Sequence-Specific Alterations of Epitope Production by HIV Protease Inhibitors

Abstract

Antigen processing by intracellular proteases and peptidases and epitope presentation are critical for recognition of pathogen-infected cells by CD8+ T lymphocytes. First generation HIV protease inhibitors (PIs) alter proteasome activity, but the effect of first or second generation PIs on other cellular peptidases, the underlying mechanism and impact on antigen processing and epitope presentation to CTL are still unknown. Here we demonstrate that several HIV PIs altered not only proteasome but also aminopeptidase activities in PBMC. Using an in vitro degradation assay involving PBMC cytosolic extracts we showed that PIs altered the degradation patterns of oligopeptides and peptide production in a sequence-specific manner, enhancing the cleavage of certain residues and reducing others’. PIs affected the sensitivity of peptides to intracellular degradation, altered the kinetics and amount of HIV epitopes produced intracellularly. Accordingly the endogenous degradation of incoming virions in the presence of PIs led to variations in CTL-mediated killing of HIV-infected cells. By altering host protease activities and the degradation patterns of proteins in a sequence-specific manner, HIV PIs may diversify peptides available for MHC-I presentation to CTL, alter the patters of CTL responses, and may provide a complementary approach to current therapies for the CTL-mediated clearance of abnormal cells in infection, cancer or other immune disease.

HIV Protease Inhibitor–Induced Cathepsin Modulation Alters Antigen Processing and Cross-Presentation

“…The degradation was stopped with 2.5μL of 100% formic acid and peptide fragments were purified by 5% trichloroacetic acid precipitation. Degradation peptides were identified by in-house MS as previously described (8, 19). …”

“…In mice infected with lymphocytic choriomeningitis virus (LCMV) and treated with RTV, cytotoxic T cell (CTL) responses against LCMV epitopes were changed (6). We previously showed that HIV PIs altered proteasome and aminopeptidase activities in primary human cells, modified HIV protein degradation patterns in a sequence-dependent manner, epitope production and presentation, altering CTL responses (8). …”

“…We previously showed that HIV PIs alter the hydrolytic activities of non-aspartyl proteases such as proteasomes and aminopeptidases (8). However the impact of HIV PIs on cathepsins, cysteine and aspartyl proteases involved in cross-presentation, has never been assessed.…”

Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses

“…Pure peptides (2 nmol) were digested at 37°C for 30 to 240 min with 15 g of cytosolic extracts of matching cells (293T cells, B cells, and primary CD4 T cells) (20). The degradation was stopped with 2.5 l of 100% formic acid, and peptide fragments were purified by the use of 5% trichloroacetic acid (TCA) precipitation.…”

“…The degradation was stopped with 2.5 l of 100% formic acid, and peptide fragments were purified by the use of 5% trichloroacetic acid (TCA) precipitation. Degradation peptides generated in 2 to 3 independent degradation experiments were identified by in-house LC/MS-MS as previously described (20,21,28).…”