Anti-Peptide Antibody Blocks Peptide Binding to MHC Class I Molecules in the Endoplasmic Reticulum

Influenza A virus gene segment 7 encodes two proteins: the M1 protein translated from unspliced mRNA and the M2 protein produced by mRNA splicing and largely encoded by the M1 +1 reading frame. To better understand the generation of defective ribosomal products (DRiPs) relevant to MHC class I antigen presentation, we engineered influenza A virus gene segment 7 to encode the model H-2 Kb class I peptide ligand SIINFEKL at the M2 protein C-terminus. Remarkably, after treating virus-infected cells with the RNA splicing inhibitor spliceostatin A to prevent M2 mRNA generation, Kb-SIINFEKL complexes were still presented on the cell surface at levels of up to 60% of untreated cells. Three key findings indicate that SIINFEKL is produced by cytoplasmic translation of unspliced M1 mRNA initiating at CUG codons within the +1 reading frame: Synonymous mutation of CUG codons in the M2-reading frame reduced Kb-SIINFEKL generation.Kb-SIINFEKL generation was not affected by drug-mediated inhibition of AUG-initiated M1 synthesis.Kb-SIINFEKL was generated in vitro and in vivo from mRNA synthesized in the cytoplasm by vaccinia virus, and hence cannot be spliced. These findings define a viral DRiP generated by cytoplasmic non-canonical translation and demonstrate the participation of CUG-codon-based translation initiation in pathogen immunosurveillance.

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“…Immunoblotting cell lysates 12 hrs post-infection (p.i.) with a SIINFEKL-specific mAb also revealed M2 SIINFEKL expression (21) (Fig. S1B).…”

Section: Resultsmentioning

confidence: 85%

Defining Viral Defective Ribosomal Products: Standard and Alternative Translation Initiation Events Generate a Common Peptide from Influenza A Virus M2 and M1 mRNAs

Yang

Gibbs

Hickman

et al. 2016

The Journal of Immunology

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“…The superiority of endogenous loading may be explained by a high efficiency of chaperone-assisted peptide loading onto nascent MHC-I molecules in a specific loading complex in the ER (reviewed in reference 13), whereas exogenous loading requires replacement of already bound "self" or viral peptides by affinity-dependent peptide competition (59). Although SIINFEKL was found to bind with high affinity to K b , resulting in very stable K b -SIINFEKL complexes (8,30), its dissociation constant may nevertheless be too high to replace viral peptides of even higher affinity (44). Interestingly, for uninfected cells in which MHC-I molecules present only "self" peptides, exogenous loading reached a K b occupancy close to the values reached by endogenous loading after infection in the absence of immunoevasins.…”

Section: Discussionmentioning

confidence: 92%

Immune Evasion Proteins of Murine Cytomegalovirus Preferentially Affect Cell Surface Display of Recently Generated Peptide Presentation Complexes

Lemmermann

Gergely

Böhm

et al. 2010

J Virol

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For recognition of infected cells by CD8 T cells, antigenic peptides are presented at the cell surface, bound to major histocompatibility complex class I (MHC-I) molecules. Downmodulation of cell surface MHC-I molecules is regarded as a hallmark function of cytomegalovirus-encoded immunoevasins. The molecular mechanisms by which immunoevasins interfere with the MHC-I pathway suggest, however, that this downmodulation may be secondary to an interruption of turnover replenishment and that hindrance of the vesicular transport of recently generated peptide-MHC (pMHC) complexes to the cell surface is the actual function of immunoevasins. Here we have used the model of murine cytomegalovirus (mCMV) infection to provide experimental evidence for this hypothesis. To quantitate pMHC complexes at the cell surface after infection in the presence and absence of immunoevasins, we generated the recombinant viruses mCMV-SIINFEKL and mCMV-⌬m06m152-SIINFEKL, respectively, expressing the K b -presented peptide SIINFEKL with early-phase kinetics in place of an immunodominant peptide of the viral carrier protein gp36.5/m164. The data revealed ϳ10,000 K b molecules presenting SIINFEKL in the absence of immunoevasins, which is an occupancy of ϳ10% of all cell surface K b molecules, whereas immunoevasins reduced this number to almost the detection limit. To selectively evaluate their effect on preexisting pMHC complexes, cells were exogenously loaded with SIINFEKL peptide shortly after infection with mCMV-SIINFEKA, in which endogenous presentation is prevented by an L174A mutation of the C-terminal MHC-I anchor residue. The data suggest that pMHC complexes present at the cell surface in advance of immunoevasin gene expression are downmodulated due to constitutive turnover in the absence of resupply. CD8 T cells recognize infected cells by interaction of theirT-cell receptor (TCR) with a cell surface presentation complex composed of a cognate antigenic peptide bound to a presenting allelic form of a major histocompatibility complex class I (MHC-I) glycoprotein (77,85,97,98). The number of such "peptide receptors" per cell has been estimated to be on the order of 10 5 to 10 6 for each MHC-I allomorph (for a review, see reference 82). Viral antigenic peptides are generated within infected cells by proteolytic processing of viral proteins, usually in the proteasome, and associate with nascent MHC-I proteins in the endoplasmic reticulum (ER) before the peptide-MHC (pMHC) complexes travel to the cell surface with the cellular vesicular flow (for reviews, see references 13, 87, 92, and 93). CD8 T cells have long been known to protect against cytomegalovirus (CMV) infection and disease in animal models (60, 72; reviewed in references 33 and 36) and in humans (9,61,67,75,76). As shown only recently in the murine CMV (mCMV) model of infection of immunocompromised mice by adoptive transfer of epitope-specific CD8 T cells, antiviral protection against CMV is indeed TCR mediated and epitope dependent. Specifically, memory cells purified by TCR-bas...

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“…25D1.16 supernatants were obtained from hybridoma cells (kindly provided by R. Germain of the National Institutes of Health, Bethesda). 1F10 2.2 anti-SIINFEKL peptide mAb was described (30).…”

Section: Methodsmentioning

confidence: 99%

Cellular protein is the source of cross-priming antigenin vivo

Shen

Rock

2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

176

129

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Cross-priming is essential for generating cytotoxic T lymphocytes to viral, tumor, and tissue antigens that are expressed exclusively in parenchymal cells. In this process, the antigen-bearing parenchymal cells must somehow transfer their antigens to bone marrow-derived professional antigen-presenting cells. Although intact proteins, small peptides, or peptide-heat shock protein complexes can all be acquired and presented by antigen-presenting cells, the physiologically relevant form of antigen that is actually transferred from parenchymal cells and cross-presented in vivo is unknown and controversial. To address this issue we have investigated the ability of fibroblasts stably expressing chicken ovalbumin constructs targeted to different subcellular compartments to crossprime cytotoxic T lymphocytes. Although these transfectants generated similar amounts of the immunogenic ovalbumin peptide, their cross-priming activity differed markedly. Instead, the cells cross-priming ability correlated with their steady-state levels of ovalbumin protein and͞or the physical form͞location of the protein. Moreover, in subcellular fractionation experiments, the crosspriming activity colocalized with antigenic protein. In addition, depletion of intact protein antigen from these cell fractions eliminated their cross-priming activity. In contrast, the major heat shock protein candidates for cross-presentation were separable from the cell's main sources of cross-priming antigen. Therefore, cellular proteins, rather than peptides or heat shock protein͞ peptide complexes, are the major source of antigen that is transferred from antigen-bearing cells and cross-presented in vivo.

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Anti-Peptide Antibody Blocks Peptide Binding to MHC Class I Molecules in the Endoplasmic Reticulum

Cited by 16 publications

References 31 publications

Defining Viral Defective Ribosomal Products: Standard and Alternative Translation Initiation Events Generate a Common Peptide from Influenza A Virus M2 and M1 mRNAs

Defining Viral Defective Ribosomal Products: Standard and Alternative Translation Initiation Events Generate a Common Peptide from Influenza A Virus M2 and M1 mRNAs

Immune Evasion Proteins of Murine Cytomegalovirus Preferentially Affect Cell Surface Display of Recently Generated Peptide Presentation Complexes

Cellular protein is the source of cross-priming antigenin vivo

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