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Prepublished online as a Blood First Edition Paper on November 27, 2002; DOI 10.1182/blood-2002-07-2158.
RED CELLS
From the Department of Cell Research and Immunology,
The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel
Aviv, Israel; Laboratory of Viral Diseases, National Institute of
Allergy and Infectious Diseases (NIAID), National Institutes of Health
(NIH), Bethesda, MD; and Laboratory of Allergic Diseases, NIAID, NIH,
Bethesda, MD.
HFE is a nonclassical class I molecule that associates with
Human 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.4-11 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 HLA-C and -G loci.13-15 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 We have previously observed that US2, but not US11, affects the
stability of the nonclassical class I molecule, HFE, that regulates
iron metabolism.19 It is commonly accepted that the high-affinity binding of HFE to the transferrin receptor (TfR) leads to
reduced intracellular iron stores in HFE-transfected cells.20-23 A mutation in the HFE protein (Cys282Tyr),
that interferes with the proper folding, assembly, and hence cell
surface expression of the HFE molecule,24 is responsible
for most cases of the iron overload disease, hereditary hemochromatosis
(HH).25 Not surprisingly, Using a panel of human HFE monoclonal antibodies (mAbs) and
cell lines infected with recombinant vaccinia viruses (rVVs) expressing HFE, we previously showed that the assembly and stability of HFE complexes do not depend on a functional transporter of peptides and
that HFE/ In this report we demonstrate that HCMV US2 down-regulates the
expression of both class I MHC and HFE-transfected HEK 293 cells.
Furthermore, we show that in HFE overexpressing HEK 293 cells, TfR
expression is up-regulated concomitant with down-regulation in the
expression of the iron storage protein, ferritin. The introduction of
HCMV US2 expression reverses these effects. Similarly, HCMV US2
expression in HEK 293 cells results in increased ferritin levels. Thus,
a single viral protein acts to both subvert the immune response and to
interfere with basic cellular metabolic functions. Both effects may
potentiate viral replication, as well as promote damage of particular
tissues that are chronically infected with HCMV.
Cell cultures
Stable transfection
Antibodies The following antibodies were used in the course of experiments: antihuman HFE 2F5 that recognizes HFE/ 2m heterodimers and HFE
cytoplasmic tail (CT) that recognizes free HFE heavy chains (described previously19); V1-10 (antihuman TfR; a kind
gift of Dr Z. Eshhar, The Weizmann Institute of Science, Rehovot,
Israel); H68.4 (anti-TfR directed against a conserved region in the
N-terminal domain of the protein; Zymed Laboratories, CA); rabbit
antihuman ferritin (DAKO, Denmark); W6/32 (anti-HLA-A, -B,
-C30); rabbit anti-US2 and rabbit antihuman class I heavy
chain antibodies were a kind gift from Dr H. Ploegh (Harvard Medical
School, Boston, MA); DO1 (antihuman p53, a kind gift of Dr M. Oren, The
Weizmann Institute of Science).
Fluorescence-activated cell sorter (FACS) analysis Cells were harvested, incubated at 4°C with the first antibody for 60 minutes, washed, and then incubated for 45 minutes with the second antibody (fluorescein isothiocyanate [FITC]-conjugated goat antimouse immunoglobulin G [IgG]; Jackson ImmunoResearch Laboratories, PA). The cells were washed with phophate-buffered saline (PBS), and fluorescence intensity was measured with a Becton Dickinson Cell Sorter (Becton Dickinson, CA).Metabolic labeling, immunoprecipitation, and Western blot analysis Standard procedures were used.19 For metabolic labeling and immunoprecipitation, cells were split 1 day before metabolic labeling. Briefly, cells were starved for 120 minutes in methionine + cysteine-free medium, labeled in the same medium containing 3.7 MBq/mL (100 µCi/mL) 35[S]-methionine (PerkinElmer Life Sciences, MA), and chased as indicated. The protease inhibitors MG132 (30 µM; Calbiochem-Novabiochem, Germany) was added to the cells 80 minutes prior to radioactive pulsing, as well as during the pulse and the chase. The cells were lysed with a buffer containing 0.5% NP40 (Sigma Chemical, MO), 50 mM Tris (tris[hydroxymethyl]aminomethane), pH 7.5, and 150 mM NaCl and immunoprecipitated with the indicated antibodies and protein A or protein G agarose (Boehringer Mannheim GmbH, Germany). All the antibodies used in immunoprecipitation were titrated by sequential immunoprecipitations and consequently were added in access so that more than 90% of the labeled antigen was immunoprecipitated in the first round of immunoprecipitation. The total protein concentration was determined with Bradford reagent (Sigma Chemical), and equivalent protein amounts were immunoprecipitated. The immunoprecipitates were fractionated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as indicated, and the dried gels were exposed to X-OMAT AR X-ray films (Eastman Kodak Company, NY). For Western blot analysis, cell lysates were fractionated, and the proteins were transferred to nitrocellulose Hybond-C membrane (Amersham Pharmacia Biotech, United Kingdom). The membrane was incubated with 5% dry milk/PBS, probed with the specific antibody, and detected with the appropriate horseradish peroxidase-conjugated secondary antibodies, and enhanced chemiluminescence substrates (Amersham Pharmacia Biotech, United Kingdom).RNA analysis and RT-PCR Cytoplasmic RNA was prepared as previously described.29 Reverse transcription (RT) of RNA was carried out for 1 hour at 42°C, in 20 µL RT buffer containing 1 µg total RNA, 0.5 µg 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 µL cDNA, 20 to 25 pmol of the relevant primers, 0.8 mM nucleotide mixture, and 1.5 units Taq DNA polymerase (Biological Industries). The following primers were used: HFE sense, 5'-TGGCAAGGGTAAACAGATCC-3'; -sense,
5'-CTCAGGCACTCCCTCTCAACC-3'; HCMV US2 sense,
5'-CGGAATTCCGCGCATGAACAATCTCTGGAAAGC-3'; -sense, 5'-GCTCTAGAGCTCAGCACACGAAAAACCG-3'; actin sense,
5'-GTTTGAGACCTTCAACACCCC-3'; -sense,
5'-GTGGCCATCTCTTGCTCGAAGTC-3'. After appropriate number of PCR
cycles (MiniCycler; MJ Research, MA), 10 µL reaction mix was
fractionated on agarose gel.
TfR synthesis is induced and ferritin synthesis is reduced in HFE-transfected HEK 293 cells HEK 293 cells were transfected with HFE, and selected clones were further analyzed for cell surface expression of HFE and HLA class I complexes. The expression of HFE/2m heterodimers was analyzed with
anti-HFE mAb, 2F5, that recognizes an epitope on the HFE/2m
heterodimer that is masked following association with
TfR.19 The expression of HLA was analyzed with mAb W6/32 that recognizes a public epitope on HLA-A, -B, and -C complexes. Figure
1A demonstrates that 293/HFE cells
express high levels of HFE/2m complexes; however, the expression of
HLA complexes is reduced relative to the parental HEK 293 cells. A
possible explanation for these observations is that HFE and HLA class I heavy chains compete for binding with the available 2m. Indeed, comparison of the relative amounts of conformed HLA complexes with free
HLA heavy chains (Figure 1B) in 293/HFE versus HEK 293 cells by a
quantitative immunoprecipitation revealed that the relative amount of
conformed HLA-A, -B, and -C complexes is reduced, whereas that of free
class I heavy chains is increased in 293/HFE cells, supporting the
hypothesis that there is competition for 2m.
Next, we analyzed whether the overexpression of HFE affects
intracellular iron homeostasis by determining the levels of the iron-transporter receptor, TfR, and the iron-storage protein, ferritin,
in 5 individual HFE-expressing clones. The levels of newly synthesized
ferritin were reduced, whereas TfR levels were increased in all clones
(Figure 2). These data suggest that HEK 293 cells overexpressing functional HFE complexes have reduced intracellular iron pools, leading to induced translation of TfR and
reduced translation of ferritin. Similar data were obtained with
HFE-transfected HeLa cells22,31-33 but not in
TfR-deficient Chinese hamster ovary (CHO) cells that have been
reconstituted for the expression of human HFE, human TfR, and human
HCMV US2 down-regulates the expression of HFE and class I molecules via a similar mechanism One of the HFE-transfected HEK 293 clones was further transfected with HCMV US2 cDNA. Three individual clones that expressed the US2 gene as determined by RT-PCR (Figure 3A) and immunoprecipitation (Figure 3B) were chosen for further studies. Cell surface expression of HFE and HLA class I complexes was analyzed and revealed a complete or a partial reduction in HFE expression but no significant changes in the level of HLA expression (Figure 4A). Most likely, the absence of change in cell surface HLA class I expression results from the assembly of residual HLA class I heavy chains (Figure 1B) with2m made available by HFE degradation. Otherwise, US2 is effective in
reduction of class I as demonstrated by the 3-fold reduction in cell
surface HLA class I complexes expressed by US2-transfected HEK 293 cells (Figure 4B). The decrease in cell surface HLA class I is
correlated with reduced level of newly synthesized free HLA class I
heavy chains (Figure 4C, top) and total HLA molecules (Figure 4C,
bottom) in these cells. Next, the intracellular levels of HFE and HLA
class I molecules were analyzed in 293/HFE/US2 cells. Figure
5A demonstrates that the levels of newly
synthesized free HFE chains, HFE/2m, TfR-associated HFE/2m
complexes are dramatically reduced. The clonal variation in
US2-mediated reduction of intracellular HFE (Figure 5) was consistent
with that observed for cell surface HFE reduction (Figure 4A). Figure
5B shows that the level of newly synthesized free HLA class I heavy
chains was greatly reduced, whereas the level of conformed class I
complexes was unchanged. The latter is in agreement with results shown
in Figure 4A. The detection of comparable levels of conformed class I
complexes in immunoprecipitates from cell lysates and on the cell
surface agrees with the idea that US2-mediated HFE degradation in
293/HFE/US2 cells promotes class I complex formation by increasing the
supply of 2m. Whereas US2 mediated reduction in the levels of total
HFE and total HLA, it did not affect the level of a nonrelevant protein, p53, in US2-transfected 293/HFE cells (Figure 5C),
demonstrating that US2 is not functioning nonspecifically. The overall
data presented in Figures 4 and 5 suggest that both HFE and HLA class I
molecules are down-regulated in US2-expressing HEK 293 cells. The data
demonstrate that, despite the similar effect of US2 on HFE and class I
molecules, the outcome, in this system, is not identical and that US2
appears to have a more dramatic effect on free HFE chains and conformed
HFE complexes than on HLA molecules.
HCMV US2 targets HFE to proteasome-dependent degradation We have previously demonstrated that US2 HCMV targets HFE to proteasome-mediated degradation in HeLa cells coinfected with rVV-HFE and rVV-US2. However, although previous publications demonstrated the accumulation of a deglycosylated class I heavy chain in some US2-transfected cell lines that have been treated with proteasome inhibitors,35 we could not detect a smaller HFE fragment in infected cells that have been treated with MG132 or lactacystin. A better analysis of the mechanism of US2-mediated HFE degradation could be performed in 293/HFE/US2 cells. Figure 6 demonstrates that the expression of free HFE and HFE/2m complexes was reconstituted following treatment
of cells with the proteasome inhibitor MG132. The same results were
obtained following treatment with lactacystin (data not shown). Similar
to our previous results,19 it appears that the conformed
complexes are not retranslocated to the cytosol in MG132-treated cells
as indicated by the strong signal observed in the 2F5
immunoprecipitate. However, a smaller band that corresponds in size to
N-glycanase-treated HFE was observed in the free HFE immunoprecipitate
(Figure 6B), whereby the mechanism of US2-mediated degradation of HFE
is similar to that of classical class I heavy chains. The heavy chains
are translocated to the cytosol, deglycosylated, and, subsequently,
degraded. Therefore, in the presence of a proteasome inhibitor,
although a deglycosylated form of HFE is stabilized, most of the
molecules are glycosylated and about half of the molecules remain
conformed as demonstrated previously.19
Expression of US2 HCMV reconstitutes TfR and ferritin levels Because HFE overexpression results in an iron-starved phenotype (Figure 2), we expected that the degradation of HFE because of US2 expression would lead to reconstitution of intracellular iron homeostasis. Figure 7A shows that indeed, in the 3 US2-expressing clones, the synthesis of TfR is reduced and the synthesis of ferritin is increased as compared with the 293/HFE cells. Quantitative analysis of the total ferritin and TfR levels in US2-expressing cells by sequential immunoprecipitations with anti-TfR and antiferritin antibodies revealed that the effect on ferritin was more dramatic than the effect on TfR expression (data not shown). Thus, the US2-mediated degradation of HFE leads to a complete reconstitution of intracellular iron homeostasis. We observed that in the US2 transfectants ferritin levels were higher than in HEK 293 cells, suggesting that US2 was affecting more than the transfected HFE. To investigate this further we analyzed the level of TfR (Figure 7B, top), and ferritin (Figure 7B, bottom) in US2-transfected HEK 293 cells. The results demonstrate that US2-expressing cells have increased levels of ferritin in comparison to the parental cells but not consistent changes in TfR. These changes in ferritin are unlikely to be due to a direct effect of US2 on ferritin because these 2 proteins exist in different cellular compartments; however, they may be due to US2-mediated degradation of endogenous HFE. Thus, it appears that ferritin regulation is more sensitive than TfR regulation to changes in the level of intracellular iron pools, and that US2 expression results in altered intracellular iron homeostasis in the HEK 293 cell line despite the low expression level of endogenous HFE.
HCMV is a ubiquitous herpesvirus that infects a variety of tissues and cell types, in particular epithelial and endothelial cells, but also fibroblasts, smooth muscle cells, and macrophages.36,37 HCMV infection causes a mild disease that can be associated with hepatitis in immunocompetent hosts,38,39 but it is a life-threatening disease in immunosuppressed patients, including transplantation and AIDS patients. It is commonly accepted that viruses have evolved a variety of means of interfering with immune responses. Specifically, HCMV encodes several proteins that interfere with class I MHC-mediated antigen presentation, leading to evasion from cellular immune responses.35,40 Because HCMV infection is often associated with diseases of the gastrointestinal tract,41 the site of expression of HFE, and because HFE is closely related to classical class I MHC molecules that are targets for some HCMV encoded molecules, we postulated that these viral-encoded proteins may have a broader scope of action. We have previously demonstrated19 that coinfection of
rVV-expressing HFE and US2 prevents the expression of free HFE,
HFE/ US2-mediated regulation of HFE has wide-reaching implications for iron metabolism. Expression of HFE in transfected cells results in decreased levels of ferritin and increased levels of TfR (Figure 2), changes that are consistent with an iron-starved phenotype. This situation is not surprising because HFE is thought to down-regulate intracellular iron.22,31-33 In US2-transfected cells HFE levels are reduced. Consequently, we observed that the expression of ferritin and TfR are reversed, such as ferritin synthesis is enhanced and TfR synthesis is inhibited (Figure 7A). US2 induced ferritin not only in cells overexpressing HFE but also in parental HEK 293 cells. In parallel to reduction of HFE, US2 induced reduction of HLA class I proteins (Figures 4B-C and 5B-C) as previously reported.40 Thus, one viral protein may be involved in the subversion of immune responses and regulation of factors involved in iron hemostasis. The 2 effects might contribute to viral maintenance, to survival of virus-infected cells, and also to the damage of chronically infected tissues because of iron accumulation that results from the reduction in HFE expression and induced iron uptake. The effect of US2 on HFE and on HLA heavy chains is similar but not
identical. Although US2 almost completely abolishes intracellular and
cell surface expression of HFE heavy chains and HFE complexes, it
affects mainly free HLA heavy chains and does not significantly change
the overall expression of conformed class I complexes. There are
several possible explanations for these observations: US2 has higher
affinity for HFE than for HLA molecules. The high concentration of HFE
in the transfected cells may preferentially facilitate HFE association
with US2 and its consequential degradation. In this case free HFE
chains are no longer available for assembly with Current data show that HCMV US2 targets HLA-A and HLA-B locus products, but not HLA-C and HLA-G locus products for proteasome-dependent degradation despite the high homology between these genes at the predicted US2-binding site.13,43 However, in specific systems, US2 targets class II DR and DM complexes for degradation, despite their very low homology to HLA-A and HLA-B locus products.16 These observations raised the possibility that US2 was specifically selected for its ability to block class I and class II presentation pathways. Hence, MHC molecules that are expressed in specific tissues and might have specific functions such as HLA-G (expressed in the trophoblasts and protects from natural killer [NK]-mediated activity44) or HLA-C (known to have reduced surface stability45) may be somehow resistant to this effect. However, the data presented in this article show that US2 has a more dynamic effect. It targets to degradation a nonclassical class I molecule that is not homologous to class I at the predicted US2 binding site43 and does not play any direct role in the classical pathway of antigen presentation. How a viral protein can associate with completely different sequence elements remains an enigma. However, Chevalier et al17 demonstrated recently that the US2 residues involved in binding to MHC class I differ subtly from those involved in binding to class II proteins. The US2 binding site on HFE was not characterized yet, but it might be HFE specific. Thus, the virus uses particular sequence elements in the same protein for altering functions that might promote its survival and replication. On one hand, it interferes with classical antiviral immune responses (mediated by class I and II complexes), and, on the other hand, it induces cellular iron uptake that might support its growth. The fact that HCMV infection is often associated with tissue injury in
the same tissues that HFE is highly expressed is particularly interesting in view of observations that iron supports viral
replication and chronic infections. Hepatic iron concentration has
consistently been observed as being directly correlated with the
response to interferon therapy in the treatment of chronic hepatitis C
virus infection.46 Moreover, treatment of patients with
hepatitis C and iron overload with iron chelators improved their
response to interferon therapy.47 In vitro studies
demonstrated that iron enhances hepatitis C virus replication in
cultured human hepatocytes.48 Replication of human
immunodeficiency virus type 1 (HIV-1) can be influenced by iron as
demonstrated by the fact that iron chelators inhibit virus
replication.49,50 Iron chelators also inhibit CMV
infection and CMV-induced pathogenic changes.51 Zoll et
al52 showed recently that the Mengovirus leader protein interferes with antiviral immune responses through inhibition of
iron/ferritin-mediated activation of nuclear factor
We thank Dr C. Enns (Oregon Health Sciences University, Oregon) for the fruitful discussions; Drs Z. Eshhar, M. Oren (The Weizmann Institute of Science, Rehovot, Israel), and H. Ploegh (Harvard Medical School, Boston, MA) for the antibodies; and Dr N. Smorodinsky and M. Yaacubovicz for their collaboration in the generation of the antihuman HFE mAbs.
Submitted July 18, 2002; accepted November 13, 2002.
Prepublished online as Blood First Edition Paper, November 27, 2002; DOI 10.1182/blood-2002-07-2158.
Supported by the United States-Israel Binational Science Foundation (BSF), the EC Contract No. QLG1-CT-1999-00665, the Israel Ministry of Health, and by the Mozelsio Fund for Pediatric Cancer Research. S.V.-B.A. is a recipient of an ICRETT fellowship (International Union Against Cancer UICC, Switzerland).
Correspondence: Rachel Ehrlich, Department of Cell Research and Immunology, Tel Aviv University, Ramat Aviv 69978, Israel; e-mail: rachele{at}post.tau.ac.il.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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© 2003 by The American Society of Hematology.
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  D. N. Hebert and M. Molinari In and Out of the ER: Protein Folding, Quality Control, Degradation, and Related Human Diseases Physiol Rev, October 1, 2007; 87(4): 1377 - 1408. [Abstract] [Full Text] [PDF]
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 S. F. de Almeida, J. V. Fleming, J. E. Azevedo, M. Carmo-Fonseca, and M. de Sousa Stimulation of an Unfolded Protein Response Impairs MHC Class I Expression J. Immunol., March 15, 2007; 178(6): 3612 - 3619. [Abstract] [Full Text] [PDF]
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 C. Thilo, P. Berglund, S. E. Applequist, J. W. Yewdell, H.-G. Ljunggren, and A. Achour Dissection of the Interaction of the Human Cytomegalovirus-derived US2 Protein with Major Histocompatibility Complex Class I Molecules: PROMINENT ROLE OF A SINGLE ARGININE RESIDUE IN HUMAN LEUKOCYTE ANTIGEN-A2 J. Biol. Chem., March 31, 2006; 281(13): 8950 - 8957. [Abstract] [Full Text] [PDF]
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 S. F. de Almeida, I. F. Carvalho, C. S. Cardoso, J. V. Cordeiro, J. E. Azevedo, J. Neefjes, and M. de Sousa HFE cross-talks with the MHC class I antigen presentation pathway Blood, August 1, 2005; 106(3): 971 - 977. [Abstract] [Full Text] [PDF]
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 S. Pascolo, F. Ginhoux, N. Laham, S. Walter, O. Schoor, J. Probst, P. Rohrlich, F. Obermayr, P. Fisch, O. Danos, et al. The non-classical HLA class I molecule HFE does not influence the NK-like activity contained in fresh human PBMCs and does not interact with NK cells Int. Immunol., February 1, 2005; 17(2): 117 - 122. [Abstract] [Full Text] [PDF]
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R. Salerno-Goncalves, M. Fernandez-Vina, D. M. Lewinsohn, and M. B. Sztein Identification of a Human HLA-E-Restricted CD8+ T Cell Subset in Volunteers Immunized with Salmonella enterica Serovar Typhi Strain Ty21a Typhoid Vaccine J. Immunol., November 1, 2004; 173(9): 5852 - 5862. [Abstract] [Full Text] [PDF]
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 W. E. Crowe, L. M. Maglova, P. Ponka, and J. M. Russell Human cytomegalovirus-induced host cell enlargement is iron dependent Am J Physiol Cell Physiol, October 1, 2004; 287(4): C1023 - C1030. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
B
(NF