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Volume 271, Number 16, Issue of April 19, 1996 pp. 9851-9857
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Induction of Ferritin Synthesis in Cells Infected with Mengo Virus (*)

(Received for publication, December 4, 1995; and in revised form, January 30, 1996)

Michael R. Mulvey (1), (§),  Lukas C. Kühn (2),  Douglas G. Scraba (1)

From the  (1)Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and the (2)Swiss Institute for Experimental Cancer Research, CH-1006 Epalingess/Lausanne, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have recently identified ferritin as a cellular protein particle whose synthesis is stimulated in mouse or human cells infected by the picornavirus Mengo(1) . Immunoprecipitation of the particle from infected murine L929 cells showed a 4- and 6-fold increase in the intracellular concentrations of H and L apoferritin subunits, respectively. This differential expression altered the H/L subunit ratio from 3.0 in uninfected cells to 2.2 in Mengo virus-infected cells. The induction is not due to an increase in transcription of the apoferritin L and H genes, nor is it due to an increase in stability of the apoferritin mRNAs. At the level of translation, the iron regulatory protein (IRP) remained intact, with similar amounts being detected in uninfected and infected cells. The Mengo virus RNA genome does not compete with the iron regulatory element (IRE) for the binding of IRP, and sequence analysis confirmed that there are no IREs in the virus RNA. The IRE binding activity of IRP in infected cells decreased approximately 30% compared with uninfected cells. The decrease in binding activity could be overcome by the addition of Desferal (deferoxamine mesylate; CIBA) an intracellular iron chelator, which suggests that virus infection causes an increase in intracellular free iron. Electron paramagnetic resonance (EPR) studies have confirmed the increase in free iron in Mengo virus infected cells. The permeability of cells for iron does not change in virus infected cells, suggesting that the induction of ferritin by Mengo virus is due to a change in the form of intracellular iron from a bound to a free state.

INTRODUCTION

Free ferrous or ferric iron in biological systems can catalyze a number of reactions which generate free radicals and can lead to protein, DNA, and/or lipid damage. To prevent these deleterious reactions from occurring, iron is usually associated with proteins. Ferritin serves as the major eukaryotic intracellular iron storage protein. Murine ferritin consists of 24 subunits of the L (25 kDa) and H (18 kDa) apoferritin proteins in various ratios, depending on the type of tissue and physiological state of the cells(2) . The two subunits combine in the cytoplasm to form a hollow shell (molecular weight 450,000) capable of binding as many as 4500 atoms of iron (reviewed in (2) ).

The regulation of apoferritin biosynthesis is complex and involves control mechanisms that function both at the level of transcription and translation. Specific regulatory mechanisms also appear to be dependent on cell species and type. Several factors have been shown to increase transcription of the apoferritin genes. Treatment of cells with tumor necrosis factor-alpha results in the selective increase of H apoferritin mRNA in certain murine (3) and human (4, 5) cell lines. However, when mouse fibroblast (L929) cells were so treated, apoferritin mRNA synthesis was not induced(6) . Interleukin 1-alpha, thyrotropin and cAMP have also been shown to activate preferentially the synthesis of H apoferritin(7, 8, 9) . Other compounds such as phorbol esters or dimethyl sulfoxide alter both L and H apoferritin mRNA levels(10, 11) . The role of iron or hemin in the induction of transcription of the two apoferritin genes is also dependent on the particular cell line tested: free iron has been shown to increase the levels of L and H apoferritin mRNA in human myoblasts(4) , human K562 cells(12) , mouse fibroblasts, and Chinese hamster ovary cells (13) ; however, this did not occur in human HeLa cells(13) .

The translational control of apoferritin is mediated by a 98-kDa ``iron regulatory protein-1'' (IRP-1), (^1)previously referred to as the ``iron regulatory factor'' (IRF; (14) ), the ``iron response element-binding protein'' (IRE-BP; (15) ), or the ``ferritin repressor protein'' ((16) ). It recognizes an ``iron regulatory element'' (IRE; (17) and (18) ), which is a stem-loop forming sequence in the 5`-untranslated region of L and H apoferritin mRNAs. IREs are also located in the 5`-untranslated region of the mRNAs for erythroid 5-aminolevulinic acid synthetase and mitochondrial aconitase(19, 20) . The 3`-untranslated region of the transferrin receptor mRNA contains five copies of the IRE in tandem (14, 21) . When intracellular levels of iron are low, the IRP exhibits a high affinity for the IRE. Binding of IRP to IRE results in the repression of translation of mRNAs containing the IREs in the 5`-untranslated region and stabilization of mRNAs containing the IREs in the 3`-untranslated region. When intracellular levels of iron increase, the IRP binds iron as an iron-sulfur [4Fe-4S]-cluster. This produces a conformational change that reduces its IRE binding activity and creates an aconitase activity(22, 23, 24, 25, 26) . Thus, the cell is provided with a mechanism for dealing rapidly with potentially damaging concentrations of free iron: under conditions of high intracellular concentrations of iron, L and H apoferritins are synthesized to store the excess iron, while the transferrin receptor mRNA rapidly degrades and a diminished number of transferrin receptors are produced.

A second IRE-binding protein, called IRP-2 (previously IRP(B) or IRE-BP2), has recently been characterized(27, 28, 29) . IRP-2 has a molecular mass of 105 kDa and is structurally related to, but distinct from, IRP-1(27, 29) . It has similar affinity for IREs but lacks aconitase activity and, unlike IRP-1, is rapidly degraded in cells supplemented with iron(28, 29, 30, 31, 32) . The tissue distribution of IRP-2 is somewhat different from that of IRP-1(27, 28) , and IRP-2 has been shown to be activated specifically during liver regeneration in the absence of changes in total tissue iron content(33) .

Infection of cells with viruses can also affect the synthesis of apoferritin. The adenovirus E1A protein was shown to repress the synthesis of apoferritin H mRNA but to have no effect on the synthesis of L apoferritin(34) . Human immunodeficiency virus infection of three different permissive cell lines resulted in a decrease in total cytoplasmic ferritin levels(35) ; however, no attempt was made to determine if the decrease was a result of transcriptional or translational inhibition. Other experiments have found that there is an increase in serum and red blood cell ferritin levels in patients infected with human immunodeficiency virus(36) . The picornaviruses Mengo and Theiler's murine encephalitis have been shown to induce the synthesis of both L and H apoferritin proteins in infected mouse L929 fibroblasts and human HeLa cells(1, 37, 38) . In contrast, no increase in ferritin production in HeLa cells infected with poliovirus or L929 cells infected with reovirus was observed.

In this communication we present the results of investigations into the mechanism of apoferritin induction during Mengo virus infection of murine fibroblast cells. Induction is not due to transcriptional activation of apoferritin genes nor is it due to the destruction or inactivation of the IRP. We find that induction is a result of a change in form of the intracellular iron from a bound to a free state which in turn leads to the derepression of translation of apoferritin mRNAs.

MATERIALS AND METHODS

Tissue Culture and Media

Mouse L929 fibroblasts were grown in 199 medium (Hyclone) supplemented with 5% fetal calf serum (Hyclone). Confluent monolayers were infected with Mengo virus at a multiplicity of 100 plaque forming units/cell in medium 199 supplemented with 1% fetal calf serum. After 1 h the media was changed to 199 with 5% fetal calf serum and the incubation was continued. In experiments involving uninfected control cells, the identical procedure was followed but omitting the virus inoculum. Where indicated, cells were treated with 50 µM deferoxamine mesylate (Desferal, CIBA) for 24 h prior to the start of the experiment.

Immunoprecipitation and Western Blotting

L929 fibroblasts were mock-infected or infected with Mengo virus and radiolabeled 5 h post-infection with [S]methionine (ICN) as described previously(1) . Total protein synthesis was measured by trichloroacetic acid precipitation(37) . Ferritin was immunoprecipitated from cell extracts (1.0 times 10^6 cpm) with anti-human ferritin (Sigma) or anti-rabbit heme oxygenase I (StressGen) and protein A-Sepharose (Phamacia Biotech Inc.), using the method of Sambrook et al.(39) . Proteins were separated by SDS-PAGE (40) in 12% gels. These were dried, exposed to a phosphorimaging plate (Fuji), and radioactivity distribution analyzed with a Fujix Bio-imaging analyzer BAS 1000. The amount of [S]methionine incorporated into the immunoprecipitated proteins was determined using the MacBAS version 1.0 software provided with the phosphorimager.

Western blot analysis of L and H apoferritin subunits and IRP-1 was carried out essentially as described by Mulvey et al.(1) . Briefly, proteins separated by SDS-PAGE were blotted onto polyvinylidine difluoride membranes in an electroblotting device (Bio-Rad) at 300 mA constant current for 30 min at 4 °C. After blocking with BSA, the membranes were incubated with rabbit polyclonal anti-human spleen ferritin (Sigma), which recognizes both mouse and human ferritins(41) , or rabbit polyclonal anti-human IRP-1, which recognizes mouse IRP-1, but not IRP-2(27) . Goat anti-rabbit IgG conjugated with alkaline phosphatase (Sigma) was used for detection of the antigen-antibody complexes.

RNA Isolation and Northern Blot Analysis

Total cytoplasmic RNA from uninfected and Mengo virus-infected cells was extracted at the indicated times by the method of Sambrook et al.(39) . RNA isolated from equivalent numbers of cells was fractionated by electrophoresis on a 0.8% agarose gel containing 0.66% formaldehyde and blotted onto a nylon membrane (Hybond-N, Amersham Corp.) according to the manufacturer's directions. RNA was quantitated by absorbance at A.

Apoferritin L cDNA for probing Northern blots was synthesized using 1 µg of L cell mRNA, the reverse primer 5`-CTTTCCAGGAAGTCACAGAG-3`, which corresponds to base numbers 550-569 of the murine L apoferritin cDNA(42) , and 200 units of Superscript reverse transcriptase (Life Technologies, Inc.). The cDNA was amplified by polymerase chain reaction with the addition of the forward primer 5`-CTTGCTTCAACAGTGTTTGC-3`, which corresponds to base numbers 1-20 of the murine L apoferritin. The apoferritin H probe was prepared as above using the forward primer 5`-CTTGTTATTTTGACCGAGATG-3` (base numbers 236-256) and the reverse primer 5`-GGGGATCATTCTTGTCAGTA-3`, which corresponds to base numbers 486-505 of the apoferritin H coding sequence(42) . The amplified apoferritin L and H DNAs were purified by agarose gel electrophoresis and the labeled with [alpha-P]dCTP (3000 Ci/mmol, DuPont NEN) by random priming (Life Technologies, Inc.). The murine ribosomal protein L32 cDNA probe (43) and the cDNA clone of Mengo virus (pMC24; generously provided by Dr. Ann Palmenberg, University of Wisconsin) were labeled by random priming. Hybridizations were carried out at 42 °C in 50% formamide for 18 h, and blots were washed as described by Sambrook et al.(39) .

Preparation of Cytoplasmic Extracts

Cytoplasmic protein extracts for mobility shift experiments were obtained by the following procedure described by Leibold and Munro(44) . Briefly, 5.0 times 10^7 cells were harvested and lysed at 4 °C in 415 µl of lysis buffer (10 mM HEPES, pH 7.6, 3 mM MgCl(2), 40 mM KCl, 5% glycerol, 1.0 mM dithiothreitol) supplemented with 0.2% Nonidet P-40. After lysis, samples were diluted 3-fold with lysis buffer, and nuclei were removed by centrifugation at 10,000 times g for 1 min. After aliquoting into 50-µl volumes, the supernatants were stored at -70 °C. Protein concentrations were determined by a modified version of the Lowry assay (45) using the set of reagents supplied by Bio-Rad.

Preparation of IRE Transcripts

Transcription reactions were performed using the plasmid pSPT-fer, which contains the 5`-untranslated region of the human apoferritin heavy chain (bases 31-58) downstream from the promoter for T7 RNA polymerase(14) . Reactions were carried out with 2 µg of HindIII-digested plasmid DNA, 100 µCi of [alpha-P]CTP (650 Ci/mmol, ICN), 2.5 mM ATP, GTP, UTP (Sigma) and 50 units of T7 RNA polymerase (Life Technologies, Inc.) in a final volume of 20 µl. Samples were incubated at 38.5 °C for 2 h before the reaction was stopped by the addition of 40 µl of H(2)O and 1 µl of 0.5 M EDTA, pH 8.0. Carrier yeast tRNA (100 µg; Sigma) was added, and RNA transcripts were precipitated with 2.5 M ammonium acetate, and 2.5 volumes of 100% ethanol. The identical procedure was used to produce unlabeled transcripts, except that 2.5 mM CTP (Sigma) was added instead of the radionucleotide.

IRE Binding Studies

Binding reactions were carried out essentially as described by Müllner et al.(14) . Approximately 0.1 ng of P-labeled transcript (30,000 cpm) was incubated with 20 µl of a cytoplasmic extract at room temperature for 30 min. After the incubation, 1 unit of T1 RNase was added, and the incubation was continued for an additional 10 min. Heparin was added to a final concentration of 5 mg/ml, and the incubation was continued. After 10 min, 40 µl of loading buffer (30 mM Tris-HCl, pH 7.5, 40% sucrose, 0.2% bromphenol blue) was added, and the samples were loaded onto a 6% native polyacrylamide gel and electrophoresed in 0.3 times TBE (27 mM Tris borate, 0.6 mM EDTA) for 2 h at 200 V. Gels were dried on a nylon membrane, exposed to a phosphorimager plate and analyzed as described previously.

Iron Uptake Experiments

Determination of Fe uptake has been described previously by Lambert and Husain(46) . Briefly, confluent L cell monolayers were mock- or Mengo virus-infected as described above. After infection, 5 µCi of Fe as ferrous citrate (DuPont NEN) was added, and cells were incubated at 37 °C. The cells were harvested at various times post-infection, washed three times in incomplete phosphate-buffered saline, and counted in a LKB 1270 Rackgamma II.

EPR Studies

Control and Mengo virus-infected cells were harvested from roller bottles at various times after infection and lysed by homogenization in the presence of 1.0 mM Desferal (deferoxamine mesylate; CIBA). Chelated iron was determined by EPR in a Bruker Spectrospin ESP 300 equipped with an Oxford Instruments ESR 900 flowing helium cryostat at 12 K 20 mW as described previously (47) .

RESULTS

Mengo Virus Infection Induces the Synthesis of Apoferritin

To determine the amounts of synthesis of each of the two apoferritin subunits that were induced by Mengo virus, L929 fibroblasts were infected, metabolically labeled with [S]methionine 4 h after infection, and lysed 1 h later. Ferritin was immunoprecipitated using human anti-ferritin antibodies and protein A-Sepharose. The immunocomplex was denatured by boiling in SDS, electrophoresed in a denaturing 12% polyacrylamide gel, and exposed to a phosphorimaging plate (Fig. 1, inset). Visual comparison of the phosphorimages indicated an increase in both L and H apoferritin levels in Mengo virus-infected cells and quantitation of the phosphorimages demonstrated that there was a 4.3-fold increase in the synthesis of H apoferritin and a 5.8-fold increase in L apoferritin synthesis (Fig. 1). This also resulted in a decrease in H/L subunit ratio in Mengo virus-infected cells (3:1) compared with uninfected cells (2.2:1).

Figure 1: Induction of ferritin H and L subunits in Mengo virus-infected cells. L929 cells were harvested 5 h post-infection after being metabolically labeled with [S]Met for 1 h. Protein equivalent to 1 times 10^6 precipitable cpm was immunoprecipitated with anti-human ferritin antibodies, analyzed by SDS-PAGE, and exposed to a phosphorimaging plate (inset). Densitometric quantitation of mock-infected control (C) or Mengo virus-infected (V) L929 cells is shown from the average of three independent experiments.


Apoferritin L and H Gene Transcription Is Not Increased during Mengo Virus Infection

To determine if the increase in apoferritin synthesis during Mengo virus infection was the result of an increase in the transcription rate or stability of the corresponding mRNAs, total cytoplasmic RNA was isolated from both infected and mock-infected cells at various times and analyzed by Northern blotting. The amounts of cytoplasmic RNA isolated from control or infected L cells is shown in Fig. 2as a function of time after infection. Cytoplasmic RNA levels from Mengo virus-infected cells remained constant for up to 3 h after infection. Between 3 and 9 h, there was a dramatic (50%) decrease, after which the levels remained constant for an additional 2 h. The decrease in RNA synthesis in the virus-infected cell cultures was not due to cell lysis, because it occurred before virus release into the medium (Fig. 2). In mock-infected cells the amount of cytoplasmic RNA continued to increase with time.

Figure 2: Effect of Mengo virus infection on cytoplasmic RNA levels in L cells. Total cytoplasmic RNA was isolated from uninfected (box) and Mengo virus-infected (circle) L cells at the various times indicated. Cytoplasmic RNA was isolated from culture dishes each containing 1.3 times 10^7 cells. The production of infectious Mengo virus (plaque-forming units/ml) in the supernatant of infected L cell cultures at various times is shown ().


The cytoplasmic RNA obtained from this experiment was separated under denaturing conditions by agarose gel electrophoresis and blotted onto a nylon membrane. This blot was then probed with cDNA for H apoferritin and exposed to a phosphorimaging plate. The relative quantities of the control and virus-infected cytoplasmic H apoferritin RNA were then determined using the digitizing program supplied with the phosphorimager. The apoferritin H probe was then stripped from the membrane, and the blot was reprobed with the cDNA specific for the RNAs of L apoferritin, ribosomal protein L32 (RPL32), or Mengo virus. The phosphorimages are shown in Fig. 3A, and the intensity of each band is graphically represented in Fig. 3B. The H and L apoferritin and RPL32 mRNA levels remained constant for up to 3 h post-infection and then began to decline to about 20% by 7 h post-infection. There was no increase in either of the apoferritin mRNAs in infected cells; instead there was a decrease in the amounts of both after 5 h. The H/L apoferritin mRNA ratio was consistently 1. This result is similar to that of Tsuji et al.(34) with mouse 3T3 cells. Mengo virus RNA was not detected until 3 h post-infection, increased to a maximum at 5 h, and then declined slightly by 7 h post-infection. That the Mengo virus RNA appears as a smear on the Northern blot images is probably due to the presence of intermediate replicating complexes.

Figure 3: Mengo virus infection does not induce the transcription of ferritin genes. A, Northern blot analysis of 1 mg of cytoplasmic RNA isolated at various times after infection or mock-infection. The blot was sequentially hybridized with ferritin H cDNA, ferritin L cDNA, ribosomal protein L32 cDNA, and Mengo virus cDNA. The position of the ribosomal 18 and 26 S subunits are shown in the Mengo virus blot. B, graphical representation of the Northern blots in A showing mRNA levels in uninfected (box) and Mengo virus-infected (circle) cells.


IRP Remains Intact during Mengo Virus Infection

Since the transcription of mRNA for the two apoferritin subunits does not increase in infected L cells, the induction of apoferritin synthesis observed must occur at the level of translation. The Mengo virus genome encodes a proteinase, designated 3C, which is involved in processing the viral precursor polyprotein into functional proteins(48) . To examine the possibility that the IRP is cleaved by this proteinase or by an activated cellular proteinase resulting from infection, proteins from an equal number of mock-infected and Mengo virus-infected cells were electrophoresed in denaturing polyacrylamide gels and transferred electrophoretically to polyvinylidine difluoride membranes for Western blot analysis. The blots were incubated with antibodies to rat liver ferritin or to mouse IRP-1 (Fig. 4). Ferritin levels in infected cells were increased approximately 3-fold as compared with uninfected cells. However, when the same amounts of cellular protein were tested for IRP-1 protein levels, there was no difference found between control and infected cells.

Figure 4: The IRP remains intact in Mengo virus-infected cells. Cells were either mock (C)- or Mengo virus-infected (V), harvested after 5 h, and lysed by homogenization. Protein from an equivalent number of cells was subjected to SDS-PAGE and blotted onto polyvinylidine difluoride. Blots were treated either with rabbit anti-human spleen ferritin antibodies or rabbit anti-human IRP-1 antibodies. Western blots were visualized by treatment with goat anti-rabbit IgG conjugated with alkaline phosphatase as described under ``Materials and Methods.''


Mengo Virus RNA Does Not Inhibit IRP Complex Formation

One explanation for the increase in apoferritin synthesis observed in Mengo virus-infected cells could be that there is a competition for the IRP protein between the IREs in the apoferritin mRNAs and IRE-like elements in the viral RNA. The IRP binds to two major classes of loop sequences, 5`-CAGUGN-3` and 5`-UAGUAN-3`, both of which contain an extrahelical cytidine in the stem 5 bp from the loop(49) . A search of the entire Mengo RNA nucleotide sequence (^2)was undertaken to determine if elements corresponding to either of the two major IRE loop sequences existed therein. Application of the search program in Intelligenetics Suite (Release 5.4) to both the positive and negative (replicative template) strands of Mengovirus RNA did not reveal any IRE elements. It is still possible that a novel IRE sequence may exist in Mengo virus RNA. If that were the case, it would compete for IRP with an IRE RNA construct in a band shift assay. Fig. 5shows a typical band shift assay where the IRE probe forms two distinct complexes with the two species of mouse L cell IRP. The complexes formed are specific IRE-protein complexes, since addition of unlabeled IRE RNA resulted in a decrease in labeled complex formation (Fig. 5). Also, the addition of an unrelated RNA (yeast tRNA) lacking IRE sequences did not inhibit complex formation. The addition of increasing amounts of Mengo RNA did not interfere with complex formation (Fig. 5), demonstrating that Mengo RNA cannot compete with apoferritin mRNA IRE sequences for the IRP. Furthermore, no IRP complexes were observed when labeled Mengo virus RNA alone was used in this mobility shift assays (data not shown).

Figure 5: Effect of Mengo virus RNA on IRE complex formation. P-Labeled IRE (0.1 ng, 30,000 cpm) was mixed with 0-1000-fold excess unlabeled competitor RNAs prior to incubation with 5 µg of L cell lysate (5 h post-infection). The complexes were electrophoresed on a 6% nondenaturing polyacrylamide gel and exposed to a phosphorimaging plate. Free RNA and the complexes formed by IRP-1 and IRP-2 are indicated. Unlabeled RNAs were derived from pSPT-fer (IRE RNA), pMC24 (MENGO RNA), or yeast tRNA. The results shown are typical of two independent experiments.


Mengo Virus Infection Results in a Decreased IRP Binding Activity for IRE

Another explanation for the increase in ferritin protein levels in Mengo virus-infected cells could be that there is a decrease in binding activity of IRP for IRE caused by an increase in cytoplasmic free iron. To examine this possibility, an equal amount of total cellular protein from uninfected and Mengo virus infected L cells was mixed with P-labeled IRE RNA. The mixtures were electrophoresed through a nondenaturing polyacrylamide gel and exposed to a phosphorimaging plate. The images are shown in Fig. 6A and graphically represented in Fig. 6B. Treatment of extracts with 2-mercaptoethanol (2-ME; 1% solution) converts the IRP from a low to a high affinity state for RNA binding(50) . Extracts treated with 2-ME are considered to give 100% binding efficiency with IRE; however, this treatment seems to destroy the binding activity of IRP-2, the faster migrating protein (Fig. 6A). We do not know why this occurs, perhaps it may be specific to the mouse L929 strain. Comparing the RNA binding activity in the presence or absence of 2-ME indicates that only 58% of the IRP-1 has the capacity to bind IRE in Mengo virus-infected cell extracts, whereas approximately 90% of IRP-1 in uninfected cell extracts has the same activity (Fig. 6, A and B). This decrease in binding efficiency could account for the increased apoferritin L and H protein levels seen in infected cells. To determine if the observed decrease in binding efficiency was a result of higher intracellular iron levels, cells were treated with Desferal, an intracellular iron chelator, then extracts were examined for the ability of IRP to bind IRE. In uninfected cells, treatment with Desferal resulted in a small increase in the capacity of IRP-1 to bind IRE (from 90% in untreated cells to 95% in treated cells; Fig. 6, A and B). In Mengo virus-infected cells there was a large increase in IRP-1 binding: from 58 to 95%. Therefore, the decrease in binding efficiency of IRP-1 in Mengo virus-infected cells is most likely the result of an increase in intracellular free iron, and this can be neutralized by an intracellular iron chelator. It is interesting to note that treatment of the cells with Desferal also abolished the 2-ME effect on IRP-2 (Fig. 6A).

Figure 6: Mengo virus infection influences IRE-binding complex formation. A, phosphorimage of band shift assays using control or Mengo virus-infected cell extracts (5 h post-infection) and P-labeled IRE. Extracts were treated with 1% 2-ME where indicated. Cells were cultured in the presence of 50 µM Desferal for 24 h where indicated. B, graphical representation of the gel retardation assays. A binding efficiency of 100% was assigned to extracts treated with 2-ME. Efficiency was calculated by comparing 2-ME-treated extracts with the corresponding untreated extracts.


Free Iron Increases in Mengo Virus-infected Cells

Since the decreased IRE binding activity can be abolished by the intracellular iron chelator Desferal, the mechanism of induction would appear to be the result of an increase of intracellular free iron. To confirm this possibility, we used EPR spectroscopy to measure the levels of chelatable free iron in uninfected and Mengo virus-infected cell extracts. Fig. 7shows the results of a typical time course experiment. Cell lysates from various times after infection were subjected to EPR spectral analysis. Although the values obtained are not strictly quantitative, it is clear that qualitatively the level of free (chelatable) iron gradually increases in infected cell lysates from 2 to 5 h post-infection. Immunoprecipitation of ferritin from these samples showed that ferritin was induced in these extracts (data not shown).

Figure 7: Mengo virus infection increases intracellular free iron. L929 cells were infected with Mengo virus and harvested at various times indicated. Cells were lysed by homogenization in the presence of 1 mM Desferal. EPR signals were produced by subtracting the time 0 spectrum from the spectrum obtained at various times post-infection. The results shown are typical of two independent experiments.


Iron Uptake Is Not Affected by Mengo Virus Infection

Two possible explanations exist for the observed increase in intracellular free iron: the infected cells have increased the uptake of iron from the environment, or the intracellular iron has changed form from a bound to a free state. To examine the former possibility we measured the uptake of Fe from the media at various times post-infection. There was no change in the amount of Fe taken up by either of the control or Mengo virus-infected cells up to 5 h post-infection (25,000 cpm/10^6 cells). Immunoprecipitation of S-labeled ferritin from these samples demonstrated that ferritin induction had taken place as a result of Mengo virus infection (data not shown).

DISCUSSION

The first discernible metabolic effect of Mengo virus infection of mouse fibroblast cells is a rapid decline (to 30% of normal within 3 h) in protein synthesis; this is believed to be due to the ability of Mengo virus RNA to out compete the cellular mRNAs for initiation factors and 43 S ribosomal subunits(51) . Between 3 and 5 h post-infection, there is an increase in protein synthesis that is mainly due to the translation of the viral genomic RNA, which functions as a monocistronic mRNA. Late in infection there is a second decline in protein synthesis due to the phosphorylation of eIF-2 by the double-stranded RNA-activated protein kinase(52) . In contrast to general cellular protein synthesis, ferritin production begins to increase 2 h after infection, reaching a maximum 2 to 4 h later(37) . Immunoprecipitation of ferritin from uninfected and Mengo virus-infected L cells between 4 and 5 hours after infection reveals a differential increase in the synthesis of both L and H apoferritins (by approximately 6- and 4-fold, respectively). This differential induction results in the H/L ratio changing from 3.0 in uninfected L cells to 2.2 in Mengo virus-infected cells. In vitro studies have determined that L-rich ferritins are more efficient for iron incorporation and storage, whereas H-rich ferritins are probably more efficient for iron detoxification(53, 54, 55) . It is unclear if the change in Mengo virus-infected cells has any effect on intracellular iron homeostasis, because the cells begin to lyse within 6-8 h after apoferritin induction.

It is interesting to speculate as to the mechanism whereby apoferritin synthesis increases during the overall decrease in cellular protein synthesis in Mengo virus-infected cells. Perhaps the apoferritin mRNAs can successfully compete with Mengo RNA for eIF-2. It is known that apoferritin mRNAs are translated very efficiently; uncapped apoferritin mRNAs are translated at only a slightly diminished rate in vitro(56) .

We have determined that the increase in apoferritin synthesis in infected cells is not due to an increase in the transcription of the apoferritin L or H mRNAs nor in their stability. In fact, apoferritin mRNA levels appear to be slightly decreased in infected cells at a time when the apoferritin protein synthesis is induced (Fig. 3). The differential rate in L and H protein production may be a result of apoferritin L mRNA being somewhat more efficient in translation since L and H mRNA levels in infected cells are equivalent.

At the level of translation, both L and H apoferritin mRNAs are subject to a novel mode of post-translational control (reviewed in (57) ). When intracellular concentrations of iron are low, translation of the mRNAs for L and H apoferritins are repressed by IRP binding to IRE motifs in those mRNAs. We have examined the integrity of the IRP-1 using Western blot analysis and have concluded that it remains intact during the infectious process. The Mengo virus RNA genome does not contain any ``IRE-like'' motifs in either the positive or negative strand, and Mengo virus positive strand RNA does not interfere with the IRP band shift assays. However, when band shift assays were used to examine the ability of IRP to interact with IRE, there was a decrease in the IRE binding activity of IRP in infected cells. This decrease in binding is most likely caused by an increase in the concentration of intracellular free iron, because pretreatment of cells with the iron chelator Desferal inhibited the decrease in binding efficiency to IREs in Mengo virus-infected cells. We have also used EPR spectroscopy to confirm the increase of intracellular iron in infected cell extracts. The intracellular free iron levels begin to increase at 2 h post-infection and continue to increase to at least 5 h post-infection. The increase in free iron correlates with the induction of apoferritin synthesis observed in Mengo virus-infected cells(37) . Fe uptake experiments have demonstrated that the additional free iron is not the result of increased iron uptake from the exterior. This suggests that the increase in free iron in Mengo virus-infected cells is a result of a change in the form of intracellular iron from a bound state, which cannot interact with IRP, to an unbound form capable of binding IRP and inducing apoferritin synthesis.

One possible explanation for the increase in intracellular iron presumed to accompany the early stages of virus infection is that Mengo virus 3C proteinase mediates degradation of one or more iron-containing cellular proteins. The identity of heme or iron containing protein(s) which might release iron in Mengo virus-infected cells is not known. The destabilization of a heme containing protein has been shown to induce apoferritin synthesis. Cobalt protoporphyrin can lead to large decreases in cellular levels of cytochrome P450s(58) , which in turn causes an increase in apoferritin synthesis(59) . If a heme-containing protein was destabilized, then the release of heme should also induce the production of the heme-degrading enzyme heme oxygenase I; this would facilitate the release of iron from heme. Immunoprecipitation of heme oxygenase I from uninfected and Mengo virus-infected L cells did not reveal any increase in heme oxygenase I synthesis at a time when apoferritin synthesis was induced (data not shown). This suggests that the protein responsible for the release of iron in infected cells does not contain heme. Two possible non-heme iron containing proteins which may be responsible for inducing apoferritin are ribonucleotide reductase and ferrritin itself. Destabilization of either of these two proteins has previously been shown to induce apoferritin synthesis. The antitumor drug hydroxyurea causes a destabilization of the iron center in ribonucleotide reductase, which in turn leads to the induction of ferritin(41) . Treatment of cells with the glutathione-depleting drug phorone has recently been shown to initially lower intracellular ferritin content, which in turn results in the induction of ferritin synthesis(60) . In either case we believe the most likely candidate for the destabilization of the iron containing cellular proteins is the Mengo virus-encoded proteinase 3C. We are attempting to express the Mengo virus 3C proteinase alone in L929 cells to investigate its effect, if any, on apoferritin synthesis.

A number of different cells infected with viruses have been shown to synthesize increased amounts of ferritin. These include mouse L cells infected with Newcastle disease virus(61) , Hep-2 cells infected with herpes simplex virus(46) , and L cells infected with Mengo and Theiler's virus ( (1) and (38) and this study). In vivo, mice infected with mouse hepatitis virus showed increased ferritin levels in liver cells(62) , and it has been suggested that hepatitis B virus could stimulate apoferritin synthesis in human liver cells(63) . Recently, both H- and L-rich ferritins have been reported to inhibit antibody production in B lymphocytes(64) . It is interesting to speculate that the induction of ferritin by these viruses may be a defense mechanism for suppressing the host immune response directed against infection. Further work on serum ferritin levels and antibody production in Mengo virus-infected mice will have to be undertaken to explore this possibility.

FOOTNOTES

*
This work was supported by a grant from the Medical Research Council of Canada (to D. G. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

()
To whom correspondence should be addressed. Tel.: 403-492-5220; Fax: 403-492-0886.

(^1)
The abbreviations used are: IRP, iron regulatory protein; IRE, iron regulatory element; PAGE, polyacrylamide gel electrophoresis; 2-ME, 2-mercaptoethanol.

(^2)
A. Palmenberg and G. Duke, personal communication.

ACKNOWLEDGEMENTS

We are grateful to Dr. R. Rothery for his expert technical assistance with the EPR studies. We are also grateful for the technical contributions of P. Carpenter.

REFERENCES

  1. Mulvey, M. R., Fang, H., Holmes, C. F. B., and Scraba, D. G. (1994a) Virology 198, 81-91 [CrossRef][Medline] [Order article via Infotrieve]
  2. Theil, E. C. (1987) Annu. Rev. Biochem. 56, 289-315 [CrossRef][Medline] [Order article via Infotrieve]
  3. Torti, S. V., Kwak, E. L., Miller, S. C., Miller, L. L., Ringold, G. M., Myambo, K. B., Young, A. P., and Torti, F. M. (1988) J. Biol. Chem. 263, 12638-12644 [Abstract/Free Full Text]
  4. Miller, L. L., Miller, S. C., Torti, S. V., Tsuji, Y., and Torti, F. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4946-4950 [Abstract/Free Full Text]
  5. Fahmy, M., and Young, S. P. (1993) Biochem. J. 296, 175-181
  6. Warren, S., Torti, S. V., and Torti, F. M. (1993) Lymphokine Cytokine Res. 12, 75-80 [Medline] [Order article via Infotrieve]
  7. Wei, Y., Miller, S. C., Tsuji, Y., Torti, S. V., and Torti, F. M. (1990) Biochem. Biophys. Res. Commun. 169, 289-296 [CrossRef][Medline] [Order article via Infotrieve]
  8. Cox, F., Gestautas, J., and Rapoport, B. (1988) J. Biol. Chem. 263, 7060-7067 [Abstract/Free Full Text]
  9. Liau, G., Chan, L. M., and Feng, P. (1991) J. Biol. Chem. 266, 18819-18826 [Abstract/Free Full Text]
  10. Chou, C., Gatti, R. A., Fuller, M. L., Concannon, P., Wong, A., Chada, S., Davis, R. C., and Salser, W. A. (1986) Mol. Cell. Biol. 6, 566-573 [Abstract/Free Full Text]
  11. Beaumont, C., Jain, S. K., Bogard, M., Nordmann, Y., and Drysdale, J. (1987) J. Biol. Chem. 262, 10619-10623 [Abstract/Free Full Text]
  12. Mattia, E., den Blaauwen, J., Ashwell, G., and van Renswoude, J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1801-1805 [Abstract/Free Full Text]
  13. Coulson, M. R., and Cleveland, D. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7613-7617 [Abstract/Free Full Text]
  14. Müllner, E. W., Neupert, B., and Kühn, L. C. (1989) Cell 58, 373-382 [CrossRef][Medline] [Order article via Infotrieve]
  15. Rouault, T. A., Hentze, M. W., Caughman, S. W., Harford, J. B., and Klausner, R. D. (1988) Science 241, 1207-1210 [Abstract/Free Full Text]
  16. Walden, W. E., Patino, M. M., and Gaffield, L. (1989) J. Biol. Chem. 264, 13765-13769 [Abstract/Free Full Text]
  17. Aziz, N., and Munro, H. N. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8478-8482 [Abstract/Free Full Text]
  18. Hentze, M. W., Rouault, T. A., Caughman, S. W., Dancis, A., Harford, J. B., and Klausner, R. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6730-6734 [Abstract/Free Full Text]
  19. Cox, T. C., Bawden, M. J., Martin, A., and May, B. K. (1991) EMBO J. 10, 1891-1902 [Medline] [Order article via Infotrieve]
  20. Dandekar, T., Stripecke, R., Gray, N. K., Goossen, B., Constable, A., Johansson, H. E., and Hentze, M. W. (1991) EMBO J. 10, 1903-1909 [Medline] [Order article via Infotrieve]
  21. Casey, J. L., Hentze, M. W., Koeller, D. M., Caughman, S. W., Rouault, T. A., and Klausner, R. D. (1988) Science 240, 924-928 [Abstract/Free Full Text]
  22. Emery-Goodman, A., Hirling, H., Scarpellino, L., Henderson, B., and Kühn, L. C. (1993) Nucleic Acids Res. 21, 1457-1461 [Abstract/Free Full Text]
  23. Kennedy, M. C., Mende-Mueller, L., Blondin, G. A., and Beinert, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11730-11734 [Abstract/Free Full Text]
  24. Haile, D. J., Rouault, T. A., Tang, C. K., Chin, J., Harford, J. B., and Klausner, R. D. (1992a) Proc. Natl. Acad. Sci. U. S. A. 89, 7536-7540 [Abstract/Free Full Text]
  25. Haile, D. J., Rouault, T. A., Harford, J. B., Kennedy, M. C., Blondin, G. A., Beinert, H., and Klausner, R. D. (1992b) Proc. Natl. Acad. Sci. U. S. A. 89, 11735-11739 [Abstract/Free Full Text]
  26. Constable, A., Quick, S., Gray, N. K., and Hentze, M. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4554-4558 [Abstract/Free Full Text]
  27. Henderson, B. R., Seiser, C., and Kühn, L. C. (1993) J. Biol. Chem. 268, 27327-27334 [Abstract/Free Full Text]
  28. Guo, B., Yu, Y., and Leibold, E. A. (1994) J. Biol. Chem. 269, 24252-24260 [Abstract/Free Full Text]
  29. Samaniego, F., Chin, J., Iwai, K., Rouault, T. A., and Klausner, R. D. (1994) J. Biol. Chem. 269, 30904-30910 [Abstract/Free Full Text]
  30. Pantopoulos, K., Gray, N. K., and Hentze, M. W. (1995) RNA 1, 155-163 [Abstract]
  31. Henderson, B. R., and Khün, L. C. (1995) J. Biol. Chem. 270, 20509-20515 [Abstract/Free Full Text]
  32. Kim, H.-Y., Klausner, R. D., and Rouault, T. A. (1995) J. Biol. Chem. 270, 4983-4986 [Abstract/Free Full Text]
  33. Cairo, G., and Pietrangelo, A. (1994) J. Biol. Chem. 269, 6405-6409 [Abstract/Free Full Text]
  34. Tsuji, Y., Kwak, E., Saika, T., Torti, S. V., and Torti, F. M. (1993) J. Biol. Chem. 268, 7270-7275 [Abstract/Free Full Text]
  35. Ameglio, F., Tilocca, F., Arca, M. V., Alemanno, L., and Dolei, A. (1993) AIDS Res. Hum. Retroviruses 9, 795-798 [Medline] [Order article via Infotrieve]
  36. Riera, A., Gimferrer, E., Cadafalch, J., Remacha, A., and Martin, S. (1994) Haemattologica 79, 165-167
  37. Boege, U., Hancharyk, R., and Scraba, D. G. (1987) Virology 159, 358-367 [CrossRef][Medline] [Order article via Infotrieve]
  38. Mulvey, M. R., Fang, H., and Scraba, D. G. (1994b) Arch. Virol. Suppl. 9, 299-306 [Medline] [Order article via Infotrieve]
  39. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., pp. 7.12-7.15, 7.52, and 18.44-18.55, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY _
  40. Laemmli, U. K. (1970) Nature 227, 680-685 [CrossRef][Medline] [Order article via Infotrieve]
  41. Hurta, R. A. R., and Wright, J. A. (1991) Biochem. Cell Biol. 69, 635-642 [Medline] [Order article via Infotrieve]
  42. Beaumont, C., Dugast, I., Renaudie, F., Souroujon, M., and Grandchamp, B. (1989) J. Biol. Chem. 264, 7498-7504 [Abstract/Free Full Text]
  43. Dudov, K. P., and Perry, R. P. (1984) Cell 37, 457-468 [CrossRef][Medline] [Order article via Infotrieve]
  44. Leibold, E. A., and Munro, H. N. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2171-2175 [Abstract/Free Full Text]
  45. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265 [Free Full Text]
  46. Lambert, D. M., and Husain, S. S. (1973) Lab. Invest. 29, 737-742 [Medline] [Order article via Infotrieve]
  47. Yegorov, D., Kozlov, A. V., Azizova, O. A., and Vladimirov, Y. A. (1993) Free Radical Biol. Med. 15, 565-574 [CrossRef][Medline] [Order article via Infotrieve]
  48. Palmenberg, A. C. (1990) Annu. Rev. Microbiol. 44, 603-623 [CrossRef][Medline] [Order article via Infotrieve]
  49. Henderson, B. R., Menotti, E., Bonnard, C., and Kühn, L. C. (1994) J. Biol. Chem 269, 17481-17489 [Abstract/Free Full Text]
  50. Hentze, M. W., Rouault, T. A., Harford, J. B., and Klausner, R. D. (1989) Science 244, 357-359 [Abstract/Free Full Text]
  51. Abreu, S. L., and Lucas-Lenard, J. (1976) J. Virol. 18, 182-194 [Abstract/Free Full Text]
  52. DeStefano, J., Olmsted, E., Panniers, R., and Lucas-Lenard, J (1990) J. Virol. 64, 4445-4453 [Abstract/Free Full Text]
  53. Wagstaff, M., Worwood, M., and Jacobs, A. (1978) Biochem. J. 173, 969-977 [Medline] [Order article via Infotrieve]
  54. Bomford, A., Conlon-Hollingshead, C., and Munro, H. N. (1981) J. Biol. Chem. 256, 948-955 [Free Full Text]
  55. Levi, S., Luzzago, A., Cesareni, G., Cozzi, A., Franceschinelli, F., Albertini, A., and Arosio, P. (1988) J. Biol. Chem. 263, 18086-18092 [Abstract/Free Full Text]
  56. Dix, D. J., Lin, P., Kimata, Y., and Theil, E. C. (1992) Biochemistry 31, 2818-2822 [CrossRef][Medline] [Order article via Infotrieve]
  57. O'Halloran, T. V. (1993) Science 261, 715-725 [Abstract/Free Full Text]
  58. Drummond, G. S., and Kappas, A. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2384-2388 [Abstract/Free Full Text]
  59. Eisenstein, R. S., Garcia-Mayol, D., Pettingell, W., and Munro, H. N. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 688-692 [Abstract/Free Full Text]
  60. Cairo, G., Tacchini, L., Pogliaghi, G., Anzon, E., Tomasi, A., and Bernelli-Zazzera, A. (1995) J. Biol. Chem. 270, 700-703 [Abstract/Free Full Text]
  61. Husain, S. S., Mason, E., Towne, J., Yonan, J., and Changus, G. (1965) Fed. Proc. 24, 158
  62. Tiensiwakul, P., and Husain, S. S. (1979) J. Exp. Pathol. 60, 161-166
  63. Lustbader, E. D., Hann, H. L., and Blumberg, B. S. (1983) Science 220, 423-425 [Abstract/Free Full Text]
  64. Morikawa, K., Oseko, F., and Morikawa, S. (1994) Blood 83, 737-743 [Abstract/Free Full Text]
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