Identification and Functional Comparison of Seven-Transmembrane G-Protein-Coupled BILF1 Receptors in Recently Discovered Nonhuman Primate Lymphocryptoviruses

Abstract

Coevolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host antiviral immunity. BILF1 encoded by human Epstein-Barr virus (EBV) is a 7-transmembrane (7TM) G-proteincoupled receptor (GPCR) with multiple immunomodulatory functions, including attenuation of PKR phosphorylation, activation of G-protein signaling, and downregulation of major histocompatibility complex (MHC) class I surface expression. In this study, we explored the evolutionary and functional relationships between BILF1 receptor family members from EBV and 12 previously uncharacterized nonhuman primate (NHP) lymphocryptoviruses (LCVs). Phylogenetic analysis defined 3 BILF1 clades, corresponding to LCVs of New World monkeys (clade A) or Old World monkeys and great apes (clades B and C). Common functional properties were suggested by a high degree of sequence conservation in functionally important regions of the BILF1 molecules. A subset of BILF1 receptors from EBV and LCVs from NHPs (chimpanzee, orangutan, marmoset, and siamang) were selected for multifunctional analysis. All receptors exhibited constitutive signaling activity via G protein G␣i and induced activation of the NF-B transcription factor. In contrast, only 3 of 5 were able to activate NFAT (nuclear factor of activated T cells); chimpanzee and orangutan BILF1 molecules were unable to activate NFAT. Similarly, although all receptors were internalized, BILF1 from the chimpanzee and orangutan displayed an altered cellular localization pattern with predominant cell surface expression. This study shows how biochemical characterization of functionally important orthologous viral proteins can be used to complement phylogenetic analysis to provide further insight into diverse microbial evolutionary relationships and immune evasion function. IMPORTANCE Epstein-Barr virus (EBV), known as an oncovirus, is the only human herpesvirus in the genus Lymphocryptovirus (LCV). EBV uses multiple strategies to hijack infected host cells, establish persistent infection in B cells, and evade antiviral immune responses. As part of EBV's immune evasion strategy, the virus encodes a multifunctional 7-transmembrane (7TM) G-protein-coupled receptor (GPCR), EBV BILF1. In addition to multiple immune evasion-associated functions, EBV BILF1 has transforming properties, which are linked to its high constitutive activity. We identified BILF1 receptor orthologues in 12 previously uncharacterized LCVs from nonhuman primates (NHPs) of Old and New World origin. As 7TM receptors are excellent drug targets, our unique insight into the molecular mechanism of action of the BILF1 family and into the evolution of primate LCVs may enable validation of EBV BILF1 as a drug target for EBV-mediated diseases, as well as facilitating the design of drugs targeting EBV BILF1.

The Immunomodulatory Capacity of an Epstein-Barr Virus Abortive Lytic Cycle: Potential Contribution to Viral Tumorigenesis

“…Indeed, BILF1 ectopic expression resulted in increased transforming capacity in a mouse xenograft tumor model [ 65 ]. BILF1 is conserved among lymphocriptovirus isolated from non-human primates supporting an important pathological role [ 66 ]. BNLF2a prevented binding of TAP to both peptide and ATP, thereby inhibiting peptide loading onto HLA class I molecules and antigen presentation to CTLs [ 67 , 68 , 69 ].…”

Classification of human Herpesviridae proteins using Domain-architecture Aware Inference of Orthologs (DAIO)

“…In contrast to the protein families discussed so far, the evolutionary history of human Herpesviridae proteins with 7-transmembrane receptor domains is more complex ( Fig. 4 ) ( Spiess et al, 2015 ). By comparing this gene tree with a species tree for human Herpesviridae ( Fig.…”

Novel Chemokine-Based Immunotoxins for Potent and Selective Targeting of Cytomegalovirus Infected Cells

“…As such, using immunotoxins designed to target KSHV-infected cells could be a valid approach to efficiently kill KSHV-infected cells. With respect to the EBV-encoded BILF1 receptor, its pronounced cell surface expression [ 13 , 47 ], constitutive internalization [ 17 ], and expression during the lytic virus replication cycle, but also in latency [ 48 ], indicate that this receptor is a promising drug target suitable for immunotoxin targeting delivery. A drawback is that EBV-BILF1 is an orphan receptor (i.e., with no known ligands), but new technologies including nanobody and monobody design could offer highly specific ligands for immunotoxin targeting of EBV-BILF1.…”