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

Abstract

HIV proteins contain a multitude of naturally processed cytotoxic T lymphocyte (CTL) epitopes that concentrate in clusters. The molecular basis of epitope clustering is of interest for understanding HIV immunogenicity and for vaccine design. We show that the CTL epitope clusters of HIV proteins predominantly coincide with hydrophobic regions, whereas the noncluster regions are predominantly hydrophilic. Analysis of the proteasomal degradation products of full-length HIV-Nef revealed a differential sensitivity of cluster and noncluster regions to proteasomal processing. Compared with the epitope-scarce noncluster regions, cluster regions are digested by proteasomes more intensively and with greater preference for hydrophobic P1 residues, resulting in substantially greater numbers of fragments with the sizes and COOH termini typical of epitopes and their precursors. Indeed, many of these fragments correspond to endogenously processed Nef epitopes and͞or their potential precursors. The results suggest that differential proteasomal processing contributes importantly to the clustering of CTL epitopes in hydrophobic regions. C omprehensive analyses of several HIV antigens including Nef have revealed an enormous diversity of epitopes recognized by cytotoxic T lymphocytes (CTLs) of HIV ϩ patients (see the HIV Molecular Immunology Database, http:͞͞hiv-web.lanl.gov͞ content͞immunology). Moreover, HIV CTL epitopes extensively overlap in so-called epitope clusters, whereas other regions of the HIV proteins are almost completely devoid of CTL epitopes (refs. 1-6 and HIV Molecular Immunology Database). Evidence for CTL epitope clusters exists also for non-HIV antigens (7). Although CTL epitope clusters often coincide with rather conserved regions of the HIV proteins (1, 4, 6), it has been postulated that the highly nonuniform distribution of HIV CTL epitopes may be related to antigen processing and presentation (2,4,6).Proteasomes seem to participate in the generation of many if not most CTL epitopes. In the classical class I antigen-processing pathway proteasomes generate epitopes as well as precursors that are trimmed by cytosolic and endoplasmic reticulum-resident aminopeptidases. Trimming by carboxypeptidases seems to be highly unusual. Cytosolic endoproteases other than proteasomes may be involved in the production of certain epitopes, but thus far none have been unequivocally identified (for several excellent reviews, see ref. 8 and all articles in the same issue). Important information on the role of proteasomes in antigen processing has been obtained by in vitro digestion of proteins and protein fragments with isolated proteasomes. However, thus far analyses of CTL epitopecontaining full-length antigens (ovalbumin and ␤-galactosidase) (9, 10) and most antigen fragments focused on a single epitope and͞or related epitope precursors. In nearly all experiments with CTL epitope-containing substrates, 20S proteasomes were used, and excellent correlations with epitope recognition on antigenpresenting cells were often reporte...

CD8+ T Cell Epitope-Flanking Mutations Disrupt Proteasomal Processing of HIV-1 Nef

“…A previous study has shown that the proteasomal cleavage footprint of HIV Nef corresponds to the carboxyl end of the VY8 epitope eluted from the cell surface, indicating that this epitope is processed by the proteasome (55). Our data confirms this finding and further shows a lack of involvement of other proteolytic mechanisms (cysteine proteases, TPP II) in the processing and presentation of VY8.…”

“…A previous study has indicated that the HLA B*35 Nef VY8 epitope is processed by the proteasome (55). We confirmed this finding by using a rVV encoding a ubiquitinated form of Nef, rVV-UbRNef (56), which is rapidly degraded by the 26S proteasome.…”

Host Cell Gene Expression during Human Immunodeficiency Virus Type 1 Latency and Reactivation and Effects of Targeting Genes That Are Differentially Expressed in Viral Latency

“…The intermediate stage (12 to 24 h postinduction) showed maximal changes in pathways involved in immune response modulation and cell survival. These patterns correlated well with the levels of early (multiply spliced) and late (unspliced) HIV protein gene expression and known viral replication effects on the host cell (32,44,59).…”

“…Tat competes with the 11S regulatory subunit for binding to the 20S core complex due to the presence of a common binding site in Tat and the 11S regulator alpha subunit (32,59). Proteasomes are also involved in processing certain regions of HIV-1 Nef preferentially, which leads to production of Nef-specific cytotoxic T-lymphocytes (44). Many other classes of genes encoding immune response modulators, integrins, cell cycle modulators (such as Egr1), nuclear import factors, and G-protein-signaling molecules were also differentially expressed.…”

Selection, Transmission, and Reversion of an Antigen-Processing Cytotoxic T-Lymphocyte Escape Mutation in Human Immunodeficiency Virus Type 1 Infection

“…While transmitted mutations within an epitope would presumably only benefit the virus if the subsequent host expressed the same restricting HLA molecule, antigen-processing mutations that prevent proper presentation of a larger region of HIV-1 could prove beneficial regardless of the recipient's HLA molecules. Considering that some conserved regions of gag and hydrophobic stretches of nef encode substantial numbers of clustered CD8 epitopes (5,43,68), impaired antigen processing within these regions could have a significant impact on the ability of the newly infected host to mount effective CD8 responses. The impact of such mutations may be analogous to the impact that alterations in glycosylation sites have upon evasion of numerous neutralizing antibody responses (2,17,55,62).…”