Differential Proteasomal Processing of Hydrophobic and Hydrophilic Protein Regions: Contribution to Cytotoxic T Lymphocyte Epitope Clustering in HIV-1-Nef

Differential proteasomal processing of hydrophobic and hydrophilic protein regions: Contribution to cytotoxic T lymphocyte epitope clustering in HIV-1-Nef

Maria Lucchiari-Hartz*, Viv Lindo†, Niclas Hitziger*, Simone Gaedicke*, Loredana Saveanu‡, Peter M. van Endert‡, Fiona Greer†, Klaus Eichmann*, and Gabriele Niedermann*§

*Department of Cellular Immunology, Max Planck Institute of Immunobiology, Stu¨beweg 51, D-79108 Freiburg, Germany; †M-SCAN Ltd., Silwood Park, Sunninghill, Ascot SL5 7PZ, United Kingdom; and ‡Institut National de la Sante´et de la Recherche Me´dicale U25, Hoˆpital Necker, F-75743 Paris Cedex 15, France

Communicated by Richard M. Krause, National Institutes of Health, Bethesda, MD, April 15, 2003 (received for review February 20, 2003) HIV proteins contain a multitude of naturally processed cytotoxic T is located in the cytoplasm and temporarily attached to the cell lymphocyte (CTL) epitopes that concentrate in clusters. The molecular membrane via myristoylation (14, 15). It is a potent CTL antigen basis of epitope clustering is of interest for understanding HIV and reveals a typical pattern of high-density CTL epitope clusters immunogenicity and for vaccine design. We show that the CTL (refs. 1 and 16 and HIV Molecular Immunology Database). Nef- epitope clusters of HIV proteins predominantly coincide with hydro- specific CTLs may be particularly important in the control of HIV phobic regions, whereas the noncluster regions are predominantly infection, because Nef is the predominant transcript early after hydrophilic. Analysis of the proteasomal degradation products of infection and, perhaps as the only HIV protein, is expressed even full-length HIV-Nef revealed a differential sensitivity of cluster and before proviral integration (17, 18). In addition, primary HIV- noncluster regions to proteasomal processing. Compared with the infected T cells are lysed efficiently by Nef-specific CTLs (19), epitope-scarce noncluster regions, cluster regions are digested by whereas abrogation of recognition by Gag- and polymerase-specific proteasomes more intensively and with greater preference for hy- CTLs has been reported due to the Nef-mediated down-regulation drophobic P1 residues, resulting in substantially greater numbers of of HLA-A and HLA-B molecules (20). fragments with the sizes and COOH termini typical of epitopes and We recently reported that proteasome inhibitors abrogated CTL their precursors. Indeed, many of these fragments correspond to recognition of several Nef epitopes on Nef-expressing cells, sug- endogenously processed Nef epitopes and͞or their potential precur- gesting proteasome involvement in the processing of HIV-Nef (21). sors. The results suggest that differential proteasomal processing Now we have digested recombinant full-length Nef protein with contributes importantly to the clustering of CTL epitopes in hydro- isolated proteasomes, comprehensively analyzed the cleavage prod- phobic regions. ucts, and compared them with database epitope maps and a panel of naturally processed class I-binding Nef peptides determined by omprehensive analyses of several HIV antigens including Nef us. Our data suggest that the pronounced sensitivity of hydrophobic Chave revealed an enormous diversity of epitopes recognized by protein regions to proteasomal processing contributes importantly cytotoxic T lymphocytes (CTLs) of HIVϩ patients (see the HIV to the generation of an enormous multiplicity of extensively over- Molecular Immunology Database, http:͞͞hiv-web.lanl.gov͞ lapping CTL epitope peptides and to their clustering in hydrophobic content͞immunology). Moreover, HIV CTL epitopes extensively protein regions. overlap in so-called epitope clusters, whereas other regions of the HIV proteins are almost completely devoid of CTL epitopes (refs. Methods 1–6 and HIV Molecular Immunology Database). Evidence for Digestion of Recombinant Nef with Purified Proteasomes. The nef CTL epitope clusters exists also for non-HIV antigens (7). Al- gene of the HIV-1 LAI isolate was inserted into a variant (pRP261, though CTL epitope clusters often coincide with rather conserved kindly provided by B. Wollscheid, Max-Planck-Institut fu¨r Immun- regions of the HIV proteins (1, 4, 6), it has been postulated that the biologie, Freiburg, Germany) of the pGEX-3X-vectors (Amersham highly nonuniform distribution of HIV CTL epitopes may be Pharmacia) to generate a Nef-glutathione S-transferase fusion related to antigen processing and presentation (2, 4, 6). protein. Purification used glutathione-Sepharose 4B, and the Nef Proteasomes seem to participate in the generation of many if not protein was cleaved off by incubation with thrombin and purified most CTL epitopes. In the classical class I antigen-processing further by RP-HPLC. 20S proteasomes were purified from the pathway proteasomes generate epitopes as well as precursors that human lymphoblastoid cell line T1 as described (21). Recombinant are trimmed by cytosolic and endoplasmic reticulum-resident amin- Nef (60 ␮g) was digested with isolated proteasomes (2 ␮g) at 37°C opeptidases. Trimming by carboxypeptidases seems to be highly in a total volume of 300 ␮l of buffer (20 mM Hepes, pH 7.3͞1mM ͞ ͞ unusual. Cytosolic endoproteases other than proteasomes may be EGTA 5 mM MgCl2 0.5 mM 2-mercaptoethanol). The reaction involved in the production of certain epitopes, but thus far none mixture was fractionated by RP-HPLC on a Smart system (Amer- have been unequivocally identified (for several excellent reviews, sham Pharmacia) equipped with a Sephasil C18 SC2.1͞10 column see ref. 8 and all articles in the same issue). Important information with 0.1% trifluoroacetic acid (eluent A) and 80% acetonitrile on the role of proteasomes in antigen processing has been obtained containing 0.081% trifluoroacetic acid (eluent B). Degradation

by in vitro digestion of proteins and protein fragments with isolated products were separated on a gradient of 10–55% B in 50 min and IMMUNOLOGY proteasomes. However, thus far analyses of CTL epitope- eluted between 16͞17% and 52% of eluent B. Aliquots of the containing full-length antigens (ovalbumin and ␤-galactosidase) (9, HPLC fractions were analyzed by matrix-associated laser desorp- 10) and most antigen fragments focused on a single epitope and͞or tion ionization͞time-of-flight MS with a Voyager STR spectrom- related epitope precursors. In nearly all experiments with CTL eter coupled with delayed extraction (Applied Biosystems; lower epitope-containing substrates, 20S proteasomes were used, and molecular mass limit, 400–450 Da). In some cases, sequences were excellent correlations with epitope recognition on antigen- presenting cells were often reported (11–13). The HIV protein Nef is a small regulatory protein and critical for Abbreviation: CTL, cytotoxic T lymphocyte. the development of the acquired immunodeficiency syndrome. Nef §To whom correspondence should be addressed. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.1232228100 PNAS ͉ June 24, 2003 ͉ vol. 100 ͉ no. 13 ͉ 7755–7760 Downloaded by guest on September 23, 2021 Fig. 1. Map of the HIV-1-Nef protein showing CTL epitope peptides and proteasomal cleavage products. CTL epitope peptides are shown above the sequence. Black lines are those recognized by CTLs of HIV- infected subjects as recorded in the HIV Molecular Immunology Data- base. Red lines are the naturally processed peptides acid-eluted from Nef- transfected cells (see also Table 1). The 20S proteasomal cleavage products of recombinant HIV-1-Nef (LAI) protein are indicated as lines below the sequence. Fragments corresponding to acid-eluted HLA class I ligands are highlighted in red; fragments corresponding to their potential precursor peptides are highlighted in green. *, Epitopes identiﬁed by immuniza- tion of HLA-A2 transgenic mice with HIV-1-Nef (HXB3), with the sequences ALTSSNTA and MTYKAALDL instead of AITSSNTA and MTYKAAVDL (LAI); **, a 20-aa sequence (Nef161–180) where a CTL epitope of unknown HLA restriction was located; ***, sub- dominant epitope with the sequence DPEKEVLQWK (LAI sequence, DPER- EVLEWR).

confirmed by tandem-MS analysis on a Micromass Q-TOF instru- analysis, regions 68–146 and 180–206 were taken as epitope clus- ment. Aliquots of reconstituted fractions were also tested for ters, and regions 1–67 and 147–179 were taken as noncluster recognition by Nef-peptide-specific CTL in a 51Cr-release assay on regions. Although cluster and noncluster regions each cover ap- P815 murine mastocytoma cells transfected with HLA-A2, HLA- proximately half (106͞100 aa) of the Nef sequence, we detected B7, or HLA-A3. three times as many proteasomal cleavage products in the former (107 vs. 36), corresponding to twice the density of proteasomal Identification of Naturally Processed Nef Peptides and in Vitro Induc- cleavage sites in the epitope-cluster regions compared with the tion of Primary Nef-Specific CTL Lines. Naturally processed Nef noncluster regions (1͞1.5 vs. 1͞3.1 aa). peptides were identified essentially as described (21). Briefly, The sizes of the proteasomal cleavage products are given in Fig. peptides acid-eluted from Nef-transfected cells (HLA-A2.1ϩ T1, 2. Very few were shorter than 8 aa. For all peptides of 8 aa and HLA-B7.2ϩ Jurkat, or C1R cells transfected with HLA-A3) were longer, i.e., sizes potentially giving rise to MHC class I ligands, 2.6 fractionated by RP-HPLC with shallow gradients specifically de- times as many (92 vs. 35) were found in the cluster as in the signed to resolve mixtures of related peptides. CTL epitopes were noncluster regions. A substantial proportion of cleavage products determined by 51Cr-release assay with 17 polyclonal Nef-peptide- from the epitope clusters (63 of 107) fell into the approximate size specific CTL lines induced in vitro with synthetic peptides as range of MHC class I peptide ligands, i.e., 8–11 aa. Only 9 of the described (21). 36 cleavage products from the noncluster regions had this length, with the majority being longer. Results Pronounced Sensitivity of CTL Epitope Clusters to Proteasomal Diges- Enhanced Preference for Hydrophobic P1 Residues in CTL Epitope- tion. The vast majority of endogenously processed Nef-CTL Cluster Regions. At the COOH termini of the Nef proteasomal epitopes is concentrated in the central portion of the protein cleavage products we frequently found large hydrophobic amino (residues 68–146) and at the COOH terminus (residues 180–206) acids, certain charged amino acids, as well as the small amino acid (Fig. 1). Regions 68–100 and 113–146 in the central portion have alanine, with a hydrophobic side chain (Fig. 3). At the NH2 termini a particularly high epitope density, thus some authors consider of cleavage products preferences were not so pronounced, but small these as two separate epitope clusters (1). We digested the full- and certain charged amino acids were often found. Except for the length recombinant HIV-1 LAI Nef protein with 20S proteasomes acidic amino acids (which are not frequent at the COOH termini purified from T1 lymphoma cells containing both housekeeping of mammalian MHC class I ligands), particularly glutamic acid, the proteasomes and immunoproteasomes as revealed by Western blot amino acid frequencies at the COOH termini of proteasomal (data not shown). The LAI (formerly Bru) sequence was chosen cleavage fragments are in good agreement with that at the COOH because most Nef-CTL epitopes recorded in the literature are termini of mammalian MHC class I ligands (see also ref. 22). defined for LAI (HIV Molecular Immunology Database). Cleav- Unexpectedly, we found that the vast majority of cleavage age products were identified by MS. We comprehensively analyzed fragments with COOH termini suitable for MHC class I binding the peptide mixtures at Ϸ40%, 75%, and 95% of substrate con- was derived from epitope-cluster regions (Fig. 3, hatched portions sumption. Essentially all cleavage products were found at all three of the gray bars). Conversely, many fragments with COOH termini time points. Fig. 1 shows the peptides (n ϭ 143) produced when rarely found in MHC class I ligands stem from noncluster regions Ϸ40% of the substrate molecules had been consumed. To simplify (Fig. 3). These differences between fragments liberated from

7756 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1232228100 Lucchiari-Hartz et al. Downloaded by guest on September 23, 2021 acids in cluster regions (24–45%; mean 33%, Ͼ40% in positions P7, P5, P3, and P2Ј) than in noncluster regions (0–30%; mean 13%). Leucine residues in noncluster regions not used for cleavage showed a total lack of large hydrophobic amino acids in positions P8 to P3 and P2Ј. Together, these data reveal a more-pronounced preference of proteasomes for hydrophobic amino acids in P1 in cluster regions than in noncluster regions, which depends on a hydrophobic sequence environment of the cleavage site. When both the sizes and COOH termini are considered, the numbers of proteasomal cleavage fragments suitable as CTL epitopes and epitope precursors differ considerably between cluster and noncluster regions. Of the 92 cleavage fragments of Ն8aafrom the epitope clusters (see Fig. 2), 75 have a COOH-terminal amino acid frequently found in mammalian MHC class I ligands; of these, 67 have a hydrophobic and 8 have a basic COOH-terminal amino acid. In contrast, of the 35 peptides Ն8 aa from the noncluster regions, only 16 have a hydrophobic or basic COOH-terminal amino acid.

Hydrophobicity and Conservation of Cluster and Noncluster Regions. As shown in Fig. 4A, the regions that correspond to the Nef-CTL epitope clusters and that are processed intensively by proteasomes are on average more hydrophobic than the less-intensively processed noncluster regions, which are predominantly hydrophilic. To investigate whether the relationship between hydrophobicity and epitope density was unique to Nef or more general, we performed a similar analysis of Gag p17. This well studied protein contains two CTL epitope clusters (11–44 and 71–101), and a third one begins at the boundary to Gag p24 (5, 6). When the epitope clusters of Gag p17 were plotted against the hydrophobicity͞hydrophilicity profile (Fig. 4B), a striking relationship between epitope clusters and Fig. 2. Length distribution of 20S proteasomal cleavage fragments from HIV- hydrophobicity was observed, similar to that found for Nef. 1-Nef protein. Recombinant HIV-1-Nef (LAI) protein was digested with puriﬁed 20S proteasomes, and the cleavage fragments were determined by MS (see Fig. It has been shown by others that CTL epitope clusters often 1). The preferred size range of MHC class I peptide ligands is highlighted in pink. correspond to more-conserved regions of the HIV proteins (1, 3, 4, 6, 23). In fact, the central hydrophobic portion of Nef (region 68–146) encompassing the two central CTL epitope clusters is quite cluster and noncluster regions reflect pronounced differences in well conserved among the HIV-1 subtypes. However, the hydro- cleavage-site preferences. In cluster regions proteasomes cleaved phobic COOH-terminal cluster (region 180–206) is not as well after 87% (39͞45) of the hydrophobic amino acids but only after conserved. Conversely, the Nef protein contains conserved se- 37% (12͞32) in noncluster regions. For example, cleavages were quence stretches that are hydrophilic and do not correspond to observed after 83% (10͞12) of the leucine residues and after 71% epitope-cluster regions (Fig. 4A). These stretches often correspond (5͞7) of the valine residues in cluster regions and only after 20% to protein–protein interaction sites responsible for pathogenic (1͞5) of leucine and 37% (3͞8) of valine residues in noncluster effects of Nef (14). For Gag p17, both hydrophobic epitope clusters regions. We therefore analyzed whether the sequence environment show a higher degree of conservation than the noncluster regions of a cleavage site (P1-P1Ј) had an influence on the cleavage (Fig. 4B). Because hydrophobic regions are important for protein preferences. Positions P8 to P4Ј of cleavage sites with a hydrophobic folding, they may generally be quite conserved. Some correlation of P1 residue contained a higher frequency of hydrophobic amino epitope clustering to sequence conservation is therefore expected. IMMUNOLOGY

Fig. 3. Frequencies of amino acids at the COOH and NH2 termini of the 143 Nef proteasomal cleavage fragments shown in Fig. 1. The COOH-terminal amino acids are shown as gray bars, and the NH2-terminal amino acids are shown as white bars. For the COOH-terminal amino acids, the proportion of peptides from the CTL epitope clusters is indicated by the hatched parts of the bars. The frequencies (%) of individual amino acids in the Nef protein are given below the x axis.

Lucchiari-Hartz et al. PNAS ͉ June 24, 2003 ͉ vol. 100 ͉ no. 13 ͉ 7757 Downloaded by guest on September 23, 2021 cleavage products of full-length Nef (Table 1 and highlighted in red in Fig. 1). Potential NH2-terminally extended precursors were detected for the majority of but not all ligands (Table 1 and highlighted in green in Fig. 1). We also found a number of proteasomal cleavage products that correspond to CTL epitopes mapped by others (HIV Molecular Immunology Database), which may correspond to HLA ligands (see ‡ footnote in Table 1). For most of these epitopes, NH2-terminally extended precursor peptides were also among the proteasomal cleavage products. Some cleavage products correspond to both an HLA ligand and a potential precursor (see ʈ footnote in Table 1). Taken together, the data presented suggest that nearly all of the peptides excised by proteasomes from the CTL epitope-cluster regions are related to HLA class I ligands (see also Fig. 1). Discussion HIV antigens contain large numbers of endogenously processed CTL epitopes that tend to concentrate in clusters. We found that the CTL epitope clusters correspond to the hydrophobic regions of the HIV proteins and that proteasomes excise large numbers of extensively overlapping peptides with the sizes and COOH termini typical of epitope and epitope-precursor peptides, particularly from hydrophobic regions of the Nef protein. Many of these cleavage products indeed correspond to endogenously processed Nef-CTL epitope peptides and their potential precursors. We propose that this is due to four fundamental properties of proteasomes: (i) the broad cleavage-site specificity; (ii) the enhanced preference for hydrophobic P1 residues in a hydrophobic sequence context; (iii) the excision of large numbers of peptides Ն8 aa; and (iv) the versatility of proteasomal processing Fig. 4. Hydrophobicity͞hydrophilicity plots and conservation of the primary resulting in overlapping peptide patterns. Together, our data sequence of HIV-1-Nef (A) and HIV-1-Gag p17 (B). Hydrophobicity͞hydrophilicity suggest that proteasomal proteolysis contributes importantly to plots were generated with the algorithm HPLC͞retention pH 7.4; similar plots the multiplicity of endogenously processed epitope peptides and were obtained with the algorithm of Abraham and Leo (ExPASy Molecular to their clustering in hydrophobic regions. ϩ Biology Server, www.expasy.org͞cgi-bin͞protscale.pl). Plots created with win- Systematic epitope mappings with CTLs of HIV subjects thus dow sizes between 15 and 21 looked almost identical. CTL epitope clusters and far include only an incomplete set of HLA alleles (HIV Molecular single CTL epitopes are represented by boxes at the top. For Nef-CTL epitopes, see Immunology Database). Hence, the number of endogenously pro- Fig. 1; for Gag CTL epitopes, see the HIV Molecular Immunology Database. The cessed HIV CTL epitope peptides may still increase, even though variability plots (below the hydrophobicity plots) show the number of different amino acids (dots) at each position of the concensus sequences of subtypes A–D. some HLA alleles share similar peptide-binding motifs. CTL re- ͞ ͞ sponses to multiple epitopes are often observed, i.e., in different Sequence alignment was performed by using ALIGN (www2.igh.cnrs.fr bin align- ϩ guess.cgi). *, a 20-aa sequence (Nef161–180) where an HLA class I-restricted epitope studies individual HIV subjects recognized 1–13 (25), 2–17 (26), (of unknown type restriction) was mapped. and 0–13 (27) epitopes. These are minimal estimates because of the incomplete knowledge of CTL epitopes and the use of reference strain-based peptides. In addition, different subjects sharing the Relationships of Proteasomal Fragments to Naturally Processed Class same HLA allele may respond to different epitopes presentable by I-Binding Nef Peptides. To compare proteasomal cleavage fragments this allele, and the same subject may recognize different epitopes at with naturally processed MHC-presented peptides, we determined different stages of infection (25–30). class I-binding Nef peptides acid-eluted from Nef-transfected cells. HIV may not be the only virus associated with a multitude of Previously, only two naturally processed, class I-binding HIV endogenously processed CTL epitopes as well as a pronounced peptides had been identified, one from Gag and one from reverse heterogeneity of CTL responses. The hepatitis B virus surface transcriptase (24). Of the 17 eluted Nef peptides listed in Table 1, antigen harbors at least eight endogenously processed HLA-A2- 12 were identified in the present study and 5 were identified binding CTL epitopes, and patients develop CTL responses against previously (21). The peptides stem from the two central Nef-CTL multiple epitopes in the hepatitis B virus antigens (31). The few epitope clusters and are highlighted in red in Fig. 1. Due to cross hepatitis C virus proteins contain Ͼ20 different HLA-A2-binding recognition, CTL lines against the 17 Nef-HLA class I ligands epitopes, many of which have been shown to be endogenously distinguished a minimum of nine CTL epitopes. Six of the nine processed (32), and a response against multiple epitopes seems to epitopes occurred in several naturally processed length variants be characteristic of hepatitis C virus-infected individuals, particu- (four in two, and two in three length variants) including the larly during acute infection (33). Tumor antigens such as the examples presented previously (21). Recognition of the longer melanoma antigen gp100, Her-2͞neu, the PRAME protein, and variants by CTL did not depend on extracellular trimming by the MAGE antigens contain multiple epitopes recognized by CTLs carboxypeptidases present in FCS (21). The length variants were of patients; gp100 contains at least nine different endogenously predominantly but not exclusively COOH-terminal variants. These processed HLA-A2-binding epitopes (see the Peptide Database of results suggest that the presentation of COOH-terminal epitope T Cell-Defined Tumor Antigens, Ludwig Institute for Cancer length variants is a frequent phenomenon. The nine epitopes are Research, Brussels, at www.cancerimmunity.org͞peptidedatabase͞ described in the literature as being recognized by HIV-infected Tcellepitopes.htm). patients and͞or to bind to HLA class I molecules (Table 1). CTL epitope clusters may not be unique to the HIV proteins For the majority of the naturally processed Nef peptides the either. A pronounced CTL epitope cluster has been described for exactly corresponding peptides were found among the proteasomal the major outer membrane protein of Chlamydia trachomatis (7). In

7758 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1232228100 Lucchiari-Hartz et al. Downloaded by guest on September 23, 2021 Table 1. Relationships between naturally processed Nef peptides and full-length Nef proteasomal degradation products

*Eluted from Nef-transfected cells and grouped according to CTL cross recognition. †As recorded in the HIV Molecular Immunology Database. ‡Additional cleavage products found by MS corresponding to other epitopes described in the literature are 13WPTVRERM20 (HLA-B8), 79MTYKAAVDL87 and 180VLEWRFDSRL189 (HLA-A2), and 106RQDILDLWIY115 (HLA-B7); we also found 106RQDILDLWI114 and 106RQDILDLW113, which may also bind to HLA-B7, 105RRQDILDLWI114 (HLA-B27), 105RRQDILDLW113 and 105RRQDILDLWIY115, which may also bind to HLA-B27, 126NYTPGPGVRY135 (HLA-A24), 136PLTFGWCYK144 (HLA-A3), and 186DSRLAFHH193 (HLA-B35) and 186DSRLAFHHV194 (HLA-B51). §As recorded in ref. 16. ¶As recorded in ref. 1. ࿣Also an epitope͞HLA ligand; TPQVPLRPMTY (HLA-B35 epitope͞B7 ligand) and potential precursor of QVPLRPMTY and VPLRPMTY (HLA-A3 ligands͞B35 epitopes); PLRPMTYKAA (HLA-A3 ligand and potential precursor of the HLA-B7 ligand RPMTYKAA); NYTPGPGVRY (HLA-A24 epitope and precursor of the B7 ligand TPGPGVRY). **Also excised from a 35-mer Nef peptide by archaebacterial proteasomes (ref. 16). ††As recorded in ref. 25. ‡‡Detected only in very small amounts. §§Data are from ref. 21. IMMUNOLOGY

hepatitis B virus͞hepatitis C virus as well as tumor antigens several other hand, mechanisms based on evolutionary host͞virus adap- of the epitopes identified thus far (mostly HLA-A2-binding tation have been considered. HLA class I-binding motifs might have epitopes) overlap with or are close to others (refs. 31 and 32 and been eliminated in the more variable and therefore functionally Peptide Database of T Cell-Defined Tumor Antigens). The under- less-important regions due to immunoselection, a putative strategy lying basis of CTL epitope clustering in HIV proteins has been of of HIV to subvert the host defense (3). Conversely, the preferential considerable interest. On the one hand, CTL epitope clusters often presentation of more-conserved peptides might be a strategy of the coincide with conserved regions of the HIV proteins, possibly due host to combat the immune escape attempts of the virus (23). in part to the use of reference strain-based peptides (4, 6). On the Differences in antigen processing and presentation have also been

Lucchiari-Hartz et al. PNAS ͉ June 24, 2003 ͉ vol. 100 ͉ no. 13 ͉ 7759 Downloaded by guest on September 23, 2021 considered (2, 4, 6, 34). Our data suggest that such differences class I assembly is only partially sensitive to proteasome inhibitors, indeed contribute significantly to CTL epitope clustering. We a few other known endoproteases may contribute to the generation demonstrate a striking correlation between three elements of a of CTL epitopes. Tripeptidyl peptidase II might contribute to the protein sequence: the degree of hydrophobicity, the intensity of generation of CTL epitopes with basic COOH-terminal amino proteasomal processing, and the density of CTL epitopes. The acids, particularly with COOH-terminal lysine residues (39), preference of proteasomes for hydrophobic protein stretches is in whereas proteasomes usually do not cleave efficiently after lysine accord with the cleavage preferences of the proteasome for hydro- (see Fig. 3 and ‡‡ footnote in Table 1). Calpains have not been phobic P1 residues (12, 22, 35) (see Fig. 3). Moreover, our data show shown to be involved in antigen processing. The endoproteolytic that hydrophobic amino acids are used more frequently for cleavage thimet oligopeptidase seems to destroy proteasomal cleavage prod- in the hydrophobic environment of epitope clusters than in the ucts, including epitopes, in the cytosol (10). The endoplasmic hydrophilic environment of noncluster regions. Hydrophobic inter- reticulum-signal peptidase and furin in the trans-Golgi operate in actions with hydrophobic inner surfaces of the proteasome are nonclassical processing pathways (40, 41). Because of their more- probably important for translocation of substrates and for their restricted substrate and cleavage specificity, nonproteasomal endo- maintenance in an unfolded and easily degradable state (36, 37). proteases likely generate only limited subsets of class I-binding The number of proteasomal fragments potentially giving rise to peptides. Taken together, these findings and our present data CTL epitopes from the Nef clusters is 4.7-fold greater than that suggest that proteasomes perhaps are not the only but the major from the noncluster regions (75 vs. 16). Surprisingly, the difference endoprotease in charge of processing Nef. The data therefore in numbers of known CTL epitopes is even greater, namely Ͼ50 suggest an important contribution of proteasomes to epitope clus- CTL epitope peptides in cluster regions compared with 2 or 3 in tering in Nef. noncluster regions (see Fig. 1), which may be because of the CTL responses may play an important role in the control of HIV more-difficult detection of epitopes in the less-conserved nonclus- infection (2, 6). Effective HIV vaccines therefore should contain ter regions. In addition, fragments from noncluster regions that CTL epitopes restricted by many different HLA alleles including possess a suitable size and COOH terminus may lack hydrophobic those of Nef. Because hydrophobic protein regions often seem to internal or auxiliary anchor residues for MHC class I binding. be quite well conserved, they may harbor epitopes particularly Analyses of 42 individual self-MHC class I ligands from as many effective for protective host immunity. On the other hand, the different source proteins revealed that these were more hydropho- generation of a large variety of epitopes, many of which overlap and bic than the rest of their source proteins and were derived from therefore display pronounced structural similarity, may result in a evolutionarily more-conserved regions (38). Although this study did CTL response of pronounced heterogeneity and low average af- not address epitope clusters, these results suggest that our conclu- finity and therefore may be part of the immune evasion strategy of sions may not only be relevant for HIV proteins. Hydrophobic HIV. The clustering of CTL epitopes in hydrophobic regions protein regions may tend to be highly conserved because of their contrasts with Nef pathogenicity, which is based on protein–protein importance for correct protein folding. The Nef-CTL epitope interaction mostly involving hydrophilic parts of the protein (14, clusters cover virtually the entire folded core domain of the Nef 15). The preferential association of pathogenicity and immunoge- protein (see Fig. 1 and 4A; for structural information, see ref. 14). nicity with hydrophilic and hydrophobic regions, respectively, of However, proteolytic enzymes cannot recognize the conservation Nef seems particularly promising for the design of vaccines based of a protein sequence, thus it is unlikely to be causally related to on truncated versions of Nef. proteasomal processing. It is likely that Nef is predominantly processed by the classical We thank R. Escher for technical assistance and R. Cassada, I. Haidl, proteasomal pathway, in line with our finding that proteasome and B. Menz for critical reading of the manuscript. This work was inhibitors abrogated CTL recognition of several Nef epitopes on supported by a grant from the European Community (to G.N., P.M.v.E., Nef-expressing cells (21). However, because in certain cases HLA and F.G.).

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