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Qin Ning - One of the best experts on this subject based on the ideXlab platform.
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Image_1_Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression.TIF
2018Co-Authors: Yang Liu, Hongwu Wang, Xiaoyang Wan, Jiaquan Huang, Weiming Yan, Xiaoping Luo, Guanxin Shen, Qin NingAbstract:Background: Fulminant Hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses Murine Hepatitis Virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10.Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro.Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs).Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1.
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Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression
Frontiers Media S.A., 2018Co-Authors: Yang Liu, Hongwu Wang, Xiaoyang Wan, Jiaquan Huang, Weiming Yan, Xiaoping Luo, Guanxin Shen, Qin NingAbstract:Background: Fulminant Hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses Murine Hepatitis Virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10.Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro.Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs).Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1
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Table_1_Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression.DOC
2018Co-Authors: Yang Liu, Hongwu Wang, Xiaoyang Wan, Jiaquan Huang, Weiming Yan, Xiaoping Luo, Guanxin Shen, Qin NingAbstract:Background: Fulminant Hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses Murine Hepatitis Virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10.Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro.Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs).Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1.
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liver tcrγδ cd3 cd4 cd8 t cells contribute to Murine Hepatitis Virus strain 3 induced hepatic injury through a tnf α dependent pathway
Molecular Immunology, 2012Co-Authors: Xiaojing Wang, Hongwu Wang, Weiming Yan, Xiaoping Luo, Ming Wang, Lin Zhu, Qin NingAbstract:The mechanisms of each subset of immune cells contributing to the pathogenesis of viral Hepatitis remain incompletely understood. In this study, we examined the role of liver CD4(-) CD8(-) (double negative, DN) T cells during Murine Hepatitis Virus strain 3 (MHV-3)-induced Hepatitis in C3H/HeJ mice. We demonstrate that predominant population of DN T cells in the liver of healthy or MHV-3-infected mice express TCRγδ(+). The proportion of TCRγδ(+) DN T cells in liver CD3(+) T cells was markedly increased after MHV-3 infection. Adoptive transfer of TCRγδ(+) DN T cells led to dramatically decreased survival in MHV-3-infected mice, accompanied by deteriorated histopathology and elevated ALT and AST levels. It was found that these cells were hyperactivated after MHV-3 infection with a production of TNF-α, IFN-γ, IL-2 and IL-17A. Highly activated liver TCRγδ(+) DN T cells were cytotoxic to MHV-3-infected hepatocytes in vitro and this effect did not require cell-cell contact. Moreover, the cytotoxic effect of liver TCRγδ(+) DN T cells against hepatocytes involves TNF-α pathway, but not IL-17A or IFN-γ. These results indicate that liver TCRγδ(+) DN T cells play a critical role in the liver injury in MHV-3-induced Hepatitis, via a TNF-α dependent pathway.
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novel mfgl2 antisense plasmid inhibits Murine fgl2 expression and ameliorates Murine Hepatitis Virus type 3 induced fulminant Hepatitis in balb cj mice
Human Gene Therapy, 2006Co-Authors: Chuanlong Zhu, Xiaoping Luo, Weiming Yan, Yi Sun, Qin NingAbstract:Our previous reports, both experimental and human studies, have shown the importance of fibrinogen-like protein-2 (fgl2) prothrombinase in the development of fulminant viral Hepatitis, a disease with a mortality of more than 80% in cases lacking immediate organ transplantation. To interfere with this potentially effective target, a 322-bp mouse fgl2 (mfgl2) antisense plasmid complementary to the exon 1 sequence of the gene, including the translation initiation site AUG, was successfully constructed. A dose-dependent inhibitory effect on mfgl2 expression by mfgl2 antisense plasmid was observed in interferon-gamma-treated RAW 264.7 cells. On hydrodynamic delivery, mfgl2 antisense plasmid significantly reduced mfgl2 expression in vivo; markedly ameliorated inflammatory cell infiltration, fibrin deposition, and hepatocyte necrosis; prolonged the survival time period; and elevated the survival rate among BALB/cJ mice with Murine Hepatitis Virus type 3-induced fulminant Hepatitis. This study may provide an effective way to interfere with the potential therapeutic target fgl2 gene for fulminant viral Hepatitis and other diseases with similar pathological characteristics of microcirculation disorders, including acute rejection of xeno- or allograft transplantation and fetal loss syndrome, in which studies show fgl2 plays an important role.
G A Levy - One of the best experts on this subject based on the ideXlab platform.
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fibrinogen like protein 2 fibroleukin expression and its correlation with disease progression in Murine Hepatitis Virus type 3 induced fulminant Hepatitis and in patients with severe viral Hepatitis b
World Journal of Gastroenterology, 2005Co-Authors: Chuanlong Zhu, G A Levy, Weiming Yan, Xiaoping Luo, Fan Zhu, Yongfen Zhu, Deying Tian, Qin NingAbstract:AIM: To evaluate the expression of fibrinogen-like protein 2 (fgl2) and its correlation with disease progression in both mice and patients with severe viral Hepatitis. METHODS: Balb/cJ or A/J mice were infected intraperitoneally (ip) with 100 PFU of Murine Hepatitis Virus type 3 (MHV-3), liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic (AOC) Hepatitis B and 13 patients with mild chronic Hepatitis B. Fourteen patients with mild chronic Hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with severe AOC Hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinase activity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase (ALT) and total bilirubin (TBil) in serum were measured to assess the severity of liver injury. RESULTS: Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice. In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. Mouse fgl2 (mfgl2) protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC Hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 (hfgl2) was detected in 21 of 23 patients (91.30%) with severe AOC Hepatitis B, while only 1 of 13 patients (7.69%) with mild chronic Hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients (93.33%) with severe AOC Hepatitis B and 1 of 10 with mild chronic Hepatitis B had detectable hfgl2 expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2 expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC Hepatitis B. CONCLUSION: The molecular and cellular results reported here in both mice and patients with severe viral Hepatitis suggest that Virus-induced hfgl2 prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2 protein play a pivotal role in initiating severe Hepatitis. The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC Hepatitis B and a target for therapeutic intervention.
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the nucleocapsid protein of Murine Hepatitis Virus type 3 induces transcription of the novel fgl2 prothrombinase gene
Journal of Biological Chemistry, 1999Co-Authors: Qin Ning, Julian L Leibowitz, Philip A Marsden, Paul Kongkham, Jonathan Tseng, Bethany Pereira, Michail Belyavskyi, James M Phillips, G A LevyAbstract:Abstract Using a set of parental and recombinant Murine Hepatitis Virus strains, we demonstrate that the nucleocapsid protein induces transcription of the novel fgl2 prothrombinase gene and elevated procoagulant activity in those strains that produce fulminant Hepatitis. Chinese hamster ovary cells cotransfected with a construct expressing nucleocapsid protein from susceptible strains and with a luciferase reporter construct containing the fgl2promoter showed a 6-fold increase in luciferase activity compared with nontransfected cells or cells cotransfected with a construct expressing nucleocapsid protein from resistant strains. Two deletions found at coding sites 111–123 and 1143–1145 of structural domains I and III, respectively, of the nucleocapsid gene may account for the differences between pathogenic and nonpathogenic strains. Preliminary mapping of the fgl2 promoter has defined a region from −372 to −306 upstream from the ATG translation initiation site to be responsive to nucleocapsid protein. Hence, mapping of genetic determinants in parental and recombinant strains demonstrates that the nucleocapsid protein of strains that induce fulminant Hepatitis is responsible for transcription of the fgl2 prothrombinase gene. These studies provide new insights into the role of the nucleocapsid gene in the pathogenesis of viral Hepatitis.
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resistance to Murine Hepatitis Virus strain 3 is dependent on production of nitric oxide
Journal of Virology, 1998Co-Authors: M Pope, L. S. Fung, Julian L Leibowitz, Philip A Marsden, Qin Ning, Edward H. Cole, M. J. Phillips, S Sloan, J W Ding, G A LevyAbstract:The strain-specific spectrum of liver disease following Murine Hepatitis Virus type 3 (MHV-3) infection is dependent on inflammatory mediators released by macrophages. Production of nitric oxide (NO) by macrophages has been implicated in resistance to a number of Viruses, including ectromelia Virus, vaccinia Virus, and herpes simplex Virus type 1. This study was undertaken to define the role of NO in MHV-3 infection. Gamma interferon-induced production of NO inhibited growth of MHV-3 in a Murine macrophage cell line (RAW 264.7). Viral inhibitory activity was reproduced by the NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP), whereas N-acetyl-dl-pencillamine (NAP), an inactive analog of SNAP, had no effect. Electron microscopy studies confirmed the inhibitory effects of NO on viral replication. Peritoneal macrophages isolated from A/J mice known to be resistant to MHV-3 produced a fivefold-higher level of NO and higher levels of mRNA transcripts of inducible NO synthase in response to gamma interferon than macrophages from susceptible BALB/cJ mice. SNAP inhibited growth of MHV-3 in macrophages from both strains of mice to similar degrees. In vivo inhibition of NO by N-monomethyl-l-arginine resulted in loss of resistance to MHV-3 in A/J mice. These results collectively demonstrate a defect in the production of NO in macrophages from susceptible BALB/cJ mice and define the importance of endogenous NO in resistance to MHV-3 infection in resistant A/J mice.
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expression of the fgl2 and its protein product prothrombinase in tissues during Murine Hepatitis Virus strain 3 mhv 3 infection
Advances in Experimental Medicine and Biology, 1998Co-Authors: J W Ding, L. S. Fung, Qin Ning, Kevork M. Peltekian, C. Holloway, Herman Yeger, M. J. Phillips, Mingfeng Liu, A Lai, G A LevyAbstract:Murine Hepatitis Virus Strain 3 (MHV-3) produces fulminant Hepatitis with 80–90% mortality in Balb/cJ mice. Previous studies in our laboratory have shown that peritoneal macrophages from MHV-3 infected mice produce a procoagulant (PCA) which has the ability to cleave prothrombin to thrombin (prothrombinase) encoded by the gene fgl2 located on chromosome 5. PCA accounts for sinusoidal thrombosis and hepatic necrosis and the necrosis and mortality can be prevented by treatment of animals with a monoclonal antibody to PCA. These present studies were designed to examine the expression of this gene (mRNA by Northern analysis and in situ hybridization) and the gene product PCA (immunochemistry) in tissues recovered from MHV-3 infected Balb/cJ mice in an attempt to explain the liver specific nature of MHV-3 disease. Fgl2 gene expression was detected as early as 8 hours after MHV-3 infection which persisted to 48 hours in the liver, spleen and lungs whereas no gene expression was seen in the brain or kidneys despite the fact that equivalent viral titers were detected in all tissues at all times. In the liver, fgl2 gene expression was confined to endothelial and Kupffer cells with no expression in hepatocytes. Immunochemistry localized the PCA protein to Kupffer cells and endothelial cells and necrotic foci within the liver. No PCA protein was detected by immunochemistry in any other tissues at any time during the course of MHV-3 infection.
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loss of resistance to Murine Hepatitis Virus strain 3 infection after treatment with corticosteroids is associated with induction of macrophage procoagulant activity
Journal of Virology, 1996Co-Authors: R Fingerote, Julian L Leibowitz, Edward H. Cole, M. J. Phillips, Michael Abecassis, Y S Rao, G A LevyAbstract:Activation of the immune coagulation system has been implicated in the pathogenesis of liver injury following infection of inbred mice with Murine Hepatitis Virus strain 3 (MHV-3). Following MHV-3 infection, macrophages isolated from MHV-3-susceptible and -semisusceptible inbred strains of mice express increased procoagulant activity (PCA), whereas macrophages from resistant strains express no increase in PCA over basal levels. The PCA induced by MHV-3 is a prothrombinase, encoded by the gene Fgl-2, which encodes a fibrinogen-like protein (musfiblp). In this study, MHV-3-resistant A/J mice treated with methylprednisolone prior to infection with MHV-3 developed elevated levels of alanine aminotransferase in serum and died within 10 days of infection, with histological findings of fulminant Hepatitis. In vitro, macrophages isolated from A/J mice and pretreated with methylprednisolone produced a marked increase in functional PCA following infection with MHV-3. The PCA was shown to be a prothrombinase by its ability to cleave 125I-prothrombin. Northern blot analysis of RNA transcripts from these macrophages demonstrated increased transcription of the Fgl-2 gene relative to that in macrophages which had not been pretreated with methylprednisolone prior to MHV-3 infection. Methylprednisolone pretreatment of MHV-3-infected macrophages stabilized the Fgl-2 mRNA. Thus, loss of resistance to MHV-3 secondary to methylprednisolone therapy is associated with increased transcription and stability of Fgl-2 mRNA resulting in expression of the Fgl-2 gene product, musfiblp. These results provide further insight into mechanisms of PCA regulation in response to MHV-3 infection in inbred strains of mice.
Mark R Denison - One of the best experts on this subject based on the ideXlab platform.
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Murine Hepatitis Virus nsp14 exoribonuclease activity is required for resistance to innate immunity
Journal of Virology, 2018Co-Authors: James Brett Case, Susan R Weiss, Ruth Elliott, Kevin W Graepel, Nicole R Sexton, Everett Clinton Smith, Mark R DenisonAbstract:CoronaViruses (CoVs) are positive-sense RNA Viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode several innate immune antagonists that counteract the host innate immune response to facilitate efficient viral replication. CoV nonstructural protein 14 (nsp14) encodes 3'-to-5' exoribonuclease activity (ExoN), which performs a proofreading function and is required for high-fidelity replication. Outside of the order Nidovirales, arenaViruses are the only RNA Viruses that encode an ExoN, which functions to degrade double-stranded RNA (dsRNA) replication intermediates. In this study, we tested the hypothesis that CoV ExoN also functions to antagonize the innate immune response. We demonstrate that Viruses lacking ExoN activity [ExoN(-)] are sensitive to cellular pretreatment with interferon beta (IFN-β) in a dose-dependent manner. In addition, ExoN(-) Virus replication was attenuated in wild-type bone marrow-derived macrophages (BMMs) and partially restored in interferon alpha/beta receptor-deficient (IFNAR-/-) BMMs. ExoN(-) Virus replication did not result in IFN-β gene expression, and in the presence of an IFN-β-mediated antiviral state, ExoN(-) viral RNA levels were not substantially reduced relative to those of untreated samples. However, ExoN(-) Virus generated from IFN-β-pretreated cells had reduced specific infectivity and decreased relative fitness, suggesting that ExoN(-) Virus generated during an antiviral state is less viable to establish a subsequent infection. Overall, our data suggest Murine Hepatitis Virus (MHV) ExoN activity is required for resistance to the innate immune response, and antiviral mechanisms affecting the viral RNA sequence and/or an RNA modification act on Viruses lacking ExoN activity.IMPORTANCE CoVs encode multiple antagonists that prevent or disrupt an efficient innate immune response. Additionally, no specific antiviral therapies or vaccines currently exist for human CoV infections. Therefore, the study of CoV innate immune antagonists is essential for understanding how CoVs overcome host defenses and to maximize potential therapeutic interventions. Here, we sought to determine the contributions of nsp14 ExoN activity in the induction of and resistance to the innate immune response. We show that Viruses lacking nsp14 ExoN activity are more sensitive than wild-type MHV to restriction by exogenous IFN-β and that Viruses produced in the presence of an antiviral state are less capable of establishing a subsequent viral infection. Our results support the hypothesis that Murine Hepatitis Virus ExoN activity is required for resistance to the innate immune response.
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mutations across Murine Hepatitis Virus nsp4 alter Virus fitness and membrane modifications
Journal of Virology, 2015Co-Authors: Dia C Beachboard, Jordan Andersondaniels, Mark R DenisonAbstract:A common feature of infection by positive-sense RNA Virus is the modification of host cell cytoplasmic membranes that serve as sites of viral RNA synthesis. CoronaViruses induce double-membrane vesicles (DMVs), but the role of DMVs in replication and Virus fitness remains unclear. CoronaViruses encode 16 nonstructural proteins (nsps), three of which, nsp3, nsp4, and nsp6, are necessary and sufficient for DMV formation. It has been shown previously that mutations in Murine Hepatitis Virus (MHV) nsp4 loop 1 that alter nsp4 glycosylation are associated with disrupted DMV formation and result in changes in Virus replication and RNA synthesis. However, it is not known whether DMV morphology or another function of nsp4 glycosylation is responsible for effects on Virus replication. In this study, we tested whether mutations across nsp4, both alone and in combination with mutations that abolish nsp4 glycosylation, affected DMV formation, replication, and fitness. Residues in nsp4 distinct from glycosylation sites, particularly in the endoplasmic reticulum (ER) luminal loop 1, independently disrupted both the number and morphology of DMVs and exacerbated DMV changes associated with loss of glycosylation. Mutations that altered DMV morphology but not glycosylation did not affect Virus fitness while Viruses lacking nsp4 glycosylation exhibited a loss in fitness. The results support the hypothesis that DMV morphology and numbers are not key determinants of Virus fitness. The results also suggest that nsp4 glycosylation serves roles in replication in addition to the organization and stability of MHV-induced double-membrane vesicles. IMPORTANCE All positive-sense RNA Viruses modify host cytoplasmic membranes for viral replication complex formation. Thus, defining the mechanisms of Virus-induced membrane modifications is essential for both understanding Virus replication and development of novel approaches to Virus inhibition. CoronaVirus-induced membrane changes include double-membrane vesicles (DMVs) and convoluted membranes. Three viral nonstructural proteins (nsps), nsp3, nsp4, and nsp6, are known to be required for DMV formation. It is unknown how these proteins induce membrane modification or which regions of the proteins are involved in DMV formation and stability. In this study, we show that mutations across nsp4 delay Virus replication and disrupt DMV formation and that loss of nsp4 glycosylation is associated with a substantial fitness cost. These results support a critical role for nsp4 in DMV formation and Virus fitness.
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Murine Hepatitis Virus nsp4 n258t mutants are not temperature sensitive
Virology, 2013Co-Authors: Dia C Beachboard, Susan C Baker, Mark R DenisonAbstract:CoronaVirus replicase nsp4 is critical for Virus-induced membrane modifications. An nsp4 mutant (N258T) of Murine Hepatitis Virus (MHV) has been reported to be temperature-sensitive (ts) and to alter membrane targeting. We engineered and recovered all four possible codon variants of N258T in the cloned MHV-A59 background. All mutant Viruses demonstrated impaired replication compared to wildtype MHV, but no nsp4 N258T mutant Virus was ts, and all variants colocalized with viral protein markers for replication complexes, but not with markers for mitochondria. This study emphasizes that complete genome sequencing may be necessary, even with directed and confirmed reverse genetic mutants.
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Murine Hepatitis Virus nonstructural protein 4 regulates Virus induced membrane modifications and replication complex function
Journal of Virology, 2010Co-Authors: Mark Jacob Gadlage, Jennifer S Sparks, Dia C Beachboard, Joshua Doyle, Christopher C Stobart, Mark R DenisonAbstract:Positive-strand RNA Viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaViruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine Hepatitis Virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during Virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant Viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant Viruses. The nsp4 glycosylation mutants exhibited impaired Virus growth and RNA synthesis, with the N237A and N176A/N237A mutant Viruses demonstrating more profound defects in Virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of Virus-induced double-membrane vesicles (DMVs) compared to those infected with wt Virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and Virus growth of the nsp4 mutant Viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaViruses.
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a novel mutation in Murine Hepatitis Virus nsp5 the viral 3c like proteinase causes temperature sensitive defects in viral growth and protein processing
Journal of Virology, 2008Co-Authors: Jennifer S Sparks, Eric F Donaldson, Ralph S Baric, Mark R DenisonAbstract:Sequencing and reversion analysis of Murine Hepatitis Virus (MHV) temperature-sensitive (ts) Viruses has identified putative ts mutations in the replicase nonstructural proteins (nsp's) of these coronaViruses. In this study, reverse transcriptase PCR sequencing of the RNA genome of an isolate of the MHV ts Virus Alb ts6, referred to as Alb/ts/nsp5/V148A, identified a putative ts mutation in nsp5 (T10651C, Val148Ala), the viral 3C-like proteinase (3CLpro). The introduction of the T10651C mutation into the infectious MHV clone resulted in the recovery of a mutant Virus, the nsp5/V148A Virus, that demonstrated reduced growth and nsp5 proteinase activity identical to that of Alb/ts/nsp5/V148A at the nonpermissive temperature. Sequence analysis of 40 degrees C revertants of Alb/ts/nsp5/V148A identified primary reversion to Ala148Val in nsp5, as well as two independent second-site mutations resulting in Ser133Asn and His134Tyr substitutions in nsp5. The introduction of the Ser133Asn or His134Tyr substitution into the cloned nsp5/V148A mutant Virus background resulted in the recovery of Viruses with increased growth fitness and the partial restoration of nsp5 activity at the nonpermissive temperature. Modeling of the nsp5 structure of Alb/ts/nsp5/V148A predicted that the Val148Ala mutation alters residue 148 interactions with residues of the substrate binding S1 subsite of the nsp5 active-site cavity. This study identifies novel residues in nsp5 that may be important for regulating substrate specificity and nsp5 proteinase activity.
Julian L Leibowitz - One of the best experts on this subject based on the ideXlab platform.
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functional transcriptional regulatory sequence trs rna binding and helix destabilizing determinants of Murine Hepatitis Virus mhv nucleocapsid n protein
Journal of Biological Chemistry, 2012Co-Authors: Sarah C Keane, Julian L Leibowitz, David P GiedrocAbstract:Abstract CoronaVirus (CoV) nucleocapsid (N) protein contains two structurally independent RNA binding domains. These are denoted N-terminal domain (NTD) and C-terminal domain and are joined by a charged linker region rich in serine and arginine residues (SR linker). In mouse Hepatitis Virus (MHV), the NTD binds the transcriptional regulatory sequence (TRS) RNA, a conserved hexanucleotide sequence required for subgenomic RNA synthesis. The NTD is also capable of disrupting a short RNA duplex. We show here that three residues on the β3 (Arg-125 and Tyr-127) and β5 (Tyr-190) strands play key roles in TRS RNA binding and helix destabilization with Ala substitutions of these residues lethal to the Virus. NMR studies of the MHV NTD·TRS complex revealed that this region defines a major RNA binding interface in MHV with site-directed spin labeling studies consistent with a model in which the adenosine-rich 3′-region of TRS is anchored by Arg-125, Tyr-127, and Tyr-190 in a way that is critical for efficient subgenomic RNA synthesis in MHV. Characterization of CoV N NTDs from infectious bronchitis Virus and from severe acute respiratory syndrome CoV revealed that, although detailed NTD-TRS determinants are distinct from those of MHV NTD, rapid helix destabilization activity of CoV N NTDs is most strongly correlated with CoV function and Virus viability.
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Biochemical and Genetic Analyses of Murine Hepatitis Virus Nsp15 Endoribonuclease
Journal of virology, 2007Co-Authors: Hyojeung Kang, Julian L Leibowitz, Kanchan Bhardwaj, Satheesh K. Palaninathan, James C. Sacchettini, Linda A. Guarino, C. Cheng KaoAbstract:The goal of this project was to better define the relationship between the endoribonuclease activity of Murine Hepatitis Virus (MHV) Nsp15 (mNsp15) and its role in Virus infection. Molecular modeling demonstrated that the catalytic residues of mNsp15 are superimposable with its severe acute respiratory syndrome coronaVirus ortholog. Alanine substitutions at three key residues in the mNsp15 catalytic pocket (H262, H277, and G275) and a double-mutant version (H262P and H277A) generated proteins with greatly reduced but detectable endoribonuclease activities. Furthermore, these mutant proteins demonstrated lower cleavage specificities for uridylate than wild-type (WT) mNsp15. These mutations were successfully incorporated into Viruses named vH262A, vH277A, vG275A, and vH262P+H277A. All four mutant Viruses formed plaques with diameters similar to that of MHV-A59 1000 (WT Virus) on several different cell lines. Interestingly, Viruses with a mutation at a noncatalytic residue, D324A, could not be recovered despite repeated attempts, and expression of mNsp15 containing the D324A mutation in Escherichia coli resulted in an insoluble protein. Plaques derived from vH262A produced approximately 6- to 13-fold fewer PFU than those from WT Virus. Cells infected with mNsp15 mutant Viruses accumulated lesser amounts of plus- and minus-sense subgenomic RNAs and spike protein than WT Virus. The expression of mNsp15 in trans by transient transfection partially restored RNA synthesis by vH262A. These results demonstrate that mNsp15 is required for optimal infection by MHV.
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mitochondrial hsp70 hsp40 and hsp60 bind to the 3 untranslated region of the Murine Hepatitis Virus genome
Archives of Virology, 2003Co-Authors: Santosh K Nanda, Reed F Johnson, Qi Liu, Julian L LeibowitzAbstract:We have previously shown that mitochondrial-aconitase binds specifically to the 3' terminal 42 nucleotides of the Murine Hepatitis Virus (MHV) RNA along with three additional proteins of 70, 58 and 40 kDa to form a stable RNA-protein complex. Supershift and western blot assays have identified these three proteins as mitochondrial HSP70 (mtHSP70), HSP60, and HSP40. A series of co-immunoprecipitation assays have established that these four MHV RNA binding proteins are associated, even in the absence of MHV RNA. However, the presence of a synthetic RNA containing the sequence bound by these four proteins does increase the amount of co-precipitated protein, in particular the amount of HSP60 which is brought down with antibodies directed against HSP40 and mtHSP70. We have provided evidence for the interaction of these four proteins with the 3' end region of MHV RNA in infected cells by a series of immunoprecipitation RT-PCR assays. We believe it is likely that MHV RNA interacts with m-aconitase prior to its import into mitochondria in cooperation with extra-mitochondrial mtHSP70, HSP60, and HSP40.
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the nucleocapsid protein of Murine Hepatitis Virus type 3 induces transcription of the novel fgl2 prothrombinase gene
Journal of Biological Chemistry, 1999Co-Authors: Qin Ning, Julian L Leibowitz, Philip A Marsden, Paul Kongkham, Jonathan Tseng, Bethany Pereira, Michail Belyavskyi, James M Phillips, G A LevyAbstract:Abstract Using a set of parental and recombinant Murine Hepatitis Virus strains, we demonstrate that the nucleocapsid protein induces transcription of the novel fgl2 prothrombinase gene and elevated procoagulant activity in those strains that produce fulminant Hepatitis. Chinese hamster ovary cells cotransfected with a construct expressing nucleocapsid protein from susceptible strains and with a luciferase reporter construct containing the fgl2promoter showed a 6-fold increase in luciferase activity compared with nontransfected cells or cells cotransfected with a construct expressing nucleocapsid protein from resistant strains. Two deletions found at coding sites 111–123 and 1143–1145 of structural domains I and III, respectively, of the nucleocapsid gene may account for the differences between pathogenic and nonpathogenic strains. Preliminary mapping of the fgl2 promoter has defined a region from −372 to −306 upstream from the ATG translation initiation site to be responsive to nucleocapsid protein. Hence, mapping of genetic determinants in parental and recombinant strains demonstrates that the nucleocapsid protein of strains that induce fulminant Hepatitis is responsible for transcription of the fgl2 prothrombinase gene. These studies provide new insights into the role of the nucleocapsid gene in the pathogenesis of viral Hepatitis.
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resistance to Murine Hepatitis Virus strain 3 is dependent on production of nitric oxide
Journal of Virology, 1998Co-Authors: M Pope, L. S. Fung, Julian L Leibowitz, Philip A Marsden, Qin Ning, Edward H. Cole, M. J. Phillips, S Sloan, J W Ding, G A LevyAbstract:The strain-specific spectrum of liver disease following Murine Hepatitis Virus type 3 (MHV-3) infection is dependent on inflammatory mediators released by macrophages. Production of nitric oxide (NO) by macrophages has been implicated in resistance to a number of Viruses, including ectromelia Virus, vaccinia Virus, and herpes simplex Virus type 1. This study was undertaken to define the role of NO in MHV-3 infection. Gamma interferon-induced production of NO inhibited growth of MHV-3 in a Murine macrophage cell line (RAW 264.7). Viral inhibitory activity was reproduced by the NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP), whereas N-acetyl-dl-pencillamine (NAP), an inactive analog of SNAP, had no effect. Electron microscopy studies confirmed the inhibitory effects of NO on viral replication. Peritoneal macrophages isolated from A/J mice known to be resistant to MHV-3 produced a fivefold-higher level of NO and higher levels of mRNA transcripts of inducible NO synthase in response to gamma interferon than macrophages from susceptible BALB/cJ mice. SNAP inhibited growth of MHV-3 in macrophages from both strains of mice to similar degrees. In vivo inhibition of NO by N-monomethyl-l-arginine resulted in loss of resistance to MHV-3 in A/J mice. These results collectively demonstrate a defect in the production of NO in macrophages from susceptible BALB/cJ mice and define the importance of endogenous NO in resistance to MHV-3 infection in resistant A/J mice.
L. S. Fung - One of the best experts on this subject based on the ideXlab platform.
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the fgl2 fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral Hepatitis
Journal of Clinical Investigation, 2003Co-Authors: Philip A Marsden, L. S. Fung, Qin Ning, Yue Chen, Michael Mendicino, Anand Ghanekar, Jeremy A Scott, Teresa Miller, Camie W Y Chan, Mathew W C ChanAbstract:Fibrin deposition and thrombosis within the microvasculature is now appreciated to play a pivotal role in the hepatocellular injury observed in experimental and human viral Hepatitis. Importantly, the pathways by which fibrin generation is elicited in viral Hepatitis may be mechanistically distinct from the classical pathways of coagulation induced by mechanical trauma or bacterial lipopolysaccharide (LPS). In the setting of Murine Hepatitis Virus strain-3 (MHV-3) infection, a member of the Coronaviridae, activated endothelial cells and macrophages express distinct cell-surface procoagulants, including a novel prothrombinase, Fgl2/fibroleukin, which are important for both the initiation and localization of fibrin deposition. To assess the role of Fgl2/fibroleukin in Murine viral Hepatitis we generated a Fgl2/fibroleukin‐deficient mouse. Peritoneal macrophages isolated from Fgl2/fibroleukin‐/‐ mice did not generate a procoagulant response when infected with MHV-3. Fibrin deposition and liver necrosis were markedly reduced, and survival was increased in mice infected with MHV-3. To address the relevance of Fgl2/fibroleukin in human chronic viral Hepatitis we studied patients with minimal and marked chronic Hepatitis B. We detected robust expression of Fgl2/fibroleukin mRNA transcripts and protein in liver tissue isolated from patients with marked chronic Hepatitis B. Fibrin deposition was strongly associated with Fgl2/fibroleukin expression. Collectively, these data indicate a critical role for
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resistance to Murine Hepatitis Virus strain 3 is dependent on production of nitric oxide
Journal of Virology, 1998Co-Authors: M Pope, L. S. Fung, Julian L Leibowitz, Philip A Marsden, Qin Ning, Edward H. Cole, M. J. Phillips, S Sloan, J W Ding, G A LevyAbstract:The strain-specific spectrum of liver disease following Murine Hepatitis Virus type 3 (MHV-3) infection is dependent on inflammatory mediators released by macrophages. Production of nitric oxide (NO) by macrophages has been implicated in resistance to a number of Viruses, including ectromelia Virus, vaccinia Virus, and herpes simplex Virus type 1. This study was undertaken to define the role of NO in MHV-3 infection. Gamma interferon-induced production of NO inhibited growth of MHV-3 in a Murine macrophage cell line (RAW 264.7). Viral inhibitory activity was reproduced by the NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP), whereas N-acetyl-dl-pencillamine (NAP), an inactive analog of SNAP, had no effect. Electron microscopy studies confirmed the inhibitory effects of NO on viral replication. Peritoneal macrophages isolated from A/J mice known to be resistant to MHV-3 produced a fivefold-higher level of NO and higher levels of mRNA transcripts of inducible NO synthase in response to gamma interferon than macrophages from susceptible BALB/cJ mice. SNAP inhibited growth of MHV-3 in macrophages from both strains of mice to similar degrees. In vivo inhibition of NO by N-monomethyl-l-arginine resulted in loss of resistance to MHV-3 in A/J mice. These results collectively demonstrate a defect in the production of NO in macrophages from susceptible BALB/cJ mice and define the importance of endogenous NO in resistance to MHV-3 infection in resistant A/J mice.
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ribavirin inhibits viral induced macrophage production of tnf il 1 the procoagulant fgl2 prothrombinase and preserves th1 cytokine production but inhibits th2 cytokine response
Journal of Immunology, 1998Co-Authors: Qin Ning, L. S. Fung, Jinwen Ding, Edward H. Cole, Mingfeng Liu, Reginald M Gorczynski, Deron Brown, Jean Parodo, Mark S Cattral, Ori D RotsteinAbstract:Ribavirin, a synthetic guanosine analogue, possesses a broad spectrum of activity against DNA and RNA Viruses. It has been previously shown to attenuate the course of fulminant Hepatitis in mice produced by Murine Hepatitis Virus strain 3. We therefore studied the effects of ribavirin on Murine Hepatitis Virus strain 3 replication, macrophage production of proinflammatory mediators including TNF, IL-1, and the procoagulant activity (PCA), fgl2 prothrombinase; and Th1/Th2 cytokine production. Although ribavirin had inhibitory effects on viral replication (<1 log), even at high concentrations complete eradication of the Virus was not seen. In contrast, at physiologic concentrations (up to 500 μg/ml), ribavirin markedly reduced viral-induced parameters of macrophage activation. With ribavirin treatment, the concentrations of PCA, TNF-α and IL-1β all decreased to basal concentrations: PCA from 941 ± 80 to 34 ± 11 mU/106 cells; TNF-α from 10.73 ± 2.15 to 2.74 ± 0.93 ng/ml; and IL-1β from 155.91 ± 22.62 to 5.74 ± 0.70 pg/ml. The inhibitory effects of ribavirin were at the level of gene transcription as evidenced by Northern analysis. Both in vitro and in vivo, ribavirin inhibited the production of IL-4 by Th2 cells, whereas it did not diminish the production of IFN-γ in Th1 cells. In contrast, ribavirin had no inhibitory effect on TNF-α and IL-1β production in LPS-stimulated macrophages. These results suggest that the beneficial effects of ribavirin are mediated by inhibition of induction of macrophage proinflammatory cytokines and Th2 cytokines while preserving Th1 cytokines.
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ribavirin inhibits viral induced macrophage production of tnf il 1 the procoagulant fgl2 prothrombinase and preserves th1 cytokine production but inhibits th2 cytokine response
Journal of Immunology, 1998Co-Authors: Qin Ning, L. S. Fung, Edward H. Cole, Mingfeng Liu, J W Ding, R M Gorczynski, Deron Brown, Jean Parodo, Mark S Cattral, Ori D RotsteinAbstract:Ribavirin, a synthetic guanosine analogue, possesses a broad spectrum of activity against DNA and RNA Viruses. It has been previously shown to attenuate the course of fulminant Hepatitis in mice produced by Murine Hepatitis Virus strain 3. We therefore studied the effects of ribavirin on Murine Hepatitis Virus strain 3 replication, macrophage production of proinflammatory mediators including TNF, IL-1, and the procoagulant activity (PCA), fgl2 prothrombinase; and Th1/Th2 cytokine production. Although ribavirin had inhibitory effects on viral replication (<1 log), even at high concentrations complete eradication of the Virus was not seen. In contrast, at physiologic concentrations (up to 500 microg/ml), ribavirin markedly reduced viral-induced parameters of macrophage activation. With ribavirin treatment, the concentrations of PCA, TNF-alpha and IL-1beta all decreased to basal concentrations: PCA from 941 +/- 80 to 34 +/- 11 mU/10(6) cells; TNF-alpha from 10.73 +/- 2.15 to 2.74 +/- 0.93 ng/ml; and IL-1beta from 155.91 +/- 22.62 to 5.74 +/- 0.70 pg/ml. The inhibitory effects of ribavirin were at the level of gene transcription as evidenced by Northern analysis. Both in vitro and in vivo, ribavirin inhibited the production of IL-4 by Th2 cells, whereas it did not diminish the production of IFN-gamma in Th1 cells. In contrast, ribavirin had no inhibitory effect on TNF-alpha and IL-1beta production in LPS-stimulated macrophages. These results suggest that the beneficial effects of ribavirin are mediated by inhibition of induction of macrophage proinflammatory cytokines and Th2 cytokines while preserving Th1 cytokines.
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expression of the fgl2 and its protein product prothrombinase in tissues during Murine Hepatitis Virus strain 3 mhv 3 infection
Advances in Experimental Medicine and Biology, 1998Co-Authors: J W Ding, L. S. Fung, Qin Ning, Kevork M. Peltekian, C. Holloway, Herman Yeger, M. J. Phillips, Mingfeng Liu, A Lai, G A LevyAbstract:Murine Hepatitis Virus Strain 3 (MHV-3) produces fulminant Hepatitis with 80–90% mortality in Balb/cJ mice. Previous studies in our laboratory have shown that peritoneal macrophages from MHV-3 infected mice produce a procoagulant (PCA) which has the ability to cleave prothrombin to thrombin (prothrombinase) encoded by the gene fgl2 located on chromosome 5. PCA accounts for sinusoidal thrombosis and hepatic necrosis and the necrosis and mortality can be prevented by treatment of animals with a monoclonal antibody to PCA. These present studies were designed to examine the expression of this gene (mRNA by Northern analysis and in situ hybridization) and the gene product PCA (immunochemistry) in tissues recovered from MHV-3 infected Balb/cJ mice in an attempt to explain the liver specific nature of MHV-3 disease. Fgl2 gene expression was detected as early as 8 hours after MHV-3 infection which persisted to 48 hours in the liver, spleen and lungs whereas no gene expression was seen in the brain or kidneys despite the fact that equivalent viral titers were detected in all tissues at all times. In the liver, fgl2 gene expression was confined to endothelial and Kupffer cells with no expression in hepatocytes. Immunochemistry localized the PCA protein to Kupffer cells and endothelial cells and necrotic foci within the liver. No PCA protein was detected by immunochemistry in any other tissues at any time during the course of MHV-3 infection.
