From the Department of Infectious Disease, Tongji
Hospital, Institute of Immunology, Tongji Medical College of Huazhong
University of Science and Technology, Wuhan, 430030, China and
Multi-Organ Transplant Program and Department of Medicine and
Pathology, the Toronto General Hospital, University of Toronto,
Toronto M5G 2C4, Canada
Received for publication, December 16, 2002, and in revised form, January 23, 2003
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ABSTRACT |
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Fibrinogen-like protein 2/fibroleukin
(Fgl2) plays a pivotal role in the pathogenesis of both
experimental and human fulminant hepatic failure. We have reported
recently that the nucleocapsid (N) protein from strains of murine
hepatitis virus (MHV-3, MHV-A59), which cause massive hepatocellular
necrosis but not from strains (MHV-JHM, MHV-2) which do not produce
serious liver disease, induces transcription of fgl2. The
purpose of the present study was to characterize both viral and host
factor(s) necessary for viral induced transcription of
fgl2. Mutation of residues Gly-12, Pro-38, Asn-40, Gln-41,
and Asn-42 within domain 1 of the N protein of MHV-A59 to their
corresponding residues found in MHV-2 abrogated fgl2
transcription, whereas mutation of other N protein domains, including a
protein expressed from an internal reading frame (I protein), did not
affect fgl2 gene transcription. We then examined the Tissue-selective expression of the novel
fgl21
prothrombinase gene in liver, where it is localized specifically to the
endothelium of intrahepatic veins and hepatic sinusoids, has been shown
to play a critical role in the development of acute hepatitis and fulminant hepatic failure (FHF) induced by mouse hepatitis virus type 3 (MHV-3) (1-3). Following MHV-3 infection, fgl2
prothrombinase mRNA transcripts and protein are expressed by
macrophages and endothelial cells within the liver resulting in direct
cleavage of prothrombin to thrombin followed by fibrin deposition in
the intrahepatic veins and hepatic sinusoids, which then culminates in
confluent hepatocellular necrosis (4). Evidence that fgl2 prothrombinase is implicated in the pathogenesis of MHV-induced viral
hepatitis is supported by the observation that levels of prothrombinase
activity correlate with disease severity (2, 5, 6) and that treating
mice with a neutralizing monoclonal antibody to fgl2
prothrombinase prevents the lethality of MHV disease (2).
Previous work from our laboratory has shown that not all MHV strains
induce transcription of fgl2. Strains of MHV that cause massive hepatocellular necrosis, including MHV-3 and MHV-A59, induce
transcription of fgl2, whereas strains such as MHV-2 and MHV-JHM that do not lead to severe liver injury fail to induce transcription (7). By using a set of parental and recombinant murine
hepatitis virus strains, we have reported recently (8) that the
nucleocapsid (N) protein induced transcription of fgl2 leading to increased functional prothrombinase activity in strains of
mice, which develop fulminant hepatitis.
The MHV N protein is a phosphoprotein that interacts with the genomic
viral RNA to form a helical nucleocapsid (9, 10). It is made up of
conserved structural domains 1-3. Domains 1 and 2 are rich in basic
amino acids, whereas domain 3 has an abundance of acidic residues.
Variable spacer regions denoted as A and B separate the structural
domains. They have no known biological function, and deletion
experiments have shown that they are dispensable (10). The RNA-binding
function has been localized to domain 2; the exact functions of domains
1 and 3 have yet to be elucidated (10, 11). It has been shown that
there is an internal (I) gene appearing in most of the N genes of
different MHV strains including MHV-3, MHV-A59, and MHV-2. The internal
I gene is in the +1 reading frame relative to the N gene and encodes a
largely hydrophobic polypeptide of 203-220 amino acids. I protein has been shown to be expressed in MHV-infected cells and is not essential for the replication of MHV either in tissue culture or in its natural
host (12).
A region located between nucleotides The aim of the current study was to characterize the viral and host
factors involved in the transcription of fgl2. Here we report that domain 1 of the N protein from strains of MHV that cause FHF (MHV-A59) in contrast to that from strains that do not cause
FHF induced fgl2 gene transcription. We have excluded the importance of the I protein in induction of fgl2
transcription. Mutation of Gly-12, Pro-38, and
40NQN42 in domain 1 of the nucleocapsid
protein from MHV-A59 to the cognate sequence of MHV-2 abrogated
fgl2 gene transcription. The data further show that the N
protein indirectly stimulates fgl2 transcription through the
liver-enriched transcription factor HNF4 Mice--
Female BALB/cJ mice, 6-8 weeks of age (Charles
River Laboratories, Laval, Quebec, Canada), were kept in micro-isolated
cages and housed in the animal facilities at the Toronto General
Hospital, University of Toronto, and fed a standard lab chow diet and
water ad libitum.
Virus--
The parental viruses MHV-A59, MHV-JHM, and MHV-2 have
been described previously (14). MHV-A59 I mutant virus, Alb 110, and its isogenic wild type counterpart, Alb 111, were described previously (15). The Alb 110 mutant has a disrupted start codon and a stop codon
introduced in the I protein reading frame of the nucleocapsid gene from
MHV-A59. Viruses were grown in mouse 17 CL1 cells, and plaque titers
and plaque purifications were performed in mouse L2 cells as described
previously (1).
Cells--
Peritoneal macrophages were harvested from BALB/cJ
mice 4 days after intraperitoneal administration of 1.5 ml of 4%
thioglycollate (Difco) as described previously (7). Macrophages were
resuspended in RPMI 1640 (Invitrogen) supplemented with 2 mM L-glutamine (Sigma) and 2% heat-inactivated
fetal calf serum (Invitrogen). Macrophages were greater than 95% in
purity as determined by morphology and nonspecific esterase stain.
Viability was greater than 95% by trypan blue exclusion. The CHO cell
line (ATCC) was maintained in F-12K medium (Invitrogen) supplemented
with 10% heat-inactivated fetal calf serum and 10 mM
penicillin/streptomycin (Invitrogen).
Generation of Constructs--
The entire coding region of the N
gene and 3'-untranslated regions of MHV-A59 and MHV-2 was amplified by
reverse transcriptase-PCR and then subcloned into a 5.0-kb expression
vector (Invitrogen) under the control of the cytomegalovirus promoter
and bovine growth hormone 3'-processing signals as described before
(14). A 1.3-kb DNA fragment flanking the 5' end of mouse
fgl2 was released from the subcloned pBluescript-m166 (pm
166) P1 plasmid and then inserted into the SmaI and
XhoI sites of the pGL2-basic luciferase vector (Promega,
Nepean, Canada) to form fgl2p(
Expression constructs bearing mutant gene variants of the MHV-A59 N
protein were generated by PCR using the wild type MHV-A59 N protein
expression construct as a template according to the manufacturer's
protocol in the QuikChangeTM Site-directed Mutagenesis kit
(Stratagene, La Jolla, CA). The N protein cDNA sequences from
MHV-A59 and MHV-2 were determined by our laboratory and compared with
the sequences in GenBankTM. The MHV-A59 N protein cDNA
was identical to that in GenBankTM (accession number
M35256). The cDNA sequence for the MHV-2 N protein was submitted to
GenBankTM under the accession number AF061835 (15). The
cDNA sequences for the N protein from MHV-3 and MHV-JHM were
obtained from GenBankTM (accession numbers M35254 and
M25875). The cDNA sequences were converted into amino acids and
simultaneously compared using DNasis software. Residues in domains 1 and 2 of the MHV-A59 sequence were mutated so they would be identical
to their corresponding residues in the protein from MHV-2. Forward and
reverse primers were designed according to the specifications in the
Stratagene QuikChangeTM Site-directed Mutagenesis
kit. The four-way comparison is depicted in Fig. 1, and a summary of
the N protein mutations and the primers used for their construction is
shown in Table I.
The following primers were used to construct the single and double
mutations in the HNF4 Transfection--
CHO cells were cultured in 6-well plates until
50-80% confluent. 1 µg of N gene construct DNA, 0.5 µg of
fgl2p/LUC reporter gene construct DNA or pGL2-basic, and 0.25 µg of
Confocal Microscopy--
Thioglycollate-elicited BALB/cJ
peritoneal macrophages were implanted as a monolayer on glass slide
flasks (VWR Scientific, San Diego) and infected with MHV-A59 or MHV-2
at an m.o.i. of 2.5 for 208 h in RPMI 1640 supplemented with 10%
fetal bovine serum and 200 mM glutamine. Mock-infected
macrophages and MHV-3-infected macrophages represented negative and
positive controls, respectively. The cells were fixed in 100% cold
methanol at 4 °C, air-dried, and stored at Fgl2 Prothrombinase Activity--
Thioglycollate-elicited
peritoneal BALB/cJ macrophages, infected with wild type MHV or mutant
MHV Alb 100 at a multiplicity of infection (m.o.i.) of 2.5, were
incubated for 8 h in RPMI 1620 supplemented with 20% fetal bovine
serum and 200 mM glutamine. Mock-infected macrophages and
MHV-3-infected macrophages represented negative and positive controls.
Macrophages were evaluated for functional fgl2
prothrombinase activity in a one-stage clotting assay, as described
previously (1). After incubation, samples were washed three times with
un-supplemented RPMI 1640 and resuspended to a final concentration of
106 cell/ml. Samples were assayed for the ability to
shorten the spontaneous clotting time of normal citrated human
platelet-poor plasma. Milliunits of procoagulant activity were
assigned by reference to a standard curve generated with serial log
dilutions of a standard of rabbit brain thromboplastin (Dade Division
American Hospital Supply Co., Guelph, Canada).
Cytoplasmic and Nuclear Extract Preparation--
Nuclear and
cytoplasmic extracts were prepared as described previously (2).
Briefly, 107 cells were resuspended in 1 ml of cold Buffer
A (10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 50 mM DTT, and 50 mM phenylmethylsulfonyl fluoride). After brief
centrifugation, the cells were resuspended in 20 µl of cold Buffer A
with 0.1% Nonidet P-40 and incubated on ice for 10 min. Cells were
then gently vortexed and centrifuged at maximum speed; the supernatant,
representing the cytoplasmic extract, was collected, snap-frozen in
liquid nitrogen, and stored at Western Blot Analysis--
Nuclear and cytoplasmic extracts were
obtained from uninfected and MHV-infected macrophages obtained from the
peritoneal cavity of BALB/cJ mice. In order to detect the presence of
putative transcription factors, 20 µg of cytoplasmic and nuclear
extracts were boiled for 5 min in 2× SDS buffer containing 20% DTT
and resolved by SDS-PAGE. The resolved proteins were transferred to a
nitrocellulose membrane. The membrane was blocked with 5% non-fat
milk, 0.05% Tween 20, PBS for 2 h at room temperature with
shaking and then probed for 1 h at room temperature with shaking
with one of the following antibodies: rabbit polyclonal anti HNF-4 Electrophoretic Mobility Shift Assays (EMSA)--
The probes
used in EMSA were chemically synthesized oligonucleotides (see below).
200 ng of sense and antisense probe were mixed in annealing buffer
(Invitrogen) in a total volume of 30 µl and incubated at 75 °C for
5 min and then gradually cooled to room temperature to form
double-stranded DNA probes. The probes were labeled with
[ Nucleotide Sequence of the EMSA Probes--
Double-stranded
probes corresponding to the different binding sites upstream from the
murine fgl2 gene were obtained by annealing the following
forward and reverse synthetic oligonucleotides: HNF4 Statistical Analysis--
Data are expressed as means ± S.E. (S.E.) where applicable. Statistics were done with one-way
analysis of variance using the SigmaStat advisory statistical software
(Jandel Corp.).
Domain 1 of the MHV-A59 N Protein Is Responsible for Enhanced
Transcription of fgl2--
To determine the domain(s) of the N protein
from strains of MHV that increased fgl2 transcription, a
number of in-frame internal point mutations and deletions were
generated based upon the 4-way comparison of the N protein amino acid
sequences (Fig. 1). Transient transfection of CHO cells with mutant MHV-A59 N protein expression constructs revealed that 4 of 5 residues mutated in domain 1 (A59G12S, A59P38L, A59P38del, and A59NQN40-42del) as described under
"Materials and Methods" resulted in decreased fgl2
transcription to less than 10% activity observed with the wild type
MHV-A59 N protein construct (Fig. 2).
Mutations E85Q within domain 1 and V321A in domain 2 in the MHV-A59 N
protein had no effect on fgl2 gene transcription. Based on
these results, differences in residues in domain 1 of the MHV-A59 N
protein as compared with MHV-2 were shown to be responsible for the
differences in transcription of fgl2.
The Internal (I) Protein Does Not Induce fgl2 Gene
Transcription--
To determine whether the I protein is responsible
for enhanced transcription of fgl2, macrophages from BALB/cJ
mice were infected with the MHV-A59 I mutant virus A1b 110, which does
not express the I gene or the isogenic wild type virus, A1b 111, as a
control at an m.o.i. of 2.5. Macrophages were harvested 8-10 h
post-infection, and functional fgl2 prothrombinase activity
was measured in a one-stage clotting assay. The results in Fig.
3 revealed no significant difference in
fgl2 prothrombinase activity induced by MHV-A59, the MHV-A59
I mutant virus A1b 110, or the control A1b 111. To exclude further the
involvement of the I gene, CHO cells were co-transfected with an I gene
expression vector and fgl2p( Cellular Localization and Binding of N Protein Does Not Account for
fgl2 Gene Transcription--
To determine whether differences in
domain 1 of the N protein from MHV-A59 and MHV-2 resulted in
differences in nuclear localization, thioglycollate-elicited BALB/cJ
peritoneal macrophages were infected with either MHV-A59 or MHV-2, and
the cellular localization of the MHV N protein was studied as described
above. No obvious differences in amount of N protein or cellular
localization could be detected. At 1 h post-infection, N protein
was seen in the cytoplasm of both MHV-A59- and MHV-2-infected cells.
The N protein was first detected in the nuclei of both MHV-A59 and
MHV-2-infected cells 2 h post-infection, and cytoplasmic staining
was also seen. Controls showed no labeling (Fig.
4). Although more N protein was detected in the nuclear compartment 4 and 6 h post-infection in
MHV-A59-infected cells compared with that seen in MHV-2-infected cells,
this was not significant (data not shown). These results suggest that
differences in the subcellular localization of the N protein from
MHV-A59 and MHV-2 does not account for the difference seen in domain 1 of the N protein and the difference in terms of fgl2 gene
transcription.
As the N protein is known to bind to nucleic acids, the residues in
domain 1 from the MHV-A59 N protein may facilitate an interaction with
the Host Nuclear Proteins Bind to HNF4 The Putative HNF4
Although the data above strongly implicate the putative HNF4-binding
site in transcription of fgl2, on further analysis of the
HNF4
To determine whether HNF4 The present study defines the viral and host factors involved in
the transcription of the fgl2 gene, previously implicated in
the pathogenesis of both experimental and human viral induced fulminant
hepatic failure (1, 4, 8, 13, 17). Previously, we have shown that the N
protein from strains of MHV (MHV-A59 and MHV-3) that cause massive
liver necrosis, in contrast to the N protein from strains of MHV (MHV-2
and MHV-JHM) that do not produce massive liver injury, induce
fgl2 transcription (8, 9). It is known that the MHV
nucleoprotein binds to the viral genome to form a helical nucleocapsid
in the virion (11). However, aside from its role in viral RNA
encapsidation, the N protein may also enhance viral RNA translation and
partake in viral transcription and replication (12). Based on the amino
acid sequence, the MHV N protein putatively consists of three highly
conserved domains (domains 1-3) separated by two variable spacers (A
and B) (18). Although it is known that domain 2 binds to the viral
genome, the biological functions and significance of domains 1 and 3 have yet to be elucidated (18-20). Interspersed in the 3' end of many Coronavirus genomes, where the structural proteins are encoded, are
several small open reading frames (15, 21-23). One open reading frame
is encoded entirely within the 5' end of the MHV-A59 N gene and is
referred to as the internal (I) gene (15, 18). The I gene reading frame
is +1 relative to the N protein reading frame; it has been identified
in 10 of 11 N genes from different MHV strains that have been sequenced
to date (15, 18, 21, 23, 24). The I gene encodes a predominantly
hydrophobic protein ranging from 203 to 220 amino acids long. Although
a study suggests that the I protein is part of the virion structure,
further studies have shown that it is not needed for viral genome
replication, tissue tropism, or for viral dissemination (15).
The first set of experiments in the current study was designed to
characterize the strain-specific nature of N protein enhancement of
fgl2 gene expression. The data demonstrated that the I
protein does not account for the increased fgl2 promoter
transcription observed but implicated domain 1 of the MHV-A59 N protein
in the increased gene transcription of fgl2. When the
residues Gly-12, Pro-38, Asn-40, Gln-41, and Asn-42 in domain 1 of
MHV-A59 were mutated to the corresponding amino acids found in MHV-2
and MHV-JHM, fgl2 transcription was lost. In contrast,
residues in domains 2 and 3 of the N protein from MHV-A59 did not
affect fgl2 gene transcription, and thus we can conclude
that the differences in domain 1 of the nucleocapsid protein accounts
for fgl2 gene transcription.
To determine whether the N protein from the pathogenic strains in
contrast to non-pathogenic strains differentially localized to the
nucleus of infected cells, we performed confocal microscopy. The
nucleocapsid from both the pathogenic and non-pathogenic strains entered the nuclei of infected BALB/cJ macrophages in a similar time
frame and concentration fashion consistent with other observations of
nuclear localization of the MHV nucleoprotein (25). These results were
not surprising because the putative nuclear localization signals of the
nucleoprotein are located in domain 3; in the 4-way N protein amino
acid comparison, no strain-specific differences were found in domain 3. The possibility that MHV-A59 N protein bound to the fgl2
promoter and directly induced transcription was examined, but this
possibility was dismissed through EMSA analysis. Thus, the data
strongly indicate that the MHV-A59 N protein increases fgl2
gene transcription through an indirect mechanism that involves other factors.
The focus of the next set of experiments was the identification of
putative host factor(s) through which the N protein induced fgl2 transcription. Preliminary analysis of the sequence,
which spans HNF4 HNF4 HNF4 HNF4 Our EMSA experiments demonstrated that in an uninfected state, an
unidentified protein occupies the HNF4 Whereas it is known that HNF4 The current study provides a model with which to study the pathogenesis
of human hepatitis infection. Previous findings have also suggested
that the human FGL2 is involved in the pathogenesis of chronic
HCV and HBV infection in humans (17, 52). Liver tissue and
peripheral blood mononuclear cells from patients with chronic HCV and
HBV infection have increased mRNA transcripts for FGL2. Following
treatment with a combination of interferon- The present study demonstrated that the MHV-A59 N protein indirectly
initiates transcription of the murine fgl2 gene in
susceptible hosts. A host factor, HNF4372
to
306 sequence within the 1.3-kb fgl2 promoter region upstream from the transcription start site that was previously identified as necessary for N protein-induced gene transcription. We
demonstrated that the
331/
325 HNF4 cis-element and its cognate transcription factor, HNF4
, are necessary for virus-induced
fgl2 gene transcription. In uninfected macrophages and
macrophages infected with MHV-2, an unidentified protein occupies the
HNF4 cis-element. Following stimulation with MHV-A59, it was shown by
electrophoretic mobility shift assay that HNF4
binds the HNF4 cis-element in the fgl2 promoter. We further report the
unprecedented presence of HNF4
in peritoneal macrophages.
Collectively, the results of this study define both viral and host
factors necessary for induction of fgl2 prothrombinase gene
transcription in MHV infection and may provide an explanation for the
hepatotrophic nature of MHV-induced fulminant hepatic failure.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
372 and
306 in the
fgl2 promoter has been identified as being necessary for N
protein-induced transcription of fgl2 (8). Preliminary
mapping of this region has defined a number of candidate cis-elements
upstream from the ATG translation initiation site, which include
hepatocyte nuclear factor 4
(HNF4
)/liver factor A1 (LF-A1),
cytomegalovirus immediate early gene 1.2 (IE1.2) regulatory element,
and granulocyte macrophage colony-stimulating factor-binding element
(GM-CSF) (13).
. We have shown further that
HNF
is expressed in macrophages, an observation never reported
previously. These results collectively define both viral and host
factors necessary for MHV-induced fgl2 gene transcription and provide further insights into the pathogenesis of FHF.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1328)/LUC. The Rous sarcoma virus
-galactosidase vector was purchased from Promega.
Primers used to generate the MHV-A59 N protein mutant constructs
and IE1.2 putative cis-elements located within
372 to
306 of the murine fgl2 promoter:
Fgl2pHNF4
mut(
350/
294)t, 5'-CCA ACT CTT TCC CCA CTA
GCG TCG
ACA GTA TAT AAT ATG GTA
TCT TTT GGG CAC TGG-3'; Fgl2pIE1.2mut(
377/
325), 5'-GAA GAA GCT CAC
AGA CAT TTA GAC GTT CAA ACG
GAT CCA CCA CTA GTG GAC
CAA G-3'; Fgl2pHNF4
/IE1.2mut(
377/
334), 5'-GAA GAA GCT CAC AGA
CAT 1TTA GAC GTT CAA ACG
GAT CCA CCA CTA GCG TCG
ACA G-3'.The boldface letters indicate mutated sequences, and the underlined nucleotides represent convenient restriction endonuclease sites for screening. Insert orientation and sequence were verified by sequencing.
-galactosidase DNA were mixed with 3.5 µl of
LipofectAMINETM (Invitrogen) (2 ml/mg DNA) in 200 µl of
OPTI-MEM serum-free medium (Invitrogen) by vortexing. After a 30-min
incubation at room temperature, 1.8 ml of OPTI-MEM serum-free medium
was added to bring the final volume to 2 ml. One ml of this mixture was
distributed into each of the duplicated wells containing the CHO cells.
The transfection was carried out at 37 °C and 5% CO2
for 44-48 h. Cells were harvested in lysis buffer and freeze-thawed 3 times in liquid nitrogen. Aliquots of supernatant were assayed for
luciferase activity to measure promoter activation; values were
normalized with
-galactosidase levels.
80 °C until
probed. Cells were re-hydrated with 0.1 M PBS, pH 7.4, and
blocked with 10% normal horse serum in PBS at room temperature for
2 h. The slides were first incubated with a monoclonal antibody
against the MHV N protein (provided by Dr. P. Masters) at room
temperature for 2 h followed by 5 washes in PBS with 0.05% Tween
20. Cells were then incubated with a fluorescent isothiocyanate-conjugated goat IgG fraction against the mouse IgG Fc
fragment at room temperature for 1 h, followed by 5 washes with
0.05% Tween 20/PBS. Slides were then air-dried, mounted with 90%
glycerol, and viewed under a confocal microscope.
80 °C until needed. The remaining
pellet, containing nuclei, was resuspended in 15 µl of cold Buffer B
(20 mM HEPES, pH 7.9, 25% glycerol, 20 mM
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, pH
8.0, and proteinase inhibitor mixture) and incubated on ice for 15 min before centrifugation at maximum speed. Supernatants and the nuclear extracts were collected and diluted with 75 ml of cold Buffer C (20 mM HEPES, pH 7.9, 20% glycerol, 0.2 mM EDTA,
pH 8.0, 50 mM KCl, 0.05 mM DTT, 0.05 mM phenylmethylsulfonyl fluoride, and proteinase inhibitor
mixture). Aliquots were quickly frozen in liquid nitrogen and stored at
80 °C. Protein concentrations were quantified using the Bradford
method according to the protocol accompanying the Bio-Rad protein
concentrate; bovine
-globulin (Bio-Rad) was used to generate a
standard curve.
(kindly provided by Dr. Sladek), rabbit polyclonal anti-C/EBP
(Santa
Cruz Biotechnology, Santa Cruz, CA), or rabbit polyclonal anti-HNF3
(Santa Cruz Biotechnology). The membranes were rinsed twice with PBS
and washed 6 times with 0.05% Tween 20/PBS for 5 min with shaking. The
membrane was probed with a horseradish peroxidase-labeled goat
anti-rabbit secondary antibody (Santa Cruz Biotechnology) for 1 h
with shaking before being rinsed twice with PBS and washed 6 times in
0.05% Tween 20/PBS for 5 min. The blots were developed by
chemiluminescence and exposed to Kodak X-Omat blue film.
-32P]ATP (Amersham Biosciences) and T4 polynucleotide
kinase (Invitrogen) at 37 °C for 1 h. The labeled probes were
purified with ProbeQuantTM G-50 micro-columns (Amersham
Biosciences) according to the manufacturer's protocol; 2 ml of probe
was counted with a
scintillation counter (Beckman Instruments,
Mississauga, Canada). For each EMSA reaction, 5-15 µg of nuclear
extracts were preincubated for 15 min at room temperature with binding
buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 10% glycerol, 0.05% Nonidet P-40, 5 µg of
poly(dI-dC), 50 mM NaCl and 5 mM
MgCl2). For competition studies, 100-fold molar excess of
cold specific competitor was allowed to incubate with the nuclear
extracts for 30 min prior to the addition of labeled probe. For
supershift reactions, the extracts were incubated with 1 µg of
antibody for 30 min at room temperature prior to probe addition. 1 × 104 dpm of probe was added before the incubation was
allowed to proceed for an additional 30 min at room temperature. For
controls, the probe was allowed to incubate in the absence of nuclear
extracts or in the presence of antibody without the nuclear extracts.
After the addition of loading dye, DNA-protein-antibody
complexes were resolved by electrophoresis in 5% non-denaturing
polyacrylamide gels in Tris-glycine buffer. The gels were dried for
1 h at 80 °C and exposed to Kodak X-Omat blue film at
70 °C.
fgl2(
338/
316), 5'-CAC TAG TGG ACC AAG TAT ATA AT-3' and 5'-AT TAT ATA CTT GGT CCA CTA GTC-3'; IE1.2fgl2(
353/
336), 5'-TTC CAA CTC TTT CCC AC-3' and 5'-AAT TGT GGG AAA GAG TTG CAA-3';
GM-CSFfgl2(
368/
351), 5'-ACA GAC ATT TAG AGG TTC-3' and 5'-AAT TGA
ACG TCT AAT GTC TGT-3'; fgl2(
338/
306), 5'-CCA CTA GT G GAC CAA GTA
TAT AAT ATG GTA TCT-3'; C/EBP
-binding oligonucleotide, 5-TTT GTA GTG
TTT CCC AAC TCA GAT TCT GAG T-3' (3); HNF3
-binding oligonucleotide,
5'-GAT CGT TGA CTA AGT CAA TAA TCA G-3' (5); HNF4
-binding
oligonucleotide, 5'-AAA GGT CCA AAG GGC GCC T-3' (6); Prx2-binding
oligonucleotide, 5'-TAA CTA ATT AAC TAA CTA ATT AAC TAA CTA ATT AAC-3'
(16). The double-stranded oligonucleotide 5'-CGC CTG AGT CAG GCG GCG GTG GC-3' was used as a nonspecific competitor.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (100K):
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Fig. 1.
Four-way comparison of the N protein amino
acid sequences from MHV-A59, MHV-3, MHV-2, and MHV-JHM. The
reference sequence (in single letter code) is from MHV-A59
(top), and amino acids that differ from the other strains
are indicated. Domains 1 (amino acids 1-139), 2 (amino acids
163-379), and 3 (amino acids 406-444) are shaded, and the
spacer regions A (amino acids 140-162) and B (amino acids 380-405)
are underlined with fine lines.
Asterisk represents the stop codon. Residues targeted for
mutation in the MHV-A59 sequence are underlined with
heavy lines. The nature of the mutations are further
described in Table I.
View larger version (31K):
[in a new window]
Fig. 2.
Relative activation of the fgl2
promoter by wild type N proteins or MHV-A59 N protein mutant
variants in transfected CHO cells. N gene expression constructs
from MHV-A59 and MHV-2 and a series of N gene mutants from MHV-A59 were
co-transfected with the wild type fgl2 promoter/LUC
construct into CHO cells. Relative luciferase activity is expressed in
fold increase relative to CHO cells co-transfected with the MHV-2 N
protein expression construct and the fgl2 promoter/LUC
construct. The PGL2-basic vector was used as a negative control. Values
represent the mean ± S.E. of five separate experiments done in
triplicate. Asterisk indicates a p < 0.01 compared with cells co-transfected with MHV-2 N construct. # indicates
a p < 0.01 compared with cells co-transfected with N
gene construct from wide type MHV-A59.
1328)/LUC. No increase in luciferase
activity was observed (data not shown). These data demonstrate that the
I protein is not involved in the enhanced transcription of
fgl2, either alone or with the N protein.
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Fig. 3.
Expression of BALB/cJ macrophage functional
procoagulant activity induced by MHV and the I mutant viral
strains. Macrophages from BALB/cJ were infected with MHV and
MHV-A59 I mutant (Alb 110 and its isogenic wide type Alb 111) at an
m.o.i. of 2.5 for 8-10 h and harvested for measurement of
procoagulant activity. Values represent the mean ± S.E.
of three separate experiments done in triplicate. * represents a
p < 0.01 compared with uninfected macrophages.
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Fig. 4.
Subcellular localization of the nucleocapsid
protein. The N protein from the pathogenic and non-pathogenic MHV
strains enters the nuclei of infected cells in vitro.
BALB/cJ peritoneal macrophages were infected with MHV-A59 or MHV-2 for
1, 2, 4, or 6 h. Fixed cells were probed for the subcellular
localization of the MHV N protein by indirect antibody labeling.
Depicted is a confocal section of a single macrophage uninfected
(A), infected with MHV-2 (B), and infected with
MHV-A59 (C) for 2 h and labeled for the N protein. The
N protein can be detected in both the cytoplasm and nucleus of MHV-2-
and MHV-A59-infected macrophages. These results are representative of
three independent experiments.
338/
306 sequence in the murine fgl2 promoter region
that was identified previously as being necessary for virus-induced transcription (15). This possibility was explored through EMSA analysis. Nuclear extracts from BALB/cJ peritoneal macrophages infected
with MHV-A59 and 32P-labeled fgl2(
338/
306)
probe were incubated in the presence or absence of anti-MHV
nucleocapsid antibody. Two bands were observed, neither of which was
specifically shifted upon addition of anti-N protein antibody (Fig.
5A). Furthermore, additional
experiments demonstrated the absence of a band corresponding to N
protein binding to the fgl2 promoter (Fig. 5B).
Based on the data provided here, one could conclude that
fgl2 gene transcription was not due to direct interaction
between the N protein of MHV and the fgl2 promoter.
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Fig. 5.
The MHV-A59 N protein does not bind to the
338/
306 region of the murine fgl2 promoter
in vitro. Supershift analysis was performed with
nuclear extracts from MHV-A59-stimulated BALB/cJ peritoneal macrophages
(M
/MHV-A59 nucl extr) (A) or uninfected
BALB/cJ peritoneal macrophages (M
nucl extr)
(B) incubated with 32P-labeled
338/
306 probe
in the presence or absence of anti-MHV N protein antibody as described
under "Experimental Procedures."
and IE1.2 Cis-elements within
a Region Spanning
372/
306 in the fgl2
Promoter--
Preliminary characterization of the
372 to
306
sequence using DNasis software identified three non-overlapping
candidate cis-elements. The cis-elements include binding sites for
GM-CSF, cytomegalovirus IE1.2, and HNF4
(
331/
325) (15). To
determine the relevance of these cis-elements, EMSAs were performed
using nuclear extracts from MHV-A59-infected BALB/cJ macrophages and 32P-labeled oligonucleotides of the putative binding sites.
Binding was observed to the HNF4
probe (Fig.
6) and to the IE1.2 probe but not to the
GM-CSF probe (Fig. 6). To ascertain whether the observed band shifts
were due to specific binding, we performed competition experiments
using a 100-fold molar excess of unlabeled, specific, and nonspecific
double-stranded oligonucleotides. Binding of nuclear extracts from
MHV-A59-infected macrophages to the labeled HNF4
fgl2 and
IE.1.2 fgl2 probes were competed with a 100-fold molar
excess of cold specific oligonucleotides but not with an excess of
nonspecific oligonucleotides (Fig. 6). Based on the results of these
assays, either or both of the putative HNF4
- and IE1.2-binding sites
but not GM-CSF-binding site are implicated in viral induced
fgl2 gene transcription.
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Fig. 6.
EMSA analysis revealed that nuclear
proteins bind to the HNF4 and IE1.2
cis-elements located in the fgl2 promoter upon MHV-A59
infection. 32P-Labeled oligonucleotides were incubated
with nuclear extracts in the presence and absence of a 100-fold molar
excess of cold competitor oligonucleotides as described under
"Experimental Procedures."
(
331/
325) Cis-element Is
Responsible for the Activation of the fgl2 Gene in Response to the
MHV-A59 N Protein--
Mutational analysis was then used to determine
which of the two identified binding sites are necessary for viral
induced transcription of the fgl2 gene. CHO cells were
transfected with either the single (HNF4
mut/LUC and
fgl2pIE1.2mut/LUC) or double (fgl2HNF4
/IE1.2mut/LUC) mutant promoter constructs or wild type fgl2 promoter
construct with the wild type MHV-A59 N protein expression construct.
Mutation of the HNF4
consensus sequence resulted in a 75% decrease
of fgl2 transcriptional activity relative to the wild type
fgl2p(
1328)/LUC construct (Fig.
7). In contrast, mutation of the
IE1.2-binding site alone had no statistical effect on
transcriptional activity (Fig. 7).
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Fig. 7.
Transient expression of luciferase activity
induced by the wild type fgl2 promoter or the mutant
variants in response to the MHV-A59 N protein in transfected CHO
cells. 0.5 µg of N gene constructs from MHV-A59, MHV-2, was
co-transfected with 0.25 µg of wild type (WT)
pfgl2( 1328)/LUC or its mutants for candidate cis-elements HNF4
(HNF4mut), IE1.2 (IE1.2mut), or HNF4 and IE1.2 double mutation
(HNF4/IE1.2mut), respectively, in CHO cells for 40-44 h; cells were
harvested and freeze-thawed three time for measurement of luciferase
activity. PGL2-basic vector was used as a negative control. Values
represent the mean ± S.E. of four separate experiments done in
triplicate. Asterisk represents p < 0.01 compared with cells co-transfected with MHV-2 N construct. # represents
p < 0.01 compared with cells co-transfected with wide
type pfgl2(
1328)/LUC.
338/
306 promoter region using TFSEARCH software
(cbrc.jp/research/db/TFSEARCH.html) and Transfac data base,
additional putative cis-elements were identified including sites for
C/EBP
, Prx2, and HNF3
(Fig.
8A). Initially, Western blot
analyses were performed on extracts from CHO cells to assess the
presence of the putative transcription factors. Previously, a number of
investigators have shown that HNF4
is present in CHO cells; however,
the antibodies that were available for this present study did not react
with the CHO cell extracts. Therefore, primary macrophages from BALB/cJ
mice that are known to express fgl2 and that reacted with
the available antibodies were used for all further studies. Western
blot analysis was performed to determine which of the candidate
transcription factors were present in unstimulated and MHV-A59-infected
BALB/cJ peritoneal macrophages. The extracts could not be probed for
the expression of Prx2 as no known antibody is available. The results show that C/EBP
is present in the nuclear extracts of uninfected and
MHV-A59-infected BALB/cJ peritoneal macrophages (Fig. 8B). Similarly, HNF4
was detected in the nuclear extracts from uninfected and MHV-A59-infected macrophages (Fig. 8C). In contrast,
HNF3
was not detected in either the cytoplasmic or nuclear extracts of MHV-A59 infected or uninfected macrophages (Fig. 8C).
These data provide evidence for the putative role of HNF4
or
C/EBP
or both in MHV-A59-induced transcription of
fgl2.
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Fig. 8.
A, locations of the putative host
transcription factors involved in MHV-A59-N protein-induced
transcription of the murine fgl2 gene. The region spanning
338/
306 of the fgl2 promoter was analyzed using the
TFSEARCH program and the Transfac data base. The lines
below the
338/
306 nucleotide sequence depict the
schematic locations of the transcription factor binding sites; the
exact locations of the cis-elements are in parentheses. The
numbering of the promoter nucleotides is relative to the ATG
translation start site, which is designated (+1). Promoter
distances are not to scale. B-D, Western blot analyses
depict the expression of the candidate transcription factors involved
in the MHV-A59-induced transcription of the murine fgl2
gene. Equal amounts of protein from nuclear and cytoplasmic
extracts from uninfected and MHV-A59 infected (2.5 h) BALB/cJ
peritoneal macrophages were resolved by SDS-PAGE, transferred to
nitrocellulose membranes, and probed for the presence of C/EBP
(B), HNF
(C), and HNF3
(D).
Lane 1, MHV-A59-infected BALB/cJ peritoneal
macrophage cytoplasmic extracts; lane 2, MHV-A59-infected
BALB/cJ peritoneal macrophage nuclear extracts; lane 3,
uninfected BALB/cJ peritoneal macrophage cytoplasmic extracts;
lane 4, uninfected BALB/cJ peritoneal macrophage nuclear
extracts.
Binds to Its Cis-element
332/
325 in the
Murine fgl2 Promoter in MHV-A59-stimulated Macrophages--
EMSAs were
performed to determine whether HNF4
or C/EBP
in nuclear extracts
from MHV-A59-infected macrophages bound to the
338/
306
fgl2 promoter region. Nuclear extracts from MHV-A59 infected
macrophages were incubated with a 32P-labeled
338/
306
oligonucleotide probe in the presence or absence of 100-fold molar
excess of cold competitor oligonucleotides. Addition of excess cold
338/
306 oligonucleotide probe competed with protein binding (Fig.
9A). Addition of excess cold
oligonucleotide bearing a sequence known to bind HNF4
competed with
the labeled
336/
308 probe for binding to nuclear proteins, thus
demonstrating the presence of an HNF4
cis-element in the
338/
306
region of the fgl2 promoter (Fig. 9A). In
contrast, an excess of cold oligonucleotide known to bind C/EBP
,
HNF3
, and Prx2 transcription factors failed to compete with labeled
338/
306 probe for binding to nuclear proteins, thereby excluding
the involvement of these transcription factors in fgl2 gene
transcription following MHV-A59 infection (Fig. 9A).
Supershift analyses were then performed to determine whether the
protein binding to the fgl2 promoter was HNF4
. Nuclear extracts from uninfected, MHV-A59-infected, and MHV-2-infected BALB/cJ
macrophages were incubated with a 32P-labeled
338/
306
probe in the presence or absence of HNF4
antibody. The HNF4
antibody produced a specific shifted complex when added to nuclear
extracts from MHV-A59-infected cells (Fig. 9B). In contrast,
no supershift was detected when the HNF4
antibody was incubated with
nuclear extracts from uninfected macrophages, suggesting that in the
resting state, the HNF4
cis-element was occupied by a factor
distinct from HNF4
(Fig. 9B). In MHV-2-infected BALB/cJ
peritoneal macrophages, HNF4
antibody also failed to specifically
shift the protein-DNA complex demonstrating that the HNF4
cis-element was occupied by an unidentified protein, similar to what
was seen in uninfected resting macrophages (Fig. 9B). A
32P-labeled oligonucleotide probe incubated with
HNF4
-specific antibody or an irrelevant antibody in the absence of
nuclear extracts failed to produce a supershift thus verifying that the
observed shifted complex was not due to an interaction between the
oligonucleotide and the antibody and that the interaction was specific
(data not shown).
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Fig. 9.
A, bandshift demonstrates that HNF4
binds to its cognate site
332/
325 in the murine fgl2
promoter in vitro. Nuclear extracts from MHV-A59-infected
(2.5 h) BALB/cJ peritoneal macrophages (M
/A59 nucl extr)
were incubated with 32P-labeled
338/
306 in the presence
or absence of a 100-fold molar excess of cold competitors that include
cold
338/
306 and cold oligonucleotides known to bind C/EBP
,
HNF3
, HNF4
, and Prx2. The arrowhead denotes the
HNF4
-specific band. B, supershift analyses verify that
HNF4
binds to its cognate cis-element in the
338/
306 region in
the murine fgl2 promoter in MHV-A59 infection. Nuclear
extracts from uninfected (M
), MHV-A59-infected (2.5 h) (M
/A59),
and MHV-2-infected (2.5 h) (M
/V2) BALB/cJ peritoneal macrophages
were incubated with 32P-labeled probes in the presence or
absence of HNF4
-specific antibody (Ab) as described under
"Experimental Procedures." The HNF4
antibody-specific shift is
denoted by an arrow, and the HNF
-specific band is
indicated by an arrowhead. C, HNF4
is
expressed in the nuclear extracts of uninfected and MHV-infected
BALB/cJ peritoneal macrophages. Macrophages were infected with MHV-A59
or MHV-2 for 1, 2, or 4 h. Nuclear extracts from each sample were
resolved by SDS-PAGE and probed for the presence of HNF4
by Western
blot analysis. Lane 1, uninfected macrophages;
lane 2, MHV-A59-infected macrophages (1 h); lane
3, MHV-A59-infected macrophages (2 h); lane 4,
MHV-A59-infected macrophages (4 h); lane 5, space;
lane 6, MHV-2-infected macrophages (1 h); lane 7,
MHV-2-infected macrophages (2 h); lane 8, MHV-2-infected
macrophages (4 h).
protein expression was altered during MHV
infection, Western blot analyses were performed using nuclear and
cytoplasmic extracts from uninfected and MHV-A59- and MHV-2-infected
BALB/cJ macrophages. The peritoneal macrophages were infected with
either MHV-A59 or with MHV-2 for 2, 4, and 6 h; cells were
harvested and nuclear and cytoplasmic extracts isolated as described.
Western blot analysis verified that there were no obvious differences
in the time course or concentration of HNF4
in the cytoplasm and
nucleus in uninfected and MHV-2- and MHV-A59-infected cells. (Fig.
9C). Collectively, these data demonstrate that binding to
the HNF4
cis-element
332/
325 and subsequent fgl2 gene
transcription is specific to the MHV strain, MHV-A59, that causes
massive hepatocellular necrosis. In contrast, in uninfected macrophages
and in macrophages infected with MHV-2, a strain that does not cause
significant liver disease, the HNF4
cis-element is occupied by an
unidentified factor and does not bind HNF4
.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
372 to
306 in the fgl2 gene promoter that is
necessary for MHV-A59-induced transcription, revealed the presence of
three non-overlapping cis-elements, including those for IE1.2, GM-CSF, and HNF4
. Based on bandshift and competition EMSA analyses, only the
region
338/
306 encompassing the putative HNF4
cis-element was
required for viral induced transcription of fgl2. Because cis-elements can bind to various transcription factors with varying affinities, the possibility that factors other than HNF4
might be
involved was explored. Extensive characterization of the
338/
306 region revealed the presence of cis-elements for C/EBP
, HNF3
, and
Prx2 (26, 27). Bandshift analyses ruled out the direct involvement of
C/EBP, HNF3
, and Prx2. The identity of the factor binding to the
fgl2 promoter was confirmed by supershift analysis. In
uninfected and MHV-2-infected macrophages, as shown by Western blot
analysis, HNF4
was present. EMSA analyses using nuclear extracts
from these uninfected or MHV-2-infected macrophages demonstrated binding to the
338/
306 region, but a specific shift was not observed upon addition of HNF4
-specific antibody. Thus, in
uninfected and MHV-2-infected macrophages an unidentified protein
occupies the HNF4 site. In contrast, in MHV-A59 infection, an
HNF4
-specific supershift was seen, demonstrating HNF4
binding to
the HNF4 consensus site.
is a constitutively expressed transcription factor that resides
in the nucleus of cells (28-31). It is expressed predominantly in the
liver although it is also found in the kidneys and intestine (31, 32),
thus it is referred to as a "liver-enriched" transcription factor.
HNF4
may regulate gene transcription in conjunction with other
liver-enriched transcription factors, such as HNF1, HNF3, and C/EBP
(31). HNF3
- and C/EBP
-binding sites have been detected in the
fgl2 promoter, and HNF4
may require these factors in the cascade of events leading to gene transcription. By Western blot analysis, C/EBP
was detected in the nuclear extracts from
MHV-A59-infected cells; however, HNF3
was not detected in either
uninfected or MHV-A59-infected cells. Whether C/EBP
is indirectly
involved in the hepatotrophic transcription of fgl2 has yet
to be determined.
regulates expression of HNF1, a transcription factor important
in the transcription of several hepatic genes (31, 33). HNF4
is
highly conserved and is essential for differentiation and development
of the liver and kidney during embryogenesis; targeted disruption of
the HNF4
gene is embryonic lethal (34), and in adults, HNF4
regulates the expression of liver-specific genes as well as genes
involved in the metabolism of cholesterol, amino acids, carbohydrates,
glucose, and lipids (6, 27, 35-37). Dysregulation of HNF4
is
implicated in several diseases, including maturity onset diabetes of
the young (38), hepatitis B infection (39), hemophilia (40), and
atherosclerosis (41). Of interest, HNF4
is involved in the
regulation of genes encoding coagulation factors VII, IX, X, and XII
and the anti-thrombin gene (5, 42-45). Moreover, HNF4
, through
HNF1, indirectly regulates the expression of fibrinogen (46).
exists in solution as a stable homodimer and also binds to DNA
as a homodimer (47). Originally, the HNF4
-binding site was
identified as a consensus sequence of AGGTCAXAGGTCA
(i.e. a direct repeat, DR1, where X is any
nucleotide) (33, 48). However, as more genes were identified with
HNF4
enhancer elements, it has been shown that HNF4
sites tend to
be variable and show a high degree of flexibility (5, 6, 37, 40,
42-44, 49). In the murine fgl2 promoter, HNF4
binds to
the sequence TGGACCAA (
332/
325), which has 86% sequence identity
with an HNF4
consensus binding element identified by Ramji et
al. (49).
activity is regulated at several levels. Phosphorylation of
HNF4
regulates subnuclear localization, DNA binding affinity, and
transcription activation activity (28, 50). Tyrosine phosphorylation affects the nuclear localization of HNF4
; phosphorylated protein displays a compartmental distribution within the nucleus (28). Upon
treatment with genistein, which removes phosphate groups from tyrosine,
HNF4
displays a diffuse localization within the nucleus (28). With
respect to DNA binding and transcription activation potential, one
group reported that tyrosine phosphorylation of 1 (or more) of 12 putative tyrosine phosphorylation sites in HNF4
is important for DNA
binding and transactivation potential (28). In contrast, others (50)
have shown that phosphorylation of any of 13 putative serine and
threonine residues increases DNA binding affinity and transcription
activation potential; evidence of tyrosine phosphorylation was not
detected in this investigation. Two independent investigations further
reported that serine phosphorylation by cAMP-dependent
protein kinase or protein kinase C inhibits both DNA binding and
transcription activation by HNF4
(51). The different observations
may reflect cell type- and target gene-specific differences and methods used.
site. Following MHV-A59 but
not MHV-2 infection, the HNF4
site is occupied by HNF4
. At
present the reason why MHV-A59 as opposed to MHV-2 results in binding
of HNF4
is not known. Initial studies in our laboratory have shown
that MHV-A59 infection resulted in increased phosphorylation of
tyrosine residues of HNF4
, although this did not occur following MHV-2 infection. The enhanced phosphorylation of HNF4
may result in
displacement of an inhibitor promoting enhanced binding of HNF4
to
its cis-element and resulting in fgl2 gene transcription. To
confirm this hypothesis, additional studies will need to be performed
to examine the phosphorylation status of not only tyrosine but also
serine and threonine residues of HNF4
following MHV-A59 and MHV2
infection. The ability to block phosphorylation, prevent binding of
HNF4
, and transcription of fgl2 will confirm the
importance of differential phosphorylation to the biology of
fgl2. These studies are now underway.
is constitutively expressed in the
liver, among other tissues, we have shown for the first time the
expression of HNF4
in macrophages. Several implications stem from
this observation. First, the macrophage is a source of human and
murine FGL2 protein, which is pivotal to the pathogenesis of FHF in
infected hosts. Second, the liver has the highest population of
resident macrophages, the Kupffer cell. Finally, macrophages can act as
a reservoir of disease, as in the case of human immunodeficiency virus
infection. The circulation of macrophages that have been stimulated by
viral infection to produce and express fgl2 can account for the spotty thrombosis and necrosis observed in extra hepatic tissues, such as the brain and lungs.
and the nucleoside
analog Ribavirin, in those patients who developed a sustained
anti-viral response, FGL2 transcripts could not be detected (52).
Preliminary studies have been performed using protein expression
constructs from HCV genotype 1b Taiwanese strain (HCV(Tw)1b) (kindly
provided by Dr. Michael Lai) and a construct with 1.3 kb of the
fgl2 promoter upstream the luciferase reporter gene to
determine whether any viral proteins regulated human FGL2 gene
transcription. The results suggest that the HCV core protein, which is
functionally analogous to the MHV nucleocapsid protein and a known
activator of host gene transcription (53-55), initiates human FGL2
gene transcription. Further studies are currently in progress to
identify the putative cis-elements in the promoter region responsive to
the HCV(Tw)1b core protein. Of interest is the determination of whether
there are genotype-specific differences in fgl2 gene
transcription; genotypes of interest include HCV-2 and HCV-3, both of
which are common in North America and, unlike HCV 1, produce less
severe disease and are more susceptible to antiviral regimens (56).
, was identified as directly
causing fgl2 transcription. Involvement of HNF4
, a
liver-enriched factor now also identified in peritoneal macrophages,
may explain the liver-specific fgl2 transcription and
hepatotrophic nature of tissue injury in MHV infection. The
investigation also provided a strategy with which to study the
pathogenesis of human viral hepatitis and highlight possible
therapeutic targets.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Paul Masters who provided the I wild type and mutant viruses and antibodies to the I protein and Charmaine Beal for technical assistance in the preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Canadian Institutes for Health and Research Grant MOP 37780.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Supported by National Science Foundation of China (NSFC) Grant NSFC 30170846 and NSFC for Distinguished Young Investigator Grant NSFC 30225040. To whom correspondence may be addressed: Tongji Hospital, Research Institute of Immunology, Tongji Medical College, Wuhan 430030, China. Tel.: 86-27-83662391; E-mail: qning@tjh. tjmu.edu.cn.
** To whom correspondence may be addressed: Toronto General Hospital, 621 University Ave., NU 10-116, Toronto, Ontario M5G 2C4, Canada. Tel.: 416-340-5166; Fax: 416-340-3378; E-mail: glfgl2@attglobal.net.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M212806200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: fgl2, mouse fgl2; FGL2, human FGL2; FHF, fulminant hepatic failure; MHV, murine hepatitis virus; LUC, luciferase; CHO, Chinese hamster ovary cells; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; N, nucleocapsid; HNF, hepatocyte nuclear factor; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; GM-CSF, granulocyte macrophage colony-stimulating factor; IE1.2, immediate early protein 1.2; HCV, hepatitis C virus; HBV, hepatitis B virus.
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---|
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