Human Immunodeficiency Virus Type 1 Gag p24 Alters the Composition of Immunoproteasomes and Affects Antigen Presentation

Abstract

Proteasomes are the major source of proteases responsible for the generation of peptides bound to major histocompatibility complex class I molecules. Antigens, adjuvants, and cytokines can modulate the composition and enzymatic activity of proteasomes and thus alter the epitopes generated. In the present study, we examined the effect of human immunodeficiency virus type 1 (HIV-1) p24 on proteasomes from a dendritic cell line (JAWS II), from a macrophage cell line (C2.3), and from murine primary bone marrow-derived macrophages and dendritic cells. HIV-1 p24 downregulated PA28␤ and the ␤2i subunit of the immunoproteasome complex in JAWS II cells but did not decrease the immunoproteasome subunits in macrophages, whereas in primary dendritic cells, PA28␣, ␤2i, and ␤5i were downregulated. Exposure of JAWS II cells and primary dendritic cells to HIV-1 p24 for 90 min significantly decreased the presentation of ovalbumin to a SIINFEKL-specific CD8 ؉ T-cell hybridoma. The decrease in antigen presentation and the downmodulation of the immunoproteasome subunits in JAWS II cells and primary dendritic cells could be overcome by pretreating the cells with gamma interferon for 6 h or by exposing the cells to HIV-1 p24 encapsulated in liposomes containing lipid A. These results suggest that early antigen processing kinetics could influence the immunogenicity of CD8 ؉ T-cell epitopes generated.Detection and elimination of cells infected with intracellular pathogens such as viruses or parasites are controlled by cytotoxic T lymphocytes (CTLs) that specifically recognize antigenic peptides (approximately 8 to 10 amino acids) bound to major histocompatibility complex (MHC) class I molecules on the surface of the cell (23,43,68,71). It is the interaction of the T-cell receptor and the peptide-bound MHC class I molecule that determines recognition of self or foreign peptide (17,27). Virtually all of the peptides bound to MHC class I molecules are derived from processing of endogenous proteins by the proteasome complex (22, 51). Peptides ranging between 2 and 25 amino acids in length are generated by the proteasome cleavage step (24,52,56,66). These peptides are further trimmed and transported across the membrane of the endoplasmic reticulum and the cis-Golgi apparatus in an ATPdependent manner by specific transporters associated with antigen processing (4, 40).A key component in MHC class I processing is the 26S multicatalytic proteasome complex, which is thought to be responsible for generating 95% of the peptides (32). The proteasome complex is barrel shaped and consists of a catalytic 20S core and two 19S regulatory complexes that bind and unfold ubiquinated proteins (31). The 20S core consists of four stacked rings, with each ring composed of seven subunits. The two outer rings consist of seven different but homologous ␣ subunits (␣1 to ␣7), which bind to the regulatory complexes of the core. The two inner rings consist of seven different ␤ subunits (␤1 to ␤7) (25). The active centers of the constitutive proteasomes (c20S; consti...

Computational and Experimental Validation of B and T-Cell Epitopes of the In Vivo Immune Response to a Novel Malarial Antigen

“…The concentration of the isolated proteasomes was determined using the (BCA) (Pierce, Rockford, IL). The composition of the isolated proteasomes was determined by two-dimensional isoelectrophoresis/SDS-PAGE (2-D IEF) followed by Western Blot detection of either the constitutive proteasome subunits or the immunoproteasome subunits (ENZOLifeSciences) [30], [31].…”

“…Based on previously described methods [30], [31] 10 µg of CelTOS were incubated with purified proteasomes (1 µg) or with each of the respective CAT (0.5 µg) for 16 hrs at 37°C unless otherwise stated. To ensure the specificity of proteasomes, epoxomicin was used as an inhibitor (EnzoLifeSciences, Farmingdale, NY).…”

Cell Type-Specific Proteasomal Processing of HIV-1 Gag-p24 Results in an Altered Epitope Repertoire

“…The concentration of proteasomes was determined using the bicinchoninic acid (BCA) assay. The constitutive and immunoproteasome subunits were identified by two-dimensional (2-D) isoelectrophoresis/SDS-PAGE followed by Western blotting as previously described (59,60). The dried blots were scanned using UMAX VistaScan software.…”

“…This results in a more efficient generation and presentation of subdominant CTL epitopes (20). We have previously demonstrated that Gag-p24 downregulates the PA28␤ subunit in murine DC and therefore interferes with PA28␣/␤ cap formation, resulting in a decrease in antigen presentation (60).…”

“…There are several HIV-1 proteins capable of disrupting and altering antigen processing, including Nef (61), Tat (21), and Gag-p24 (60). Gavioli et al (21) demonstrated that Tat modifies the catalytic subunit compositions and activities of immunoproteasomes in B and T cells.…”