Dynamics of SARS-CoV-2 host cell interactions inferred from transcriptome analyses
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Abstract
The worldwide spread of severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) caused an urgent need for an in-depth understanding of interactions between the virus and its host. Here, we dissected the dynamics of virus replication and the host cell transcriptional response to SARS-CoV-2 infection at a systems level by combining time-resolved RNA sequencing with mathematical modeling. We observed an immediate transcriptional activation of inflammatory pathways linked to the anti-viral response followed by increased expression of genes involved in ribosome and mitochondria function, thus hinting at rapid alterations in protein production and cellular energy supply. At later stages, metabolic processes, in particular those depending on cytochrome P450 enzymes, were downregulated. To gain a deeper understanding of the underlying transcriptional dynamics, we developed an ODE model of SARS-CoV-2 infection and replication. Iterative model reduction and refinement revealed that a negative feedback from virus proteins on the expression of anti-viral response genes was essential to explain our experimental dataset. Our study provides insights into SARS-CoV-2 virus-host interaction dynamics and facilitates the identification of druggable host pathways supporting virus replication.
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SciScore for 10.1101/2021.07.04.450986: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: Cells have been tested negative for mycoplasma infection (MycoAlert Plus; Lonza, Basel, Switzerland).
Authentication: Microscopy: To analyze SARS-CoV-2 N protein expression in Caco-2 cells by immunofluorescence, microscopic images were taken at 0, 4, 24 and 48 hpi using a Nikon Eclipse Ti-S fluorescence microscope (Nikon, Tokio, Japan).Table 2: Resources
Antibodies Sentences Resources Reagents: We used antibodies to detect the SARS-CoV-2 nucleoprotein (mouse monoclonal; Sino Biologicals, Hong Kong, China) and GFP (mouse monoclonal; … SciScore for 10.1101/2021.07.04.450986: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: Cells have been tested negative for mycoplasma infection (MycoAlert Plus; Lonza, Basel, Switzerland).
Authentication: Microscopy: To analyze SARS-CoV-2 N protein expression in Caco-2 cells by immunofluorescence, microscopic images were taken at 0, 4, 24 and 48 hpi using a Nikon Eclipse Ti-S fluorescence microscope (Nikon, Tokio, Japan).Table 2: Resources
Antibodies Sentences Resources Reagents: We used antibodies to detect the SARS-CoV-2 nucleoprotein (mouse monoclonal; Sino Biologicals, Hong Kong, China) and GFP (mouse monoclonal; Roche Diagnostics). GFPsuggested: NoneFor microscopy, the secondary antibody Alexa Fluor goat anti-mouse 568 (Thermo Fisher Scientific, Waltham, MA, USA) was applied. anti-mouse 568suggested: NoneA Horseradish peroxidase-conjugated anti-mouse antibody (Southern Biotech, Birmingham, AL, USA) was used for immunoblotting. anti-mousesuggested: NoneSecondary antibody (anti-mouse CW800) and DNA dye Draq5 (Abcam, Cambridge, UK) were diluted 1/10,000 in blocking buffer and incubated for 1 h at RT. anti-mouse CW800suggested: NoneDraq5suggested: (Biostatus Cat# DR50050, RRID:AB_2314341)Experimental Models: Cell Lines Sentences Resources Caco-2, Vero E6 and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (Biochrom, Berlin, Germany), penicillin and streptomycin (100μg/ml; Invitrogen). Caco-2suggested: NoneVero E6suggested: NoneHEK293T cells were transfected with X-tremeGENE HP (Roche Diagnostics, Rotkreuz, Switzerland) following the manufacturer’s instructions. HEK293Tsuggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)Recombinant DNA Sentences Resources To generate a construct for co-expressing mCherry and SARS-CoV-2 main protease (3CLpro), a fragment encoding 3CLpro was first obtained as a double-stranded DNA fragment (gBlock; IDT, San José, CA, USA) and cloned into pcDNA3.1(−) via unique NheI/NotI restriction sites. pcDNA3.1suggested: RRID:Addgene_79663)The 3CLpro encoding vector was then linearized 5’ of the 3CLpro start codon via NheI and the mCherry-P2A fragment was inserted via Gibson Assembly, hence yielding a vector encoding mCherry-P2A-3CLpro. mCherry-P2A-3CLprosuggested: NoneSoftware and Algorithms Sentences Resources Paired-end sequencing of 2×150bp was performed at GeneWiz Inc. using an Illumina NovaSeq 6000 instrument (Illumina, San Diego CA, USA). GeneWizsuggested: (GENEWIZ, RRID:SCR_003177)Live-cell experiments were performed on a Nikon Ti inverted microscope, equipped with a CSU-22 Yokogawa confocal spinning disc slider (Yokogawa Electric Corporation, Tokyo, Japan), a 60× Plan Apo NA 1.4 objective lens (Nikon), a Hamamatsu C9100-02 EMCCD camera (Hamamatsu Photonics, Hamamatsu, Japan), and the Volocity software (PerkinElmer; Waltham, MA, USA). Volocitysuggested: (Volocity 3D Image Analysis Software, RRID:SCR_002668)Microscopic images were evaluated with ImageJ software (NIH, Bethesda, MA, USA). ImageJsuggested: (ImageJ, RRID:SCR_003070)For read alignment, the STAR software was used with default settings (Dobin et al, 2013). STARsuggested: (STAR, RRID:SCR_004463)Subsequently, BAM files were split by their reference using the SAMtools software suite (Li et al, 2009) and counted separately using the featureCounts function from the Subread package (Liao et al, 2019). SAMtoolssuggested: (SAMTOOLS, RRID:SCR_002105)featureCountssuggested: (featureCounts, RRID:SCR_012919)Overrepresented GO terms were inferred using the ‘goana’ and ‘topGO’ function from the limma package (Ritchie et al, 2015). limmasuggested: (LIMMA, RRID:SCR_010943)The MATLAB toolbox PottersWheel was used for model fitting (Maiwald & Timmer, 2008). MATLABsuggested: (MATLAB, RRID:SCR_001622)To this end, the following six model simplifications were tested: (1) description of A turnover by one turnover parameter instead of separate parameters for synthesis and degradation of A (Variant 4.0.1), (2) description of the synthesis of A by mass-action instead of Michaelis-Menten (MM) kinetics (Variant 4.0.2), (3) mass-action instead of MM-kinetics for describing synthesis of P (Variant 4.0.3), (4) mass-action kinetics for describing synthesis and degradation of A by single turnover parameter (Variant 4.0.4), (5) single parameter for describing turnover of A and mass-action instead of MM-kinetics for synthesis of P (Variant 4.0.5), (6) mass-action kinetics for synthesis of A as well as P. Variantsuggested: (VARIANT, RRID:SCR_005194)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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