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The Journal of Immunology, 1999, 162: 1324-1332.
Copyright © 1999 by The American Association of Immunologists

Processing of HIV-1 Envelope Glycoprotein for Class I-Restricted Recognition: Dependence on TAP1/2 and Mechanisms for Cytosolic Localization1

Robert L. Ferris*,{dagger}, Christopher Hall*, Nikolaos V. Sipsas{ddagger}, Jeffrey T. Safrit§, Alicja Trocha{ddagger}, Richard A. Koup, R. Paul Johnson{ddagger},|| and Robert F. Siliciano2,*

* Department of Medicine, Johns Hopkins University School of Medicine, Baltimore MD, 21205; {dagger} Immunology Graduate Program and Department of Otolaryngology/Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, MD 21205; {ddagger} AIDS Research Service and Infectious Disease Unit, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129; § Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30329; Department of Medicine, Division of Infectious Diseases, University of Texas Southwestern Medical School, Dallas, TX 75235; and || New England Primate Research Center, Harvard Medical School, Southborough, MA 01772


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Processing of viral proteins for recognition by CTL involves degradation of the proteins in the cytosol of an infected cell followed by transport of the resulting peptides into the endoplasmic reticulum (ER) by the TAP1/2 complex. Uncertainty exists over the site of processing of viral envelope (env) proteins since the extracellular domains of env proteins are not present in the cytosol where the class I Ag-processing pathway begins. Rather, the ectodomains of env proteins are cotranslationally translocated into the ER during biosynthesis. To analyze env protein processing, we used the herpes simplex virus protein ICP47 to block peptide transport by TAP1/2 and examined the effects of TAP blockade on the processing of the HIV-1 env protein. For the majority of env-specific CD8+ CTL, the processing pathway required TAP1/2-mediated transport of cytosolic peptides into the ER. To determine how env peptides are generated in the cytosol, we analyzed the processing of two TAP1/2-dependent epitopes containing N-linked glycosylation sites. In each case, processing involved glycosylation-dependent posttranslational modification of asparagine residues to aspartic acid. These results are consistent with cotranslational translocation of env into the ER, where glycosylation occurs. This is followed by export of a fraction of the newly synthesized protein into the cytosol, where it is deglycosylated, with conversion of the asparagines to aspartic acid residues. Following cytoplasmic proteolysis, env peptides are retransported by TAP1/2 into the ER, where association with class I occurs. Thus, the env protein can enter the class I pathway through multiple distinct processing mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The recognition of virally infected cells by CD8+ CTL is dependent upon a complex series of degradation, transport, and association reactions that permit peptide fragments of endogenously synthesized viral proteins to be displayed on the surfaces of infected cells in association with class I MHC molecules see Refs. (1, 2, 3) for comprehensive reviews). For viral proteins synthesized on free ribosomes in the cytoplasm of an infected cell, processing involves the degradation of some fraction of the viral protein in the cytoplasm (4, 5, 6) and transport of the resulting peptides into the endoplasmic reticulum (ER)3 by the TAP1/2 complex (7, 8, 9, 10, 11, 12, 13, 14, 15, 16). Following transport into the ER, peptides can bind newly synthesized MHC class I molecules, which are noncovalently associated with the TAP1/2 heterodimer (17, 18, 19, 20). The peptide-MHC complexes then progress through the exocytic pathway to the cell surface for recognition by CD8+ T cells.

While the processing of viral proteins localized to the cytosol or nucleus has been relatively well defined, the processing of viral envelope (env) proteins is less clearly understood. The ectodomains of viral env proteins are cotranslationally translocated into the ER during biosynthesis. Uncertainty exists over the site of processing of viral env proteins, since these proteins are not normally present in intact form in the cytosol, the site where the class I-restricted Ag-processing pathway begins (reviewed in 21 .

Recent analyses of other membrane and secreted proteins have shown that reverse translocation of newly synthesized protein to the cytosol may occur. This provides a quality control mechanism for degrading misfolded cellular proteins following their cotranslocational translocation into the ER (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). It is not clear whether such a mechanism has an important role in the processing of viral proteins to generate class I-restricted epitopes. Mosse et al. (34) have reported that processing of an epitope in the melanocyte protein, tyrosinase, involves conversion of an asparagine residue in the epitope to aspartic acid (35). This asparagine is part of an N-linked glycosylation site in the protein, suggesting that processing involves cotranslational translocation of full-length tyrosinase into the ER before extrusion back to the cytosol for deglycosylation and degradation. Consistent with this conclusion, Bacik et al. (36) engineered an N-linked glycosylation site into an influenza virus nucleoprotein (NP) epitope. This protein was processed in a TAP1/2-dependent fashion, whether NP was expressed as a cytosolic protein or artificially as a secreted protein via addition of a signal sequence. The recognition of this epitope was selectively reduced for secreted NP, suggesting that the epitope was exposed to the glycosylation machinery in the ER lumen before its export from the secretory pathway back to the cytosol for processing. However, it is unclear whether viral env proteins normally undergo such reverse translocation and deglycosylation steps during processing.

HIV-1 env-specific CTL appear to be an important component of the initial cellular immune response to acute HIV-1 infection (37). Recent studies suggest a correlation between env-specific memory CTL and control of viral replication and slower declines in CD4+ cell counts (38, 39). Late in the asymptomatic phase of infection, viral escape mutations may enable the evolution of strains that are able to resist this anti-HIV-1 env CTL response, contributing to the development of immunodeficiency (40).

We have previously analyzed the processing of the HIV-1 env protein for recognition by CTL. Both TAP1/2-independent and TAP1/2-dependent pathways have been identified (41, 42). TAP1/2-dependent pathways are of particular interest because they require a mechanism of generating env peptides in the cytosol. We have studied the processing of a TAP1/2-dependent HIV-1 env epitope containing a normally utilized N-glycosylation site and used the structure of the minimal epitope to infer the processing mechanism that resulted in its generation (43). An HLA B*3501-restricted CTL clone preferentially (>5 logs) recognized a form of the epitope containing a free asparagine (N form) that had never been modified by the ER glycosylation machinery, suggesting that a fraction of HIV-1 env may be translated on free ribosomes in the cytosol to generate this B*3501-restricted epitope.

The current study demonstrates that most class I-restricted epitopes in the HIV-1 env protein are generated by TAP1/2-dependent mechanisms. In addition to the cytosolic translation mechanism described above, we show that the env protein can also be processed by a second TAP1/2-dependent mechanism. This pathway involves cotranslational translocation into the ER, glycosylation, export back to the cytosol, and deglycosylation for processing and retransport into the ER by TAP1/2 for association with class I molecules. These studies of HIV-1 env processing, using the same method of analysis to detect access of the protein to the ER lumen, show that two distinct mechanisms of TAP1/2-dependent processing can occur for the same protein, HIV-1 env. Our findings suggest that glycosylation-dependent posttranslational modification of particular asparagine residues after exposure to the ER results in optimal recognition of certain epitopes. This suggests that cytoplasmic mislocalization of membrane proteins to enhance presentation of class I-restricted epitopes (44, 45) may not be beneficial for certain epitopes. These findings have implications for designing strategies to enhance the CTL response against viral env proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
CTL clones and cell lines

CTL clones used in this study are described in Table IGo and have been previously described (46, 47, 48, 49, 50, 51). The HLA Cw*08-restricted CD8+ CTL clone LWF A5 was isolated from an HIV-1 seropositive individual (LWF) who was exposed to the HIV-1 IIIB strain in 1985 (48). CTL clone 11/1e was isolated from an HIV-1 seropositive donor within weeks of seroconversion (49). All CTL clones were restimulated with PHA or anti-CD3 mAb 12F6 and irradiated allogeneic PBMC at intervals of 14–28 days, as described (42). The use of TAP1/2-negative T2 cell line has been described (42), with the appropriate restriction element expressed either by stable transfection or by recombinant vaccinia virus (rVV), as indicated in Table IGo. CTL clones tested in this study were also characterized for TAP1/2-dependence using autologous B-lymphoblastoid cell line (B-LCL) infected with rVV expressing the herpes simplex virus (HSV) protein ICP47 for 1 h at 37°C before coinfection with HIV-1 vaccinia vectors expressing env for an additional 2 h, as detailed in Fig. 1Go.


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  		Table I. Summary of CD8+ CTL clones described in this study

 


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  		FIGURE 1. HSV ICP47 selectively blocks peptide transport by the TAP1/2 complex. The TAP1/2-independent CTL clone A42.46 (A) and the TAP1/2-dependent CTL clone LWF A5 (B) were used in a standard 51Cr-release assay to determine the role of TAP1/2-mediated peptide transport in presentation of their respective epitopes in target cells expressing HSV ICP47. Autologous B-LCL were either infected with a control vaccinia vector (vSC8, open symbols) or vvICP47 (closed symbols) for 1 h at 37°C. Target cells infected with vSC8 or vvICP47 were then coinfected with a vaccinia vector encoding full-length HIV env protein (vPE16, circles) or a vaccinia vector encoding a signal sequence minus form of the HIV env protein (vPE11, squares) for 2 h at 37°C. Negative control targets were either mock-infected (open diamonds), infected with a control vaccinia vector (vSC8, closed triangles), or co-infected with vvICP47 and vSC8 (crosses). Additional controls included targets infected with vSC8 or vvICP47 and pulsed with epitopic peptide. All targets were incubated for 14–16 h and a standard 51Cr-release assay was performed at the indicated E:T ratios.

 
rVV vectors

The vector vPE7 contains the full-length env gene of LAI isolate (BH8 clone) of HIV-1; the vector vPE16 contains the same env gene with silent mutations that remove cryptic VV transcription termination sequences. The vector vPE11 possesses a 30-amino acid deletion eliminating the signal peptide of the HIV-1 env gene. These vectors were kindly provided by Drs. Patricia Earl and Bernard Moss of the National Institutes of Health and have been previously described (52). The vector vvICP47 (53) was a gift of Dr. Barry Rouse (University of Tennessee, Knoxville, TN).

Synthetic peptides

Synthetic peptides corresponding to env sequences from the BH8 clone of the LAI isolate of HIV-1 were purchased from Bio-Synthesis (Lewisville, Texas). Glycosylated env156–165N*N* and env 298–307N* were made using conventional solid-phase synthesis, except that an activated aspartic acid derivative bound to N-acetyl D-glucosamine was used during the coupling reaction in place of asparagine (43). Peptide concentrations were measured by amino acid analysis or bicinchoninic acid (BCA) kit (Pierce, Rockford, IL). After reverse phase-HPLC purification to >90% purity, peptide sequences were confirmed by amino acid analysis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Lyophilized peptides were reconstituted at 1–2 mg/ml in 10% DMSO and water or in 100% DMSO and diluted into culture medium (RPMI 1640, 10% FCS, 4 mM L-glutamine, and 100 µg/ml of penicillin and streptomycin) before use with or without DTT at 1–2 mM.

Cytotoxicity assays

Cytolytic activity was measured in conventional 4-h 51Cr-release assays as described (42). For peptide titrations, autologous EBV-transformed B-LCL were incubated with indicated peptides after 51Cr labeling. Effector cells were added at an effector/target ratio as indicated, and specific lysis was measured after 4 h at 37°C in a standard 51Cr-release assay. Background lysis was <25% of maximal lysis.

Proteasome inhibitors

Lactacystin (54) was purchased from Dr. E. J. Corey (Harvard University). ZL3VS (55, 56) was a generous gift of Dr. Hidde Ploegh (Harvard University).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TAP1/2-dependent processing of the HIV-1 env protein

Previous studies have shown that the HIV-1 env protein can be processed by both TAP1/2-dependent and TAP1/2-independent mechanisms (41, 42). To determine which mechanism represents the major pathway by which this protein is processed for recognition by CTL, we used a panel of env-specific CD8+ CTL clones recognizing defined epitopes in the env protein. To investigate whether processing of each epitope was TAP1/2-dependent, we coexpressed the env protein in target cells already expressing HSV ICP47, a 7-kDa cytosolic protein that interacts with the cytosolic peptide binding site on the TAP1/2 heterodimer with a much higher affinity than most transported peptides (57, 58, 59). Results obtained in CTL assays using ICP47 were concordant with previous studies using TAP1/2-defective target cells. Thus, for an A*0301-restricted clone, no inhibition by ICP47 was observed (Fig. 1GoA), consistent with previous conclusions that this epitope can be generated by a novel TAP1/2-independent mechanism localized to the ER (41). As an additional control to demonstrate the ability of ICP47 to block maximal levels of TAP1/2-dependent transport, a cytosolic form of env (vPE11) was expressed without a signal sequence in separate targets, so that all of the epitope generated would require TAP1/2 for presentation. In targets expressing cytosolic env, all presentation of the epitope was blocked when vvICP47, but not the control vaccinia, vSC8, was used to preinfect the targets. Similar results were obtained with clones, including a nef-specific CTL clone (data not shown). The inhibition by ICP47 did not reflect a nonspecific inhibition of lysis since vvICP47-infected targets were readily lysed when they were pulsed with the appropriate epitopic peptide (Fig. 1GoA).

Using this system, the TAP1/2-dependence of a series of additional clones was evaluated. In contrast to results obtained with the A*0301-restricted clone, prior expression of ICP47 in env-expressing targets prevented recognition by the Cw*08-restricted clone LWF A5 (Fig. 1GoB). As expected, ICP47 also completely inhibited lysis of targets cells expressing a signal sequence minus form of the env protein. These results indicate that the processing of the env protein to generate the epitope seen by this clone is strictly TAP1/2-dependent. When env was expressed in targets preinfected with a control vaccinia (vSC8), however, lysis was readily detectable.

Table IGo lists information regarding the CTL clones that were tested using vvICP47. Five CD8+ clones specific for different env epitopes and previously uncharacterized with respect to TAP1/2-dependence were all found to be TAP1/2-dependent. These data, combined with six previously characterized env-specific CTL clones demonstrate that most epitopes appear to require TAP1/2 for presentation.

Polyclonal CTL cultures generated by stimulation of PBMC from an HIV-1-infected donor (60) were also assayed using ICP47 to corroborate the clonal data and to control for bias when isolating individual clones. One HIV-1+ donor who had a consistently detectable CTL response against HIV-1 env with minimal background of vaccinia-specific CTL was used as a source of PBMC. This donor had the class I MHC genotype A2, A3, B7, B14. Thus, the donor expressed two alleles that are associated with TAP-independent presentation at the clonal level. A representative experiment is shown in Fig. 2Go, demonstrating exclusively TAP1/2-dependent presentation of all env epitopes recognized by the bulk CTL culture from an HIV+ individual. Taken together, these results lead us to conclude that the TAP1/2-dependent pathways represent the primary mechanism of processing for HIV-1 env.



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  		FIGURE 2. Polyclonal CD8+ CTL from an HIV+ individual are TAP1/2-dependent. CTL were derived from PBMC from an HIV+ volunteer. PBMC were subjected to env-specific stimulation for 10 days (60), before depletion of CD4+ cells by mAb-conjugated beads on the day of the CTL assay. Autologous B-LCL were either mock-infected or infected with vSC8 or vvICP47 for 1 h at 37°C. As indicated on the x-axis, target cells infected with vSC8 or vvICP47 were then coinfected with either vSC8, vPE16, or vPE11 for 2 h at 37°C. Bulk CTL effectors were added to these targets at an E:T ratio of 50:1 for 4 h at 37°C. Vaccinia specific background lysis was subtracted from y-axis values.

 
Optimal form of the HLA Cw*08 epitope in HIV-1 gp120 recognized by clone LWF A5

Previous studies have characterized in detail an HLA Cw*08-restricted epitope recognized by clone LWF A5, which was isolated from a laboratory worker who was exposed to HIV-1 in 1985 (48). This epitope is located in the extracellular domain of the gp120 subunit of the HIV-1 env protein (Fig. 3Go). As shown above, processing of this epitope is TAP1/2-dependent. The Cw*08-restricted epitope represents residues 156–165 of the HIV-1LAI env protein and contains two potential N-linked glycosylation sites (Fig. 3Go). Both glycosylation sites have been carefully studied and have been shown to be utilized in vivo (61). Gregory and colleagues (61) have confirmed the presence of exclusively complex-type oligosaccharides at both asparagine residues in the epitope. Deglycosylation by the only known mammalian deglycosylation enzyme, peptide:N-glycanase (PNGase F), would yield a form of the epitope in which both asparagine residues are converted to aspartic acid residues (62, 63, 64). The cysteine residue at position 157 is disulfide-linked to the cysteine at residue 101 in full-length env (61).



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  		FIGURE 3. Location of the HLA A*0301, HLA Cw*08, HLA B*07, and HLA B*3501 epitopes in the extracellular domain of the HIV-1 env protein (black boxes). Also indicated are the hydrophobic signal (S), fusion (F), and transmembrane (T) domains. Residues in N-linked glycosylation sites are underlined. Numbering of amino acid residues is based on the HXB2 isolate starting with the first Met residue. The glycosylation sites have been defined biochemically by Gregory and colleagues (61). The numbering system used by Gregory starts at the N terminus of the mature protein (position 30).

 
To determine what form of this epitope is recognized by Cw*08-restricted CTL, synthetic peptides representing various forms of this epitope were tested for the capacity to sensitize uninfected autologous target cells for lysis by clone LWF A5. Initial studies demonstrated that autologous B-LCL target cells pulsed with a synthetic peptide representing the sequence encoded by the HIV-1 env gene (NCSFNISTSI, NN form) were lysed by clone LWF A5 only when high concentrations (10–100 µM) of peptide were used to pulse the target cells (Fig. 4Go and 48 . Since most optimal class I-restricted epitopes sensitize target cells for lysis in the pM to nM range, this requirement for high peptide concentrations suggested that another form of the epitope might represent the peptide actually recognized by this clone in vivo.



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  		FIGURE 4. Dose-response curves for synthetic peptides representing various possible forms of the minimal epitope recognized by the Cw*08-restricted CTL clone LWF A5. Autologous B-LCL were labeled with 51Cr and pulsed with the indicated concentrations of synthetic peptide for 30 min at 25°C. Assays were performed using peptides reconstituted in the absence (A) or presence (B) of 1 mM DTT. Effector cells were added at an E:T ratio of 5:1 for 4 h at 37°C, and lysis was measured in a standard 51Cr release assay. Peptides were not toxic to target cells at the concentrations used (not shown).

 
To characterize the optimal epitope recognized by clone LWF A5 in more detail, we synthesized a form of this epitope containing a single N-acetyl D-glucosamine attached to each asparagine residue (N*N*) and compared the immunologic properties of the synthetic nonglycosylated (NN) and glycosylated (N*N*) forms of this epitope. This result demonstrated that even a single monosaccharide attached to each asparagine residue was sufficient to prevent recognition even at high peptide concentrations (data not shown). In light of the poor recognition of the genetically encoded NN form and the glycosylated N*N* forms of the epitope, we postulated that a deglycosylated form of the epitope containing aspartic acids in place of asparagines might represent the naturally processed epitope.

Clone LWF A5 was tested for recognition of forms of the epitope containing aspartic acid residues at position 1 or position 5, or at both positions (DN, ND, and DD forms, respectively; Fig. 4Go). As stated above, conversion of the asparagine residue to aspartic acid is characteristic of the reaction conducted by the cytosolic enzyme, PNGase F. The presence of an aspartic acid at position 5 (NCSFDISTSI), or at positions 1 and 5 (DCSFDISTSI), enabled recognition at the peptide concentrations that were 1000-fold lower than those required for the NN and also the DN forms (Fig. 4Go). Therefore, this clone preferentially recognizes posttranslationally modified aspartic acid forms of the epitope. This may be due to the failure of the NN or DN forms of the epitope to bind strongly to Cw*08 or to rapid off rates or to poor recognition of the NN and DN forms by the TCR on clone LWF A5.

Another structural feature in this epitope is the presence of a cysteine residue at position 157, which participates in a disulfide linkage in full-length env (61). We tested whether subjecting the synthetic peptides to reducing conditions had a measurable effect on their recognition by clone LWF A5. As shown in Fig. 4Go, there was no significant difference in the recognition of each form of the epitope when exposed to DTT before pulsing targets in a CTL assay.

Mechanism of processing of the HLA B*07-restricted epitope in HIV-1 gp120

A second TAP1/2-dependent env epitope containing an N-glycosylation site has been described (42, 49). This epitope represents residues 298–307 of the HIV-1LAI env protein (see Table IGo and Fig. 3Go). TAP1/2-dependence was previously demonstrated using TAP1/2-defective T2 cells transfected with HLA B*07 (42). Similar to the results presented above for the LWF A5 epitope, Fig. 5Go shows recognition by CTL clone 11/1e of T2-B7 cells pulsed with nonglycosylated (RPNNNTRKSI, N form) and deglycosylated (RPNDNTRKSI, D form) peptides representing potential forms of the minimal epitope. Target cells pulsed with the D form of the peptide were recognized over 100-fold more efficiently than the N form, indicating that the D epitope was the optimal peptide recognized by clone 11/1e. In a separate experiment, a mono-N-glycosylated (N*) form of the peptide was recognized at similar concentrations as the nonglycosylated peptide (N form).



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  		FIGURE 5. Dose-response curves for synthetic peptides representing various possible forms of the minimal epitope recognized by the B*07-restricted CTL clone 11/1e. Autologous B-LCL were labeled with 51Cr and pulsed with the indicated concentrations of synthetic peptide for 30 min at 25°C. Effector cells were added at an E:T ratio of 5:1 for 4 h at 37°C, and lysis was measured in a standard 51Cr release assay. Note: Env298–307N* was tested in a separate experiment. Peptides were not toxic to target cells at the concentrations used (not shown).

 
In other experiments, there appeared to be no significant preferential stabilization of surface HLA B*07 molecules on T2-B7 cells pulsed with each peptide (data not shown). This result is consistent with reports that the asparagine or aspartic acid at position 4 in the epitope would not be involved in anchoring the peptide to a binding pocket in the MHC molecule (65). Rather, this residue and the monosaccharide attached synthetically may be solvent-exposed and may participate in recognition of the peptide-MHC complex by the 11/1e TCR. For this reason, it is particularly interesting that the monosaccharide (N* form) of the peptide was recognized in some experiments with similar efficiency as the nonglycosylated (N form) peptide.

Such preferential recognition of forms of these epitopes that are not encoded in the env gene can be most readily explained by postulating that processing of these epitopes involves cotranslational translation of the ectodomain of env into the ER, glycosylation of env in the ER, followed by reverse translocation back to the cytosol, where deglycosylation and TAP1/2-dependent transport into the ER would occur. The TAP1/2-dependence of the processing reaction is consistent with this notion.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The mechanisms by which membrane proteins enter the class I Ag-processing pathway are still controversial. TAP1/2-independent processing in the ER or a related compartment generates a subset of epitopes that can also by processed in a TAP1/2-dependent manner. Two mechanisms for this more general TAP1/2-dependent pathway have been identified. Failure of signal sequence-mediated translocation of HIV-1 env into the ER results in the translation of full-length nonglycosylated env in the cytosol and its rapid degradation into antigenic peptides (43). In the current study, we show that a second TAP1/2-dependent pathway consists of dislocation of full-length, N-glycosylated env from the ER to the cytoplasm, where deglycosylation and degradation occur before peptides are transported back into the ER by TAP1/2. Recent analyses of some membrane and secreted proteins have shown that reverse translocation of full-length proteins to the cytosol may be a quality control mechanism for degrading misfolded or mistranslated cellular proteins (23, 24, 25, 26, 27, 28, 29, 30). It had not been clear whether such a mechanism had an important role in the processing of viral proteins to generate class I-restricted epitopes.

Our analysis of the HIV-1 env protein has shown that most class I-restricted epitopes in the extracellular domain of this protein require the TAP1/2 heterodimer for transport into the ER. Using HSV ICP47 expression to selectively block TAP1/2, we have shown here that the main processing pathway for the env protein requires this transporter. Using targets in which TAP1/2 is either deleted (42) or functionally blocked by ICP47, we have also shown that a subset of these epitopes can also be generated in a TAP1/2-independent fashion.

Previous studies have identified one potential mechanism for TAP1/2-dependent processing, which involves failure of the nascent env protein to be translocated into the ER (43). In the current study, we also present evidence for the second mechanism of TAP1/2-dependent env processing using two interesting epitopes presented by HLA Cw*08 and B*07. These epitopes contain well-characterized N-linked glycosylation sites (61), and we reasoned that the minimal forms of these epitopes recognized by CTL might provide insights into how class I epitopes are generated from membrane proteins.

HLA Cw*08-positive target cells were lysed by clone LWF A5 only when the targets were pulsed with high concentrations (10–100 µg/ml) of a synthetic peptide representing the actual sequence (NCSFNISTSI) encoded by the HIV-1 env gene (Fig. 4Go). A glycosylated form (N*N*) of the epitope did not appear to be recognized even at high peptide concentrations (not shown). These results suggested that the naturally processed peptide recognized by this clone might have a different structure. Therefore, we also determined whether lysis of target cells occurred when they were loaded with forms of the epitope containing aspartic acid residues at position 1 or position 5, or at both positions. Conversion of the N-linked asparagine residue to aspartic acid is characteristic of the reaction conducted by the only known mammalian deglycosylation enzyme, peptide:N-glycanase (62, 63, 64). Therefore, preferential (>3 logs) recognition of the ND and DD epitopes containing aspartic acid instead of asparagine residue(s) strongly suggests prior translocation of env into the ER and glycosylation, followed by reverse translocation back to the cytosol, where deglycosylation, processing, and TAP1/2-dependent transport into the ER would occur. The DN and ND forms may be intermediates in the conversion of the NN form to the DD form. The poor recognition of the DN and NN forms may reflect poor binding to or rapid dissociation from HLA Cw*08 or poor recognition of bound forms of these peptides by the TCR. In any event, it is clear that posttranslational modification of the epitope is necessary for efficient recognition by this clone.

Similar results using an HLA B*07-restricted epitope (Fig. 5Go) show that this second TAP1/2-dependent processing pathway may be a general mechanism for generating class I-restricted epitopes from viral env proteins. The 100-fold more efficient recognition and lysis of targets pulsed with env 298–307D over env298–307N or N*, despite similar class I stabilization by N and D containing peptides suggests that the recognition of the MHC-peptide complex by the 11/1e TCR is much more efficient for the env298–307D form. In part, this conclusion arises from the position of the new acidic residue (D) in the middle of the epitope, at a site not felt to be an MHC-binding anchor residue (65). We presume this residue to be solvent-exposed and to contribute to the topological surface encountered by the 11/1e TCR. It is also possible that the difference is attributable to differences in the off rates for the three peptides, although we did not see differential stabilization of surface B7 molecules on T2-B7 cells pulsed with comparable concentrations of each peptide. In any event, the critical point is that conversion of asparagine to aspartic acid is necessary for optimal recognition via this TAP-dependent pathway. These results provide the first evidence that a single membrane protein can be processed by two distinct TAP1/2-dependent mechanisms in vivo.

Selby et al. (66) have studied a TAP1/2-dependent epitope in the Hepatitis C virus (HCV) env glycoprotein. This epitope contains an N-linked glycosylation site. CTL from HCV-infected chimpanzees did not recognize the nonglycosylated (N) form of the epitope even at high peptide concentrations, whereas 6/6 HCV-infected chimpanzees possessed T cells capable of lysing autologous cells pulsed with the deglycosylated (D) form of the minimal epitope (66). In addition to our current report of two HIV-1 env epitopes, this result provides further evidence that this TAP1/2-dependent processing pathway may be a generally utilized processing pathway for class I-restricted CTL responses to viral env proteins. The conversion of asparagine to aspartic acid during enzymatic deglycosylation is the most plausible explanation for the presence of CTL that recognize nontemplated, D forms, of viral env epitopes.

While deamidation may occur spontaneously by hydrolysis, this reaction appears to be very slow, with half-times at pH 7.4 of 6–500 days (67, 68, 69). Also, an N-terminal asparagine deaminase has been described recently (70), but this enzyme had no detectable activity on internal asparagine residues. This property would preclude any role for this enzyme in either epitope studied in this report, since the D forms of each optimally recognized epitope require conversion of an internal, not N-terminal, asparagine residue (Figs. 4Go and 5Go). Therefore, it is very unlikely that slow, spontaneous, or enzymatic deamination reactions could have biological relevance to account for the phenomena reported here.

Taken together with our previous findings showing preferential recognition of a nonglycosylated (N form) epitope (43), our results suggest that these various processing mechanisms may occur in the same cell during HIV-1 infection. The fate of the env protein may be dependent on many factors (Fig. 6Go). A temporary excess of signal sequence containing nascent polypeptides over signal recognition particles may result in a fraction of the nascent env protein being synthesized and degraded in the cytosol. For env, which is normally translocated into the ER during translation, a fraction of misfolded env may be subject to the ER "quality control" process, resulting in the extrusion of full-length protein back to the cytosol, where processing and TAP1/2-dependent transport would occur. Some investigators have shown that defective ribosomal products (DRiPs) may account for the processing and presentation of certain cytosolic epitopes in a TAP1/2-dependent fashion (71, 72, 73). TAP1/2-independent processing appears to be another potential, though perhaps minor, pathway for generation of class I-restricted epitopes. This processing mechanism may involve ER- or Golgi-resident proteases and may generate epitopes in contexts that allow liberation by these proteases.



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  		FIGURE 6. Model for synthesis and degradation of HIV-1 env to generate class I-restricted CTL epitopes.

 
Our results demonstrate that the particular processing pathway utilized to generate each epitope may have a qualitative as well as quantitative impact on the presentation of that epitope. Intentional mislocalization of viral env proteins to the cytosol for rapid degradation has been shown to increase their immunogenicity (44, 45). Evidence presented in this report suggests that this increase in epitope presentation may have undesired effects on the presentation of certain epitopes, since qualitative differences in particular epitope sequences may result from posttranslational modifications that we and others have shown to occur. Our findings suggest that all of these pathways are potential mechanisms of class I-restricted epitope presentation and, therefore, should be considered in the design of strategies to enhance the CD8+ CTL response against viral env proteins.


   Acknowledgments
 
We thank Dr. R. Bollinger, M. Lubaki, and H. Hon for providing two CTL clones, and Dr. B. Rouse for providing vvICP47.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI28108 and AI32871 (to R.F.S.) and by National Institutes of Health Contract N01-05061. R.L.F. was supported by a research training fellowship from the National Institute of Deafness and Communicative Disorders and by a grant from the American Academy of Otolaryngology/Head and Neck Surgery-American Academy of Otolaryngic Allergy. Back

2 Address correspondence and reprint requests to Dr. Robert F. Siliciano, Johns Hopkins University School of Medicine, Ross Building Room 1049, 720 Rutland Avenue, Baltimore, MD 21205. E-mail address: Back

3 Abbreviations used in this paper: ER, endoplasmic reticulum; env, envelope; VV, vaccinia virus; B-LCL, B-lymphoblastoid cell line; HSV, herpes simplex virus. Back

Received for publication July 10, 1998. Accepted for publication October 15, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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