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 pro- tein 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 pro- cessed peptides acid-eluted from Nef- transfected cells (see also Table 1). The 20S proteasomal cleavage prod- ucts of recombinant HIV-1-Nef (LAI) protein are indicated as lines below the sequence. Fragments correspond- ing to acid-eluted HLA class I ligands are highlighted in red; fragments cor- responding to their potential precur- sor 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