Analysis of TLR3 pathway in macrophages.

<p>A) Schematic representation of the determined TLR3 pathway topology in macrophages. dsRNA or poly (I∶C) stimulated TLR3 triggers TRIF dependant response by the recruitment of TRIF to the cytoplasmic domain of the receptor which then allows RIP1, TRAF6, TBK1 and TRAF3 to bind with TRIF. This results in the activation of MAP kinases (MKK1/2, MKK3/6 and MKK4/7) and IκB kinase complex; MKK1/2, MKK3/6 and MKK4/7 activate ERK, JNK and p38, respectively and IκBα degradation releases NF-κB. TBK1 phosphorylates IRF-3 and 7. ERK, JNK and p38 translocate to the nucleus and activate the transcription factor AP-1, and NF-κB, IRF-3 and IRF-7 translocate to the nucleus. AP-1 and NF-κB bind to the promoter regions of cytokine genes such as <i>Tnf</i> and <i>Il6</i> while IRF-3, IRF-7 together with NF-κB bind to the promoter region of chemokine genes such as <i>Cxcl10</i> and <i>Ccl5</i> and induce their transcription. Protein-protein interactions between molecules at the two signaling branches analyzed are highlighted in brown and blue. The dotted lines indicate weak activation (see maintext). B), C) and D) show simulations of NF-κB, JNK and p38 activation, respectively, in wildtype (WT). The <i>x</i>-axis represents the time in minutes and the <i>y</i>-axis represents the relative activation profile.</p

A novel pathway is crucial for MAP kinases activation in poly (I∶C) stimulated macrophages.

<p>A) Schematic representation of the final TLR3 model after adding novel pathway (blue) from TRIF activates MAP kinases. B), C) and D) show simulations of NF-κB, JNK and p38 activation, respectively, of the final macrophages TLR3 model with novel pathway, in wildtype (WT) (black), TRAF6 KO (green) and TRADD KO (orange). The <i>x</i>-axis represents the time in minutes and the <i>y</i>-axis represents the relative activation profile.</p

The modeling strategy.

<p>See maintext for details.</p

NF-κB and JNK experimental activation profiles.

<p>A), B) and C) show experimental activation profiles of NF-κB, JNK and p38, respectively in (WT) (black), TRAF6 KO (green), TRADD KO (orange) obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004661#pone.0004661-Gohda1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004661#pone.0004661-Pobezinskaya1" target="_blank">[12]</a>. The activation levels were quantified from the western blots using ImageJ [<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>]. The <i>x</i>-axis represents the time in minutes and the <i>y</i>-axis represents the relative activation profile. Note: As TRADD KO data is unavailable at 10 min (earliest at 15 min), we could not observe delayed activation of as noted for WT and TRAF6 KO. Therefore, we used dotted line to connect 0 min and 15 min time points.</p

The final <i>in silico</i> TLR3 model reactions and parameter values.

<p>The final <i>in silico</i> TLR3 model reactions and parameter values.</p

Prediction of missing steps prior to poly (I∶C)/TLR3 binding.

<p>A) Schematic representation of TLR3 model after adding three signaling intermediates upstream of TLR3 representing uncharacterized cellular processes (blue) and TLR3-ectodomain dimerization (red dotted) (see maintext). Note: from our model, it is not possible to equate the three intermediary steps to represent exactly three actual biological events, since spatial transport processes might be one of candidates for the time delay. B), C) and D) show simulations of NF-κB, JNK and p38 activation, respectively, in the wildtype (WT) (black), TRAF6 KO (green), TRADD KO (orange). The <i>x</i>-axis represents the time in minutes and the <i>y</i>-axis represents the relative activation profile.</p

Table_1_Interferon regulatory factor 3 mediates effective antiviral responses to human coronavirus 229E and OC43 infection.pdf

Interferon regulatory factors (IRFs) are key elements of antiviral innate responses that regulate the transcription of interferons (IFNs) and IFN-stimulated genes (ISGs). While the sensitivity of human coronaviruses to IFNs has been characterized, antiviral roles of IRFs during human coronavirus infection are not fully understood. Type I or II IFN treatment protected MRC5 cells from human coronavirus 229E infection, but not OC43. Cells infected with 229E or OC43 upregulated ISGs, indicating that antiviral transcription is not suppressed. Antiviral IRFs, IRF1, IRF3 and IRF7, were activated in cells infected with 229E, OC43 or severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). RNAi knockdown and overexpression of IRFs demonstrated that IRF1 and IRF3 have antiviral properties against OC43, while IRF3 and IRF7 are effective in restricting 229E infection. IRF3 activation effectively promotes transcription of antiviral genes during OC43 or 229E infection. Our study suggests that IRFs may be effective antiviral regulators against human coronavirus infection.</p

Image_4_Interferon regulatory factor 3 mediates effective antiviral responses to human coronavirus 229E and OC43 infection.tif

Interferon regulatory factors (IRFs) are key elements of antiviral innate responses that regulate the transcription of interferons (IFNs) and IFN-stimulated genes (ISGs). While the sensitivity of human coronaviruses to IFNs has been characterized, antiviral roles of IRFs during human coronavirus infection are not fully understood. Type I or II IFN treatment protected MRC5 cells from human coronavirus 229E infection, but not OC43. Cells infected with 229E or OC43 upregulated ISGs, indicating that antiviral transcription is not suppressed. Antiviral IRFs, IRF1, IRF3 and IRF7, were activated in cells infected with 229E, OC43 or severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). RNAi knockdown and overexpression of IRFs demonstrated that IRF1 and IRF3 have antiviral properties against OC43, while IRF3 and IRF7 are effective in restricting 229E infection. IRF3 activation effectively promotes transcription of antiviral genes during OC43 or 229E infection. Our study suggests that IRFs may be effective antiviral regulators against human coronavirus infection.</p

Image_2_Interferon regulatory factor 3 mediates effective antiviral responses to human coronavirus 229E and OC43 infection.tif

Interferon regulatory factors (IRFs) are key elements of antiviral innate responses that regulate the transcription of interferons (IFNs) and IFN-stimulated genes (ISGs). While the sensitivity of human coronaviruses to IFNs has been characterized, antiviral roles of IRFs during human coronavirus infection are not fully understood. Type I or II IFN treatment protected MRC5 cells from human coronavirus 229E infection, but not OC43. Cells infected with 229E or OC43 upregulated ISGs, indicating that antiviral transcription is not suppressed. Antiviral IRFs, IRF1, IRF3 and IRF7, were activated in cells infected with 229E, OC43 or severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). RNAi knockdown and overexpression of IRFs demonstrated that IRF1 and IRF3 have antiviral properties against OC43, while IRF3 and IRF7 are effective in restricting 229E infection. IRF3 activation effectively promotes transcription of antiviral genes during OC43 or 229E infection. Our study suggests that IRFs may be effective antiviral regulators against human coronavirus infection.</p

Image_3_Interferon regulatory factor 3 mediates effective antiviral responses to human coronavirus 229E and OC43 infection.tif

Interferon regulatory factors (IRFs) are key elements of antiviral innate responses that regulate the transcription of interferons (IFNs) and IFN-stimulated genes (ISGs). While the sensitivity of human coronaviruses to IFNs has been characterized, antiviral roles of IRFs during human coronavirus infection are not fully understood. Type I or II IFN treatment protected MRC5 cells from human coronavirus 229E infection, but not OC43. Cells infected with 229E or OC43 upregulated ISGs, indicating that antiviral transcription is not suppressed. Antiviral IRFs, IRF1, IRF3 and IRF7, were activated in cells infected with 229E, OC43 or severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). RNAi knockdown and overexpression of IRFs demonstrated that IRF1 and IRF3 have antiviral properties against OC43, while IRF3 and IRF7 are effective in restricting 229E infection. IRF3 activation effectively promotes transcription of antiviral genes during OC43 or 229E infection. Our study suggests that IRFs may be effective antiviral regulators against human coronavirus infection.</p