doi: 10.1016/j.molcel.2020.01.003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib52] 52.Alhebshi A., Sideri T.C., Holland S.L., Avery S.V. The essential iron-sulfur protein Rli1 is an important target accounting for inhibition of cell growth by reactive oxygen species. Mol. Biol. Cell. 2012;23:3582–3590. doi: 10.1091/mbc.E12-05-0413. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib53] 53.Manjunath H., Zhang H., Rehfeld F., Han J., Chang T.C., Mendell J.T. Suppression of ribosomal pausing by eIF5A is necessary to maintain the fidelity of start codon selection. Cell Rep. 2019;29:3134–3146.e6. doi: 10.1016/j.celrep.2019.10.129. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

CRISPR screening reveals a dependency on ribosome recycling for efficient SARS-CoV-2 programmed ribosomal frameshifting and viral replication

Frederick Rehfeld

Jennifer L Eitson

Maikke B Ohlson

Tsung-Cheng Chang

John W Schoggins

Joshua T Mendell

Abstract

Graphical abstract

Introduction

Figure 1.

Results

A genome-wide CRISPR-Cas9 screen for regulators of SARS-CoV-2 frameshifting

Host factors that inhibit SARS-CoV-2 frameshifting

Figure 2.

Ribosome recycling is required for efficient SARS-CoV-2 frameshifting

Figure 3.

SARS-CoV-2 stop codon position confers dependency of frameshifting on ribosome recycling

Figure 4.

Reduced ribosomal load abolishes the dependency of frameshifting on ribosome recycling

Figure 5.

Ribosome recycling is required for SARS-CoV-2 viral replication and frameshifting

Figure 6.

Discussion

Limitations of the study

STAR★Methods

Key resources table

Resource availability

Lead contact

Materials availability

Experimental model and subject details

Cell lines

Method details

Generation of SARS-CoV-2 PRF reporter cell lines

CRISPR-Cas9 screens

CRISPR-Cas9 mediated gene knockout

Flow cytometry analysis

Protein isolation and immunoblotting

Dual-luciferase frameshifting assays

In vitro transcription and transfection of dual-luciferase mRNAs

SARS-CoV-2 virus production and infection

RNA isolation and qRT-PCR

Quantification and statistical analysis

Statistical analysis

Acknowledgments

Author contributions

Declaration of interests

Inclusion and diversity

Footnotes

Supplemental information

Data and code availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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