A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates - PubMed (original) (raw)

A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates

Andrew D Hislop et al. J Exp Med. 2007.

Abstract

gamma 1-Herpesviruses such as Epstein-Barr virus (EBV) have a unique ability to amplify virus loads in vivo through latent growth-transforming infection. Whether they, like alpha- and beta-herpesviruses, have been driven to actively evade immune detection of replicative (lytic) infection remains a moot point. We were prompted to readdress this question by recent work (Pudney, V.A., A.M. Leese, A.B. Rickinson, and A.D. Hislop. 2005. J. Exp. Med. 201:349-360; Ressing, M.E., S.E. Keating, D. van Leeuwen, D. Koppers-Lalic, I.Y. Pappworth, E.J.H.J. Wiertz, and M. Rowe. 2005. J. Immunol. 174:6829-6838) showing that, as EBV-infected cells move through the lytic cycle, their susceptibility to EBV-specific CD8(+) T cell recognition falls dramatically, concomitant with a reductions in transporter associated with antigen processing (TAP) function and surface human histocompatibility leukocyte antigen (HLA) class I expression. Screening of genes that are unique to EBV and closely related gamma 1-herpesviruses of Old World primates identified an early EBV lytic cycle gene, BNLF2a, which efficiently blocks antigen-specific CD8(+) T cell recognition through HLA-A-, HLA-B-, and HLA-C-restricting alleles when expressed in target cells in vitro. The small (60-amino acid) BNLF2a protein mediated its effects through interacting with the TAP complex and inhibiting both its peptide- and ATP-binding functions. Furthermore, this targeting of the major histocompatibility complex class I pathway appears to be conserved among the BNLF2a homologues of Old World primate gamma 1-herpesviruses. Thus, even the acquisition of latent cycle genes endowing unique growth-transforming ability has not liberated these agents from evolutionary pressure to evade CD8(+) T cell control over virus replicative foci.

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Figures

Figure 1.

Figure 1.

Diagrammatic alignment of the right-hand ends of the sequenced γ1-herpesviruses. ORFs of EBV, the rhesus LCV, and marmoset LCV are shown as boxes; closed symbols represent ORFs with homologues in other herpesviruses; open symbols represent genes found only in γ1-herpesviruses; and hatched symbols represent latent genes.

Figure 2.

Figure 2.

Representative cytotoxicity assays testing recognition of target cells coexpressing EBV-unique genes and target proteins. (A) Epithelial cells (top) were infected with vaccinia viruses expressing EBNA3A (vAntigen) and combinations of the indicated viruses expressing genes unique to EBV or a control vaccinia virus lacking an inserted gene (vTk−), before being incubated with HLA-B*0801–restricted CD8+ T cell clones specific for EBNA3A. EBV-transformed B cells (middle and bottom) were coinfected with modified vaccinia Ankara expressing invariant chain–targeted EBNA3C (vAntigen) and the indicated combinations of vaccinia viruses. In parallel assays, the infected B cells were incubated with either HLA-B*2705–restricted CD8+ T cell clones specific for EBNA3C (middle) or CD4+ T cell clones restricted by HLA DQ5 specific for EBNA3C (bottom). (B) EBV-transformed B cells were infected with vaccinia viruses expressing BNLF2a, and the different EBV antigens indicated (vAntigen) encoding epitopes presented through a range of HLA types. Infected cells were incubated with cognate CD8+ T cells specific for an HLA-A*0201 epitope encoded by BMLF1, an HLA-A*2402 epitope encoded by BMRF1, an HLA-B*38 epitope encoded by EBNA2, an HLA-B*2705 epitope encoded by EBNA3B, an HLA-B*0801 epitope encoded by BZLF1, or an HLA-C*0101 epitope encoded by BGLF4. Error bars represent means ± SD.

Figure 3.

Figure 3.

Expression of BNLF2a prevents CD8+ T cell recognition by disrupting antigen presentation. (A) MJS cells were retrovirally transduced to express BNLF2aHA (bottom) or control GFP (top). These cells were either infected with vaccinia viruses expressing BZLF1 or a control TK− virus, or sensitized with RAKFKQLL peptide or DMSO as a control before being incubated with CD8+ T cells specific for the BZLF1-encoded RAKFKQLL epitope in 5-h cytotoxicity assays. Error bars represent means ± SD. (B) Flow cytometry histograms of the same cell lines show surface staining for HLA class I (B9.12.1) and HLA class II (L243), or staining with an isotype control. (C) Lysates of the two cell lines were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for the HA tag (12CA5), TAP1 (148.3), TAP2 (435.4), tapasin (7F6), HLA class I heavy chains (HC10), and HLA class II DRα chains (DA6-147).

Figure 4.

Figure 4.

EBV BNLF2a blocks peptide transport by TAP. (A) TAP-dependent peptide transport in BNLF2aHA-expressing and control MJS cells was assessed by permeabilizing the cells with streptolysin O and incubating them with a fluoresceinated peptide in the presence or absence of ATP. Translocated peptides that had become glycosylated in the endoplasmic reticulum were recovered by adsorption to concanavalin A–sepharose beads. After elution, the recovered peptide was quantitated by fluorometry in arbitrary units. Error bars represent the SEM of triplicates in a representative experiment. (B) Digitonin lysates of BNLF2aHA- expressing and control MJS-GFP cells were subjected to immunoprecipitation (IP) with antibodies specific for TAP1 (148.3), TAP2 (435.4), tapasin (R.gp46C), HLA class I heavy chains (HC10), and HLA class II DRα chains (DA6-147). Cell lysates and immune complexes were separated by SDS-PAGE, followed by Western blot analysis and staining with anti-HA antibody (12CA5) to detect HA-tagged BNLF2a.

Figure 5.

Figure 5.

EBV BNLF2a inhibits peptide and ATP binding to TAP. (A) Microsomes prepared from BNLF2aHA-expressing and control MJS cells were incubated with a radiolabeled model peptide; where indicated, an excess of ICP47 competitor peptide was included in this incubation. After UV cross-linking, microsomes were lysed, and the proteins were separated by SDS-PAGE and exposed to a phosphoimaging screen. The position where TAP1 migrated in parallel immunoprecipitation experiments is shown by the arrowhead. The asterisk denotes a nonspecific background band. (B) Quantification of triplicate bands representing TAP-bound peptide. Results are shown as the percentage of peptide binding relative to peptide binding in control MJS cells (set as 100%). Error bars represent means ± SD. (C) Digitonin and NP-40 lysates of MJS-BNLF2aHA and control MJS-GFP cells were incubated with ATP-agarose beads. ATP-agarose–bound (pellet) and unbound (supernatant) protein fractions were separated by SDS-PAGE and immunoblotted. Membranes were probed with antibodies specific for TAP1 (148.3), TAP2 (435.4), tapasin (7F6), HLA class I heavy chain (HC10), and the HA tag (12CA5).

Figure 6.

Figure 6.

Old World primate γ1-herpesvirus BNLF2a sequence and function. (A) The BNLF2 regions from the indicated herpesviruses were sequenced, and the predicted amino acid sequence of the BNLF2a genes are shown. Black regions represent regions of homology between the different BNLF2a species, whereas gray-shaded regions represent conservative amino acid changes. (B) BNLF2a genes were subcloned into plasmid expression vectors that coexpressed GFP, and these were transiently transfected into MJS cells. At 48 h, surface levels of HLA class I and class II were assessed on GFP-positive cells by staining with the relevant antibody and analyzing the cells by flow cytometry. Black histograms represent surface marker intensity of cells transfected with the relevant BNLF2a, whereas open histograms represent the intensity of cells transfected with the empty vector plasmid.

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