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. 2022 Dec;11(1):1717-1729.
doi: 10.1080/22221751.2022.2093133.

Modulation of viral replication, apoptosis and antiviral response by induction and mutual regulation of EGR and AP-1 family genes during coronavirus infection

Lixia Yuan  1   2 , To Sing Fung  1 , Jiawen He  1   2 , Rui Ai Chen  2 , Ding Xiang Liu  1   2
Affiliations

Modulation of viral replication, apoptosis and antiviral response by induction and mutual regulation of EGR and AP-1 family genes during coronavirus infection

Lixia Yuan et al. Emerg Microbes Infect. 2022 Dec.

Abstract

Coronaviruses have evolved a variety of strategies to exploit normal cellular processes and signalling pathways for their efficient reproduction in a generally hostile cellular environment. One immediate-early response gene (IEG) family, the AP-1 gene family, was previously shown to be activated by coronavirus infection. In this study, we report that another IEG family, the EGR family, is also activated in cells infected with four different coronaviruses in three genera, i.e. gammacoronavirus infectious bronchitis virus (IBV), alphacoronaviruses porcine epidemic diarrhoea virus (PEDV) and human coronavirus-229E (HCoV-229E), and betacoronavirus HCoV-OC43. Knockdown of EGR1 reduced the expression of cJUN and cFOS, and knockdown of cJUN and/or cFOS reduced the expression of EGR1, demonstrating that these two IEG families may be cross-activated and mutual regulated. Furthermore, ERK1/2 was identified as an upstream kinase, and JNK and p38 as inhibitors of EGR1 activation in coronavirus-infected cells. However, upregulation of EGR family genes, in particular EGR1, appears to play a differential role in regulating viral replication, apoptosis and antiviral response. EGR1 was shown to play a limited role in regulation of coronavirus replication, and an anti-apoptotic role in cells infected with IBV or PEDV, but not in cells infected with HCoV-229E. Upregulation of EGR1 may also play a differential role in the regulation of antiviral response against different coronaviruses. This study reveals a novel regulatory network shared by different coronaviruses in the immediate-early response of host cells to infection.

Keywords: AP-1; Coronavirus; EGR1; ERK1/2; apoptosis; cross-activation; cytokines; immediate-early genes.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Upregulation of EGR family genes in IBV-infected cells and chicken embryos. (a) Upregulation of EGR family genes at the mRNA level in IBV-infected H1299, Vero and DF1 cells. Cells were infected with IBV (MOI∼2), and harvested at indicated time points. The levels of virus genomic RNA (gRNA) and the mRNA levels of EGR family genes (EGR1/EGR2/EGR3/EGR4) were determined by qPCR. (b) Upregulation of EGR family genes at the mRNA level in IBV-infected chicken embryos. Ten-day-old SPF chicken embryos were inoculated with 200 µL of IBV (500 PFU). At 60 hpi, chicken embryo viscera were collected. The EGR family genes (EGR1/EGR2/EGR3/EGR4) at the mRNA level as well as IBV gRNA were determined by qPCR. Shown are the results of three repeated experiments, as indicated. (c) Upregulation of EGR1 at the protein level in IBV-infected H1299 and Vero cells. H1299 and Vero cells were infected with IBV (MOI∼2), or UV-IBV. Cell lysates were harvested at the indicated time points and subjected to Western blot analysis using indicated antibodies. Sizes of protein ladders in kDa were indicated on the left.
Figure 2.
Figure 2.
Functional significance of EGR1 upregulation on coronavirus replication and apoptosis (a) Promotion of IBV-induced apoptosis by knockdown of EGR1. H1299 cells were transfected with siEGFP and siEGR1, before infected with IBV (MOI∼2). Cells were harvested at the indicated time points and subjected to RT-qPCR and Western blot analysis, respectively. The mRNA levels of EGR1 and IBV gRNA were determined by qPCR. Western blot analysis was performed using the indicated antibodies. Sizes of protein ladders in kDa were indicated on the left. Percentage of PARP cleavage [PARP Clv. (%)] was calculated as the intensity of cleaved PARP (Cl) divided by the total intensities of the full-length PARP (FL) + Cl. (b) Inhibition of IBV-induced apoptosis by overexpression of EGR1. H1299 cells were transfected with pXJ40-Flag and pXJ40-Flag-EGR1, respectively before being infected with IBV (MOI∼2). Cell lysates were prepared and analysed as (a). The mRNA levels of EGR1 and IBV gRNA were determined by qPCR. Western blot was performed using antibodies against EGR1, IBV N, and PARP. (c) Effects of EGR1-knockdown on PEDV-induced apoptosis. H1299 cells were treated as (a), before infected with PEDV (MOI∼2). Cell lysates were prepared and analysed as (a). (d) Effects of EGR1-knockdown on HCoV-229E-induced apoptosis. H1299 cells were treated, before infected with HCoV-229E (MOI∼2), lysates prepared and analysed as (a).
Figure 3.
Figure 3.
Mutual regulation of the coronavirus infection-induced EGR1 and cFOS/cJUN expression. (a) Down-regulation of cFOS/cJUN expression by knockdown of EGR1 in IBV-infected cells. H1299 cells were transfected with siEGFP and siEGR1, before infected with IBV. Cells were harvested at the indicated time points and subjected to RT-qPCR and Western blot analysis, respectively. The mRNA levels of cJUN and cFOS were determined by qPCR. Western blot analysis was performed using the indicated antibodies. Sizes of protein ladders in kDa were indicated on the left. (b) Down-regulation of cFOS/cJUN expression by knockdown of EGR1 in PEDV-infected cells. H1299 cells were treated before infected with PEDV, lysates prepared and analysed as (a). (c) Down-regulation of cFOS/cJUN expression by knockdown of EGR1 in HCoV-229E-infected cells. H1299 cells were treated before infected with HCoV-229E, lysates prepared and analysed as (a). (d) Down-regulation of EGR1 expression by knockdown of cFOS and/or cJUN in IBV-infected cells. H1299 cells were transfected with siEGFP, sicJUN, sicFOS and sicJUN&sicFOS, before infected with IBV. Cell lysates were prepared and analysed as (a). The mRNA levels of EGR family genes (EGR1/EGR2/EGR3/EGR4) were determined by qPCR. Western blot analysis was performed as (a).
Figure 4.
Figure 4.
Identification of ERK1/2 as an upstream kinase(s) for EGR1 induction in IBV-infected cells. (a/b/c) Identification of the upstream kinase(s) MAPK for EGR1 induction in IBV-infected cells. H1299 cells were infected with IBV and treated with MAPK/ERK1/2/JNK/p38 inhibitor U0126, SP600125 and SB03580 at the indicated concentrations or with the same volume of DMSO at 2 hpi, respectively, Cells were harvested at the indicated time points. Western blot analysis was performed using the indicated antibodies. Sizes of protein ladders in kDa were indicated on the left. (d/e/f) Effects of ERK1/2-, JNK- or p38-knockdown on the expression of EGR1 in IBV-infected cells. H1299 cells were transfected with siEGFP and siERK1/2/JNK/p38, before infected with IBV. Cells were harvested at the indicated time points and subjected to Western blot analysis as (a/b/c). (g) Effects of inhibitors on cell viability. H1299 cells were treated with 10 µM U0126, 8 µM sp600125 and 20 µM SB203580 for 24 h, respectively. The cell viability rate was determined by measuring the absorbance value at 450 nm after adding the CCK solution for 2 h.
Figure 5.
Figure 5.
Regulation of the expression of cytokines and chemokines by coronavirus infection-induced EGR1 upregulation. (a) Differential effects of EGR1-knockdown on the expression of cytokines and chemokines in coronavirus-infected cells. H1299 cells were transfected with siEGFP and siEGR1, before infected with IBV/PEDV/229E at MOI∼2. Cell lysates were harvested at the indicated time points for RT-qPCR. The mRNA levels of EGR1, CXCL2, ISG15, IL-8 and IFN-β were determined by qPCR. Viral gRNA levels were determined as an indicator for IBV/PEDV/229E replication efficiency. (b) Effects of EGR1-knockdown on the expression of EGR2, EGR3 and EGR4 in IBV-infected cells. H1299 cells were treated as (a), Cells were harvested and analysed as (a). The mRNA levels of EGR1, EGR2, EGR3 and EGR4 were determined. (c) Effects of EGR1-overexpression on the expression of cytokines and chemokines in IBV-infected cells. H1299 cells were transfected with pXJ40-Flag and pXJ40-Flag-EGR1 before being infected with IBV or mock-infected (Mock). Cells were harvested and analysed as (a).
Figure 6.
Figure 6.
Current working model. The working model showing the activation of the MKKs-ERK1/2-EGR1 pathway and mutual regulation of ERG and AP-1 family genes during coronavirus infection. The functional impact of EGR1 activation on coronavirus-induced apoptosis and proinflammatory response is illustrated. Pointed and blunt arrows denote activation and suppression, respectively. “P” denotes phosphorylation. Dotted lines denote processes that are not fully characterized.
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Grants and funding

This work was partially supported by National Natural Science Foundation of China [grant numbers 31972660, 31900135 and 32170152], the Zhaoqing Xijiang Innovative Team Foundation of China [grant number P20211154-0202].