Epstein-Barr Virus-Encoded BILF1 Orthologues From Porcine Lymphotropic Herpesviruses Display Common Molecular Functionality

Abstract

Infection of immunosuppressed transplant patients with the human γ-herpesvirus Epstein-Barr virus (EBV) is associated with post-transplant lymphoproliferative disease (PTLD), an often fatal complication. Immunosuppressed miniature pigs infected with γ-herpesvirus porcine lymphotropic herpesvirus 1 (PLHV1) develop a similar disease, identifying pigs as a potential preclinical model for PTLD in humans. BILF1 is a G protein-coupled receptor (GPCR) encoded by EBV with constitutive activity linked to tumorigenesis and immunoevasive function downregulating MHC-I. In the present study, we compared BILF1-orthologues encoded by the three known PLHVs (PLHV1-3) with EBV-BILF1 to determine pharmacological suitability of BILF1 orthologues as model system to study EBV-BILF1 druggability. Cell surface localization, constitutive internalization, and MHC-I downregulation as well as membrane proximal constitutive Gαi signaling patterns were conserved across all BILFs. Only subtle differences between the individual BILFs were observed in downstream transcription factor activation. Using Illumina sequencing, PLHV1 was observed in lymphatic tissue from PTLD-diseased, but not non-diseased pigs. Importantly, these tissues showed enhanced expression of PLHV1-BILF1 supporting its involvement in PTLD infection.

Patterns of human and porcine gammaherpesvirus-encoded BILF1 receptor endocytosis

“…1 a, b). These results are consistent with our previously reported data for EBV-BILF1 and PLHV1-2-BILF1 [ 15 ]. Furthermore, EBV-BILF1 also demonstrated the fastest internalization kinetics, with an estimated t 1/2 of 20 min, whereas PLHV1 and PLHV2-BILF1 showed slower internalization with t 1/2 values of ~ 37 and ~ 45 min, respectively (Fig.…”

“…Constitutive internalization occurs when a membrane receptor transports to the cytoplasm independently of an extracellular ligand. This has been described for several vGPCRs [ 8 , 15 , 35 , 62 , 63 ] and endogenous GPCRs [ 53 , 62 ]. Although constitutive internalization is a common feature in vGPCRs, its functional role remains poorly understood.…”

“…Here, we have confirmed the constitutive internalization of EBV-BILF1 and PLHV1-2 BILF1 orthologs using a novel real-time FRET-based internalization assay, which allowed us to study the endocytic properties of these receptors in live HEK-293A cells. Low levels of receptor expression and low internalization ratios [ 15 ] observed for PLHV3-BILF1 made further interpretation of the endocytic properties difficult—and consequently, this receptor was not included in this study.…”

“…A role of constitutive internalization for EBV-BILF1 has been proposed previously, suggesting that the receptor forms a complex with MHC-I molecules at the plasma membrane and induces its internalization, resulting in hindered immunorecognition by CD8 + T cells [ 12 ]. The kinetics of the BILF1 constitutive internalization reported here correspond to the ability of these receptors to downregulate MHC-I molecules [ 15 ], where EBV-BILF1 showed the highest impact on MHC-I expression in HEK-293 cells. However, to assign the biological function of constitutive internalization to the BILFs, further studies will be required.…”

“…The mechanism underlying this process has not yet been studied in detail, but the importance of receptor trafficking has been suggested in this regard [ 14 ]. We have recently shown that BILF1 orthologs encoded by porcine lymphotropic herpesviruses (PLHVs) exhibit conserved signaling, constitutive internalization, and immunomodulatory properties, indicating the applicability of a porcine model to study EBV-related diseases and BILF1 as a potential therapeutic target [ 15 ]. Here, we further studied the endocytic mechanisms used by BILF1 receptors to describe their trafficking from the plasma membrane to the cell interior.…”