(Received for publication, December 4, 1995; and in revised form, January 30, 1996)
From the
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.
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- 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-
, 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), ()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 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.
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.
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
[-
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) .
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
10
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.
Figure 2:
Effect of Mengo virus infection on
cytoplasmic RNA levels in L cells. Total cytoplasmic RNA was isolated
from uninfected () and Mengo virus-infected (
) L cells at
the various times indicated. Cytoplasmic RNA was isolated from culture
dishes each containing 1.3
10
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
() and Mengo virus-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.''
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.
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.
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.
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.