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• W3174568143 abstract "•3.2-Å cryo-EM structure of the Epstein-Barr virus GPCR BILF1 in complex with human Gi•Global remodeling of class A GPCR microswitches leads to constitutive Gi signaling•Unexpected dissimilarity to chemokine receptors with an occluded extracellular face•Topological similarity to lipid GPCRs with a density observed between TM helices Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1’s constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR “microswitches” stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV. Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1’s constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR “microswitches” stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV. Epstein-Barr virus (EBV), also known as human herpesvirus 4, is a ubiquitous virus that primarily targets human B cells and endothelial cells and is estimated to infect almost 90% of the human population (Young et al., 2016Young L.S. Yap L.F. Murray P.G. Epstein-Barr virus: more than 50 years old and still providing surprises.Nat. Rev. Cancer. 2016; 16: 789-802Crossref PubMed Scopus (357) Google Scholar). During EBV infection, there is an interplay between EBV and the host that promotes viral persistence, including a variety of strategies to manipulate the host immune response. Although most EBV infections are asymptomatic, EBV causes widespread immunosuppression and is known to be the causative agent of mononucleosis. EBV has oncogenic activity on B cells and is implicated in endemic Burkitt’s lymphoma as well as other cancers (Farrell, 2019Farrell P.J. Epstein-Barr Virus and Cancer.Annu. Rev. Pathol. 2019; 14: 29-53Crossref PubMed Scopus (149) Google Scholar; Mundo et al., 2020Mundo L. Del Porro L. Granai M. Siciliano M.C. Mancini V. Santi R. Marcar L. Vrzalikova K. Vergoni F. Di Stefano G. et al.Frequent traces of EBV infection in Hodgkin and non-Hodgkin lymphomas classified as EBV-negative by routine methods: expanding the landscape of EBV-related lymphomas.Mod. Pathol. 2020; 33: 2407-2421Crossref PubMed Scopus (14) Google Scholar). The EBV genome encodes various immune modulators; among them, BILF1 is a putatively “orphan” G protein-coupled receptor (GPCR) (Beisser et al., 2005Beisser P.S. Verzijl D. Gruijthuijsen Y.K. Beuken E. Smit M.J. Leurs R. Bruggeman C.A. Vink C. The Epstein-Barr virus BILF1 gene encodes a G protein-coupled receptor that inhibits phosphorylation of RNA-dependent protein kinase.J. Virol. 2005; 79: 441-449Crossref PubMed Scopus (83) Google Scholar; Paulsen et al., 2005Paulsen S.J. Rosenkilde M.M. Eugen-Olsen J. Kledal T.N. Epstein-Barr virus-encoded BILF1 is a constitutively active G protein-coupled receptor.J. Virol. 2005; 79: 536-546Crossref PubMed Scopus (104) Google Scholar). BILF1 is expressed predominantly in the early lytic phase, contributing to immune evasion of EBV-infected cells during viral replication (Nijmeijer et al., 2010Nijmeijer S. Leurs R. Smit M.J. Vischer H.F. The Epstein-Barr virus-encoded G protein-coupled receptor BILF1 hetero-oligomerizes with human CXCR4, scavenges Gαi proteins, and constitutively impairs CXCR4 functioning.J. Biol. Chem. 2010; 285: 29632-29641Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar; Zuo et al., 2009Zuo J. Currin A. Griffin B.D. Shannon-Lowe C. Thomas W.A. Ressing M.E. Wiertz E.J.H.J. Rowe M. The Epstein-Barr virus G-protein-coupled receptor contributes to immune evasion by targeting MHC class I molecules for degradation.PLoS Pathog. 2009; 5: e1000255Crossref PubMed Scopus (118) Google Scholar), but also detected during viral latency (Tierney et al., 2015Tierney R.J. Shannon-Lowe C.D. Fitzsimmons L. Bell A.I. Rowe M. Unexpected patterns of Epstein-Barr virus transcription revealed by a high throughput PCR array for absolute quantification of viral mRNA.Virology. 2015; 474: 117-130Crossref PubMed Scopus (52) Google Scholar). The EBV lytic replication cycle and periodic reactivation allow the virus to produce infectious particles and spread. BILF1 associates physically with major histocompatibility complex (MHC) class I molecules to enhance internalization and depletion of MHC class I from the host cell surface to escape surveillance by cytotoxic CD8+ T cells (Fares et al., 2019Fares S. Spiess K. Olesen E.T.B. Zuo J. Jackson S. Kledal T.N. Wills M.R. Rosenkilde M.M. Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation.MBio. 2019; 10: 1-15Crossref Scopus (6) Google Scholar; Griffin et al., 2013Griffin B.D. Gram A.M. Mulder A. Van Leeuwen D. Claas F.H.J. Wang F. Ressing M.E. Wiertz E. EBV BILF1 evolved to downregulate cell surface display of a wide range of HLA class I molecules through their cytoplasmic tail.J. Immunol. 2013; 190: 1672-1684Crossref PubMed Scopus (46) Google Scholar; Zuo et al., 2009Zuo J. Currin A. Griffin B.D. Shannon-Lowe C. Thomas W.A. Ressing M.E. Wiertz E.J.H.J. Rowe M. The Epstein-Barr virus G-protein-coupled receptor contributes to immune evasion by targeting MHC class I molecules for degradation.PLoS Pathog. 2009; 5: e1000255Crossref PubMed Scopus (118) Google Scholar). An important feature of BILF1 is that it appears to exhibit constitutive GPCR activities despite the fact that the existence of a natural ligand has not been ascertained (De Groof et al., 2021De Groof T.W.M. Elder E.G. Siderius M. Heukers R. Sinclair J.H. Smit M.J. Viral G Protein-Coupled Receptors: Attractive Targets for Herpesvirus-Associated Diseases.Pharmacol. Rev. 2021; 73: 828-846Crossref PubMed Scopus (5) Google Scholar). Thus, mere expression of BILF1 on the infected cell surface is enough to initiate a cascade of downstream events to enhance its virulence and pathogenesis in an unregulated fashion. BILF1 is coupled with inhibitory G protein (Gi) (Beisser et al., 2005Beisser P.S. Verzijl D. Gruijthuijsen Y.K. Beuken E. Smit M.J. Leurs R. Bruggeman C.A. Vink C. The Epstein-Barr virus BILF1 gene encodes a G protein-coupled receptor that inhibits phosphorylation of RNA-dependent protein kinase.J. Virol. 2005; 79: 441-449Crossref PubMed Scopus (83) Google Scholar; Lyngaa et al., 2010Lyngaa R. Nørregaard K. Kristensen M. Kubale V. Rosenkilde M.M. Kledal T.N. Cell transformation mediated by the Epstein-Barr virus G protein-coupled receptor BILF1 is dependent on constitutive signaling.Oncogene. 2010; 29: 4388-4398Crossref PubMed Scopus (36) Google Scholar; Paulsen et al., 2005Paulsen S.J. Rosenkilde M.M. Eugen-Olsen J. Kledal T.N. Epstein-Barr virus-encoded BILF1 is a constitutively active G protein-coupled receptor.J. Virol. 2005; 79: 536-546Crossref PubMed Scopus (104) Google Scholar), the major intracellular signaling effector for human chemokine receptors (Flock et al., 2017Flock T. Hauser A.S. Lund N. Gloriam D.E. Balaji S. Babu M.M. Selectivity determinants of GPCR-G-protein binding.Nature. 2017; 545: 317-322Crossref PubMed Scopus (189) Google Scholar; Murphy, 1994Murphy P.M. The molecular biology of leukocyte chemoattractant receptors.Annu. Rev. Immunol. 1994; 12: 593-633Crossref PubMed Scopus (1116) Google Scholar). One effect of BILF1-mediated Gi activation is to attenuate Gi signaling of other endogenous chemokine receptors expressed on the infected cell by hijacking the host signal transduction pathway (Nijmeijer et al., 2010Nijmeijer S. Leurs R. Smit M.J. Vischer H.F. The Epstein-Barr virus-encoded G protein-coupled receptor BILF1 hetero-oligomerizes with human CXCR4, scavenges Gαi proteins, and constitutively impairs CXCR4 functioning.J. Biol. Chem. 2010; 285: 29632-29641Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In this scenario, BILF1 exhausts the intracellular Gi pool via its constitutive Gi coupling, leading to impaired signaling by the human CXC chemokine receptor (CXCR) 4 upon stimulation by the CXC chemokine ligand 12 (CXCL12). The “cis repression” phenomenon is also observed with endogenous human GPCRs, where prolonged activity of one GPCR reduces signaling from another GPCR that shares the same intracellular signaling pathway (Canals and Milligan, 2008Canals M. Milligan G. Constitutive activity of the cannabinoid CB1 receptor regulates the function of co-expressed Mu opioid receptors.J. Biol. Chem. 2008; 283: 11424-11434Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar; Ziffert et al., 2020Ziffert I. Kaiser A. Babilon S. Mörl K. Beck-Sickinger A.G. Unusually persistent Gαi-signaling of the neuropeptide Y2 receptor depletes cellular Gi/o pools and leads to a Gi-refractory state.Cell Commun. Signal. 2020; 18: 49Crossref PubMed Scopus (7) Google Scholar). In addition to its role in immune evasion, BILF1’s constitutive Gi coupling likely enhances its neoplastic activity and tumorigenesis (Lyngaa et al., 2010Lyngaa R. Nørregaard K. Kristensen M. Kubale V. Rosenkilde M.M. Kledal T.N. Cell transformation mediated by the Epstein-Barr virus G protein-coupled receptor BILF1 is dependent on constitutive signaling.Oncogene. 2010; 29: 4388-4398Crossref PubMed Scopus (36) Google Scholar). BILF’s role in promoting EBV infection and oncogenesis is enabled by its constitutive activities and makes the BILF1-Gi signaling axis an attractive target for therapeutic intervention (De Groof et al., 2021De Groof T.W.M. Elder E.G. Siderius M. Heukers R. Sinclair J.H. Smit M.J. Viral G Protein-Coupled Receptors: Attractive Targets for Herpesvirus-Associated Diseases.Pharmacol. Rev. 2021; 73: 828-846Crossref PubMed Scopus (5) Google Scholar). Despite their importance in viral pathogenesis, immune evasion, and cancer, structural information about viral GPCRs (vGPCRs) (Montaner et al., 2013Montaner S. Kufareva I. Abagyan R. Gutkind J.S. Molecular mechanisms deployed by virally encoded G protein-coupled receptors in human diseases.Annu. Rev. Pharmacol. Toxicol. 2013; 53: 331-354Crossref PubMed Scopus (45) Google Scholar) encoded by β- and γ-herpesviruses is limited compared with mammalian GPCRs. We showed previously how US28, a chemokine receptor encoded by human cytomegalovirus (HCMV), acts as a “chemokine trap” that binds to and is activated by human CX3C chemokine ligand 1 (CX3CL1; fractalkine) to subvert the host immune system (Burg et al., 2015Burg J.S. Ingram J.R. Venkatakrishnan A.J. Jude K.M. Dukkipati A. Feinberg E.N. Angelini A. Waghray D. Dror R.O. Ploegh H.L. et al.Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor.Science. 2015; 347: 1113-1117Crossref PubMed Scopus (203) Google Scholar). In contrast, EBV-encoded BILF1 immunosuppresses the host through its constitutive activity, but the identity of a putative ligand and its role(s) in the immunomodulatory functions of BILF1 remain undefined despite considerable efforts (Beisser et al., 2005Beisser P.S. Verzijl D. Gruijthuijsen Y.K. Beuken E. Smit M.J. Leurs R. Bruggeman C.A. Vink C. The Epstein-Barr virus BILF1 gene encodes a G protein-coupled receptor that inhibits phosphorylation of RNA-dependent protein kinase.J. Virol. 2005; 79: 441-449Crossref PubMed Scopus (83) Google Scholar); BILF1 is assumed to possess a chemokine-type ligand. The absence of a known ligand has also prevented a full understanding of the mechanisms underlying the robust constitutive BILF1-Gi signaling. We took a structural approach to examine the basis of constitutive BILF1-Gi signaling. Through cryoelectron microscopy (cryo-EM), we solved the structure of BILF1 in complex with a Gi heterotrimer at 3.2-Å resolution. Structural and functional analysis revealed that BILF1 has occluded access to the typical soluble GPCR ligand-binding site and assumes an atypical ligand-independent “on state” compared with canonical class A GPCRs. The BILF1-dependent Gi activities were highly tolerant to mutagenesis, suggesting that interactions throughout the entire protein are important to retain robust constitutive activity and negate the need for a host ligand. Thus, the constitutive activity of BILF1, which enables its major immunoevasive and oncogenic activities, results from an evolved ability to signal in the absence of a natural host or viral ligand. This constitutive activity results in unregulated host immune suppression and oncogenic transformation. Wild-type full-length BILF1, a vGPCR (Figure 1A), was expressed in human embryonic kidney (HEK) 293S GnTI− cells and extracted in a buffer containing the detergent lauryl maltose neopentyl glycol (LMNG) with cholesterol hemisuccinate (CHS). The purified receptor was assembled with a Gi heterotrimer in the presence of a G protein-stabilizing antibody, scFv16 (Maeda et al., 2018Maeda S. Koehl A. Matile H. Hu H. Hilger D. Schertler G.F.X. Manglik A. Skiniotis G. Dawson R.J.P. Kobilka B.K. Development of an antibody fragment that stabilizes GPCR/G-protein complexes.Nat. Commun. 2018; 9: 3712Crossref PubMed Scopus (59) Google Scholar), in a buffer containing LMNG, CHS, and glyco-diosgenin (GDN), yielding a monodisperse holocomplex (Figures S1A and S1B). We performed single-particle cryo-EM for the sample, which yielded a 3D reconstruction of the BILF1 signaling complex at a global indicated resolution of 3.2 Å (Figures 1B, S1C–S1F, and S2; Table S1). The receptor has the typical seven-transmembrane (7TM) GPCR topology with an α helix 8 (H8) that runs along the cell membrane plane (Figure 1C). The BILF1-Gi complex adopts a single canonical nucleotide-free state (Figure S1E), with the overall docking mode similar to mammalian GPCR-G protein complexes (Wang et al., 2020Wang J. Hua T. Liu Z.J. Structural features of activated GPCR signaling complexes.Curr. Opin. Struct. Biol. 2020; 63: 82-89Crossref PubMed Scopus (27) Google Scholar). BILF1 interacts exclusively with the Gαi1 subunit and has limited contact with the Gβ1γ2 subunits, as seen in other GPCR-Gi/o complexes, with interface areas of 1,180 Å2 and 50 Å2, respectively. A striking feature of BILF1 is that extracellular loop (ECL) 2 forms a lid that caps the extracellular vestibule (Figures 2A and 2B). This feature differs from the chemokine-binding GPCRs CXCR2, CXCR4, and US28 (Figures 2C and S3), where the extracellular vestibule is open and accessible for chemokine recognition. BILF1-ECL3 also undergoes an inward movement, collectively blocking the typical orthosteric ligand binding pocket and restricting access of soluble ligands to the extracellular pocket. BILF1 has not been functionally paired with chemokines, or any ligand, for signaling or ligand sequestration (De Groof et al., 2021De Groof T.W.M. Elder E.G. Siderius M. Heukers R. Sinclair J.H. Smit M.J. Viral G Protein-Coupled Receptors: Attractive Targets for Herpesvirus-Associated Diseases.Pharmacol. Rev. 2021; 73: 828-846Crossref PubMed Scopus (5) Google Scholar), so the occluded pocket supports the hypothesis that BILF1 is likely not an “orphan” but, rather, has evolved to not require binding to chemokines or other ligands. Comparison of the current active-state structure of BILF1 with the inactive state of the closest human homolog, CXCR4, shows that the TM helices are structurally different, yielding a root-mean-square deviation (RMSD) of ∼8.1 Å (Figure S3B). The disposition of ECL2 in viral and chemokine GPCRs may provide clues about function. Among the vGPCR family, BILF1 belongs to a branch that is distinct from HCMV-encoded US28 and is closer in evolutionary relatedness to the Kaposi’s sarcoma herpesvirus (KSHV) chemokine receptor ORF74 and orphan HCMV GPCRs UL33 and UL78 (Montaner et al., 2013Montaner S. Kufareva I. Abagyan R. Gutkind J.S. Molecular mechanisms deployed by virally encoded G protein-coupled receptors in human diseases.Annu. Rev. Pharmacol. Toxicol. 2013; 53: 331-354Crossref PubMed Scopus (45) Google Scholar). In the human (e.g., CXCR2 and CXCR4) and viral chemokine receptors, ECL2 forms a β-hairpin (Figure 2C) and plays key roles in their ligand-binding functions (Burg et al., 2015Burg J.S. Ingram J.R. Venkatakrishnan A.J. Jude K.M. Dukkipati A. Feinberg E.N. Angelini A. Waghray D. Dror R.O. Ploegh H.L. et al.Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor.Science. 2015; 347: 1113-1117Crossref PubMed Scopus (203) Google Scholar; Liu et al., 2020Liu K. Wu L. Yuan S. Wu M. Xu Y. Sun Q. Li S. Zhao S. Hua T. Liu Z.J. Structural basis of CXC chemokine receptor 2 activation and signalling.Nature. 2020; 585: 135-140Crossref PubMed Scopus (48) Google Scholar; Qin et al., 2015Qin L. Kufareva I. Holden L.G. Wang C. Zheng Y. Zhao C. Fenalti G. Wu H. Han G.W. Cherezov V. et al.Crystal structure of the chemokine receptor CXCR4 in complex with a viral chemokine.Science. 2015; 347: 1117-1122Crossref PubMed Scopus (261) Google Scholar). In human class A GPCRs, ECL2 is connected to TM helix 3 by a conserved disulfide bond to maintain the architecture of the ligand binding pocket and contributes to agonist recognition. In the structure of the CX3CL1-US28 complex, the agonist:ECL2 interaction is mediated via the chemokine’s β1-β2 turn (the 30 s loop) (Burg et al., 2015Burg J.S. Ingram J.R. Venkatakrishnan A.J. Jude K.M. Dukkipati A. Feinberg E.N. Angelini A. Waghray D. Dror R.O. Ploegh H.L. et al.Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor.Science. 2015; 347: 1113-1117Crossref PubMed Scopus (203) Google Scholar; Miles et al., 2018Miles T.F. Spiess K. Jude K.M. Tsutsumi N. Burg J.S. Ingram J.R. Waghray D. Hjorto G.M. Larsen O. Ploegh H.L. et al.Viral GPCR US28 can signal in response to chemokine agonists of nearly unlimited structural degeneracy.eLife. 2018; 7: e35850Crossref PubMed Scopus (22) Google Scholar). In the BILF1 structure, ECL2 (G162–P178), which includes three glycine-proline (GP) repeats, sits on top of the extracellular ligand binding pocket, acting like a self ‘ligand’ that is closely packed by ECL1 and ECL3 (Figure 2B). The extracellular architecture is further stabilized by the extra disulfide bond between the N-terminal loop and ECL3 via the C28-C258 bridge, a signature of chemokine receptors. The ECL2 sequence is highly conserved among BILF1 sequences encoded by nonhuman primate lymphocryptoviruses (Spiess et al., 2015Spiess K. Fares S. Sparre-Ulrich A.H. Hilgenberg E. Jarvis M.A. Ehlers B. Rosenkilde M.M. Identification and functional comparison of seven-transmembrane G-protein-coupled BILF1 receptors in recently discovered nonhuman primate lymphocryptoviruses.J. Virol. 2015; 89: 2253-2267Crossref PubMed Scopus (14) Google Scholar; Figure S4), strongly implying that these BILF1 homologs also have occluded ligand binding pockets. We initially hypothesized that ECL2 may play a crucial role in its constitutive activity, perhaps acting as a “self-agonist,” as seen in the GPR52 structure (Lin et al., 2020Lin X. Li M. Wang N. Wu Y. Luo Z. Guo S. Han G. Li S. Yue Y. Wei X. et al.Structural basis of ligand recognition and self-activation of orphan GPR52.Nature. 2020; 579: 152-157Crossref PubMed Scopus (32) Google Scholar). However, radical substitutions of BILF1 ECL2 residues did not abolish the constitutive Gi signaling (Figures 2D and S5), although they substantially reduced the protein expression levels (Figure 2E). The Gi signaling activity of BILF1 mutants was assessed by a membrane-proximal cell-based assay in HEK293 cells expressing BILF1 constructs together with a chimeric Gi protein (GαΔ6qi4myr) that recognizes Gi-coupled GPCRs but elicits Gq-dependent phospholipase C activation. We measured the accumulation of inositol triphosphate (IP3) as a readout of this chimeric Gi signaling (Figure 2D). To validate the system, we first compared the signaling between the wild-type BILF1 and the K122A3.50 mutant. K1223.50 locates at the DR3.50Y-like EK3.50T motif (superscripts donate generic numbering for class A GPCRs [Isberg et al., 2015Isberg V. de Graaf C. Bortolato A. Cherezov V. Katritch V. Marshall F.H. Mordalski S. Pin J.P. Stevens R.C. Vriend G. Gloriam D.E. Generic GPCR residue numbers - aligning topology maps while minding the gaps.Trends Pharmacol. Sci. 2015; 36: 22-31Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar]; for BILF1, the numbering is based on structural comparison with CXCR4; Figures S3 and S4), which is typically important for conformational activation of class A GPCRs and direct interaction with the G protein (Zhou et al., 2019Zhou Q. Yang D. Wu M. Guo Y. Guo W. Zhong L. Cai X. Dai A. Jang W. Shakhnovich E.I. et al.Common activation mechanism of class A GPCRs.eLife. 2019; 8: e50279Crossref PubMed Scopus (112) Google Scholar). Alanine substitution of this residue is known to silence BILF1’s Gi-cyclic AMP (cAMP) signaling and reduce its ability to transform mammalian cells (Lyngaa et al., 2010Lyngaa R. Nørregaard K. Kristensen M. Kubale V. Rosenkilde M.M. Kledal T.N. Cell transformation mediated by the Epstein-Barr virus G protein-coupled receptor BILF1 is dependent on constitutive signaling.Oncogene. 2010; 29: 4388-4398Crossref PubMed Scopus (36) Google Scholar). Consistent with this, the K122A3.50 mutant showed significantly lower Gi signaling (∼20% activities) with even ∼50% higher protein expression than the wild-type BILF1. We note that the wild-type and mutant BILF1 showed a gene-dose-dependent increase in signaling up to 10 ng DNA/well and a decrease at higher gene concentrations. Because similar behaviors were observed for most other mutants tested in this study regardless of their expression levels, we chose the 10 ng DNA/well condition for normalization and analysis of all cell-based signaling experiments. We also noticed that a few mutants showed a trend to reach the maximum signaling at lower gene doses, where we performed an additional analysis at 5 ng DNA/well. For comparison of protein expression levels, we used the 50 ng DNA/well conditions because of limited detection sensitivity at lower concentrations (Figure 2E). The relative membrane-proximal signaling and protein expression are summarized in Table 1.Table 1Summary of the relative Gi signaling and total expression of BILF1 variantsBILF1 VariantRelative Gi signaling (GαΔ6qi4myr-IP3, 10 ng DNA/well)Relative expression (ELISA, 50 ng DNA/well)Wild-type100%100%K122A16.0% ± 4.5% (∗∗)151.3% ± 16.4% (∗∗)ECL2GGG63.8% ± 2.8% (∗∗)25.2% ± 7.3% (∗∗)ECL2CXCR4a63.9% ± 6.4% (∗∗)32.1% ± 2.0% (∗∗)ECL2CXCR4b58.5% ± 5.7% (∗∗)37.3% ± 0.7% (∗∗)ECL2CXCR4ab59.5% ± 4.4% (∗∗)27.1% ± 4.1% (∗∗)TM6x7-4A89.1% ± 8.8% (∗)111.1% ± 1.4% (∗)TM6x7-4A1F78.0% ± 5.1% (∗∗)108.5% ± 2.7% (ns)A271F84.3% ± 13.2% (∗∗)66.4% ± 13.5% (∗∗)M240F112.6% ± 7.5% (∗)92.9% ± 9.0% (ns)L241F91.5% ± 2.8% (∗)91.7% ± 9.0% (ns)Y282A41.8% ± 4.2% (∗∗)129.7% ± 7.1% (∗∗)E121A55.1% ± 2.0% (∗∗)58.7% ± 3.2% (∗∗)H115A58.1% ± 4.9% (∗∗)139.4% ± 7.2% (∗∗)L75A50.4% ± 10.4% (∗∗)97.8% ± 7.5% (ns)NPxxY59.1% ± 3.8% (∗∗)84.8% ± 7.8 (∗)CWxM76.8% ± 3.2% (∗∗)100.7% ± 4.9% (ns)CWxP77.8% ± 3.7% (∗∗)98.2% ± 10.6% (ns)SWxP92.0% ± 8.6% (ns)90.5% ± 3.6% (ns)A125F76.8% ± 0.8% (∗∗)88.2% ± 3.2% (∗)H288A98.9% ± 5.7% (ns)91.5% ± 7.1% (ns)F286-H288A87.1% ± 4.2% (∗∗)85.0% ± 9.3% (∗)H66A-F286A-H288A76.0% ± 2.3% (∗∗)79.7% ± 6.1% (∗∗)ns: not significant, ∗: p < 0.05, ∗∗: p < 0.01 Open table in a new tab ns: not significant, ∗: p < 0.05, ∗∗: p < 0.01 We next assessed the ECL2 chimeras using the same system. Replacement of residues L167–N172 with a triglycine linker (ECLGGG) maintained ∼60% of Gi signaling but with ∼30% of protein expression compared with the wild type. The same outcome was observed by exchanging 7 amino acid residues before or 6 amino acids after Cys174 in ECL2, termed ECL2a or ECL2b (Figure S5A), respectively, with the corresponding CXCR4 residues as well as for combined exchange of ECL2a and ECL2b with those of CXCR4, confirming that the conserved residues in ECL2 are not essential for Gi signaling. We also determined inhibition of forskolin-induced cAMP response element (CRE) activity (Figure S5), where the differences among mutants were smaller than in the membrane-proximal assay. Compared with CRE inhibition by wild-type BILF1 at 10 ng DNA/well, K122A3.50 showed ∼75% inhibition, giving only a small range to rank the activities of mutants. The result was analogous to the IP3 assay, and these findings indicate that ECL2 has only supportive effects on BILF1’s Gi signaling but is more critical for its protein expression and stability. This is in contrast to the orphan GPR52, whose ECL2 is deeply plugged into the major ligand binding pocket, works as a built-in “agonist,” and is indispensable for receptor coupling to Gs protein (Lin et al., 2020Lin X. Li M. Wang N. Wu Y. Luo Z. Guo S. Han G. Li S. Yue Y. Wei X. et al.Structural basis of ligand recognition and self-activation of orphan GPR52.Nature. 2020; 579: 152-157Crossref PubMed Scopus (32) Google Scholar). Although thought to be a chemokine GPCR homolog (de Munnik et al., 2015de Munnik S.M. Smit M.J. Leurs R. Vischer H.F. Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors.Front. Pharmacol. 2015; 6: 40Crossref PubMed Scopus (31) Google Scholar), the overall structural topology of BILF1 is more similar to lipid GPCRs (prostaglandin receptors and thromboxane A2 receptor) (Audet et al., 2019Audet M. White K.L. Breton B. Zarzycka B. Han G.W. Lu Y. Gati C. Batyuk A. Popov P. Velasquez J. et al.Crystal structure of misoprostol bound to the labor inducer prostaglandin E2 receptor.Nat. Chem. Biol. 2019; 15: 11-17Crossref PubMed Scopus (24) Google Scholar; Fan et al., 2019Fan H. Chen S. Yuan X. Han S. Zhang H. Xia W. Xu Y. Zhao Q. Wu B. Structural basis for ligand recognition of the human thromboxane A2 receptor.Nat. Chem. Biol. 2019; 15: 27-33Crossref PubMed Scopus (28) Google Scholar; Morimoto et al., 2019Morimoto K. Suno R. Hotta Y. Yamashita K. Hirata K. Yamamoto M. Narumiya S. Iwata S. Kobayashi T. Crystal structure of the endogenous agonist-bound prostanoid receptor EP3.Nat. Chem. Biol. 2019; 15: 8-10Crossref PubMed Scopus (30) Google Scholar; Toyoda et al., 2019Toyoda Y. Morimoto K. Suno R. Horita S. Yamashita K. Hirata K. Sekiguchi Y. Yasuda S. Shiroishi M. Shimizu T. et al.Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface.Nat. Chem. Biol. 2019; 15: 18-26Crossref PubMed Scopus (56) Google Scholar; Figure 3A). Prostanoid receptors share a similar ECL2 structure capping the TM helical bundle that blocks access of soluble ligands, but they accommodate lipidic agonists and antagonists in the major ligand binding groove from the gap between TM1 and TM7 at the side of the receptors buried in the plasma membrane. Although BILF1 shows tight TM1-TM7 packing, we observed a gap between TM6 and TM7 that might be large enough to accommodate aliphatic small molecules or lipids. Indeed, we observed a prominent EM density embedded in the gap (Figures 3B and S2E). Because the shape of the density does not resemble any components used during purification, we surmised that it could be an endogenous lipidic compound co-purified with BILF1 from the HEK293 cell membrane, although we do not have biochemical evidence of the identity of this density. Additionally, we observed a weak cryo-EM density at the tip of the Y2677.37 side chain that could be a sulfate or phosphate modification site (Figure 3B, right). To probe the potential importance of this density occupying the TM6-TM7 gap, a selection of residues in TM6 (M2406.50, L2416.51, and L2436.53) and TM7 (Y2677.37 and A2717.42) that make contact with the unassigned density were selected for substitution with alanine or phenylalanine (Figures 3C–3F and S5). However, the effects of these mutations on Gi signaling were small or insignificant. The single “bulky” point mutant A271F7.42 reduced expression by ∼30%–40%, likely because of its effect on protein stabilities, whereas M240F6.50 and L241F6.51 maintained protein expression at the level of wild-type BILF1. M240F6.50 and L241F6.51 mutants achieved wild-type-like s" @default.
• W3174568143 created "2021-07-05" @default.
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• W3174568143 date "2021-07-01" @default.
• W3174568143 modified "2023-09-28" @default.
• W3174568143 title "Structural basis for the constitutive activity and immunomodulatory properties of the Epstein-Barr virus-encoded G protein-coupled receptor BILF1" @default.
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• W3174568143 doi "https://doi.org/10.1016/j.immuni.2021.06.001" @default.
• W3174568143 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8282746" @default.
• W3174568143 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/34216564" @default.
• W3174568143 hasPublicationYear "2021" @default.
• W3174568143 type Work @default.

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