Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation

ABSTRACT The Epstein-Barr virus (EBV) BILF1 gene encodes a constitutively active G protein-coupled receptor (GPCR) that downregulates major histocompatibility complex (MHC) class I and induces signaling-dependent tumorigenesis. Different BILF1 homologs display highly conserved extracellular loops (E...

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Autores principales:	Suzan Fares, Katja Spiess, Emma T. B. Olesen, Jianmin Zuo, Sarah Jackson, Thomas N. Kledal, Mark R. Wills, Mette M. Rosenkilde
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	EBV-BILF1 Epstein-Barr virus GPCR major histocompatibility complex signaling tumor immunology Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/dee11feb9d2349f88db72d5cfb52eacb
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spelling	oai:doaj.org-article:dee11feb9d2349f88db72d5cfb52eacb2021-11-15T15:55:13ZDistinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation10.1128/mBio.01707-182150-7511https://doaj.org/article/dee11feb9d2349f88db72d5cfb52eacb2019-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01707-18https://doaj.org/toc/2150-7511ABSTRACT The Epstein-Barr virus (EBV) BILF1 gene encodes a constitutively active G protein-coupled receptor (GPCR) that downregulates major histocompatibility complex (MHC) class I and induces signaling-dependent tumorigenesis. Different BILF1 homologs display highly conserved extracellular loops (ECLs) including the conserved cysteine residues involved in disulfide bridges present in class A GPCRs (GPCR bridge between transmembrane helix 3 [TM-3] and ECL-2) and in chemokine receptors (CKR bridge between the N terminus and ECL-3). In order to investigate the roles of the conserved residues in the receptor functions, 25 mutations were created in the extracellular domains. Luciferase reporter assays and flow cytometry were used to investigate the G protein signaling and MHC class I downregulation in HEK293 cells. We find that the cysteine residues involved in the GPCR bridge are important for both signaling and MHC class I downregulation, whereas the cysteine residues in the N terminus and ECL-3 are dispensable for signaling but important for MHC class I downregulation. Multiple conserved residues in the extracellular regions are important for the receptor-induced MHC class I downregulation, but not for signaling, indicating distinct structural requirements for these two functions. In an engineered receptor containing a binding site for Zn+2 ions in a complex with an aromatic chelator (phenanthroline or bipyridine), a ligand-driven inhibition of both the receptor signaling and MHC class I downregulation was observed. Taken together, this suggests that distinct regions in EBV-BILF1 can be pharmacologically targeted to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation. IMPORTANCE G protein-coupled receptors constitute the largest family of membrane proteins. As targets of >30% of the FDA-approved drugs, they are valuable for drug discovery. The receptor is composed of seven membrane-spanning helices and intracellular and extracellular domains. BILF1 is a receptor encoded by Epstein-Barr virus (EBV), which evades the host immune system by various strategies. BILF1 facilitates the virus immune evasion by downregulating MHC class I and is capable of inducing signaling-mediated tumorigenesis. BILF1 homologs from primate viruses show highly conserved extracellular domains. Here, we show that conserved residues in the extracellular domains of EBV-BILF1 are important for downregulating MHC class I and that the receptor signaling and immune evasion can be inhibited by drug-like small molecules. This suggests that BILF1 could be a target to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation, thereby facilitating virus recognition by the immune system.Suzan FaresKatja SpiessEmma T. B. OlesenJianmin ZuoSarah JacksonThomas N. KledalMark R. WillsMette M. RosenkildeAmerican Society for MicrobiologyarticleEBV-BILF1Epstein-Barr virusGPCRmajor histocompatibility complexsignalingtumor immunologyMicrobiologyQR1-502ENmBio, Vol 10, Iss 1 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	EBV-BILF1 Epstein-Barr virus GPCR major histocompatibility complex signaling tumor immunology Microbiology QR1-502
spellingShingle	EBV-BILF1 Epstein-Barr virus GPCR major histocompatibility complex signaling tumor immunology Microbiology QR1-502 Suzan Fares Katja Spiess Emma T. B. Olesen Jianmin Zuo Sarah Jackson Thomas N. Kledal Mark R. Wills Mette M. Rosenkilde Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation
description	ABSTRACT The Epstein-Barr virus (EBV) BILF1 gene encodes a constitutively active G protein-coupled receptor (GPCR) that downregulates major histocompatibility complex (MHC) class I and induces signaling-dependent tumorigenesis. Different BILF1 homologs display highly conserved extracellular loops (ECLs) including the conserved cysteine residues involved in disulfide bridges present in class A GPCRs (GPCR bridge between transmembrane helix 3 [TM-3] and ECL-2) and in chemokine receptors (CKR bridge between the N terminus and ECL-3). In order to investigate the roles of the conserved residues in the receptor functions, 25 mutations were created in the extracellular domains. Luciferase reporter assays and flow cytometry were used to investigate the G protein signaling and MHC class I downregulation in HEK293 cells. We find that the cysteine residues involved in the GPCR bridge are important for both signaling and MHC class I downregulation, whereas the cysteine residues in the N terminus and ECL-3 are dispensable for signaling but important for MHC class I downregulation. Multiple conserved residues in the extracellular regions are important for the receptor-induced MHC class I downregulation, but not for signaling, indicating distinct structural requirements for these two functions. In an engineered receptor containing a binding site for Zn+2 ions in a complex with an aromatic chelator (phenanthroline or bipyridine), a ligand-driven inhibition of both the receptor signaling and MHC class I downregulation was observed. Taken together, this suggests that distinct regions in EBV-BILF1 can be pharmacologically targeted to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation. IMPORTANCE G protein-coupled receptors constitute the largest family of membrane proteins. As targets of >30% of the FDA-approved drugs, they are valuable for drug discovery. The receptor is composed of seven membrane-spanning helices and intracellular and extracellular domains. BILF1 is a receptor encoded by Epstein-Barr virus (EBV), which evades the host immune system by various strategies. BILF1 facilitates the virus immune evasion by downregulating MHC class I and is capable of inducing signaling-mediated tumorigenesis. BILF1 homologs from primate viruses show highly conserved extracellular domains. Here, we show that conserved residues in the extracellular domains of EBV-BILF1 are important for downregulating MHC class I and that the receptor signaling and immune evasion can be inhibited by drug-like small molecules. This suggests that BILF1 could be a target to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation, thereby facilitating virus recognition by the immune system.
format	article
author	Suzan Fares Katja Spiess Emma T. B. Olesen Jianmin Zuo Sarah Jackson Thomas N. Kledal Mark R. Wills Mette M. Rosenkilde
author_facet	Suzan Fares Katja Spiess Emma T. B. Olesen Jianmin Zuo Sarah Jackson Thomas N. Kledal Mark R. Wills Mette M. Rosenkilde
author_sort	Suzan Fares
title	Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation
title_short	Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation
title_full	Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation
title_fullStr	Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation
title_full_unstemmed	Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation
title_sort	distinct roles of extracellular domains in the epstein-barr virus-encoded bilf1 receptor for signaling and major histocompatibility complex class i downregulation
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/dee11feb9d2349f88db72d5cfb52eacb
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Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation

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