The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism

ABSTRACT Most of the intracellular pattern recognition receptors (PRRs) reside in either the endolysosome or the cytoplasm to sense pathogen-derived RNAs, DNAs, or synthetic analogs of double-stranded RNA (dsRNA), such as poly(I:C). However, it remains elusive whether or not a pathogen-derived prote...

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Autores principales:	Yi Wang, Li Liu
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/8f4b76d71301487ab62c82f108b62a79
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spelling	oai:doaj.org-article:8f4b76d71301487ab62c82f108b62a792021-11-15T15:49:39ZThe Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism10.1128/mBio.01872-152150-7511https://doaj.org/article/8f4b76d71301487ab62c82f108b62a792016-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01872-15https://doaj.org/toc/2150-7511ABSTRACT Most of the intracellular pattern recognition receptors (PRRs) reside in either the endolysosome or the cytoplasm to sense pathogen-derived RNAs, DNAs, or synthetic analogs of double-stranded RNA (dsRNA), such as poly(I:C). However, it remains elusive whether or not a pathogen-derived protein can function as a cytosolic pathogen-associated molecular pattern (PAMP). In this study, we demonstrate that delivering the membrane gene of severe acute respiratory syndrome coronavirus (SARS-CoV) into HEK293T, HEK293ET, and immobilized murine bone marrow-derived macrophage (J2-Mφ) cells significantly upregulates beta interferon (IFN-β) production. Both NF-κB and TBK1-IRF3 signaling cascades are activated by M gene products. M protein rather than M mRNA is responsible for M-mediated IFN-β induction that is preferentially associated with the activation of the Toll-like receptor (TLR) adaptor proteins MyD88, TIRAP, and TICAM2 but not the RIG-I signaling cascade. Blocking the secretion of M protein by brefeldin A (BFA) failed to reverse the M-mediated IFN-β induction. The antagonist of both TLR2 and TLR4 did not impede M-mediated IFN-β induction, indicating that the driving force for the activation of IFN-β production was generated from inside the cells. Inhibition of TRAF3 expression by specific small interfering RNA (siRNA) did not prevent M-mediated IFN-β induction. SARS-CoV pseudovirus could induce IFN-β production in an M rather than M(V68A) dependent manner, since the valine-to-alanine alteration at residue 68 in M protein markedly inhibited IFN-β production. Overall, our study indicates for the first time that a pathogen-derived protein is able to function as a cytosolic PAMP to stimulate type I interferon production by activating a noncanonical TLR signaling cascade in a TRAF3-independent manner. IMPORTANCE Viral protein can serve as a pathogen-associated molecular pattern (PAMP) that is usually recognized by certain pathogen recognition receptors (PRRs) on the cell surface, such as Toll-like receptor 2 (TLR2) and TLR4. In this study, we demonstrate that the membrane (M) protein of SARS-CoV can directly promote the activation of both beta interferon (IFN-β) and NF-κB through a TLR-related signaling pathway independent of TRAF3. The driving force for M-mediated IFN-β production is most likely generated from inside the cells. M-mediated IFN-β induction was confirmed at the viral infection level since a point mutation at the V68 residue of M markedly inhibited SARS-CoV pseudovirally induced IFN-β production. Thus, the results indicate for the first time that SARS-CoV M protein may function as a cytosolic PAMP to stimulate IFN-β production by activating a TLR-related TRAF3-independent signaling cascade.Yi WangLi LiuAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 7, Iss 1 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Yi Wang Li Liu The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism
description	ABSTRACT Most of the intracellular pattern recognition receptors (PRRs) reside in either the endolysosome or the cytoplasm to sense pathogen-derived RNAs, DNAs, or synthetic analogs of double-stranded RNA (dsRNA), such as poly(I:C). However, it remains elusive whether or not a pathogen-derived protein can function as a cytosolic pathogen-associated molecular pattern (PAMP). In this study, we demonstrate that delivering the membrane gene of severe acute respiratory syndrome coronavirus (SARS-CoV) into HEK293T, HEK293ET, and immobilized murine bone marrow-derived macrophage (J2-Mφ) cells significantly upregulates beta interferon (IFN-β) production. Both NF-κB and TBK1-IRF3 signaling cascades are activated by M gene products. M protein rather than M mRNA is responsible for M-mediated IFN-β induction that is preferentially associated with the activation of the Toll-like receptor (TLR) adaptor proteins MyD88, TIRAP, and TICAM2 but not the RIG-I signaling cascade. Blocking the secretion of M protein by brefeldin A (BFA) failed to reverse the M-mediated IFN-β induction. The antagonist of both TLR2 and TLR4 did not impede M-mediated IFN-β induction, indicating that the driving force for the activation of IFN-β production was generated from inside the cells. Inhibition of TRAF3 expression by specific small interfering RNA (siRNA) did not prevent M-mediated IFN-β induction. SARS-CoV pseudovirus could induce IFN-β production in an M rather than M(V68A) dependent manner, since the valine-to-alanine alteration at residue 68 in M protein markedly inhibited IFN-β production. Overall, our study indicates for the first time that a pathogen-derived protein is able to function as a cytosolic PAMP to stimulate type I interferon production by activating a noncanonical TLR signaling cascade in a TRAF3-independent manner. IMPORTANCE Viral protein can serve as a pathogen-associated molecular pattern (PAMP) that is usually recognized by certain pathogen recognition receptors (PRRs) on the cell surface, such as Toll-like receptor 2 (TLR2) and TLR4. In this study, we demonstrate that the membrane (M) protein of SARS-CoV can directly promote the activation of both beta interferon (IFN-β) and NF-κB through a TLR-related signaling pathway independent of TRAF3. The driving force for M-mediated IFN-β production is most likely generated from inside the cells. M-mediated IFN-β induction was confirmed at the viral infection level since a point mutation at the V68 residue of M markedly inhibited SARS-CoV pseudovirally induced IFN-β production. Thus, the results indicate for the first time that SARS-CoV M protein may function as a cytosolic PAMP to stimulate IFN-β production by activating a TLR-related TRAF3-independent signaling cascade.
format	article
author	Yi Wang Li Liu
author_facet	Yi Wang Li Liu
author_sort	Yi Wang
title	The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism
title_short	The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism
title_full	The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism
title_fullStr	The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism
title_full_unstemmed	The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism
title_sort	membrane protein of severe acute respiratory syndrome coronavirus functions as a novel cytosolic pathogen-associated molecular pattern to promote beta interferon induction via a toll-like-receptor-related traf3-independent mechanism
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/8f4b76d71301487ab62c82f108b62a79
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The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism

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