Targeting HIV-1 Gag into the Defective Ribosomal Product Pathway Enhances MHC Class I Antigen Presentation and CD8+ T Cell Activation

Abstract

The main source for endogenous peptides presented by the MHC class I (MHC-I) pathway are de novo-synthesized proteins which are degraded via the ubiquitin proteasome pathway. Different MHC-I Ag pools can be distinguished: first, short-lived defective ribosomal products, which are degraded in concert with or shortly after their synthesis, and, second, functional proteins that enter the standard protein life cycle. To compare the contribution of these two Ag sources to the generation of MHC-I-presented peptides, we established murine cell lines which express as a model Ag the HIV-1 Gag polyprotein fused to ubiquitin (Ub) carrying the epitope SIINFEKL (SL). Gag was expressed either in its wild-type form (UbMGagSL) or as a variant UbRGagSL harboring an N-end rule degron signal. Although UbRGagSL displayed wild-type protein stability, its inherent defective ribosomal products rate observed after proteasome shutdown was increased concomitant with enhanced presentation of the SL epitope. In addition, UbRGagSL induces enhanced T cell stimulation of SL-specific B3Z hybridoma cells as measured in vitro and of adoptively transferred TCR-transgenic OT-1 T cells in vivo. Furthermore, an elevated frequency of SL-specific T cells was detected by IFN-γ ELISPOT after immunization of naive C57BL/6 mice with UbRGagSL/EL4 cells. These results further underline the role of the defective ribosomal product pathway in adaptive immunity.

The PTAP Sequence within the p6 Domain of Human Immunodeficiency Virus Type 1 Gag Regulates Its Ubiquitination and MHC Class I Antigen Presentation

“…We have shown previously that Gag can artificially be targeted into the DRiP pathway by introduction of an N-end rule degradation signal (14). The increased DRiP rate of Gag harboring a destabilizing Arg residue at its N terminus correlated with enhanced class I presentation of a Gag-derived epitope and, consequently, an enhanced CD8 + T cell activation, as shown in vitro and in vivo (14).…”

“…The increased DRiP rate of Gag harboring a destabilizing Arg residue at its N terminus correlated with enhanced class I presentation of a Gag-derived epitope and, consequently, an enhanced CD8 + T cell activation, as shown in vitro and in vivo (14). Based on this finding, it was intriguing to look for naturally existing sequence motifs within Gag that might regulate its DRiP rate and, thus, its entry into the MHC-I pathway.…”

“…DRiPs have been demonstrated for a number of viral Ags (10-13), among them the HIV-1 structural protein Gag (8,14). Gag is synthesized in the cytoplasm as the polyprotein precursor Pr55 that is both essential and sufficient for the release of virus like particles (VLPs).…”

Melanoma cells present high levels of HLA‐A2‐tyrosinase in association with instability and aberrant intracellular processing of tyrosinase

“…Defectively processed proteins are thought to be the major source of substrates for MHC class I peptide presentation, as altering the processing/degradation rate of a protein, is associated with significant changes in peptide presentation [5,13,15,16]. For example, adding a degradation signal or retargeting a protein from the exocytic to the cytosolic pathway was shown to enhance epitope production [17] and generation of peptides from a misfolded protein, with a half-life of approximately 70-180 min was demonstrated to be more efficient than the generation of peptides from a ubiquitin-conjugated protein (half-life of ∼10 min) or from a stable protein [5].…”

Manipulation of MHC-I/TCR Interaction for Immune Therapy

“…When the degron signal linked with N terminal of protein galactosidase, the MHC-I presentation of the protein was enhanced and elevated T cell activation evoked (110). Andreas Goldwith et al demonstrated that an antigen conjugated to a degron signal was lead into defective ribosomal product pathway and the antigen was efficiently processed and specific CD8 + T cell activation against a dominant MHC-I epitope in the protein was elevated both in vitro and in vivo (111).…”