(PDF) 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 (2021) | Marica Grossegesse (2022)

(PDF) 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 (2021) | Marica Grossegesse (1)

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(which was not certified by peer review) is the author/funder.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

(PDF) 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 (2021) | Marica Grossegesse (2)

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

(PDF) 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 (2021) | Marica Grossegesse (3)

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

(PDF) 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 (2021) | Marica Grossegesse (4)

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

(PDF) 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 (2021) | Marica Grossegesse (5)

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

FAQs

What is the SARS-CoV-2 protein? ›

In addition to these genes, SARS-CoV-2 contains genes that encode four structural proteins which are involved in infectious virus assembly: (S)pike, (E)nvelope), (M)embrane, and (N)ucleocapsid) proteins.

What is the anatomy of SARS-CoV-2? ›

In most SARS-CoV-2 sequences, a furin protease cleavage site is inserted between the S1 and S2 subunits, and mutation of the cleavage site attenuates disease in animal models (Johnson et al., 2020). The S1 fragment, at the membrane distal tip of S, includes an N-terminal domain (NTD) and receptor binding domain (RBD).

Does SARS-CoV-2 infect alveolar cells? ›

Infection of the alveolar epithelium: potential for structural and functional damage and severe illness. SARS-CoV-2 infection of the alveolar epithelium (A) SARS-CoV-2 enters the alveoli, infects ACE2 expressing alveolar type II cells and replicates.

What cells are affected by SARS-CoV-2? ›

The inhaled virus SARS-CoV-2 likely binds to epithelial cells in the nasal cavity and starts replicating. ACE2 is the main receptor for both SARS-CoV2 and SARS-CoV [2, 3]. In vitro data with SARS-CoV indicate that the ciliated cells are primary cells infected in the conducting airways [4].

Can spike proteins be removed? ›

A recent in-vitro study demonstrated near complete the removal of the SARSCoV2 spike protein by a mini-Hemopurifier within 30 min (Figure 1).

What type of virus is Covid? ›

COVID-19 is caused by a virus called SARS-CoV-2. It is part of the coronavirus family, which include common viruses that cause a variety of diseases from head or chest colds to more severe (but more rare) diseases like severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS).

Is SARS-CoV-2 an RNA or DNA virus? ›

Hence, it was termed SARS-CoV-2 by experts of the International Committee on Taxonomy of Viruses. The single-stranded RNA genome of SARS-CoV-2 contains 29891 nucleotides, encoding for 9860 amino acids.

How do you get SARS-CoV-2? ›

SARS-CoV-2 was first known to infect people in 2019. The virus is thought to spread from person to person through droplets released when an infected person coughs, sneezes, or talks. It may also be spread by touching a surface with the virus on it and then touching one's mouth, nose, or eyes, but this is less common.

How does Covid spike protein work? ›

It enters the muscle cells and uses the cells' machinery to produce a harmless piece of what is called a spike protein. The spike protein is found on the surface of the virus that causes COVID-19.

How does SARS-CoV-2 infect the respiratory system? ›

These cells mediate mucociliary clearance, which is the primary innate defense of the lungs. SARS-CoV-2 infects the ACE2-expressing ciliated cells, replicates, and then infects the neighboring cells upon apical release, which occurs within six hours from the initial infection.

Do lung cells regenerate? ›

Normally lungs have the ability to repair and regenerate as they are constantly exposed to pollution and microbes from the external environment.

Which cell type reduces infection? ›

NK cells are derived from the bone marrow and are present in relatively low numbers in the bloodstream and in tissues. They are important in defending against viruses and possibly preventing cancer as well. NK cells kill virus-infected cells by injecting them with a killer potion of chemicals called cytotoxic granules.

Top Articles

You might also like

Latest Posts

Article information

Author: Kieth Sipes

Last Updated: 07/30/2022

Views: 6098

Rating: 4.7 / 5 (47 voted)

Reviews: 86% of readers found this page helpful

Author information

Name: Kieth Sipes

Birthday: 2001-04-14

Address: Suite 492 62479 Champlin Loop, South Catrice, MS 57271

Phone: +9663362133320

Job: District Sales Analyst

Hobby: Digital arts, Dance, Ghost hunting, Worldbuilding, Kayaking, Table tennis, 3D printing

Introduction: My name is Kieth Sipes, I am a zany, rich, courageous, powerful, faithful, jolly, excited person who loves writing and wants to share my knowledge and understanding with you.