MHC Class I Heavy Chain mRNA Must Exceed a Threshold Level for the Reconstitution of Cell Surface Expression of Class I MHC Complexes in Cells Transformed by the Highly Oncogenic Adenovirus 12

Fromm, Sharon Vigodman; Mey-Tal, Sigal Winograd; Coligan, John E.; Schechter, Chana; Ehrlich, Rachel

doi:10.1074/jbc.273.24.15209

Cited by 9 publications

(11 citation statements)

References 54 publications

(34 reference statements)

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“…29 Reverse transcription (RT) of RNA was carried out for 1 hour at 42°C, in 20 L RT buffer containing 1 g total RNA, 0.5 g Oligo dT primer (Promega, WI), nucleotide mixture (1 mM), and 15 units AMV reverse transcriptase (Promega), and 24 units RNasin ribonuclease inhibitor (Promega). Polymerase chain reaction (PCR) was carried out in the appropriate buffer with 1 L cDNA, 20 to 25 pmol of the relevant primers, 0.8 mM nucleotide mixture, and 1.5 units Taq DNA polymerase (Biological Industries).…”

Section: Rna Analysis and Rt-pcrmentioning

confidence: 99%

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A single viral protein HCMV US2 affects antigen presentation and intracellular iron homeostasis by degradation of classical HLA class I and HFE molecules

Arieh

Laham

Schechter

et al. 2003

Blood

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HFE is a nonclassical class I molecule that associates with ␤2-microglobulin (␤2m) and with the transferrin receptor. HFE accumulates in transferrin-containing endosomes, and its overexpression in human cell lines correlates with decreased transferrin receptor (TFR)-mediated iron uptake and decreased intracellular iron pools. A mutation that interferes with proper folding and assembly of HFE complexes results in a severe iron-overload disease hereditary hemochromatosis. We previously suggested that viruses could also interfere with iron metabolism through the production of proteins that inactivate HFE, similarly to classical class I proteins. In particular, we demonstrated in a transient expression system that human cytomegalovirus (HCMV) US2 targeted HFE for proteasomal degradation. Here we demonstrate that the stable expression of HCMV US2 in HEK 293 cells constitutively expressing HFE leads to loss of HFE expression both intracellularly and on the cell surface, and the significant reduction of classical class I expression. Both HFE and classical class I molecules are targeted to degradation via a similar pathway. This HCMV US2-mediated degradation of HFE leads to increased intracellular iron pools as indicated by reduced synthesis of TfR and increased ferritin synthesis. Whether this interference with regulation of iron metabolism potentiates viral replication and/or promotes damage of HCMV-infected tissues remains to be determined. Nevertheless, the deleterious effect of US2 on the expression of HFE and classical class I major histo-compatibility complexes ( IntroductionHuman cytomegalovirus (HCMV) down-regulates the surface expression of major histocompatibility complex (MHC) class I molecules. 1-3 Analysis of HCMV deletion mutants led to the identification of genes within the short segment of the HCMV genome, ie, US2, US3, US6, and US11 that independently mediate this effect. [4][5][6][7][8][9][10][11] Two of these gene products, US2 and US11, target class I heavy chains for dislocation from the endoplasmic reticulum (ER) to the cytosol. 6,7 The heavy chains are deglycosylated by N-glycanase and, subsequently, degraded by the proteasome. The general mode of action of the 2 proteins seem to be similar, but they differ in their ability to attack allelic class I heavy chain gene products. 12 It has been suggested that US2 and US11 specifically down-regulate molecules that are associated with classical antigen presentation pathways because they preferentially promote the degradation of HLA-A and -B loci products relative to those encoded by Supporting the association of HCMV US2 with reduced antigen presentation by HCMV-infected cells and their escape from specific immune responses are data showing that US2 also down-regulates class II MHC DM␣ and DR␣ molecules. 16,17 Most effects of US2 were demonstrated using an astrocytoma cell line; however, recently both US2 and US11 have been shown to down-regulate class I, but not class II, MHC molecules in human dendritic cells. 18 We have previously observed that US...

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Section: Rna Analysis and Rt-pcrmentioning

confidence: 99%

“…29 HEK 293 cells and one of the HFE-transfected clones were further cotransfected with HCMV US2/pcDNA3.1 Zeo (Invitrogen, The Netherlands). Individual clones were selected and expanded.…”

Section: Stable Transfectionmentioning

confidence: 99%

A single viral protein HCMV US2 affects antigen presentation and intracellular iron homeostasis by degradation of classical HLA class I and HFE molecules

Arieh

Laham

Schechter

et al. 2003

Blood

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“…In view of this finding we analyzed the fate of another established substrate for proteasomal degradation from the ER, free class I MHC heavy chains (Hughes et al, 1997). Free class I MHC heavy chains, like ASGPR H2a, are retained in the ER in an endo H sensitive state and are targeted for proteasomal degradation (Machold et al, 1995;Fromm et al, 1998). Although upon proteasomal inhibition we find a small portion of the heavy chains in the cytosol, most of them are associated with the membrane fraction in a subcellular region similar to H2a.…”

Section: Introductionmentioning

confidence: 99%

A Novel Quality Control Compartment Derived from the Endoplasmic Reticulum

Kamhi-Nesher

Shenkman

Tolchinsky

et al. 2001

MBoC

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Degradation of proteins that, because of improper or suboptimal processing, are retained in the endoplasmic reticulum (ER) involves retrotranslocation to reach the cytosolic ubiquitin-proteasome machinery. We found that substrates of this pathway, the precursor of human asialoglycoprotein receptor H2a and free heavy chains of murine class I major histocompatibility complex (MHC), accumulate in a novel preGolgi compartment that is adjacent to but not overlapping with the centrosome, the Golgi complex, and the ER-to-Golgi intermediate compartment (ERGIC). On its way to degradation, H2a associated increasingly after synthesis with the ER translocon Sec61. Nevertheless, it remained in the secretory pathway upon proteasomal inhibition, suggesting that its retrotranslocation must be tightly coupled to the degradation process. In the presence of proteasomal inhibitors, the ER chaperones calreticulin and calnexin, but not BiP, PDI, or glycoprotein glucosyltransferase, concentrate in the subcellular region of the novel compartment. The “quality control” compartment is possibly a subcompartment of the ER. It depends on microtubules but is insensitive to brefeldin A. We discuss the possibility that it is also the site for concentration and retrotranslocation of proteins that, like the mutant cystic fibrosis transmembrane conductance regulator, are transported to the cytosol, where they form large aggregates, the “aggresomes.”

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“…However, even small changes in MHC class I gene expression (i.e. 2-fold) can have a profound effect on MHC class I antigen cell surface expression and the resultant effect on cytotoxic T-cell function (35)(36)(37)(38). For this reason, it is likely that overexpression of CDP/cut in tumor cells, which produces up to a 3.5-fold decrease in expression driven by the HLA-B7 promoter (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…However, even small changes in the level of constitutive MHC class I mRNA levels have been shown to result in much larger changes in MHC class I surface antigen expression and cytotoxic T-cell function (35)(36)(37)(38). It is likely that the large locus-specific variations in MHC class I gene expression observed in many human tumors are the result of net changes in the relative levels of several individual MHC class I transcriptional activators and suppressors in a given tumor type.…”

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confidence: 99%

Identification of CCAAT Displacement Protein (CDP/cut) as a Locus-specific Repressor of Major Histocompatibility Complex Gene Expression in Human Tumor Cells

Snyder¹,

Wang²,

Waring³

et al. 2001

Journal of Biological Chemistry

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The human MHC 1 class I genes encode the highly polymorphic HLA class I antigen heavy chains, membrane-spanning proteins that combine with the invariant ␤ 2 -microglobulin to form the HLA class I surface antigen (1). Classical MHC class I (HLA-A, -B, and -C) antigen expression is essential for processing and presentation of peptide antigens to cytotoxic CD8ϩ lymphocytes (2). In addition, recent evidence suggests that certain classical and nonclassical class I antigens play an important role in inhibition of natural killer (NK) cell function (3-6) and certain subsets of cytotoxic T-cells (7). Therefore, changes in MHC class I gene expression can have profound effects on the overall susceptibility to NK cell and cytotoxic T-cell-mediated lysis, the net effect depending on the specific MHC Class I genes involved. Down-regulation of MHC class I gene expression is observed in many human tumors and transformed cell lines, resulting in decreased susceptibility to cytotoxic T-cell-mediated lysis (8 -13). Global down-regulation of surface class I expression can arise by several mechanisms, including loss of ␤ 2 -microglobulin or peptide transporter gene expression (9, 14). More frequently, loss of MHC class I antigen expression in tumor cells occurs at a single locus. For example, selective down-regulation of HLA-B locus gene expression has been observed in many cell lines derived from patients with metastatic melanoma (15) and colon cancer (16). The specific MHC class I alleles in a particular tumor that activate cytotoxic T-cells may be different from those that inhibit NK cell function. Therefore, understanding how MHC class I antigen expression is controlled at the level of specific loci and alleles is an important goal with possible implications for the development of vaccine-based cancer therapeutics.The mechanisms that mediate locus-specific down-regulation of MHC class I gene expression are incompletely understood. Transcriptional mechanisms appear to be involved in many cases (17-19) but have not been well characterized. The promoter and 5Ј-flanking regions of most MHC class I genes contain several highly conserved, well characterized DNA sequence elements involved in maintenance of constitutive expression (20 -22). Down-regulation of MHC class I expression due to decreased binding of members of the rel family of transcription factors to the highly conserved enhancer A element has been reported in human tumor cell lines (23) and in adenovirus-12-transformed cell lines (24). Our laboratory has previously shown that another highly conserved element, the interferon-stimulated response element, functions as a locusspecific activator of HLA-A gene expression in several

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MHC Class I Heavy Chain mRNA Must Exceed a Threshold Level for the Reconstitution of Cell Surface Expression of Class I MHC Complexes in Cells Transformed by the Highly Oncogenic Adenovirus 12

Cited by 9 publications

References 54 publications

A single viral protein HCMV US2 affects antigen presentation and intracellular iron homeostasis by degradation of classical HLA class I and HFE molecules

A single viral protein HCMV US2 affects antigen presentation and intracellular iron homeostasis by degradation of classical HLA class I and HFE molecules

A Novel Quality Control Compartment Derived from the Endoplasmic Reticulum

Identification of CCAAT Displacement Protein (CDP/cut) as a Locus-specific Repressor of Major Histocompatibility Complex Gene Expression in Human Tumor Cells

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