Dysregulation in mTOR/HIF-1 signaling identified by proteo-transcriptomics of SARS-CoV-2 infected cells

Abstract

How Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remain largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected HuH7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro . Western blot validation of the downstream effector molecules of these pathways revealed a significant reduction in activated S6K1 and 4E-BP1 at 72 hours post infection. Unlike other human respiratory viruses, we found a significant inhibition of HIF-1α through the entire time course of the infection, suggesting a crosstalk between the SARS-CoV-2 and the mTOR/HIF-1 signaling. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe COVID-19 patients.

SciScore for 10.1101/2020.04.30.070383: (What is this?)

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Antibodies
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Subsequent antibody detection was performed at 4°C over-night or 2h at room temperature for β-Actin.	β-Actin suggested: None
Membranes were washed using 0.1% PBSt and secondary antibody incubated 1h at room-temperature using Dako Polyconal Goat Anti-Rabbit or Anti-Mouse Immunoglobulins/HRP (Aglient Technologies, Santa Clara, CA, USA) washed using 0.1% PBSt and visualized using ECL or ECL Select (GE Healthcare, Chicago, IL, USA) on ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA).	Anti-Rabbit suggested: None Anti-Mouse suggested: None
Experimental Models: Cell Lines
Sentences	Resources
, …

SciScore for 10.1101/2020.04.30.070383: (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

Antibodies
Sentences	Resources
Subsequent antibody detection was performed at 4°C over-night or 2h at room temperature for β-Actin.	β-Actin suggested: None
Membranes were washed using 0.1% PBSt and secondary antibody incubated 1h at room-temperature using Dako Polyconal Goat Anti-Rabbit or Anti-Mouse Immunoglobulins/HRP (Aglient Technologies, Santa Clara, CA, USA) washed using 0.1% PBSt and visualized using ECL or ECL Select (GE Healthcare, Chicago, IL, USA) on ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA).	Anti-Rabbit suggested: None Anti-Mouse suggested: None
Experimental Models: Cell Lines
Sentences	Resources
, Philipps-Universität Marburg, Marburg, Germany fully matching the STR reference profile of HuH-7.	HuH-7 suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
27 SARS-CoV-2 infection of Huh7 cells: Huh7 cells were plated in 6 well plates (2,5×105 cells/well) in DMEM (Thermo Fisher Scientific, US) supplemented with 10% heat-inactivated FBS (Thermo Fisher, US).	Huh7 suggested: None
Software and Algorithms
Sentences	Resources
The raw sequence data were first subjected to quality check using FastQC tool kit version 0.11.8	FastQC suggested: (FastQC, RRID:SCR_014583)
Illumina adapter sequences and low-quality bases were removed from the raw reads using the tool Trim Galore version 0.6.1.	Trim Galore suggested: (Trim Galore, RRID:SCR_011847)
Short read aligner STAR version 2.7.3a was used for the alignment.	STAR suggested: (STAR, RRID:SCR_015899)
Normalization factors were calculated using the R package edgeR 28 from read counts matrix to scale the raw library sizes.	edgeR suggested: (edgeR, RRID:SCR_012802)
Peptide identification and preprocessing: The raw files were imported to Proteome Discoverer v2.4 (Thermo Scientific) and searched against the Homo sapiens SwissProt (2020_01 release with 20,595 entries) and the pre-leased SARS-CoV-2 UniProt (completed with 14 SARS-CoV2 sequences of COVID-19 UniProtKB release 2020_04_06) protein databases with Mascot v 2.5.1 search engine (MatrixScience Ltd., UK).	Proteome Discoverer suggested: (Proteome Discoverer, RRID:SCR_014477) UniProtKB suggested: (UniProtKB, RRID:SCR_004426) Mascot suggested: (Mascot, RRID:SCR_014322)
Moderated paired-t-test using limma with adjustment for replicates was applied.	limma suggested: (LIMMA, RRID:SCR_010943)
Gene set enrichment analysis was performed on each community (n > 30) through Enrichr for KEGG Human 2019 where backgrounds were selected based on the node number of each network.	KEGG suggested: (KEGG, RRID:SCR_012773)
All analyses were performed in Python 3.7.	Python suggested: (IPython, RRID:SCR_001658)
Protein interaction network is created using Cytoscape version 3.6.1 35	Cytoscape suggested: (Cytoscape, RRID:SCR_003032)
Scatter plots produced using ggplot2 represent the bivariate relationship between proteins and time.	ggplot2 suggested: (ggplot2, RRID:SCR_014601)
Membranes were washed using 0.1% PBSt and secondary antibody incubated 1h at room-temperature using Dako Polyconal Goat Anti-Rabbit or Anti-Mouse Immunoglobulins/HRP (Aglient Technologies, Santa Clara, CA, USA) washed using 0.1% PBSt and visualized using ECL or ECL Select (GE Healthcare, Chicago, IL, USA) on ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA).	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)

Results from OddPub: Thank you for sharing your code.

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

There are some limitations of our study. First, we only used the HuH7 cell line but the SARS-CoV-2 can be also cultured in Vero E6, Vero CCL81, or HEK-293T cells. Whereas SARS-CoV-2 exerts rapid cytopathic effects in Vero E6 cells (within 24hpi), viral replication is slower in HuH7 and HEK293T cells, allowing to study host-cellular responses for 3 days after viral challenge 25. Moreover, an earlier study used HuH7 cells to identify the transcriptomics signature of early cellular responses to SARS-CoV and HCoV-229E infections 26. However, the observed effects could be cell type-specific and thus, we are currently assessing the effect of SARS-CoV-2 infection in other cells lines. Moreover, SARS-CoV-2 has a propensity to mutate and our experiments were performed with only one virus strain isolated from a Swedish patient. Of note, our virus isolate has close sequence similarity to the initial strains circulating in Wuhan, China. In conclusions, we observed marked alterations of mTOR/HIF-1 signaling at the proteo-transcriptomic levels in response to SARS-CoV-2 infections, though the exact mechanistic role of these changes remains to be elucidated. Targeting mTOR/HIF-1 signaling could be an attractive candidate as a potential therapy, alone or preferably combined with antivirals, for the management of COVID-19 patients. Moreover, mTOR inhibition could be used to reduce the cytokine storm syndrome in severe cases of COVID-19.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 20. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

Results from rtransparent:

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Dysregulation in mTOR/HIF-1 signaling identified by proteo-transcriptomics of SARS-CoV-2 infected cells

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