Genes with 5′ terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 Nsp1 protein

Abstract

Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a nonstructural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation levels. We discover that a functionally coherent subset of human genes is preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5′ terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5′ UTRs from TOP transcripts can drive preferential expression in the presence of Nsp1. Finally, we found that LARP1, a key effector protein in the mTOR pathway, may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine-tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.

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Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
For the MeTAFlow Assay, HEK293T cells were plated at a density of 3×105 cells in a 6-well plate.	HEK293T suggested: None
Among the 166 genes with relatively increased translation efficiency upon Nsp1 expression, 144 were quantified in QTI-Seq experiments in HEK293 cells.	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
Software and Algorithms
Sentences	Resources
Data was later analyzed by Flowjo 10.6.1.	Flowjo suggested: (FlowJo, RRID:SCR_008520)
The remaining reads were mapped to principal isoforms obtained from the APPRIS database (Rodriguez et al., 2018) for the human …

SciScore for 10.1101/2020.09.13.295493: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
For the MeTAFlow Assay, HEK293T cells were plated at a density of 3×105 cells in a 6-well plate.	HEK293T suggested: None
Among the 166 genes with relatively increased translation efficiency upon Nsp1 expression, 144 were quantified in QTI-Seq experiments in HEK293 cells.	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
Software and Algorithms
Sentences	Resources
Data was later analyzed by Flowjo 10.6.1.	Flowjo suggested: (FlowJo, RRID:SCR_008520)
The remaining reads were mapped to principal isoforms obtained from the APPRIS database (Rodriguez et al., 2018) for the human transcriptome.	APPRIS suggested: (APPRIS, RRID:SCR_012019)
We note that deduplication via UMI-tools is an experimental feature of RiboFlow as of this study and was not a feature of the stable release at the time of its publication.	UMI-tools suggested: (UMI-tools, RRID:SCR_017048)
Gene set enrichment analyses for gene ontology terms was carried out using FuncAssociate (http://llama.mshri.on.ca/funcassociate/) with default settings (Berriz et al., 2009).	http://llama.mshri.on.ca/funcassociate/ suggested: (FuncAssociate: The Gene Set Functionator, RRID:SCR_005768)
R packages cowplot, pheatmap, EnhancedVolcano, ggpubr, ggplot2, and reshape2 were used for analyses and plotting (Blighe et al., 2019; Kassambara, 2018; Kolde, 2012; Wickham, 2012, 2011; Wilke, 2016)	ggplot2 suggested: (ggplot2, RRID:SCR_014601)
Therefore, we determined differential CDS occupancy of ribosome footprints across transcripts using EdgeR with an adjusted p-value cutoff 5x10 −2.	EdgeR suggested: (edgeR, RRID:SCR_012802)
To generate a control set with a similar distribution of RNA expression, we used the MatchIt R package (Ho et al., 2007).	MatchIt suggested: None
Sequence feature analysis and statistical testing: Gene names from the high-TE, low-TE, and non-DE genesets were converted to Ensembl gene IDs with the Bioconductor Ensembl v79 release version 2.99.0 (Johannes Rainer, 2017).	Bioconductor suggested: (Bioconductor, RRID:SCR_006442) Ensembl suggested: (Ensembl, RRID:SCR_002344)
Region sequences were extracted using bedtools v2.29.1 (Quinlan and Hall, 2010) and nucleotide content was computed via the Bioconductor Biostrings package v2.54.0 (Pagès et al., 2017).	bedtools suggested: (BEDTools, RRID:SCR_006646) Biostrings suggested: (Biostrings, RRID:SCR_016949)
Minimum free energy (MFE) was computed via the PyPi package seqfold (https://pypi.org/project/seqfold/), which uses a nearest-neighbor dynamic programming algorithm for the minimum energy structure (Zuker and Stiegler, 1981); (Turner and Mathews, 2010).	PyPi suggested: None
To analyze experimental eCLIP data, RBP narrowpeak BED files (Van Nostrand et al., 2020) were downloaded from ENCODE project website (	ENCODE suggested: (Encode, RRID:SCR_015482)

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One caveat with the MeTAFlow method is the potential difference in sensitivity between the modalities used for detection of polypeptide synthesis and mRNA abundance namely fluorescence signal from labelled OPP and the molecular beacon, respectively. For instance, subtle changes in cellular mRNA levels might robustly increase nascent protein production, making detection of newly synthesized proteins more sensitive than changes in the cellular mRNA levels. Further, the sensitivity of the molecular beacon to detect total mRNA can be affected by differences in accessibility or length of the mRNA poly (A) tail. One caveat of using HEK293T cells as a model is that they are not the primary cell type infected by SARS-CoV-2. However, HEK293T cells are permissive to SARS-CoV-2 infection (Harcourt et al., 2020). Moreover, proteins identified in SARS-CoV-2 interactome studies using HEK293T cells were shown to have their highest expression in the lung tissue compared to others, indicating the relevance of this model for such studies (Gordon et al., 2020). MetaFlow in HEK293T cells revealed global changes in translation and mRNA abundance but did not give insight into gene-specific responses. To illuminate any potential gene-specific changes, we further analyzed Nsp1 effects on the translation and transcriptome of the host cells by ribosome profiling and RNA sequencing studies. A recent ribosome profiling study of SARS-CoV-2 infected Vero and Calu-3 cells revealed the high resolution map o...

Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from JetFighter: We did not find any issues relating to colormaps.

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Genes with 5′ terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 Nsp1 protein

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