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Presentation by Class I MHC Molecules Requires Cytoplasmic Hepatitis C Virus Envelope Glycoprotein E1 Originates in the Endoplasmic Reticulum and Requires Cytoplasmic Processing for Presentation by Class I MHC Molecules This information is current as of September 26, 2021. Mark Selby, Ann Erickson, Christine Dong, Stewart Cooper, Peter Parham, Michael Houghton and Christopher M. Walker J Immunol 1999; 162:669-676; ; http://www.jimmunol.org/content/162/2/669 Downloaded from References This article cites 45 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/162/2/669.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Hepatitis C Virus Envelope Glycoprotein E1 Originates in the Endoplasmic Reticulum and Requires Cytoplasmic Processing for Presentation by Class I MHC Molecules1 Mark Selby,* Ann Erickson,* Christine Dong,* Stewart Cooper,† Peter Parham,† Michael Houghton,* and Christopher M. Walker2* We investigated whether hepatitis C virus envelope glycoprotein E1 is transported from the endoplasmic reticulum (ER) to the cytoplasm of infected cells for class I MHC processing. Target cells expressing E1 were killed by CTL lines from a hepatitis C virus-infected chimpanzee, and synthetic peptides were used to define an epitope (amino acids 233-GNASRCWVA-241) presented by the Patr-B*1601 class I MHC molecule. An unusually high concentration (>100 nM) of this nonameric peptide was required 234 for target cell lysis, but this could be reduced at least 1000-fold by replacing the asparagine at amino acid position 234 (Asn ) Downloaded from with aspartic acid (Asp), the anticipated anchor residue for NH2-terminal peptide binding to Patr-B*1601. Conspicuously, position 234 is part of an N-glycosylation motif (Asn-Xaa-Ser/Thr), suggesting that the Asn234 to Asp substitution might occur naturally within the cell due to deglycosylation/deamidation of this amino acid by the cytosolic enzyme peptide N-glycanase. In support of this model, we demonstrate that presentation of the epitope depended on 1) cotranslational synthesis of E1 in the ER, 2) glyco- sylation of the E1 molecule, and 3) a functional TAP transporter to shuttle peptide from the cytosolic to ER compartment. These results indicate for the first time that during infection of the host, viral envelope glycoproteins originating in the ER are processed http://www.jimmunol.org/ in the cytoplasm for class I MHC presentation. That a posttranslational change in amino acid sequence from Asn to Asp alters the repertoire of peptides presented to CD81 CTL has implications for the design of antiviral vaccines. The Journal of Immu- nology, 1999, 162: 669–676. pitopes presented by class I MHC molecules are gener- Processing pathways for transmembrane glycoproteins that are ated in a multistep process that begins with attachment of not normally present in the cytoplasm remain undefined. Under E ubiquitin (Ub)3 to cytoplasmic or nuclear proteins that are some circumstances processing can occur in the ER rather than the then targeted to the 26S proteasome for degradation into peptides. cytoplasm. For instance, signal peptidase (5–8) or other unidenti- by guest on September 26, 2021 Translocation of peptides from the cytoplasm into the endoplasmic fied proteases (6, 9) can generate peptides that bind directly to reticulum (ER) is mediated by TAP, a heterodimer encoded by class I MHC molecules in the ER, thus bypassing the cytoplasmic TAP-1 and TAP-2 genes located in the MHC (1). Class I MHC Ub/proteasome/TAP pathway. Most epitopes from transmembrane molecules in the ER are closely associated with TAP and various glycoproteins are TAP dependent, however, suggesting that at chaperones that facilitate loading of antigenic peptides (2). A sta- least some processing events occur in the cytoplasm. b ble complex consisting of a class I molecule, 2m, and a peptide At least two different mechanisms could account for cytoplas- of about 8–10 amino acids, is transported to the cell surface via the 1 mic processing of proteins that normally localize to the ER. In the exocytic pathway for surveillance by CD8 CTL (3). This classi- first mechanism, transmembrane glycoproteins may occasionally cal processing pathway might not, however, generate all class I 1 be synthesized on free cytoplasmic rather than membrane-bound MHC-associated peptides displayed by a cell. CD8 CTL also ribosomes, and thus targeted for ubiquitination and destruction by recognize transmembrane glycoproteins, including viral envelope the proteasome. Studies of an HLA-B35-restricted epitope from proteins, tumor Ags, and certain alloantigens, that are cotransla- the HIV-1 gp120 envelope indicate that this pathway is functional tionally synthesized in the ER on membrane-bound ribosomes (4). in virus-infected cells (9, 10). The gp120 envelope contains a num- ber of signal motifs for Asn-linked glycosylation (Asn-Xaa-Ser/ *Chiron Corp., Emeryville, CA 94608; and †Department of Structural Biology, Stan- Thr, where Xaa is any amino acid except Pro), and all are known ford University, Stanford, CA 94305 to be modified by glycans during synthesis of the polyprotein in Received for publication August 10, 1998. Accepted for publication September the ER (11). One of these Asn residues is contained in the epitope, 24, 1998. but surprisingly was not glycosylated before presentation by the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance HLA-B35 molecules (10). This result suggests that gp120 polypro- with 18 U.S.C. Section 1734 solely to indicate this fact. tein produced in the cytoplasm because of an error in translation 1 This work was supported by Chiron Corp., National Institutes of Health Grant (12–14) or possibly signal peptide-mediated targeting to the ER (9, AI31168 (to P.P.), and a postdoctoral fellowship from the Arthritis Foundation (to S.C.). 10) was the substrate for class I MHC processing. A second mechanism appears to involve export of defective 2 Address correspondence and reprint requests to Dr. Christopher Walker, Department of Pediatrics, Children’s Hospital, Room W503, 700 Children’s Dr., Columbus, OH transmembrane glycoproteins from the ER to the cytoplasm for 43205. E-mail address: [email protected] proteasome-mediated destruction (15–20). This pathway is sup- 3 Abbreviations used in this paper: Ub, ubiquitin; ER, endoplasmic reticulum; ported by recent studies of class I MHC-restricted epitopes in the PNGase, peptide:N-glycanase; E1, envelope glycoprotein 1 of hepatitis C virus; HCV, hepatitis C virus; NS3, nonstructural 3; moi, multiplicity of infection; LCL, lympho- influenza virus nucleoprotein (21) and the cellular glycoprotein blastoid cell line. tyrosinase that is expressed in melanocytes (22, 23). In the case of Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 670 AN ER TO CYTOSOL Ag PROCESSING PATHWAY FOR VIRAL GLYCOPROTEINS tyrosinase, it was demonstrated that efficient presentation of one der of E1 to amino acid 383. The internal primers were designated E1-M5 TAP-dependent epitope by HLA-A2 required posttranslational and E1-M3 (CGCGAGGGCGACGCCTCTAGATGTTGG and CGCCAC- conversion of an encoded Asn residue to Asp (22). This required CCAACATCTAGAGGCGTCGCC). The identity of the resulting clones was confirmed by sequencing. Subsequently, the E1-coding sequences cotranslational glycosylation of tyrosinase in the ER and subse- were transferred into pSC11 for generation of recombinant vaccinia vi- quent translocation of the Ag to the cytoplasm where it was deg- ruses. An 80% confluent monolayer of African green monkey BSC-40 cells lycosylated (23), probably by peptide:N-glycanase (PNGase) (24, on a 60-mm dish was infected with 0.05 plaque-forming units of wild-type vaccinia virus (WR strain)/cell. After2hofinfection at 37°C, the cells 25). Removal of glycans by this cytoplasmic enzyme causes a 1 1 were transfected with 12.5 mg of pSC11 E1s1, pSC11 E1s2, and coding change from Asn to Asp by a process of deamidation, and 1 pSC11 E1s2D234 DNA using lipofectin reagent (Life Technologies, it is this modified form of tyrosinase that is processed by the Ub/ Grand Island, NY). Virions were harvested 2 days later, and serial dilutions proteasome/Tap pathway. Whether this processing pathway ap- (from 1021 to 1024) of each were used to infect 80% confluent monolayers plies generally to other glycoproteins is not yet known. of thymidine kinase-deficient human osteosarcoma 143 B cells on 60-mm In this study we demonstrate for the first time that the ER to dishes. After2hat37°C the inoculum was aspirated off the cells. An overlay that included 1% sea plaque agarose and 50 mg/ml 5-bromode- cytosol processing pathway is not restricted to cellular glycopro- oxyuridine was added to infected monolayers, and 3 days later the dishes teins such as tyrosinase, but is also operative for viral glycopro- were overlaid with 1% sea plaque agarose containing 0.03% 5-bromo-4- teins produced in infected cells. Class I MHC-restricted CTL spe- chloro-3-indolyl-b-D-galactoside. Each blue recombinant plaque was cific for envelope glycoproteins E1 and E2 of hepatitis C virus picked from a dish as an agarose plug and transferred into DMEM.
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