Chen et al. Cell Death Discovery (2021) 7:114
https://doi.org/10.1038/s41420-021-00487-z
Cell Death Discovery
ARTICLE Open Access
Type-I interferon signatures in SARS-CoV-2 infected
Huh7 cells
Xi Chen
1
, Elisa Saccon
1
,K.Sofia Appelberg
2
, Flora Mikaeloff
1
, Jimmy Esneider Rodriguez
3
, Beatriz Sá Vinhas
1
,
Teresa Frisan
4
, Ákos Végvári
3
,AliMirazimi
1,2
, Ujjwal Neogi
1,5
and Soham Gupta
1
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) has
caused a global health emergency. A key feature of COVID-19 is dysregulated interferon-response. Type-I interferon
(IFN-I) is one of the earliest antiviral innate immune responses following viral infection and plays a significant role in
the pathogenesis of SARS-CoV-2. In this study, using a proteomics-based approach, we identi fied that SARS-CoV-2
infection induces delayed and dysregulated IFN-I signaling in Huh7 cells. We demonstrate that SARS-CoV-2 is able to
inhibit RIG-I mediated IFN-β production. Our results also confirm the recent findings that IFN-I pretreatment is able to
reduce the susceptibility of Huh7 cells to SARS-CoV-2, but not post-treatment. Moreover , senescent Huh7 cells, in spit e
of showing accentuated IFN-I response were more susceptible to SARS-CoV-2 infection, and the virus effectively
inhibited IFIT1 in thes e cells. Finally, proteomic comparis on between SARS-CoV-2, SARS-CoV, and MERS-CoV revealed a
distinct differential regulatory signature of interferon-related proteins emphasizing that therapeutic strategies based
on observations in SARS-CoV and MERS-CoV should be used with caution. Our findings provide a better understanding
of SARS-CoV-2 regulation of cellular interferon response and a perspective on its use as a treatment. Investigation of
different interferon-stimulated genes and their role in the inhibition of SARS-CoV-2 pathogenesis may direct novel
antiviral strategies.
Introduction
The novel severe acute respiratory syndrome cor-
onavirus 2 (SARS-CoV-2) caused a major ongoing pan-
demic with more than a million deaths worldwide by the
end of 2020
1
. SARS-CoV-2 shares similar clinical features
to two other well-known coronavirus infections, namely
SARS-CoV and MERS-CoV, but it presents a lower case
fatality compared to them
2,3
. However, the most severe
forms of coronavirus diseases are often associated with a
dysregulated type-I interferon (IFN-I) response
4
.
IFN-I response, majorly IFN-α and IFN-β, is one of the
first lines of defense against viruses
5
. The early activation
of IFN responses against coronaviruses is initiated by
recognition of viral products by the host pattern recog-
nition receptors like Toll-like receptors (TLRs) and RIG-
I-like receptors (RLRs). RLRs can recognize the viral RNA
that promotes their oligomerization and subsequent
activation of a signaling cascade leading to the production
of IFNα and IFNβ
6
. Through autocrine and paracrine
signaling, the secreted IFN binds to IFN-α/β membrane
receptors, activating the JAK-STAT signaling cascade that
triggers the transcription of several interferon-stimulated
genes (ISGs) with diverse antiviral properties
7
. Cor-
onaviruses have evolved mechanisms to evade the host ’s
antiviral immune response. Several structural and non-
structural proteins in SARS-CoV
8
, in MERS-CoV
8,9
, and
in SARS-CoV-2
10,11
have been shown to be strong IFN-
antagonists. The dynamics of the IFN response varies
between these three coronaviruses
12–14
. Distinct virus-
specific patterns in host cell response were also noted in
transcriptomics analysis
15
. Thus, a deeper understanding
of the SARS-CoV-2 mediated regulation of IFN response
© The Author(s) 2021
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Correspondence: Soham Gupta (soham.gupta@ki.se)
1
Division of Clinical Microbiology, Department of Laboratory Medicine,
Karolinska Institutet, ANA Futura, Campus Flemingsberg, Stockholm, Sweden
2
Public Health Agency of Sweden, Solna, Sweden
Full list of author information is available at the end of the article
These authors contributed equally: Xi Chen, Elisa Saccon
Edited by Chiara Agrati
Official journal of the Cell Death Differentiation Association
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is necessary to develop rationale and novel therapeutic
approaches for SARS-CoV-2.
In this study, we characterized the SARS-CoV-2 medi-
ated dysregulation of IFN-signaling in Huh7 infected cells
using quantitative proteomics. We show a delayed acti-
vation of IFN-signaling with the ability of the virus to
evade RIG-I mediated IFN-signaling during early infec-
tion. In line with recent studies, susceptibility of Huh7
cells to SARS-CoV-2 decreased upon IFN-pretreatment,
but not post-treatment. We also determined the IFN-
signaling response pattern of SARS-CoV and MERS-CoV
infection in Huh7 cells using proteomics and show a
distinction compared to SARS-CoV-2. Together, the
results provide a perspective of immune regulation by
coronaviruses.
Results
Quantitative proteomics and transcriptomics of SARS-CoV-
2 infected Huh7 cells identifies dysregulation in IFN-I
signaling pathways
To understand the modulation of IFN responses fol-
lowing SARS-CoV-2 infection, we reused the proteomics
and transcriptomics datasets from our earlier study
16
.We
first analyzed the quantitative proteomics data on Huh7
cells that were either mock-infected or infected with
SARS-CoV-2 at a multiplicity of infection (MOI) of 1,
over a period of 24 and 48 h post infection (hpi). Genes
associated with the interferon response, including the
IFN-α/β signaling (Pathway:R-HSA-909733), IFN-γ sig-
naling (Pathway:R-HSA-877300), and the antiviral
mechanism by ISGs (Pathway:R-HSA-1169410) were
extracted from the data. For mock-infected, we con-
sidered the data for two replicates as the third one was a
major outlier as shown in the PCA plot (Fig. S1). No
major changes were observed in the ISGs at 24 hpi and
significant modulation was only observed at 48 hpi after
infection as represented in the heatmap (Fig. 1A). Of the
94 proteins studied, a number of proteins showed a sig-
nificant reduction in abundance (n = 20), while a major
cluster of proteins showed an increase (n = 26) (LIMMA,
false-discovery rate (FDR) < 0.05). The log2fold change of
the significantly regulated genes is represented as a vol-
cano plot (Fig. 1B). The protein-protein interaction net-
work of the significantly changed genes showed two
definite clusters: cluster-1 involved proteins associated
with the RIG-I/DDX58 and IFN-I signaling, while cluster-
2 consisted of transporter proteins belonging to the
components of nucleoporin complex and karyopherin
family (Fig. 1C).
We also looked into the IFN-signaling genes in the
transcriptomics dataset and observed no major changes in
the differential expression of the transcripts related to this
pathway except for EIF4A2, STAT2, TRIM10 (upregu-
lated), and FLNA, JAK1, GBP2, MT2A, TRIM26
(downregulated) at 48 hpi (Fig. 2A). Of the genes corre-
sponding to the proteins that were altered in the pathway
(Fig. 2B) only EIF4A2, STAT2, JAK1, GBP2, and FLNA
showed transcript levels correlating with protein expres-
sion (Fig. 2C).
SARS-CoV-2 induces delayed and low-level activation of
RIG-I signaling and inhibits IFN-β in Huh7 cells
In our proteomics data, we observed a delayed activa-
tion of RIG-I and dysregulation of ISGs. RIG-I, a key
cytosolic receptor is responsible for the activation of IFN-
β (Fig. 3A). We next studied the effect of SARS-CoV-2 in
the induction of IFN-β. We did not observe any sig-
nificant changes in the levels of IFN-β specific messenger
RNAs (mRNAs) in SARS-CoV-2 infected Huh7 cells both
at 24 and 48 hpi, with a marginal increase with MOI 0.1 at
48 hpi (Fig. 3B). This effect was concomitant with a
marginal suppression of RIG-I and MDA-5 protein
expression at 24hpi and an observable increase at 48 hpi
detected in western blots probed with specific antibodies
(Fig. 3C, D). The Western blot data were in line with our
proteomics data.
Since we did not observ e any IFN-β induction or RIG-I
activation at 24 hpi, we next investigated whether SARS-
CoV-2 is abl e to inhibit IFN -β activation in Huh7 cells.
To determine this Huh7 cells were either mock-infected
or infected with SARS-CoV- 2 at MOI 0.1, followed by
IFN-β induction by treating with RIG-I agonists, aci-
tretin or polyI:C for 24 h. Treatment with acitretin or
polyI:C post infection did not inhibit the production of
the virus (Fig. 3E, G). SARS-CoV-2 was able to efficiently
inhibit the IFN-β production i n the RIG-I activated cells
(Fig. 3F, H).
Effect of SARS-CoV-2 on ISGs
IFN-β can stimulate the expression of several ISGs with
antiviral properties using JAK-STAT signaling pathway
(Fig. 4A). Similar to our transcriptomics data, qPCR
analysis to detect IFIT1, RIG-I (DDX58), and MX2
expression in SARS-CoV-2 infected Huh7 cells did not
show any significant changes compared to uninfected cells
(Fig. 4B). On contrary, our proteomics data showed an
increase in the protein level of several ISGs, including
ISG15 at 48 hpi. ISG15 can behave as an antiviral cytokine
in its free form and also can conjugate to diverse cellular
and viral proteins and regulate their functions
17,18
. The
mRNA levels of ISG15 in SARS-CoV-2 infected Huh7
cells at 24 and 48 hpi did not change significantly (Fig.
4C). However, at the protein level, it was interesting to
note an observable decrease in the conjugated ISG15 at 24
hpi and a marked increase in host-protein ISGylation at
48 hpi (Fig. 4D, E) in a dose-dependent manner, sug-
gesting the virus can modulate protein ISGylation to alter
the cellular environment
11
.
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Fig. 1 SARS-CoV-2 induced a delayed and dysregulated IFN signaling response identified in proteomics data. A Heatmap of IFN-stimulated
proteins before infection and at 24 and 48 hpi. Data were quantile normalized and Z-score transformed. Lower values are represented in yellow and
higher values in purple. Significant differentially expressed proteins between time points are indicated in blue if downregulated and in red if
upregulated. B Volcano plots of proteins with differential abundance between Mock and Huh7 cell 48 h after SARS-CoV-2 infection. Upregulated
proteins are represented in red while proteins downregulated are represented in green. FDR < 0.05. C Cytoscape network of differentially abundant
IFN-stimulated proteins. Proteins are represented as circles. Gradient color was applied on proteins depending on fold change (low = green to high
= red). The size of the circle is proportional to the fold change.
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Fig. 2 (See legend on next page.)
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SARS-CoV-2 is inhibited by IFN pretreatment
ISGs can also be stimulated in experimental models by
external treatment with IFNs. In order to evaluate the
susceptibility of SARS-CoV-2 to IFN-I, we either pre-
sensitized cells (16 h) with IFN-α2a (5000 IU) and IFN-β
(100 IU) or treated the cells with the same concentrations
of IFNs starting 1 hpi and continued for 24 h. Huh7 cells
were infected with SARS-CoV-2 at MOI 0.1 and at 24 hpi
the supernatant was collected to determine the virus
production in the presence or absence of different IFN-I
treatments. As shown in Fig. 4F, IFN pre-sensitization
lead to a significant reduction in SARS-CoV-2 production
in the supernatant as compared to levels in supernatant
from untreated cells at 24 hpi. However, IFN-I treatment
after infection did not suppress virus production (Fig. 4H).
This observation suggests firstly that the presence of a
high level of IFN-response can suppress the incoming
virus and secondly that the virus has also developed
measures to counteract these responses when it has
already established infection. Then, we further looked into
the effect of IFN-I treatment and infection in transcrip-
tional activation of a few of the ISGs that were modulated
by SARS-CoV-2 infection. For this, we selected MX2,
IFIT1, and ISG15. While SARS-CoV-2 suppressed MX2
mRNA in untreated cells, MX2 did not show any activa-
tion following IFN-treatment (not shown). Both ISG15
and IFIT1 were significantly induced following IFN-I
treatment, however, SARS-CoV-2 did not cause any sig-
nificant alterations to the mRNA levels (Fig. 4G, I).
Senescent Huh7 cells stimulate IFN-I response but
promote virus infectivity
Elderly people are more vulnerable to SARS-CoV-2
infection
19
and cellular senescence is postulated as a
factor for increased infection. Cellular senescence has
been observed to play a different role in either promoting
infection for some viruses or inhibiting infection for
others. To this end, we aimed to examine the suscept-
ibility of senescent Huh7 cells to SARS-CoV-2 and asso-
ciated IFN-I response. To induce cellular senescence
Huh7 cells were treated with 0.5 μM of etoposide for
6 days followed by 2 days without any treatment and then
infected with SARS-CoV-2 for 1 h and cells and
supernatants were harvested 24 hpi. Etoposide treatment
resulted in massive cell death and surviving cells were
large in size. Cellular senescence was determined by
detecting p21 mRNA levels (Fig. 5B). Senescent Huh7
cells showed a significant increase in SARS-CoV-2 pro-
duction in cell supernatant of senescent Huh7 cells
compared to the untreated control cells (Fig. 5A). We
next investigated the IFN-response in senescence-induced
and non-induced cells by detecting mRNA transcripts of
IFN-β and ISGs such as ISG15, IFIT1, MX2, and RIG-I.
Cellular senescence induced an increase in the IFN-
response with a significant increase in the levels of IFN-β
and other ISGs tested (Fig. 5B). SARS-CoV-2 failed to
significantly alter the levels of any tested genes except for
IFIT1, where a significant decrease in the mRNA levels
was noted upon infection (Fig. 5B). To determine if the
enhanced infectivity of senescent cells is specific to Huh7,
we tried to replicate the same experiment in Caco2 cells.
However, Caco2 cells were more resistant to 0.5 μM
etoposide treatment and did not show observable induc-
tion of senescence determined by qPCR of the p21 gene
(Fig. S2B). Most interestingly, in contrast to Huh7, even a
very low-level induction of p21 was sufficient to sig-
nificantly reduce SARS-CoV-2 susceptibility (Fig. S2A)
and among the ISGs IFIT1 showed an observable increase
upon infection (Fig. S2B). The results suggest that there is
a cell-type-specific regulation of SARS-CoV-2 and the
importance of IFIT1 as an anti-SARS-CoV-2 ISGs.
Global proteomic response to SARS-CoV-2 relative to
SARS-CoV and MERS-CoV in Huh7 cells
To explore the diff erences in pathogenicity of SARS-
CoV-2 in comparison with its predecessor human
pathogenic coronaviruses, we inf ected Huh7 cells with
SARS-CoV and MERS-CoV at MOI 1 and measured the
global proteomic changes by performing quantitative
proteomics. MERS-CoV was observed to be highly cyto-
pathic and by 48 hpi all the cells were dead restricting our
analysis t o 24 hpi, while SARS-CoV showed a slower
cytopathogenicity, and infected cells were collected both
at 24 and 48 hpi. Quanti tative proteomics was performed
utilizing a TMT-labeling strategy of mock-infected and
infected cells in triplicate as previously described by us
16
.
(see figure on previous page)
Fig. 2 SARS-CoV-2 induced transcriptional changes in the IFN-signaling genes in transcriptomics data. A Heatmap of IFN-stimulated
transcripts before infection and at 24 and 48 hpi. Data were log2 normalized and Z-score transformed. Lower values are represented in yellow and
higher values in purple. Significant differentially expressed genes between time points are indicated in blue if downregulated and in red if
upregulated. B The scheme graph of the type I interferon signaling pathways created with BioRender, in which the regulated genes expression level
trend is noted. The significantly changed proteins observed in the proteomics data are denoted by green arrows or letters (downregulated) or red
arrows or letters (upregulated). C Dot plot for each transcript that was detected as significantly altered in proteomics. For each gene, the scaled
values in triplicates are represented in mock, 24 and 48 hpi and linked by the light red line, the average value is displayed in red. The name of the
genes is indicated in a colored box based on the proteomics data. The genes corresponding to increased protein levels are in red boxes and to
decreased protein levels in green boxes.
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