Induction of Prothrombinase fgl2 by the Nucleocapsid Protein of Virulent Mouse Hepatitis Virus Is Dependent on Host Hepatic Nuclear Factor-4alpha *

Qin NingDagger §, Sophia Lakatoo§||, Mingfeng Liu||, Weiming YangDagger , Zhimo WangDagger , M. James Phillips||, and Gary A. Levy||**

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -372 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, HNF4alpha , 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 HNF4alpha binds the HNF4 cis-element in the fgl2 promoter. We further report the unprecedented presence of HNF4alpha 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
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DISCUSSION
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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 -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 4alpha (HNF4alpha )/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).

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 HNF4alpha . We have shown further that HNFalpha 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.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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(-1328)/LUC. The Rous sarcoma virus beta -galactosidase vector was purchased from Promega.

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.


                              
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Table I
Primers used to generate the MHV-A59 N protein mutant constructs
Mutations were made to the amino acid sequence of the MHV-A59 N protein by a site-directed mutagenesis protocol as described under "Experimental Procedures." Sense and antisense primers were designed to encode the desired mutations. The underlined letters indicate the mutant sequences. The letters inside the parentheses indicate the sequences being deleted. All constructs generated were sequenced to confirm the orientation and to verify the sequence.

The following primers were used to construct the single and double mutations in the HNF4alpha and IE1.2 putative cis-elements located within -372 to -306 of the murine fgl2 promoter: Fgl2pHNF4alpha 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'; Fgl2pHNF4alpha /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.

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 beta -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 beta -galactosidase levels.

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 -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.

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 -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 gamma -globulin (Bio-Rad) was used to generate a standard curve.

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-4alpha (kindly provided by Dr. Sladek), rabbit polyclonal anti-C/EBPbeta (Santa Cruz Biotechnology, Santa Cruz, CA), or rabbit polyclonal anti-HNF3beta (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.

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 [gamma -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 gamma  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.

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: HNF4alpha 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/EBPbeta -binding oligonucleotide, 5-TTT GTA GTG TTT CCC AAC TCA GAT TCT GAG T-3' (3); HNF3beta -binding oligonucleotide, 5'-GAT CGT TGA CTA AGT CAA TAA TCA G-3' (5); HNF4alpha -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.

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.).

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ABSTRACT
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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.


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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.


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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.

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(-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.

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.


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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.

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 -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 (Mphi /MHV-A59 nucl extr) (A) or uninfected BALB/cJ peritoneal macrophages (Mphi 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."

Host Nuclear Proteins Bind to HNF4alpha 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 HNF4alpha (-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 HNF4alpha 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 HNF4alpha 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 HNF4alpha - 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 HNF4alpha 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."

The Putative HNF4alpha (-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 (HNF4alpha mut/LUC and fgl2pIE1.2mut/LUC) or double (fgl2HNF4alpha /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 HNF4alpha 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.

Although the data above strongly implicate the putative HNF4-binding site in transcription of fgl2, on further analysis of the -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/EBPbeta , Prx2, and HNF3beta (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 HNF4alpha 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/EBPbeta is present in the nuclear extracts of uninfected and MHV-A59-infected BALB/cJ peritoneal macrophages (Fig. 8B). Similarly, HNF4alpha was detected in the nuclear extracts from uninfected and MHV-A59-infected macrophages (Fig. 8C). In contrast, HNF3beta 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 HNF4alpha or C/EBPbeta 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/EBPbeta (B), HNFalpha (C), and HNF3beta (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.

HNF4alpha Binds to Its Cis-element -332/-325 in the Murine fgl2 Promoter in MHV-A59-stimulated Macrophages-- EMSAs were performed to determine whether HNF4alpha or C/EBPbeta 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 HNF4alpha competed with the labeled -336/-308 probe for binding to nuclear proteins, thus demonstrating the presence of an HNF4alpha cis-element in the -338/-306 region of the fgl2 promoter (Fig. 9A). In contrast, an excess of cold oligonucleotide known to bind C/EBPbeta , HNF3beta , 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 HNF4alpha . 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 HNF4alpha antibody. The HNF4alpha 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 HNF4alpha antibody was incubated with nuclear extracts from uninfected macrophages, suggesting that in the resting state, the HNF4alpha cis-element was occupied by a factor distinct from HNF4alpha (Fig. 9B). In MHV-2-infected BALB/cJ peritoneal macrophages, HNF4alpha antibody also failed to specifically shift the protein-DNA complex demonstrating that the HNF4alpha 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 HNF4alpha -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 HNF4alpha 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 (Mphi /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/EBPbeta , HNF3beta , HNF4alpha , and Prx2. The arrowhead denotes the HNF4alpha -specific band. B, supershift analyses verify that HNF4alpha binds to its cognate cis-element in the -338/-306 region in the murine fgl2 promoter in MHV-A59 infection. Nuclear extracts from uninfected (Mphi ), MHV-A59-infected (2.5 h) (Mphi /A59), and MHV-2-infected (2.5 h) (Mphi /V2) BALB/cJ peritoneal macrophages were incubated with 32P-labeled probes in the presence or absence of HNF4alpha -specific antibody (Ab) as described under "Experimental Procedures." The HNF4alpha antibody-specific shift is denoted by an arrow, and the HNFalpha -specific band is indicated by an arrowhead. C, HNF4alpha 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 HNF4alpha 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).

To determine whether HNF4alpha 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 HNF4alpha 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 HNF4alpha 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 HNF4alpha cis-element is occupied by an unidentified factor and does not bind HNF4alpha .

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -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 HNF4alpha . Based on bandshift and competition EMSA analyses, only the region -338/-306 encompassing the putative HNF4alpha 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 HNF4alpha might be involved was explored. Extensive characterization of the -338/-306 region revealed the presence of cis-elements for C/EBPbeta , HNF3beta , and Prx2 (26, 27). Bandshift analyses ruled out the direct involvement of C/EBP, HNF3beta , 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, HNF4alpha 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 HNF4alpha -specific antibody. Thus, in uninfected and MHV-2-infected macrophages an unidentified protein occupies the HNF4 site. In contrast, in MHV-A59 infection, an HNF4alpha -specific supershift was seen, demonstrating HNF4alpha binding to the HNF4 consensus site.

HNF4alpha 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. HNF4alpha may regulate gene transcription in conjunction with other liver-enriched transcription factors, such as HNF1, HNF3, and C/EBP (31). HNF3beta - and C/EBPbeta -binding sites have been detected in the fgl2 promoter, and HNF4alpha may require these factors in the cascade of events leading to gene transcription. By Western blot analysis, C/EBPbeta was detected in the nuclear extracts from MHV-A59-infected cells; however, HNF3beta was not detected in either uninfected or MHV-A59-infected cells. Whether C/EBPbeta is indirectly involved in the hepatotrophic transcription of fgl2 has yet to be determined.

HNF4alpha regulates expression of HNF1, a transcription factor important in the transcription of several hepatic genes (31, 33). HNF4alpha is highly conserved and is essential for differentiation and development of the liver and kidney during embryogenesis; targeted disruption of the HNF4alpha gene is embryonic lethal (34), and in adults, HNF4alpha 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 HNF4alpha is implicated in several diseases, including maturity onset diabetes of the young (38), hepatitis B infection (39), hemophilia (40), and atherosclerosis (41). Of interest, HNF4alpha is involved in the regulation of genes encoding coagulation factors VII, IX, X, and XII and the anti-thrombin gene (5, 42-45). Moreover, HNF4alpha , through HNF1, indirectly regulates the expression of fibrinogen (46).

HNF4alpha exists in solution as a stable homodimer and also binds to DNA as a homodimer (47). Originally, the HNF4alpha -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 HNF4alpha enhancer elements, it has been shown that HNF4alpha sites tend to be variable and show a high degree of flexibility (5, 6, 37, 40, 42-44, 49). In the murine fgl2 promoter, HNF4alpha binds to the sequence TGGACCAA (-332/-325), which has 86% sequence identity with an HNF4alpha consensus binding element identified by Ramji et al. (49).

HNF4alpha activity is regulated at several levels. Phosphorylation of HNF4alpha regulates subnuclear localization, DNA binding affinity, and transcription activation activity (28, 50). Tyrosine phosphorylation affects the nuclear localization of HNF4alpha ; phosphorylated protein displays a compartmental distribution within the nucleus (28). Upon treatment with genistein, which removes phosphate groups from tyrosine, HNF4alpha 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 HNF4alpha 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 HNF4alpha (51). The different observations may reflect cell type- and target gene-specific differences and methods used.

Our EMSA experiments demonstrated that in an uninfected state, an unidentified protein occupies the HNF4alpha site. Following MHV-A59 but not MHV-2 infection, the HNF4alpha site is occupied by HNF4alpha . At present the reason why MHV-A59 as opposed to MHV-2 results in binding of HNF4alpha is not known. Initial studies in our laboratory have shown that MHV-A59 infection resulted in increased phosphorylation of tyrosine residues of HNF4alpha , although this did not occur following MHV-2 infection. The enhanced phosphorylation of HNF4alpha may result in displacement of an inhibitor promoting enhanced binding of HNF4alpha 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 HNF4alpha following MHV-A59 and MHV2 infection. The ability to block phosphorylation, prevent binding of HNF4alpha , and transcription of fgl2 will confirm the importance of differential phosphorylation to the biology of fgl2. These studies are now underway.

Whereas it is known that HNF4alpha is constitutively expressed in the liver, among other tissues, we have shown for the first time the expression of HNF4alpha 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.

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-alpha 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).

The present study demonstrated that the MHV-A59 N protein indirectly initiates transcription of the murine fgl2 gene in susceptible hosts. A host factor, HNF4alpha , was identified as directly causing fgl2 transcription. Involvement of HNF4alpha , 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Ning, Q., Brown, D., and Parodo, J. (1998) J. Immunol. 160, 3487-3493[Abstract/Free Full Text]
2. Nelson, G. W., and Stohlman, S. A. (1993) J. Gen. Virol. 74, 1975-1979[Abstract]
3. Attard, F. A., Wang, L., and Potter, J. J. (2000) Arch. Biochem. Biophys. 378, 57-64[CrossRef][Medline] [Order article via Infotrieve]
4. Li, C., Fung, L. S., and Crow, A. (1992) J. Exp. Med. 176, 689-697[Abstract]
5. Fernandez-Rachubinski, F., Weiner, J. H., and Blajchman, M. A. (1996) J. Biol. Chem. 271, 29502-29512[Abstract/Free Full Text]
6. Rouet, P., Raguenez, G., and Ruminy, P. (1998) Biochem. J. 334, 577-584[Medline] [Order article via Infotrieve]
7. Levy, G. A., Macphee, P., and Fung, L. S. (1983) Hepatology 3, 964-973[Medline] [Order article via Infotrieve]
8. Ning, Q., Liu, M. F., and Kongkham, P. (1999) J. Biol. Chem. 274, 9930-9936[Abstract/Free Full Text]
9. Levy, G. A., Helin, H., and Edgington, T. S. (1984) Semin. Liver Dis. 4, 59-68[Medline] [Order article via Infotrieve]
10. Lavi, E., Murray, E. M., and Makino, S. (1990) Lab. Invest. 62, 570-579[Medline] [Order article via Infotrieve]
11. Masters, P. S., Parker, M. M., Ricard, C. S., Duchala, C., Frana, M. F., Holmes, K. V., and Sturman, L. S. (1990) Adv. Exp. Med. Biol. 276, 239-246[Medline] [Order article via Infotrieve]
12. Tahara, S. M., Dietlin, T. A., and Nelson, G. W. (1998) Adv. Exp. Med. Biol. 440, 313-318[Medline] [Order article via Infotrieve]
13. Ding, J., Ning, Q., and Liu, M. F. (1997) J. Virol. 71, 9223-9230[Abstract]
14. Lai, M. C. (1992) Microbiol. Rev. 56, 61-79[Medline] [Order article via Infotrieve]
15. Fischer, F., Peng, D., and Hingley, S. T. (1997) J. Virol. 71, 996-1003[Abstract]
16. Norris, R. A., and Kern, M. J. (2001) J. Biol. Chem. 276, 26829-26837[Abstract/Free Full Text]
17. Levy, G. A., Liu, M. F., and Ding, J. W. (2000) Am. J. Pathol. 156, 1217-1225[Abstract/Free Full Text]
18. Parker, M. M., and Masters, P. S. (1990) Virology 179, 463-468[Medline] [Order article via Infotrieve]
19. Stohlman, S. A., Baric, R. S., and Nelson, G. N. (1988) J. Virol. 62, 4288-4295[Medline] [Order article via Infotrieve]
20. Baric, R. S., Nelson, G. N., and Fleming, J. O. (1988) J. Virol. 62, 4280-4287[Medline] [Order article via Infotrieve]
21. Kunita, S., Mori, M., and Terada, E. (1993) Virology 193, 520-523[CrossRef][Medline] [Order article via Infotrieve]
22. Lapps, W., Hogue, B. G., and Brian, G. A. (1987) Virology 157, 47-57[Medline] [Order article via Infotrieve]
23. Decimo, D., Philippe, H., and Hadchouel, M. (1993) Arch. Virol. 130, 279-288[Medline] [Order article via Infotrieve]
24. Peng, D., Koetzner, C. A., and Masters, P. S. (1995) J. Virol. 69, 3349-3457
25. Wurm, T., Chen, H., and Hodgson, T. (2001) J. Virol. 75, 9345-9356[Abstract/Free Full Text]
26. Yoshida, E., Aratani, S., and Itou, H. (1997) Biochem. Biophys. Res. Commun. 241, 664-669[CrossRef][Medline] [Order article via Infotrieve]
27. Dell, H., and Hadzopoulou-Cladaras, M. (1999) J. Biol. Chem. 274, 9013-9021[Abstract/Free Full Text]
28. Ktistaki, E., Ktistakis, N. T., and Papadogeorgaki, E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9876-9880[Abstract]
29. De Simone, V., and Cortese, R. (1992) Biochim. Biophys. Acta 1132, 119-126[Medline] [Order article via Infotrieve]
30. Sladek, F. M., Weimin, Z., and Lai, E. (1990) Genes Dev. 4, 2353-2365[Abstract]
31. Xanthopoulos, K. G., Prezioso, V. R., and Williams, S. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3807-3811[Abstract]
32. Simpson, K. J., Lukacs, N. W., and Colletti, L. (1997) J. Hepatol. 27, 1120-1132[CrossRef][Medline] [Order article via Infotrieve]
33. Sladek, F. M. (1993) Receptor 3, 223-232[Medline] [Order article via Infotrieve]
34. Taraviras, S., Monaghan, A. P., and Schutz, G. (1994) Mech. Dev. 48, 67-79[CrossRef][Medline] [Order article via Infotrieve]
35. Yamanda, K., Tanaka, T., and Noguchi, T. (1997) Biochem. J. 324, 917-925[Medline] [Order article via Infotrieve]
36. Reddy, S., Yang, W., and Taylor, D. G. (1999) J. Biol. Chem. 274, 33050-33056[Abstract/Free Full Text]
37. Moldrup, A., Ormandy, C., and Nagano, M. (1996) Mol. Endocrinol. 10, 661-671[Abstract]
38. Ryffel, G. (2001) J. Mol. Endocrinol. 27, 11-29[Abstract/Free Full Text]
39. Fukai, K., Takada, S., and Yokosuka, O. (1997) Virology 236, 279-287[CrossRef][Medline] [Order article via Infotrieve]
40. Crossley, M., Ludwig, M., and Stowell, K. (1992) Science 257, 377-379[Medline] [Order article via Infotrieve]
41. Jump, D., and Clarke, S. (1999) Annu. Rev. Nutr. 19, 63-90[CrossRef][Medline] [Order article via Infotrieve]
42. Erdmann, D., and Heim, J. (1995) J. Biol. Chem. 270, 22988-22996[Abstract/Free Full Text]
43. Naka, H., and Brownlee, G. (1996) Br. J. Haematol. 92, 231-240[CrossRef][Medline] [Order article via Infotrieve]
44. Miao, C. H., Leytus, S. P., and Chung, D. W. (1992) J. Biol. Chem. 267, 7395-7401[Abstract/Free Full Text]
45. Farsetti, A., Moretti, F., and Narducci, M. (1998) Endocrinology 129, 4581-4589
46. Hu, C.-H., Harris, J. E., and Davie, E. W. (1995) J. Biol. Chem. 270, 28342-28349[Abstract/Free Full Text]
47. Bogan, A. A., Dallas-Yang, Q., and Ruse, M. D., Jr. (2000) J. Mol. Biol. 302, 831-851[CrossRef][Medline] [Order article via Infotrieve]
48. Jiang, G., Lee, U., and Sladek, F. M. (1997) Mol. Cell. Biol. 17, 6546-6554[Abstract]
49. Ramji, D. P., Tadros, M. H., and Hardon, E. M. (1990) Nucleic Acids Res. 19, 1139-1146
50. Jiang, G., Nepomuceno, L., and Yang, Q. (1997) Arch. Biochem. Biophys. 340, 1-9[CrossRef][Medline] [Order article via Infotrieve]
51. Viollet, B., Kahn, A., and Raymondjean, M. (1997) Mol. Cell. Biol. 17, 4208-4219[Abstract]
52. Rasul, I., Liu, M. F., and Ning, Q. (1999) Hepatology 30, 594[CrossRef]
53. Lai, M., and Ware, C. (2000) Curr. Top. Microbiol. Immunol. 242, 117-134[Medline] [Order article via Infotrieve]
54. Large, M. C., Kittlesen, D. J., and Hahn, Y. S. (1999) J. Immunol. 162, 931-938[Abstract/Free Full Text]
55. McLauchlan, J. (2000) J. Viral Hep. 7, 2-14[CrossRef]
56. Webster, G., Brown, D., and Dusheiko, G. (2000) Baillere's Clin. Gastroenterol. 14, 229-240


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