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Deep time course proteomics of SARS-CoV and SARS-CoV-2-infected human lung epithelial cells (Calu-3) reveals strong induction of interferon-stimulated gene (ISG) expression by SARS-CoV-2 in contrast to SARS-CoV

Marica Grossegesse1, Daniel Bourquain1, Markus Neumann1, Lars Schaade1, Andreas Nitsche1, Joerg Doellinger1 •
Robert Koch Institute1
21 Apr 2021-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: In this paper, protein expression of SARS-CoV- and SARS CoV-2-infected human lung epithelial cell line Calu-3 was analyzed using data-independent acquisition mass spectrometry (DIA-MS).
Abstract: SARS-CoV and SARS-CoV-2 infections are characterized by remarkable differences, including contagiosity and case fatality rate. The underlying mechanisms are not well understood, illustrating major knowledge gaps of coronavirus biology. In this study, protein expression of SARS-CoV- and SARS-CoV-2-infected human lung epithelial cell line Calu-3 was analysed using data-independent acquisition mass spectrometry (DIA-MS). This resulted in the so far most comprehensive map of infection-related proteome-wide expression changes in human cells covering the quantification of 7478 proteins across 4 time points. Most notably, the activation of interferon type-I response was observed, which surprisingly is absent in other recent proteome studies, but is known to occur in SARS-CoV-2-infected patients. The data reveal that SARS-CoV-2 triggers interferon-stimulated gene (ISG) expression much stronger than SARS-CoV, which reflects the already described differences in interferon sensitivity. Potentially, this may be caused by the enhanced expression of viral M protein of SARS-CoV in comparison to SARS-CoV-2, which is a known inhibitor of type I interferon expression. This study expands the knowledge on the host response to SARS-CoV-2 infections on a global scale using an infection model, which seems to be well suited to analyse innate immunity.

Summary (1 min read)

Jump to: [Introduction] – [Cell culture and infection] – [Polymerase chain reaction (PCR)] and [Results]

Introduction

  • In late 2019, first cases of severe pneumonia of unknown origin were reported in Wuhan, China.
  • Shortly afterwards a new coronavirus was discovered as the causative agent and named SARS-CoV-2 and the related disease COVID-19.
  • The virus turned out to be highly contagious and caused a world-wide pandemic, which is still ongoing and has already led to the death of > 2,900,000 humans worldwide.
  • Other human lung cells lines, like A549, are only susceptible to SARS-CoV-2 infection upon overexpression of the SARS-CoV receptor ACE2 (6) which was recently found to be an interferon-stimulated gene (ISG) (7) .
  • In total, 8391 proteins were identified, 7478 of which could be reliably quantified across the experiment.

Cell culture and infection

  • Calu-3 cells (ATCC HTB-55) were cultivated in EMEM containing 10 % FCS, 2 mM L-Gln and non-essential amino acids.
  • Medium was removed and cells were infected with SARS-CoV (strain Hong Kong) or SARS-CoV-2 (hCoV-19/Italy/INMI1-isl/2020 (National Institute for Infectious Diseases, Rome, Italy, GISAID Accession EPI_ISL_410545) at an MOI of 5.
  • After one hour post infection (p.i.) cells were washed with PBS and fresh medium was added.
  • Cells were washed with PBS and prepared for proteomics as described below.
  • Additionally, triplicate mock samples per time point were taken.

Polymerase chain reaction (PCR)

  • Supernatants were extracted using the QIAamp Sample preparation for proteomics.
  • Afterwards, 200 µL of trifluoroacetic acid (TFA) (Thermo Fisher Scientific, Waltham, MA, USA) were added and cells were incubated at room temperature for 3 min.
  • For the correction of predicted peptide spectral libraries, a pooled sample was measured using gas-phase separation (8 x 100 Th) with 25 x 4 Window placement was optimised using Skyline (Version 4.2.0) (11) .
  • The single-run data were analysed using the corrected library with fixed mass tolerances of 10 ppm for MS1 and 20 ppm for MS² spectra with enabled "RT profiling" using the "robust LC (high accuracy)" quantification strategy.
  • Proteins which were not quantified in at least 2/3rd of all samples were removed, and remaining missing values were replaced from a normal distribution (width 0.3, down shift 1.8).

Results

  • Proteome analysis of SARS-CoV-and SARS-CoV-2-infected human lung epithelial cell line Calu-3 was conducted at 2, 6, 10 and 24 h p.i. including time-matched mock controls.
  • Another cluster consisting of proteins with virus-specific time-course-dependent upregulation was found to be (which was not certified by peer review) is the author/funder.
  • The low coverage of this pathway could explain at least partially the discrepancy.
  • The authors therefore hypothesize that the enhanced expression of the M protein of SARS-CoV reduces the induction of ISG expression in infected cells in comparison to SARS-CoV-2 and so contributes to the varying IFN sensitivity of both viruses.

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Figures (4)

Content maybe subject to copyright    Report

1
Deep time course proteomics of SARS-CoV- 1
and SARS-CoV-2-infected human lung 2
epithelial cells (Calu-3) reveals strong 3
induction of interferon-stimulated gene (ISG) 4
expression by SARS-CoV-2 in contrast to 5
SARS-CoV 6
Authors 7
Marica Grossegesse
1
, Daniel Bourquain
2
, Markus Neumann
1
, Lars Schaade
2
, Andreas 8
Nitsche
1
and Joerg Doellinger
1,3
* 9
10
Affiliations 11
1
Robert Koch Institute, Centre for Biological Threats and Special Pathogens: Highly 12
Pathogenic Viruses (ZBS 1) 13
2
Robert Koch Institute, Centre for Biological Threats and Special Pathogens 14
3
Robert Koch Institute, Centre for Biological Threats and Special Pathogens: 15
Proteomics and Spectroscopy (ZBS 6) 16
17
*corresponding author(s): Joerg Doellinger (Doellingerj@rki.de), phone 49-30-18754-
18
2373 19
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted April 21, 2021. ; https://doi.org/10.1101/2021.04.21.440783doi: bioRxiv preprint
2
Abstract 20
SARS-CoV and SARS-CoV-2 infections are characterized by remarkable differences, 21
including contagiosity and case fatality rate. The underlying mechanisms are not well 22
understood, illustrating major knowledge gaps of coronavirus biology. In this study, 23
protein expression of SARS-CoV- and SARS-CoV-2-infected human lung epithelial 24
cell line Calu-3 was analysed using data-independent acquisition mass spectrometry 25
(DIA-MS). This resulted in the so far most comprehensive map of infection-related 26
proteome-wide expression changes in human cells covering the quantification of 7478 27
proteins across 4 time points. Most notably, the activation of interferon type-I 28
response was observed, which surprisingly is absent in other recent proteome studies, 29
but is known to occur in SARS-CoV-2-infected patients. The data reveal that SARS-30
CoV-2 triggers interferon-stimulated gene (ISG) expression much stronger than 31
SARS-CoV, which reflects the already described differences in interferon sensitivity. 32
Potentially, this may be caused by the enhanced expression of viral M protein of 33
SARS-CoV in comparison to SARS-CoV-2, which is a known inhibitor of type I 34
interferon expression. This study expands the knowledge on the host response to 35
SARS-CoV-2 infections on a global scale using an infection model, which seems to be 36
well suited to analyse innate immunity. 37
38
KEYWORDS: SARS-CoV-2, coronavirus, interferon response, interferon-stimulated 39
gene (ISG), proteomics, data-independent-acquisition 40
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted April 21, 2021. ; https://doi.org/10.1101/2021.04.21.440783doi: bioRxiv preprint
3
Introduction 41
In late 2019, first cases of severe pneumonia of unknown origin were reported in 42
Wuhan, China. Shortly afterwards a new coronavirus was discovered as the causative 43
agent and named SARS-CoV-2 and the related disease COVID-19. The virus turned 44
out to be highly contagious and caused a world-wide pandemic, which is still ongoing 45
and has already led to the death of > 2,900,000 humans worldwide. Already in 2002, 46
another coronavirus, SARS-CoV, was discovered in China which was related to a 47
severe acute respiratory syndrome (SARS) and caused an outbreak with about 780 48
deaths (1). However, at this time the outbreak could be controlled probably due to the 49
lower contagiosity of SARS-CoV compared to SARS-CoV-2 (2). SARS-CoV and 50
SARS-CoV-2 share about 80 % of their genome sequence and protein homology 51
ranges between 40 and 94% (3, 4). Although both viruses mainly lead to respiratory 52
tract infections and can cause severe pneumonia, they are characterized by remarkable 53
differences, including contagiosity and case fatality rate (5). As the respiratory tract is 54
the first and main target of SARS-CoV and SARS-CoV-2 infections, it seems 55
conclusive to use airway epithelia cells to study differences of these two viruses. 56
However, no comparative proteomics study has been published using Calu-3 cells 57
which is the only permissive lung cell line available for SARS-CoV and SARS-CoV-2 58
(6). Other human lung cells lines, like A549, are only susceptible to SARS-CoV-2 59
infection upon overexpression of the SARS-CoV receptor ACE2 (6) which was 60
recently found to be an interferon-stimulated gene (ISG) (7). In the present study, we 61
used data-independent acquisition mass spectrometry (DIA-MS) to analyse the protein 62
expression in Calu-3 cells infected with SARS-CoV and SARS-CoV-2 over the time 63
course of 24 hours. In total, 8391 proteins were identified, 7478 of which could be 64
reliably quantified across the experiment. This results in a deep and comprehensive 65
proteome map which reflects time-dependent protein expression changes during 66
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted April 21, 2021. ; https://doi.org/10.1101/2021.04.21.440783doi: bioRxiv preprint
4
SARS-CoV and SARS-CoV-2 infections and provides deep insights into the virus-67
specific immunomodulation of human lung cells. 68
Methods 69
Cell culture and infection 70
Calu-3 cells (ATCC HTB-55) were cultivated in EMEM containing 10 % FCS, 2 mM 71
L-Gln and non-essential amino acids. A total of 5x10
5
cells per well were seeded in 6-72
well plates and incubated overnight at 37°C and 5% CO
2
in a humified atmosphere. 73
Medium was removed and cells were infected with SARS-CoV (strain Hong Kong) or 74
SARS-CoV-2 (hCoV-19/Italy/INMI1-isl/2020 (National Institute for Infectious 75
Diseases, Rome, Italy, GISAID Accession EPI_ISL_410545) at an MOI of 5. Mock 76
samples were treated with medium only. After one hour post infection (p.i.) cells were 77
washed with PBS and fresh medium was added. After 2, 6, 8, 10 and 24 h p.i. the 78
medium was removed and stored at -80 °C. Cells were washed with PBS and prepared 79
for proteomics as described below. For each time point and virus, triplicate samples 80
were taken. Additionally, triplicate mock samples per time point were taken. 81
Polymerase chain reaction (PCR) 82
The amount of SARS-CoV and SARS-CoV-2 RNA in the supernatant was analysed 83
by qPCR at 2, 6, 8, 10 and 24 h p.i.. Supernatants were extracted using the QIAamp 84
Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s 85
recommendations and eluted in 60 µL of RNase-free water. Real-time PCR targeting 86
the viral E gene was carried out as described by Michel et al. (under revision) using 87
the primers and probe published by Corman et al.(8). Quantification of viral genome 88
equivalents (GE) was done using the SARS-CoV-2 E gene WHO reference PCR 89
standard. 90
91
92
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted April 21, 2021. ; https://doi.org/10.1101/2021.04.21.440783doi: bioRxiv preprint
5
IRF-activity reporter assay 93
ACE2-A549-Dual™ cells were seeded into 96-well plates at 4x10
4
cells per well and 94
incubated overnight at 37°C and 5% CO
2
in a humified atmosphere. Cells were 95
infected with either SARS-CoV or SARS-CoV-2 at an MOI of 1.0. At 2 d p.i., 96
interferon regulatory factor (IRF)-activity was assayed using QUANTI-Luc™ 97
luminescence reagent (InvivoGen, San Diego, CA, USA) and an INFINITE 200 PRO 98
microplate reader (Tecan, Männedorf, Switzerland). 99
Sample preparation for proteomics. Samples were prepared for proteomics using 100
Sample Preparation by Easy Extraction and Digestion (SPEED) (9). At first, medium 101
was removed and cells were washed using phosphate-buffered saline. Afterwards, 200 102
µL of trifluoroacetic acid (TFA) (Thermo Fisher Scientific, Waltham, MA, USA) 103
were added and cells were incubated at room temperature for 3 min. Samples were 104
neutralized by transferring TFA to prepared reaction tubes containing 1.4 mL of 2M 105
TrisBase. After adding Tris(2-carboxyethyl)phosphine (TCEP) to a final concentration 106
of 10 mM and 2-Chloroacetamide (CAA) to a final concentration of 40 mM, samples 107
were incubated at 95°C for 5 min. 200 µL of the resulting solutions were diluted 1:5 108
with water and subsequently digested for 20 h at 37°C using 1 µg of Trypsin Gold, 109
Mass Spectrometry Grade (Promega, Fitchburg, WI, USA). Resulting peptides were 110
desalted using 200 µL StageTips packed with three Empore™ SPE Disks C18 (3M 111
Purification Inc., Lexington, USA) and concentrated using a vacuum concentrator (10, 112
11). Dried peptides were suspended in 20 µL of 0.1 % TFA and quantified by 113
measuring the absorbance at 280 nm using an Implen NP80 spectrophotometer 114
(Implen, Munich, Germany). 115
Liquid chromatography and mass spectrometry. Peptides were analysed on an 116
EASY-nanoLC 1200 (Thermo Fisher Scientific, Bremen, Germany) coupled online to 117
a Q Exactive™ HF mass spectrometer (Thermo Fisher Scientific). 1 µg of peptides 118
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted April 21, 2021. ; https://doi.org/10.1101/2021.04.21.440783doi: bioRxiv preprint
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Frequently Asked Questions (17)
Q1. How many samples were identified using a permutation-based test?

Significant protein expression differences 160 between samples were identified using an ANOVA test with a permutation-based 161 FDR of 0.05 (250 randomizations, s0 = 1). 

The 221 network revealed high connectivity among proteins related to either innate immunity 222(mainly interferon type The authorsignalling), exocytosis, including proteins related to platelet 223 degranulation (adjusted p-value: 0.01, e.g. FGB, FGG, FN1, PLG and PSAP) or 224 mitochondria-associated proteins including many members of the ribonucleoprotein 225 complex related to mtDNA expression. 

it was 282 discovered that overexpression of the M protein from SARS-CoV-2 in human cells 283 inhibits the production of type The authorand III IFNs induced by dsRNA-sensing via direct 284 interaction with RIG-I (DDX58) and reduces the induction of ISGs after Sendai virus 285 (SEV) infection and poly (I:C) transfection (33, 37). 

The expression of 2642 human proteins differed significantly between the sample 184 groups (ANOVA, FDR = 0.05), which was reduced to 261 proteins using a post-hoc 185 test (FDR = 0.05) when only proteins with at least one significant pairwise difference 186 in an infected cell with its time-matched mock control were kept. 

Peptides were ionized using 136 electrospray with a stainless steel emitter, I.D. 30 µm (Proxeon, Odense, Denmark) at 137 a spray voltage of 2.0 kV and a heated capillary temperature of 275°C. 

Out of the five 192 clusters two clusters (up-regulated 2 h p.i. and down-regulated 6 h p.i.) revealed no 193 significantly enriched GO terms but among others contained several proteins related 194 to immune response such as OAS1, INAVA and NFΚBIB. 

It must also 268 be noted that the influence of ACE2 overexpression, which was used by Stukalov et 269 al. to turn A549 into a permissive cell line, on the immune response is unknown, and 270 recently it has been shown that ACE2 is an ISG itself (7). 

The single-run data were analysed using the 149 corrected library with fixed mass tolerances of 10 ppm for MS1 and 20 ppm for MS² 150 spectra with enabled “RT profiling” using the “robust LC (high accuracy)” 151 quantification strategy. 

The authors therefore 288 hypothesize that the enhanced expression of the M protein of SARS-CoV reduces the 289 induction of ISG expression in infected cells in comparison to SARS-CoV-2 and so 290 contributes to the varying IFN sensitivity of both viruses. 

The false discovery rate was set to 0.01 for precursor 152 identifications and proteins were grouped according to their respective genes. 

The majority of viral proteins including nucleoprotein, 179 spike glycoprotein, ORF3a, ORF7a and ORF9a are expressed in equal amounts upon 180 infection with both viruses. 

The remaining infection-related proteins were grouped using hierarchical 190 clustering according to their expression profiles, and the respective main clusters were 191 analysed for enriched gene ontology terms using ClueGO (Figure 2). 

It was shown before that ISGs and IFN can be detected 260 upon infection of A549-ACE2 and Calu-3 cells with SARS-CoV-2 and that higher 261 MOIs favour interferon induction (32, 33). 

This meta-analysis 271 demonstrates that proteome coverage is still a limitation which impedes intra-study 272 cross-comparisons due to missing values. 

an interaction network of all infection-related proteins from this 220 study was constructed using STRING ((22), https://string-db.org/) 

it was 253 demonstrated that ISG expression is induced in SARS-CoV-2-infected patients in 254 general and that the increase of ISG expression, including Mx1, has a negative 255 correlation with disease severity (29). 

Gene ontology enrichment of 163 differentially expressed proteins was analysed using the ClueGO app (Version 2.5.7) 164 implemented in Cytoscape (Version 3.8.2) with a Bonferroni-adjusted p-value 165 threshold of 0.05 (12, 17, 18).