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Binding of SARS-CoV-2 protein to human TLR2. (A) Soluble recombinant human TLR2 (100 µL at 1 µg/mL) was coated in 96-well plates. After saturation, various amounts of the E-GST protein (1 ng/mL–1000 ng/mL) were added for 2 h at 37 °C. TLR2 E-GST complexes were revealed by a solution of anti-GST sera follow by anti-anti-GST conjugated to HRP. (B) Primary human monocytes were incubated with 0.1 to 10 µg/mL of GST or GST-E SARS-CoV-2 protein. Cells were stained with anti-GST (1/1000). Data were acquired using a FACScalibur. One representative experiment is shown. (C) Quantification of the SARS-CoV-2 E protein or GST control binding to human monocytes from 3 different experiments acquired using a FACScalibur. (D) Primary human macrophages were incubated with 10 µg/mL of GST or GST-E SARS-CoV-2 protein. Cells were stained with anti-GST (1/500). Images were acquired using an EVOS M700 microscope. (E) HEK-TLR2 or HEK-null (10⁶) cells were preincubated for 1 h with increasing amounts of inactivated SARS-CoV-2 (0.5–50 µg). The staining of SARS-CoV-2 complexes was performed with rabbit anti-spike antibodies used at 1/500 and donkey anti-rabbit IgG complexed with Alexa 488 used at 20 µg/mL. (F) HEK-TLR2 or HEK-null cells previously plated in 24-well plates were incubated for 1 h with various amounts of inactivated SARS-CoV-2 at RT. After 3 washes, binding of SARS-CoV-2 to HEK cells was evaluated by microscopy, and bound viral particles were detected by anti-spike antibodies. (G) HEK-TLR2 or HEK-null cells previously plated in 24-well plates were incubated for 1 h with various amounts of inactivated SARS-CoV-2 (0.1–100 µg) at RT. After washing and cell lysis, cell-associated viral particles were evaluated after SDS-PAGE and Western blotting using anti-spike antibodies. Statistical significance comparing different groups is denoted with * for p < 0.05, ** for p < 0.01 and *** for p < 0.001, while ns means non-significant.
Source publication
This paper presents a molecular characterization of the interaction between the SARS-CoV-2 envelope (E) protein and TLR2. We demonstrated that the E protein, both as a recombinant soluble protein and as a native membrane protein associated with SARS-CoV-2 viral particles, interacts physically with the TLR2 receptor in a specific and dose-dependent...
Citations
... In a prior study, it was elucidated that Toll-like receptors (TLRs) undergo activation after PDCoV infection in ST cells. Moreover, it was subsequently determined that the expression of TLR2 is augmented following PDCoV infection (43), as the activation of the TLR2 pathway during SARS-CoV-2 infection (48).In this study, HIEC-6 cells were infected with PDCoV. TLR2 on the surface of HIEC-6 cells facilitated the endocytosis of PDCoV and subsequently transported the virus to the Golgi apparatus using the ribosomal RPS16 transporter. ...
Introduction
Porcine deltacoronavirus (PDCoV) is a zoonotic pathogen with a global distribution, capable of infecting both pigs and humans. To mitigate the risk of cross-species transmission and potential outbreaks, it is crucial to characterize novel antiviral genes, particularly those from human hosts.
Methods
This research used HIEC-6 to investigate PDCoV infection. HIEC-6 cells were infected with PDCoV. Samples were collected 48 h postinfection for proteomic analysis.
Results
We discovered differential expression of MRPS6 gene at 48 h postinfection with PDCoV in HIEC-6 cells. The gene expression initially increased but then decreased. To further explore the role of MRPS6 in PDCoV infection, we conducted experiments involving the overexpression and knockdown of this gene in HIEC-6 and Caco2 cells, respectively. Our findings revealed that overexpression of MRPS6 significantly inhibited PDCoV infection in HIEC-6 cells, while knockdown of MRPS6 in Caco2 cells led to a significant increase of virus titer. Furthermore, we investigated the correlation between PDCoV infection and the expression of MRPS6. Subsequent investigations demonstrated that MRPS6 exerted an augmentative effect on the production of IFN-β through interferon pathway activation, consequently impeding the progression of PDCoV infection in cellular systems. In conclusion, this study utilized proteomic analysis to investigate the differential protein expression in PDCoV-infected HIEC-6 cells, providing evidence for the first time that the MRPS6 gene plays a restrictive role in PDCoV virus infection.
Discussion
Our findings initially provide the validation of MRPS6 as an upstream component of IFN-β pathway, in the promotion of IRF3, IRF7, STAT1, STAT2 and IFN-β production of HIEC-6 via dual-activation from interferon pathway.
... Six VLPs-based vaccines of SARS-CoV-2 are currently in clinical studies, and only one candidate co-expresses the spike (S), envelope (E), membrane (M) proteins, and nucleocapsid (N) of the virus [22]. Not only is the S protein capable of immune induction but multiple studies have shown that other structural proteins are immunogenic [25][26][27][28]. This makes homotypic SARS-CoV-2 VLPs a more attractive target than the chimeric type. ...
At times of pandemics, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the situation demands rapid development and production timelines of safe and effective vaccines for delivering life-saving medications quickly to patients. Typical biologics production relies on using the lengthy and arduous approach of stable single-cell clones. Here, we used an alternative approach, a stable cell pool that takes only weeks to generate compared to a stable single-cell clone that needs several months to complete. We employed the membrane, envelope, and highly immunogenic spike proteins of SARS-CoV-2 to produce virus-like particles (VLPs) using the HEK293-F cell line as a host system with an economical transfection reagent. The cell pool showed the stability of protein expression for more than one month. We demonstrated that the production of SARS-CoV-2 VLPs using this cell pool was scalable up to a stirred-tank 2 L bioreactor in fed-batch mode. The purified VLPs were properly assembled, and their size was consistent with the authentic virus. Our particles were functional as they specifically entered the cell that naturally expresses ACE-2. Notably, this work reports a practical and cost-effective manufacturing platform for scalable SARS-CoV-2 VLPs production and chromatographic purification.
... 14,31-34 SARS-CoV-2 E protein has been shown to physically interact with TLR2 in a dose-dependent manner. 33 TLR2 was required for SARS-CoV-2 E protein induced lung inflammation, and administration of a TLR2 inhibitor, or the use of TLR2-deficient (Tlr2 À/À ) mice protected mice from induction of inflammation. 32,34 A study of SARS-CoV-1 lacking the E protein gene Prior studies have shown that SARS-CoV-2 E protein could activate a small number of proinflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) or mouse-derived macrophages. ...
... 32 Additionally, here we show that E protein stimulation of monocyte cytokine secretion was via TLR2 signaling, which is consistent with recent studies demonstrating that E protein directly interacts with TLR2 and leads to increased production of pro-inflammatory cytokines. [31][32][33][34]44 In contrast to our data, some studies have concluded that the spike protein is the main component of SARS-CoV-2 for activating TLR2 or signaling in macrophages. [45][46][47] This could be attributed to differences in experimental design and protein administration or expression of recombinant proteins. ...
... Three prior studies demonstrated that SARS-CoV-2 E protein activated proinflammatory cytokine expression in innate immune cells and showed that this interaction could be associated with severe disease. [32][33][34] These studies also showed that through genetic knock out of TLR genes, or through blockade of TLR signaling with small molecule drugs or iScience Article monoclonal antibodies, that the viral E protein was specifically sensed through TLR2 signaling. Two of these studies further demonstrated physical protein-protein interactions of the E protein and TLR2 using immunoprecipitation and cellular immunofluorescence assays. ...
Severe COVID-19 often leads to secondary infections and sepsis that contribute to long hospital stays and mortality. However, our understanding of the precise immune mechanisms driving severe complications after SARS-CoV-2 infection remains incompletely understood. Here, we provide evidence that the SARS-CoV-2 envelope (E) protein initiates innate immune inflammation, via toll-like receptor 2 signaling, and establishes a sustained state of innate immune tolerance following initial activation. Monocytes in this tolerant state exhibit reduced responsiveness to secondary stimuli, releasing lower levels of cytokines and chemokines. Mice exposed to E protein before secondary lipopolysaccharide challenge show diminished pro-inflammatory cytokine expression in the lung, indicating that E protein drives this tolerant state in vivo. These findings highlight the potential of the SARS-CoV-2 E protein to induce innate immune tolerance, contributing to long-term immune dysfunction that could lead to susceptibility to subsequent infections, and uncovers therapeutic targets aimed at restoring immune function following SARS-CoV-2 infection.
... Additionally, platelets express a multitude of immune receptors, including CD40L, Toll-like receptors (TLRs), and the Fc receptor for IgG (FcgRIIA) (37). In line with this, a few other studies indicated that the E protein can physically interact with the TLR2 transmembrane receptor, stimulating NF-kB transcription and CXCL8 production (45,46). Additionally, the viral Spike (S) protein independently binds with CD42b and stimulates platelets (47). ...
... During viral entry and replication, SARS-CoV-2 viral components can be sensed by pattern recogni tion receptors (PRRs) located on the cell surface, in endosomes, and in the cytoplasm of immune cells including monocytes, macrophages, dendritic cells, neutrophils, and natural killer cells as well as non-immune cells such as epithelial cells. Several studies have shown that the SARS-CoV-2 envelope (E) protein is sensed by the cell surface toll-like receptor (TLR) TLR2 which forms heterodimers with TLR1 and TLR6, respectively, and has previously been shown to detect a variety of microbe-associated molecular patterns (MAMPs) including diacyl and triacyl lipopeptides, proteins, and polysaccharides from a wide array of pathogens (1)(2)(3). Additionally, the SARS-CoV-2 spike (S) protein whose main function is to bind to the host cellular entry receptor ACE2 (angiotensin-con verting enzyme 2) and mediate viral-cell membrane fusion (4) binds to and activates the cell surface PRR TLR4 that typically senses lipopolysaccharide (LPS) (5). SARS-CoV-2 also activates endosomal PRR TLR3 which recognizes double-stranded RNA (dsRNA) and is sensed by plasmacytoid dendritic cells (pDCs) via endosomal PRR TLR7 which recognizes single-stranded RNA (ssRNA) (6,7). ...
Interferons (IFNs) are essential for defense against viral infections but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, we explore the complexity of the IFN response in COVID-19, examine the effects of manipulating IFN on SARS-CoV-2 viral replication and pathogenesis, and highlight pre-clinical and clinical studies evaluating the therapeutic efficacy of IFN in limiting COVID-19 severity.
... We hypothesize that the inhibitory effects of β-escin and AH on coronavirus replication as well as cytokine production in cells infected with CCoV may be associated with the direct downregulation of the NF-κB pathway. In fact, it is proposed that NF-κB activation in coronaviruses infected cells is mediated through the recognition of viral surface proteins by TLR located at the cell surface before viral entry [28][29][30] . Hence, considering that the activation of NF-κB signaling in response to coronavirus infection is independent of viral entry and replication, β-escin and AH may affect NF-κB activation in coronavirus infected epithelial cells and macrophages, independently of their antiviral activity. ...
Respiratory viruses can cause life-threatening illnesses. The focus of treatment is on supportive therapies and direct antivirals. However, antivirals may cause resistance by exerting selective pressure. Modulating the host response has emerged as a viable therapeutic approach for treating respiratory infections. Additionally, considering the probable future respiratory virus outbreaks emphasizes the need for broad-spectrum therapies to be prepared for the next pandemics. One of the principal bioactive constituents found in the seed extract of Aesculus hippocastanum L. (AH) is β-escin. The clinical therapeutic role of β-escin and AH has been associated with their anti-inflammatory effects. Regarding their mechanism of action, we and others have shown that β-escin and AH affect NF-κB signaling. Furthermore, we have reported the virucidal and broad-spectrum antiviral properties of β-escin and AH against enveloped viruses such as RSV, in vitro and in vivo. In this study, we demonstrate that β-escin and AH have antiviral and virucidal activities against SARS-CoV-2 and CCoV, revealing broad-spectrum antiviral activity against coronaviruses. Likewise, they exhibited NF-κB and cytokine modulating activities in epithelial and macrophage cell lines infected with coronaviruses in vitro. Hence, β-escin and AH are promising broad-spectrum antiviral, immunomodulatory, and virucidal drugs against coronaviruses and respiratory viruses, including SARS-CoV-2.
... Throughout the SARS-CoV-2 life cycle, viral proteins and nucleic acids are exposed to the host-cell surface, and endosomal and cytoplasmic PRRs, conceivably triggering downstream antiviral and inflammatory signalling pathways. During viral entry, the SARS-CoV-2 E protein is sensed by Toll-like receptor 2 (TLR2), which results in the robust induction of pro-inflammatory cytokines, such as TNF, interleukin-6 (IL-6) and IFNγ, which are likely to contribute to cytokine storm production 117,118 . Growing evidence suggests roles for other TLRs in sensing SARS-CoV-2: TLR3 (double-stranded (ds)RNA in endosomes), TLR4 (S protein), TLR7 and TLR8 (guanosine-and uridine-rich single-stranded (ss)RNA) [119][120][121] . ...
The zoonotic emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing coronavirus disease 2019 (COVID-19) pandemic have profoundly affected our society. The rapid spread and continuous evolution of new SARS-CoV-2 variants continue to threaten global public health. Recent scientific advances have dissected many of the molecular and cellular mechanisms involved in coronavirus infections, and large-scale screens have uncovered novel host-cell factors that are vitally important for the virus life cycle. In this Review, we provide an updated summary of the SARS-CoV-2 life cycle, gene function and virus-host interactions, including recent landmark findings on general aspects of coronavirus biology and newly discovered host factors necessary for virus replication.
... [16] M protein, which has a molecular weight of 25-30 kDa, is also found on the envelope of the virus. [22] It is the most widely distributed and consists of three parts: an extramembrane domain, three transmembrane domains, and an intramembrane domain. M protein is mainly responsible for assembly of the coronavirus envelope. ...
The Coronavirus Disease 2019 (COVID‐19) pandemic has brought a widespread influence on the world, especially in the face of sudden coronavirus infections, and there is still an urgent need for specific small molecule therapies to cope with possible future pandemics. The pathogen responsible for this pandemic is Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), and understanding its structure and lifecycle is beneficial for designing specific drugs of treatment for COVID‐19. The main protease (Mpro) which has conservative and specific advantages is essential for viral replication and transcription. It is regarded as one of the most potential targets for anti‐SARS‐CoV‐2 drug development. This review introduces the popular knowledge of SARS‐CoV‐2 Mpro in drug development and lists a series of design principles and relevant activities of advanced Mpro inhibitors, hoping to provide some new directions and ideas for researchers.
... Based on GO analysis, E protein-up-regulated genes were also associated with the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways (Fig. 4b), two pathways that were previously suggested to be activated in COVID-19 patients to mediate the cytokine production and drive the inflammation [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] . Though these pathways were known to be mediated by a signaling cascade involving various protein kinases including the extracellular signal-regulated kinase (ERK), the c-Jun terminal kinase (JNK) and the p38 group of protein kinase (p38 MAPK) 38 , we found that E protein treatment did not seem to affect the phosphorylation of ERK or JNK ( Supplementary Fig. 5a-c), while significantly promoting the phosphorylation of p38 and NF-κB in platelets upon stimulation with low-dose of ADP (Fig. 4c-e). ...
... Together, these experiments point to a model whereby the viral E protein, presumably as part of the circulatory virions in the blood, directly engages CD36 on the platelet membrane, which leads to activation of the intracellular p38 MAPK/NF-κB pathway and then promotes platelet activation. Of note, previous studies have shown that the E protein, both as a recombinant soluble protein and as a native membrane protein associated with SARS-CoV-2 viral particles, can physically interact with the TLR2 transmembrane receptor in a specific and dose-dependent manner, and the E-TLR2 interaction can stimulate the NF-κB transcription factor and production of the CXCL8 inflammatory chemokine in cells overexpressing TLR2 (e.g., HEK-TLR2 cell line) 33,37 . ...
... Next, the samples were incubated on ice for 20 min. Equivalent amounts of samples were separated by SDS-PAGE, and protein expression was quantified by western blotting with targeted antibodies 53 Interaction of E protein with CD36 in a solid phase assay A solid-phase assay was performed as described 37 . Briefly, soluble recombinant CD36 (100 μl at 1 μg/ml, #CP94, Novoprotein) in bicarbonate buffer (PH = 9.6) was coated in 96-well plates. ...
Aberrant coagulation and thrombosis are associated with severe COVID-19 post-SARS-CoV-2 infection, yet the underlying mechanism remains obscure. Here we show that serum levels of SARS-CoV-2 envelope (E) protein are associated with coagulation disorders of COVID-19 patients, and intravenous administration of the E protein is able to potentiate thrombosis in mice. Through protein pull-down and mass spectrometry, we find that CD36, a transmembrane glycoprotein, directly binds with E protein and mediates hyperactivation of human and mouse platelets through the p38 MAPK-NF-κB signaling pathway. Conversely, the pharmacological blockade of CD36 or p38 notably attenuates human platelet activation induced by the E protein. Similarly, the genetic deficiency of CD36, as well as the pharmacological inhibition of p38 in mice, significantly diminishes E protein-induced platelet activation and thrombotic events. Together, our study reveals a critical role for the CD36-p38 axis in E protein-induced platelet hyperactivity, which could serve as an actionable target for developing therapies against aberrant thrombotic events related to the severity and mortality of COVID-19.
... 19 It is also believed to bind toll-like receptors of the host cell, causing it to play a key role in the hyperinflammation present in COVID-19 cases. 20 More recent research suggests that ectopic expression of the envelope results in translational inhibition, as the viral protein binds to the initiation factor eIF2a. 21 (which was not certified by peer review) is the author/funder. ...
Genomic surveillance is a vital strategy for preparedness against the spread of infectious diseases and to aid in development of new treatments. In an unprecedented effort, millions of samples from COVID-19 patients have been sequenced worldwide for SARS-CoV-2. Using more than 8 million sequences that are currently available in GenBank SARS-CoV-2 database, we report a comprehensive overview of mutations in all 26 proteins and open reading frames (ORFs) from the virus. The results indicate that the spike protein, NSP6, nucleocapsid protein, envelope protein and ORF7b have shown the highest mutational propensities so far (in that order). In particular, the spike protein has shown rapid acceleration in mutations in the post-vaccination period. Monitoring the rate of non-synonymous mutations (Ka) provides a fairly reliable signal for genomic surveillance, successfully predicting surges in 2022. Further, the external proteins (spike, membrane, envelope, and nucleocapsid proteins) show a significant number of mutations compared to the NSPs. Interestingly, these four proteins showed significant changes in Ka typically 2 to 4 weeks before the increase in number of human infections (surges). Therefore, our analysis provides real time surveillance of mutations of SARS-CoV-2, accessible through the project website http://pandemics.okstate.edu/covid19/. Based on ongoing mutation trends of the virus, predictions of what proteins are likely to mutate next are also made possible by our approach. The proposed framework is general and is thus applicable to other pathogens. The approach is fully automated and provides the needed genomic surveillance to address a fast-moving pandemic such as COVID-19.
