key: cord-0903520-b2i9iu78 authors: Zhang, Wenwen; Ma, Zhenling; Wu, Yaru; Shi, Xixi; Zhang, Yanyan; Zhang, Min; Zhang, Menghao; Wang, Lei; Liu, Wei title: SARS-CoV-2 3C-like protease antagonizes interferon-beta production by facilitating the degradation of IRF3 date: 2021-09-03 journal: Cytokine DOI: 10.1016/j.cyto.2021.155697 sha: eb2f79f9497e70e81bd93de5b1e384d6713cf894 doc_id: 903520 cord_uid: b2i9iu78 The prevalence of SARS-CoV-2 is a great threat to global public health. However, the relationship between the viral pathogen SARS-CoV-2 and host innate immunity has not yet been well studied. The genome of SARS-CoV-2 encodes a viral protease called 3C-like protease. This protease is responsible for cleaving viral polyproteins during replication. In this investigation, 293T cells were transfected with SARS-CoV-2 3CL and then infected with Sendai virus (SeV) to induce the RIG-I like receptor (RLR)-based immune pathway. q-PCR, luciferase reporter assays, and western blotting were used for experimental analyses. We found that SARS-CoV-2 3CL significantly downregulated IFN-β mRNA levels. Upon SeV infection, SARS-CoV-2 3CL inhibited the nuclear translocation of IRF3 and p65 and promoted the degradation of IRF3. This effect of SARS-CoV-2 3CL on type I IFN in the RLR immune pathway opens up novel ideas for future research on SARS-CoV-2. Late in 2019, a novel coronavirus strain causing a fatal respiratory disease was reported that subsequently spread worldwide [1] . The World Health Organization (WHO) gave the pathogen an interim name, novel coronavirus 2019 (2019-nCoV) [2] . Following a large number of reports describing the complete gene sequencing of this pathogen, the virus could then be quickly identified in patients by reverse transcriptionpolymerase chain reaction (RT-PCR) [3] . The WHO subsequently renamed the 2019-nCoV pathogen SARS-CoV-2 and the disease it caused coronavirus disease 2019 (COVID-19) before 12 February 2020 [4] . This was followed by the WHO officially recognizing COVID-19 as a pandemic [5] . It is worth noting that SARS-CoV-2 is an emerging virus, and a large number of studies are still underway. The spread of this pathogen involves several mechanisms, many of which are yet to be well characterized [6] . While the primary target of SARS-CoV-2 is the human immune system, the associated mechanisms remain to be characterized in detail [7] . SARS-CoV-2 is an enveloped virus with a single-stranded positive-sense RNA genome that is approximately 30 kb in length [8] . As an aetiological RNA viral pathogen, SARS-CoV-2 targets the innate immune pathway [9] . This aspect is an emergent avenue of current and future research [10, 11] . SARS-CoV-2 3CL is the 3-chymotrypsin-like protease (also known as nonstructural protein 5, NSP5) that cleaves the polyprotein at 11 different sites to produce various nonstructural proteins [12] . This makes SARS-CoV-2 3CL a vital protein in the replication of virus particles, unlike the structural or accessory protein-coding genes. Thus, SARS-CoV-2 3CL is a potential target for screening molecules that inhibit viral replication [13] . The RLR pathway, a vital part of the innate immune system, regulates the production of type I IFNs to directly or indirectly control viral replication [14, 15] . Given the essential role of SARS-CoV-2 3CL in viral replication and transcription, the protease emerges as a crucial protein [16] . Once a cell is infected by the virus, the activation of the immune system results in the expression of relevant genes in the system to mediate viral replication [17, 18] . To escape the immune system, the virus changes its unfavourable environment into a favourable environment that facilitates viral replication and its subsequent release. This involves unique mechanisms to destroy the immune defence system of the host [19] . Thus, SARS-CoV-2 3CL is a vital target of study, as it may serve as an important foundation for specific treatments [20] . In this study, SARS-CoV-2 3CL was transfected into 293T cells, followed by activation of the RLR antiviral immune pathway by Sendai virus (SeV) infection to examine the effect of the viral protease on this antiviral immune pathway. We aimed to understand the effect of SARS-CoV-2 3CL on the production of type I IFN and the possible mechanism of action. Human 293T cells were cultivated in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO). Cells lines in our study have been tested and found free of Mycoplasma. 3CL of SARS-CoV-2 SARS-CoV-2/Wuhan-Hu-1 strain) was synthesized and cloned into expression vector pCGGS with the HA-tag at N-terminus. The IFN-β promoter, ISRE promoter or NF-κB promoter, Myc-MAVS, Flag-RIG-I-N, Myc-TBK1, Flag-p65, Flag-IRF3/5D plasmids have been previously described [21] . For immunoblot analysis, the following antibodies were used: Anti-IRF3 (sc-9082, Santa Cruz), anti-p65 (8242, CST), anti-PCNA (sc-56, Santa Cruz), anti-β-Actin (4970S, CST). Once the cells were infected by SeV, the culture medium was removed from the plate and washed twice with phosphate buffered saline (PBS). Serum-free medium containing SeV ([MOI] = 1) was added at the specified time followed by replacement with the medium containing 2% fetal bovine serum. Total RNA was extracted using TRIzol (Invitrogen), and cDNA was synthesized GAPDH served as an internal control using the PCR primers, GAPDH forward, 5′-TTGTCTCCTGCGACTTCAAC-AG-3′; GAPDH reverse, 5′-GGTCTGGGATGGAAATTGTGAG-3′. Following transfection of IFN-β, ISRE, or NF-κB promoters, a β-Gal plasmid, and the relevant plasmids required for the various experiments in 24-well plates, cells were collected after infection with SeV. Twenty-four hours later, cells were lysed using lysis buffer. After centrifugation, luciferase assays were performed with a luciferase assay kit (Beyotime). Transfected cells (5 × 10 7 ) that were infected with SeV or uninfected, were washed with PBS and lysed using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology) according to the manufacturer's instructions. Due to the availability of a large amount of genomics data, along with increased knowledge of protein expression and structure relationships, codon optimization can significantly improve the level of gene expression [22] . A variety of vital factors involved in different stages of protein expression, such as GC content, mRNA secondary structure, and codon adaptability, can be considered during this process [23] . In this study, the 3CL gene of SARS-CoV-2 (SARS-CoV-2/Wuhan-Hu-1 strain) was optimized accordingly for overexpression in Escherichia coli using a commercial proprietary algorithm, NG ® Codon Optimization Technology (Synbio Technologies, NJ, USA). The codon-optimized 3CL gene of SARS-CoV-2 (SARS-CoV-2/Wuhan-Hu-1 strain) sequence was synthesized from Make Research Easy. The plasmid pET-30a (+) was linearized using the enzymes BamHI and EcoRI, and the linearized vector was purified using a DNA purification kit (Zoman Biotechnology). The SARS-CoV-2 3CL sequence was cloned into the prokaryotic expression vector pET-30a (+) with the Histag at the N-terminus using T 4 DNA ligase and incubated at 16 °C overnight. DH5α competent cells were provided by Shanghai Weidi Biotechnology. The recombinant plasmid was identified by restriction enzyme digestion and DNA sequencing and was named pET-30a-SARS-CoV-2 3CL. New Zealand rabbits were given a subcutaneous injection of 3CL protein at a to prepare the mixture. Ten days after the last immunization, the rabbits' carotid arteries were bled to collect approximately 10 mL of whole blood, and the samples were placed in a refrigerator at 4 °C overnight. The serum was collected by centrifugation and divided into 4-mL aliquots, which were stored at -80 °C for later use. Following the recovery of 293T cells showing 3CL eukaryotic expression plasmid transfection for 24 h, the transfected cell proteins were resolved by SDS-PAGE. The protein was transferred to a PVDF membrane, with 5% skimmed milk used for blocking, followed by incubation of the membrane with diluted 3CL antibodies (1:1000) at 4 °C overnight. Then, a secondary antibody labelled with horseradish peroxidase was added, and the final reaction product was visualized using an enhanced chemiluminescence (ECL) kit (Beyotime Technology). Statistical significance was determined by applying an unpaired Student's t-test using GraphPad Prism. Values of P < 0.05 were considered to be statistically significant. To study whether SARS-CoV-2 3CL inhibits the production of interferon (IFN)- S2A and B). For the analysis of the titer of the 3CL antibody in serum, rabbit serum injected with 3CL antigen was incubated at ratios of 1:100, 1:500, 1:1000, 1:5000 or 1:10,000. The results showed that the target band was observed at a titer ratio range of 1:5000 to 1:100 (supplementary Fig. S3A ). In the following studies, an anti-3CL antibody diluted 1:1000 was utilized. We next detected the expression of 3CL by western blotting analysis (Fig. 1A-D) . Reporter assays revealed that SARS-CoV-2 3CL downregulated the activation of the IFN-β promoter induced by SeV infection in 293T cells in a dose-dependent manner ( Fig. 2A) . Genes encoding type I IFNs are mainly regulated by the IRF3 and NF-κB signalling pathways [24] . Therefore, we next determined the pathways involved in IFN-β activation that were regulated by SARS-CoV-2 3CL. The SeV-induced activation of the ISRE and NF-κB promoters hypothesized to stimulate the RIG-I signalling pathway was inhibited by SARS-CoV-2 3CL in a dose-dependent manner (Figs. 2B and C) . These results demonstrated that SARS-CoV-2 3CL inhibited IRF3-and NF-κBmediated innate immune responses in a dose-dependent manner. We next examined the signalling cascade at which SARS-CoV-2 3CL blocks the antiviral innate immune response. We cotransfected the plasmid expressing SARS- IRF3 and p65 proteins translocate from the cytoplasm to the nucleus to activate the transcription of type I IFNs. We next assessed the effect of SARS-CoV-2 3CL on the nuclear translocation of IRF3 and p65. Subcellular fractionation and relative quantification analysis demonstrated that IRF3 and p65 rapidly translocated from the cytoplasm to the nuclear fraction in 293T cells compared with SARS-CoV-2 3CL-overexpressing 293T cells after SeV infection (Fig. 4A) . In an attempt to identify candidates that are direct targets for the proteolysis of SARS-CoV-2 3CL, we initially tested the effect of SARS-CoV-2 3CL on different components of the RLR pathway in terms of their stability. We found that SARS-CoV-2 3CL promoted the degradation of IRF3 (Fig. 4B) . The quantifications of the western blots are shown on the right sides of the blots. Together, these data suggest that IRF3 is the target protein of SARS-CoV-2 3CL in the suppression of RIG-I-mediated type I IFN production. The production of interferons (IFNs) is essential to eliminate invading viruses and maintain immune homeostasis [25, 26] . Moreover, type I interferon inhibits viral replication while also regulating the expression of downstream genes [27] . The production of type I interferon in the antiviral immune pathway involving RIG-I-like receptors is influenced by multiple target proteins [28, 29] . SARS-CoV-2 is a novel strain of coronavirus that cause an infectious respiratory disease and was discovered in humans in late 2019 [30] . The virus is a serious threat to life [31] . The SARS-CoV-2 3CL protease plays an important role in the replication of the virus [32] . In this study, the role of SARS-CoV-2 3CL in the innate immune antiviral pathway was examined. We found that SARS-CoV-2 3CL can inhibit the production of interferon-β and its downstream ISGs (ISG56 and Rantes) in SeV-stimulated 293T cells. Coronavirus 3C-like protease is an important protease in viral replication [33, 34] . To study the mechanisms of coronavirus infection, several extensive studies have been conducted [35] . The genetic sequence of the virus was also released [36] . It is known that the first line of defence of the immune system plays an important role in the process of resisting viral invasion [37] . Infection by an RNA virus activates the RIG-I-like receptor signalling pathway and promotes the expression of IFN-I along with several downstream genes, including ISGs [38] . In our work, 293T cells were transfected with plasmids expressing the SARS-CoV-2 3CL protein followed by infection with SeV. This was followed by detecting the expression of IFN-β and ISGs using RT-PCR. The results showed that the SARS-CoV-2 3CL protein had suppressive effects on the mRNA levels of IFNs and the ISGs studied. However, a recent report showed that no significant difference in SeV-mediated IFN-β activation was detected in the expression of 3CL (also known as NSP5) [39] . production by retaining phosphorylated IRF3 in the cytoplasm [35] . Host proteins possibly influence important target proteins in the RLR pathway, such as MAVS, RIG-I-N, TBK1, p65, and IRF3, to affect the production of type I interferon. In our study, we found that SARS-CoV-2 3CL can downregulate the production of type I interferon in the RLR pathway. Subcellular fractionation demonstrated that SARS-CoV-2 3CL inhibited the nuclear translocation of IRF3 and p65. In an attempt to identify candidates that are direct targets for proteolysis of SARS-CoV-2 3CL, we tested the effects of SARS-CoV-2 3CL on different components of the RLR pathway in terms of their stability. We found that SARS-CoV-2 3CL significantly promoted the degradation of IRF3 upon SeV infection. Taken together, these data suggested that IRF3 was the target protein of SARS-CoV-2 3CL to suppress RIG-Imediated type I IFN production. The target of the protease to inhibit the production of type I interferon and the associated mechanisms will be the subject of future research. The construction and expression of the prokaryotic expression vector SARS-CoV-2 3CL with the synthesized SARS-CoV-2 3CL protein was injected into rabbits along with an antigen adjuvant to produce a SARS-CoV-2 3CL polyclonal antibody. In this study, our results showed that the target band was observed at a titer ratio range of 1:5000 to 1:100, and we incubated the PVDF membrane with diluted 3CL antibodies (1:1000) at 4 °C overnight in immunoblot analysis. The preparation of this polyclonal antibody has also laid a foundation for future research. In our study, the production of type I interferon in the RLR antiviral immune pathway was significantly affected by SARS-CoV-2 3CL. This finding provides a foundation for future detailed studies on SARS-CoV-2, thereby making a definite contribution to research that can affect human life and health. The authors declare that they have no competing interests. Data are representative of at least three independent experiments. It is important that you return this form upon submission. We will not publish your article without completion and return of this form. Title of Paper: SARS-CoV-2 3C-like protease antagonizes interferon-beta production and a report of preparing polyclonal antibodies against 3C-like protease Please tick one of the following boxes:  √We have no conflict of interest to declare.  □We have a competing interest to declare. This statement is to certify that all authors have seen and approved the manuscript being submitted. 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