The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II

Abstract

The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8؉ cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8 ؉ cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8 ؉ cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4 ؉ cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8؉ and CD4 ؉ T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8؉ T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8 ؉ T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8 ؉ T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4 ؉ T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. E pstein-Barr virus (EBV) is a gammaherpesvirus found in more than 90% of the human population. Primary infection with EBV is usually followed by establishment of a lifelong latent infection, with occasional reactivation (1). The balance between host immune responses, including CD4 ϩ and CD8 ϩ T cells, and viral immune evasion of these responses is key to the spread and survival of EBV in human populations. Passive evasion through the ability to establish antigenically silent latent infections is an important characteristic of all herpesviruses, ...

Epstein–Barr virus microRNAs reduce immune surveillance by virus-specific CD8 + T cells

“…Some of these lytic-cycle viral proteins are immunoevasins that interfere with CD8 + T-cell recognition: the TAP inhibitor BNLF2a (14, 15); the G protein-coupled receptor BILF1 that associates with MHC class I/peptide complexes, diverts them from the exocytic pathway and the cell surface, and induces their lysomal degradation (16,17); and the protein BGLF5 that reduces MHC I expression and CD8 + T-cell recognition as a consequence of its generalized hostshutoff function (18,19). Recently, BDLF3 was identified as an additional lytic-cycle protein that targets MHC molecules for degradation (20). BNLF2a is also expressed early after infection in the prelatent phase (13 for a recent review) and reduces CD8 + T-cell recognition in the first days of infection but does not impair T-cell recognition in established latency (21,22).…”

Proteome-Wide Analysis of Cd8+ T Cell Responses to Ebv Reveals Differences Between Primary and Persistent Infection

“…With respect to the data from IM patients, our proteome-wide screening greatly strengthens the evidence for an IE > E > L hierarchy among lytic cycle antigens as targets of the EBVinduced primary response. Note that this hierarchy parallels the efficiency with which IE, E and L antigens are presented on the surface of lytically-infected cells because, as cells move through lytic cycle, endogenous antigen presentation is progressively impaired by virallycoded evasins [19][20][21][22][23][24]43] and so epitopes derived from earlier expressed antigens are preferentially displayed. This parallel leads us to suggest that the primary response to EBV may be largely driven by direct contact between the CD8+ T cell repertoire and infected cells themselves.…”

“…We reasoned that the conventional approach, stimulating PBMCs with the autologous EBV-transformed B lymphoblastoid cell line (LCL), had two disadvantages in that regard. Firstly, even in the most permissive LCLs, only a small fraction of cells enter lytic cycle; secondly, even where lytically-infected cells are present, EBV's array of immune evasins expressed as early or late phase proteins [19][20][21][22][23][24][25] increasingly retard the presentation of later-expressed antigens on the LCL cell surface. We therefore adopted an alternative protocol, adapted from studies in the HSV system [3], which avoids the phase-specific effects of evasins and renders all lytic cycle antigens available for HLA I-mediated epitope display through cross-presentation in monocyte-derived dendritic cells.…”

Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules

“…The kinetics of MHC II internalization and the appearance at the surface of MJSpuro (as a positive control), and HSV-1 or BoHV-1 gB-expressing MJS cells were assessed as described in [31], with slight modifications. Surface MHC II was stained with saturating amounts of L243 antibody in the culture medium with 2% (w/v) FBS for 1 h on ice.…”