Interactions between herpesvirus-encoded TAP-inhibitors and their target.

<p>Upper illustration: model of the TAP transporter, comprising the two subunits TAP1 and TAP2. Each subunit contains a transmembrane domain (TMD), encompassing 10 and 9 transmembrane (TM) helices for TAP1 and TAP2, respectively. The outer N-terminal helices of TAP1 and TAP2 (TMD0) form an autonomous binding platform for tapasin, whereas the core 6 TM helices are necessary for peptide transport. A peptide-binding domain is located within the cytosolic extensions of the TM helices. In addition, TAP1 and TAP2 contain a nucleotide-binding domain (NBD) in the cytosol, which harbors two ATP-binding sites. Lower illustrations: schematic representations of the interaction between the viral proteins and TAP. The sites where TAP is affected are indicated. HSV-1 ICP47 prevents peptide transport by physically obstructing the peptide-binding site. PRV, BoHV-1 and EHV (EHV-1 and EHV-4) UL49.5 leave the transporter in a transformation-incompetent conformation, thereby preventing the structural changes that are needed to translocate peptides over the ER membrane. BoHV-1 UL49.5 is known to interact with a region within the core domain of TAP, comprising the C-terminal 6 TM domains of both TAP1 and TAP2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004743#ppat.1004743.ref132" target="_blank">132</a>]. BoHV-1 UL49.5 induces the degradation of both TAP subunits, and EHV UL49.5 prevents ATP binding to TAP. HCMV US6 blocks TAP by inducing conformational changes that result in diminished ATP binding to TAP1. The protein interacts with TM domains 7–10 of TAP 1 and TM 1–4 of TAP2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004743#ppat.1004743.ref095" target="_blank">95</a>]. EBV BNLF2a inhibits peptide transport by interfering with both peptide and ATP binding to TAP.</p

Phylogenetic tree for selected members of the family Herpesviridae.

<p>The Bayesian tree is based on amino acid sequence alignments for six large, well-conserved genes, namely the orthologs of HSV-1 genes UL15, UL19, UL27, UL28, UL29, and UL30, and is derived from McGeoch and Davison [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004743#ppat.1004743.ref081" target="_blank">81</a>]. Assignments to genera and subfamilies are shown on the right. Abbreviations not mentioned in the text are: MDV-1, Marek's disease virus type 1; MDV-2, Marek's disease virus type 2; HVT, herpesvirus of turkey; ILTV, infectious laryngotracheitis virus; PsHV-1, psitticid herpesvirus 1; GTHV, green turtle herpesvirus; THV, tupaia herpesvirus; GPCMV, guinea pig cytomegalovirus; CalHV-3, callitrichine herpesvirus 3; AHV-1, alcelaphine herpesvirus 1; OHV-2, ovine herpesvirus 2; PLHV-1, porcine lymphotropic herpesvirus 1; HVS, herpesvirus saimiri; HVA, herpesvirus ateles; and RRV, rhesus rhadinovirus. Red, blue, orange, and green shading indicate viruses that encode the ICP47, UL49.5, US6, or BNLF2a TAP inhibitor genes, respectively, and corresponding coloring of virus abbreviations indicate viruses in which these genes have been shown to be functional TAP inhibitors. Light orange shading identifies all members of the <i>Cytomegalovirus</i> genus that have a US6 gene. Light blue shading indicates all members of the herpesvirus family that code for a UL49.5 gene that might be involved in chaperoning maturation of glycoprotein M.</p

Effect of BILF1 on maturation and degradation of MHC class I molecules.

<p>(A) Acquisition of endoglycosidase H (Endo H) -resistance of MHC class I heavy chain. 293 cells (2×10⁶) stably transduced with control or BILF1 retrovirus were metabolically labeled for 15 min with ³⁵S-methionine/cysteine and chased for the indicated time periods. After lysis in NP-40 detergent buffer, samples were immunoprecipitated with mAb W6/32 and treated with Endo H enzyme. Protein samples were separated by 10% acrylamide SDS/PAGE gel, dried and exposed to autoradiography. (B) Kinetics of MHC class I molecule degradation. 293 cells (2×10⁶) stably transduced with control or BILF1 retrovirus were metabolically labeled for 15 min and chased for the indicated time periods. After lysis in NP-40 detergent buffer, samples were immunoprecipitated with mAb W6/32 (HLA class I heavy chain and β₂-microglobulin) or H68.4 (TfR). Protein samples were separated by 10% SDS/PAGE gel, dried and exposed to autoradiography.</p

Characterization of cells stably transduced with a BILF1 retroviral vector.

<p>(A) 293 or MJS cells were stably transduced with control (pQCXIH) or BILF1 (pQCXIH-HABILF1) retrovirus. Surface MHC class I molecules were stained with PE-conjugated W6/32 antibodies and analyzed by flow cytometry. The solid line histograms depict the surface HLA class I staining of control cell lines, while the dashed line histogram depicts the surface HLA class I staining of cell lines expressing BILF1. The grey histogram illustrates background staining obtained with an isotype control PE-conjugated antibody. (B) Total cell lysates were generated from the retrovirus-transduced 293 and MJS cell lines, and 2×10⁵ cell equivalents were separated by SDS-PAGE and analyzed by Western Blotting with mAbs specific for BILF1 (3F10, anti-HA tag), MHC class I (HC10), MHC class II (DA6.147), TAP-1 (148.3), TAP-2 (435.3), TfR (H68.4) or with polyclonal antibodies to calregulin as a loading control.</p

BILF1 inhibits T cell recognition of endogenous EBV antigen in MJS cells.

<p>(A) MJS cells were co-transfected with 0.02 µg p509 plasmid (BZLF1 expression vector) and different amounts (0–2 µg) of pCDNA3-HABILF1-IRES-nlsGFP bulked to a constant amount of DNA with control plasmid. At 24 hr post-transfection, the MJS cells were co-cultured with CD8⁺ effector ‘RAK’ T cells for a further 18 hrs and the supernatants were tested for the release of IFN-γ as a measure of T cell recognition. All results are expressed as IFN-γ release in pg/ml and error bars indicate standard deviation of triplicate cultures. (B) Total cell lysates were generated from the above transfections, and 2×10⁵ cell equivalents were separated and analyzed by Western Blotting using antibodies specific for BZLF1, HA tag (BILF1), or calregulin as a loading control.</p

Lysosomal inhibitors block BILF1-enhanced degradation of MHC class I.

<p>(A) 293 cells stably transduced with control- (c) or BILF1- (b) retrovirus were treated with or without Bafilomycin A1, concanamycin A, or leupeptin for 20 hr. Lysates from 2×10⁵ cell equivalents were separated by SDS/PAGE gel, and analyzed by western blotting using antibodies specific for MHC class I (HC10) and calregulin. The blot is one representative of three independent experiments. The histogram shows the mean results (±S.D.) of quantification by densitometry of all the blots from 3 independent experiments, where the densities of the HC10 bands were normalized relative to their own calregulin loading control. (B) 293 cells stably transduced with control or BILF1 retrovirus were treated with concanamycin A (50 nM) for 6 hr prior to fixation and permeabilization with methanol/acetone, then stained with W6/32 primary antibodies and Alexa Fluor® 488 goat anti-mouse IgG secondary antibodies. The photographs were obtained with a conventional fluorescence microscope. (C) 293 cells stably transduced with control (c) or BILF1 (b) retrovirus were treated with or without the proteasome inhibitor, MG132, for 20 hr, and analyzed by western blot as in panel A. The additional, lower molecular weight species detected is probably deglycosylated and/or partially degraded free heavy chain that is normally targeted for proteasomal degradation.</p

BILF1 associates with MHC class I molecules at the cell surface and increases their rate of internalization.

<p>(A) BILF1 is predominantly localized at the cell surface. 293 cells stably transduced with BILF1 retrovirus were grown on glass slides, fixed and permeabilized, then stained with rat anti-HA (3F10) primary antibodies and Alexa Fluor® 594 goat anti-rat IgG. The nuclei were counterstained with DAPI. The stained slides were analyzed with a laser scanning confocal microscope, and the three photographs show different 1 micron-thick sections through representative cells. BILF1 stained red, and the nuclei stained blue. (B) BILF1 and MHC class I molecules co-precipitate at the cell surface. 293 cells (2×10⁶) stably transduced with control (c) or BILF1 (b) retrovirus were incubated with saturating concentrations of antibodies specific for MHC class I (W6/32), TfR (H68.4) or HA tagged BILF1 (3F10) on ice. After washing away excess antibody, the cells were lysed with NP40 detergent buffer, then precipitated with protein A/G beads and subjected to western-blotting as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000255#ppat-1000255-g006" target="_blank">Fig. 6B</a>, using antibodies specific for MHC class I (HC10) and HA tagged BILF1 (3F10). (C) BILF1 increases the rate of internalization of MHC class I, but not class II, from the cell surface. MJS cells stably transduced with control or BILF1 retrovirus were incubated at 0°C with saturating concentrations of mAb to MHC class I (W6/32; top graph) or MHC class II (L234; bottom graph), then washed and incubated at 37°C for different periods of time. The cells were subsequently stained with PE-conjugated goat anti-mouse IgG antibody, and analyzed by flow cytometry. The mean fluorescence intensities of staining were averaged for triplicate samples, and normalized to the initial time 0 min samples. (D) BILF1 increases the rate of internalization, but not the rate of appearance, of MHC class I at the cell surface. Top graph: 293 cells stably transduced with control or BILF1 retrovirus were incubated at 0°C with saturating concentrations of mAb to MHC class I (W6/32), then treated exactly as for the internalization assay performed with MJS cells in panel C. Bottom graph: replicate aliquots of the saturated W6/32-bound cells were harvested at the indicated time points, and the appearance of new MHC class I molecules was assayed by staining with PE-conjugated W6/32 antibody. The mean fluorescence intensities of staining were averaged for triplicate samples.</p

BILF1 downregulates surface MHC class I expression and inhibits the T cell recognition of endogenous EBV antigen in LCLs.

<p>(A) LCLs were transduced with PLZRS-HABILF1-IRES-GFP retrovirus. After 6 days, surface MHC class I was stained with PE-conjugated W6/32 mAb and MHC class II was stained with PE-conjugated anti-DR mAb, YE2/36-HLK. Two-colour flow cytometry was used to analyze staining in the untransduced, GFP⁻, population, shown as the solid line histogram, and in the transduced GFP⁺ (BILF1⁺) population, shown as the dashed line histogram. The grey histogram denotes background staining obtained with an isotype control PE-conjugated antibody. (B) LCL cultures transduced with control retrovirus or with the BILF1 retrovirus were sorted by flow cytometry to generate GFP⁺/BILF1⁻ and GFP⁺/BILF1⁺ lines to use as targets in assays with EBV-specific T cells. The control and BILF1⁺ LCL targets were incubated with HLA-matched CD8⁺ effector T cells clones specific for EBNA1 (HPV), EBNA3A (YPL), or LMP2A (CLG) peptides, or a CD4⁺ effector T cell clone specific for a BHRF1 (PYY) peptide. After 18 hrs the supernatants were tested for the release of IFN-γ as a measure of T cell recognition. All results are expressed as IFN-γ release in pg/ml and error bars indicate standard deviation of triplicate cultures.</p

<i>BILF1</i> identified as a lytic gene that downregulates surface MHC class I.

<p>293 (A) or MJS (B) cells were transfected with different EBV genes in the bi-cistronic vector, pCDNA3-IRES-nlsGFP. At 48 hr post-transfection, surface MHC class I was stained with PE-conjugated W6/32 mAb and (in MJS only) MHC class II was stained with PE-conjugated anti-DR mAb, YE2/36-HLK. Two-colour flow cytometry was used to analyse staining in the untransfected GFP⁻ population, shown as the solid line histogram, and in the transfected GFP⁺ population, shown as the dashed line histogram. The grey histogram denotes background staining obtained with an isotype control PE-conjugated antibody.</p

BILF1 is physically associated with the MHC class molecule complex.

<p>(A) 293 cells (10⁷) stably transduced with control (c) or BILF1 (b) retrovirus were metabolically labeled for 15 min and chased for 20 min. After lysis in NP-40 buffer and immunoprecipitation with mAb W6/32, the samples were dissociated by boiling in reducing sample buffer, and were re-precipitated with either 3F10 ( HA tag on BILF1) or HC10 (MHC class I heavy chain). Protein samples were separated by 10% acrylamide SDS/PAGE gel, dried and exposed to autoradiography. The arrowhead and bracket indicate the presence of 33 kD and 45–55 kD BILF1 bands, whilst the asterisks indicate probable ubiquitinated MHC class I species. (B) 293 cells (2×10⁶) stably transduced with control (c) or BILF1(b) retrovirus were treated with concanamycin A (50 nM) for 20 hr prior to lysing the cells with NP40 detergent buffer and immunoprecipitation with antibodies specific for MHC class I (W6/32), HA tagged BILF1 (12CA5), or TfR (H68.4). Cell lysates and immune complexes were separated by SDS/PAGE gel, and analyzed by western blotting using antibodies specific for MHC class I (HC10), HA tagged BILF1 (3F10), and TfR (H68.4). The first two samples on the gel are total cell lysates representing 5% of the input lysate for immunoprecipitations.</p