Cytotoxic T lymphocytes lyse target cells after T-cell-receptor-mediated recognition of class I major histocompatibility complex molecules presenting peptides. Antigenic peptides are generated in the cytoplasm by proteasomes and translocated into the lumen of the endoplasmic reticulum (ER) by peptide transporters (TAP). Herpes simplex virus (HSV) expresses a cytoplasmic protein, ICP47, which seems to interfere with such immune surveillance by mediating retention of 'empty' class I molecules in the ER. By expressing ICP47 in HeLa cells under an inducible promoter, we show that ICP47 efficiently inhibits peptide transport across the ER membrane such that nascent class I molecules fail to acquire antigenic peptides. This inhibition was overcome by transfecting murine TAP. Further, we demonstrate that ICP47 colocalizes and physically associates with TAP within the cell. Inhibition of peptide translocation by a viral protein indicates a previously undocumented potential mechanism for viral immune evasion.

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)‐mediated translocation of antigen‐derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV‐infected cells by cytotoxic T‐lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species‐specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8‐azido‐ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP‐transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100‐fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate‐binding site of TAP.

Herpes simplex virus serotype 1 (HSV-1) expresses an immediate-early protein, ICP47, that effectively blocks the major histocompatibility complex class I antigen presentation pathway. HSV-1 ICP47 (ICP47-1) binds with high affinity to the human transporter associated with antigen presentation (TAP) and blocks the binding of antigenic peptides. HSV type 2 (HSV-2) ICP47 (ICP47-2) has only 42% amino acid sequence identity with ICP47-1. Here, we compared the levels of inhibition of human and murine TAP, expressed in insect cell microsomes, by ICP47-1 and ICP47-2. Both proteins inhibited human TAP at similar concentrations, and the KD for ICP47-2 binding to human TAP was 4.8 × 10−8 M, virtually identical to that measured for ICP47-1 (5.2 × 10−8 M). There was some inhibition of murine TAP by both ICP47-2 and ICP47-1, but this inhibition was incomplete and only at ICP47 concentrations 50 to 100 times that required to inhibit human TAP. Lack of inhibition of murine TAP by ICP47-1 and ICP47-2 could be explained by an inability of both proteins to bind to murine TAP.

The nonobese diabetic (NOD) mouse, in which major histocompatibility complex genes may be involved in the susceptibility to diabetes, has been developed as a model of autoimmune diabetes. The NOD mouse expresses I-A- (20) were grown in RPMI 1640 medium supplemented with 10o fetal calf serum, 2 mM glutamine, penicillin at 100 units/ml, and streptomycin at 100 jg/ml (GIBCO/BRL). Normal islets were prepared by collagenase (Sigma) digestion of mouse pancreases as described by Lacy and Kotianovsky (21) with slight modifications (22).Western Blots. Cells (1 x 108) from each cell line were washed in Hanks' balanced saline solution, suspended in 7 ml of 0.01M Tris-buffered saline (TBS)/10 mM CaCl2/0.25 M sucrose, pH 7.4, and disrupted at 40C. The cell homogenates were centrifuged (1000 x g, 10 min), and the supernatant was subjected to ultracentrifugation (20,000 x g, 1 h). The pellet was then resuspended in 2.3% SDS/62.5 mM Tris/10%6 glycerol/5% 2-mercaptoethanol, pH 6.8. One-dimensional gel electrophoresis and electrotransfers to nitrocellulose filters were performed as described by Laemmli (22) and Burnette (23). Two-dimensional SDS/PAGE was performed as described by O'Farrell (24). The filters were soaked in 0.02 M TBS/3% gelatin, washed in 0.05% Tween 20/0.02 M TBS, and then incubated with the test sera or ascitic fluid diluted 1:50 in 1% gelatin/0.05% Tween 20/0.02 M TBS. After washing, they were incubated with biotinylated sheep antiAbbreviations: NOD, nonobese diabetic; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; NCS, newborn calf serum; MHC, major histocompatibility complex. tTo whom reprint requests should be addressed. 172The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by two transmembrane receptor subunits, IFNAR1 and IFNAR2. To elucidate the role of IFNAR1 in IFN binding and the establishment of biological activity, specific residues of IFNAR1 were mutated. Residues (62)FSSLKLNVY(70) of the S5-S6 loop of the N-terminal subdomain of IFNAR1 and tryptophan-129 of the second subdomain of IFNAR1 were shown to be crucial for IFN-alpha binding and signaling and establishment of biological activity. Mutagenesis of peptide (278)LRV in the third subdomain shows that these residues are critical for IFN-alpha-induced biological activity but not for ligand binding. These data, together with the sequence homology of IFNAR1 with cytokine receptors of known structure and the recently resolved NMR structure of IFNAR2, led to the establishment of a three-dimensional model of the human IFN-alpha/IFNAR1/IFNAR2 complex. This model predicts that following binding of IFN to IFNAR1 and IFNAR2 the receptor complex assumes a "closed form", in which the N-terminal domain of IFNAR1 acts as a lid, resulting in the activation of intracellular kinases. Differences in the primary sequence of individual IFN-alpha subtypes and resulting differences in binding affinity, duration of ligand/receptor association, or both would explain differences in intracellular signal intensities and biological activity observed for individual IFN-alpha subtypes.