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1.
Int J Mol Sci ; 21(9)2020 Apr 25.
Article in English | MEDLINE | ID: mdl-32344926

ABSTRACT

AIMS: Coxsackievirus B3 (CVB3) is known to be an important cause of myocarditis and dilated cardiomyopathy. Enterovirus-2C (E2C) is a viral RNA helicase. It inhibits host protein synthesis. Based on these facts, we hypothesize that the inhibition of 2C may suppress virus replication and prevent enterovirus-mediated cardiomyopathy. METHODS AND RESULTS: We generated a chemically modified enterovirus-2C inhibitor (E2CI). From the in vitro assay, E2CI was showed strong antiviral effects. For in vivo testing, mice were treated with E2CI intraperitoneally injected daily for three consecutive days at a dose of 8mg/kg per day, after CVB3 post-infection (p.i) (CVB3 + E2CI, n = 33). For the infected controls (CVB3 only, n = 35), mice were injected with PBS (phosphate buffered saline) in a DBA/2 strain to establish chronic myocarditis. The four-week survival rate of E2CI-treated mice was significantly higher than that of controls (92% vs. 71%; p < 0.05). Virus titers and myocardial damage were significantly reduced in the E2CI treated group. In addition, echocardiography indicated that E2CI administration dramatically maintained mouse heart function compared to control at day 28 p.i chronic stage (LVIDD, 3.1 ± 0.08 vs. 3.9 ± 0.09, p < 0.01; LVDS, 2.0 ± 0.07 vs. 2.5 ± 0.07, p < 0.001; FS, 34.8 ± 1.6% vs. 28.5 ± 1.5%; EF, 67. 9 ± 2.9% vs. 54.7 ± 4.7%, p < 0.05; CVB3 + E2CI, n = 6 vs. CVB3, n = 4). Moreover, E2CI is effectively worked in human iPS (induced pluripotent stem cell) derived cardiomyocytes. CONCLUSION: Enterovirus-2C inhibitor (E2CI) was significantly reduced viral replication, chronic myocardium damage, and CVB3-induced mortality in DBA/2 mice. These results suggested that E2CI is a novel therapeutic agent for the treatment of enterovirus-mediated diseases.


Subject(s)
Antiviral Agents/pharmacology , Coxsackievirus Infections/drug therapy , Enterovirus B, Human/enzymology , Induced Pluripotent Stem Cells/drug effects , Myocarditis/prevention & control , Myocytes, Cardiac/drug effects , RNA Helicases/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/therapeutic use , Cardiomyopathy, Dilated/etiology , Cardiomyopathy, Dilated/prevention & control , Chronic Disease , Coxsackievirus Infections/complications , Enterovirus B, Human/drug effects , Enterovirus B, Human/physiology , HeLa Cells , Humans , Induced Pluripotent Stem Cells/virology , Luciferases, Renilla/analysis , Male , Mice , Mice, Inbred DBA , Myocarditis/etiology , Myocarditis/virology , Myocytes, Cardiac/pathology , Myocytes, Cardiac/virology , Oxadiazoles/pharmacology , Oxadiazoles/therapeutic use , Oxazoles/pharmacology , Oxazoles/therapeutic use , Recombinant Fusion Proteins/metabolism , Ventricular Dysfunction, Left/etiology , Ventricular Dysfunction, Left/prevention & control , Virus Replication/drug effects
2.
PLoS One ; 12(3): e0172059, 2017.
Article in English | MEDLINE | ID: mdl-28257427

ABSTRACT

Seasonal influenza is caused by two influenza A subtype (H1N1 and H3N2) and two influenza B lineage (Victoria and Yamagata) viruses. Of these antigenically distinct viruses, the H3N2 virus was consistently detected in substantial proportions in Korea during the 2010/11-2013/14 seasons when compared to the other viruses and appeared responsible for the influenza-like illness rate peak during the first half of the 2011/12 season. To further scrutinize possible causes for this, we investigated the evolutionary and serological relationships between the vaccine and Korean H3N2 strains during the 2011/12 season for the main antigenic determinants of influenza viruses, the hemagglutinin (HA) and neuraminidase (NA) genes. In the 2011/12 season, when the number of H3N2 cases peaked, the majority of the Korean strains did not belong to the HA clade of A/Perth/16/2009 vaccine, and no Korean strains were of this lineage in the NA segment. In a serological assay, post-vaccinated human sera exhibited much reduced hemagglutination inhibition antibody titers against the non-vaccine clade Korean H3N2 strains. Moreover, Korean strains harbored several amino acid differences in the HA antigenic sites and in the NA with respect to vaccine lineages during this season. Of these, the HA antigenic site C residues 45 and 261 and the NA residue 81 appeared to be the signatures of positive selection. In subsequent seasons, when H3N2 cases were lower, the HA and NA genes of vaccine and Korean strains were more phylogenetically related to each other. Combined, our results provide indirect support for using phylogenetic clustering patterns of the HA and possibly also the NA genes in the selection of vaccine viruses and the assessment of vaccine effectiveness.


Subject(s)
Evolution, Molecular , Hemagglutinins/genetics , Influenza, Human/genetics , Neuraminidase/genetics , Antigens, Viral/genetics , Hemagglutination Inhibition Tests , Humans , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/pathogenicity , Influenza, Human/virology , Phylogeny , RNA, Viral/genetics , Republic of Korea , Seasons
3.
Sci Rep ; 7: 40675, 2017 01 13.
Article in English | MEDLINE | ID: mdl-28084423

ABSTRACT

Human infection with an avian influenza virus persists. To prepare for a potential outbreak of avian influenza, we constructed a candidate vaccine virus (CVV) containing hemagglutinin (HA) and neuraminidase (NA) genes of a H5N1 virus and evaluated its antigenic stability after serial passaging in embryonated chicken eggs. The passaged CVV harbored the four amino acid mutations (R136K in PB2; E31K in PA; A172T in HA; and R80Q in M2) without changing its antigenicity, compared with the parental CVV. Notably, the passaged CVV exhibited much greater replication property both in eggs and in Madin-Darby canine kidney and Vero cells. Of the four mutations, the PA E31K showed the greatest effect on the replication property of reverse genetically-rescued viruses. In a further luciferase reporter, mini-replicon assay, the PA mutation appeared to affect the replication property by increasing viral polymerase activity. When applied to different avian influenza CVVs (H7N9 and H9N2 subtypes), the PA E31K mutation resulted in the increases of viral replication in the Vero cell again. Taken all together, our results suggest the PA E31K mutation as a single, substantial growth determinant of avian influenza CVVs and for the establishment of a high-yield avian influenza vaccine backbone.


Subject(s)
Influenza A virus/genetics , Influenza A virus/immunology , Influenza Vaccines/genetics , Influenza Vaccines/immunology , Mutation , RNA-Dependent RNA Polymerase/genetics , Viral Proteins/genetics , Amino Acid Substitution , Animals , Chick Embryo , Chlorocebus aethiops , Enzyme Activation , Female , Genetic Engineering , Genetic Vectors/genetics , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/immunology , Mice , Models, Molecular , Protein Conformation , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , Structure-Activity Relationship , Vaccinia virus/genetics , Vero Cells , Viral Proteins/chemistry , Viral Proteins/metabolism , Virus Replication
4.
Biochem Biophys Res Commun ; 479(2): 192-197, 2016 10 14.
Article in English | MEDLINE | ID: mdl-27613087

ABSTRACT

Avian influenza H7N9 virus has posed a concern of potential human-to-human transmission by resulting in seasonal virus-like human infection cases. To address the issue of sustained human infection with the H7N9 virus, here we investigated the effects of hemagglutinin (HA) and neuraminidase (NA) N-linked glycosylation (NLG) patterns on influenza virus transmission in a guinea pig model. Based on the NLG signatures identified in the HA and NA genetic sequences of H7N9 viruses, we generated NLG mutant viruses using either HA or NA gene of a H7N9 virus, A/Anhui/01/2013, by reverse genetics on the 2009 pandemic H1N1 virus backbone. For the H7 HA NLG mutant viruses, NLG pattern changes appeared to reduce viral transmissibility in guinea pigs. Intriguingly, however, the NLG changes in the N9 NA protein, such as a removal from residue 42 or 66 or an addition at residue 266, increased transmissibility of the mutant viruses by more than 33%, 50%, and 16%, respectively, compared with a parental N9 virus. Given the effects of HA-NA NLG changes with regard to viral transmission, we then generated the HA-NA NLG mutant viruses harboring the H7 HA of double NLG addition and the N9 NA of various NLG patterns. As seen in the HA NLG mutants above, the double NLG-added H7 HA decreased viral transmissibility. However, when the NA NLG changes occurred by a removal of residue 66 and an addition at 266 were additionally accompanied, the HA-NA NLG mutant virus recovered the transmissibility of its parental virus. These demonstrate the effects of specific HA-NA NLG changes on the H7N9 virus transmission by highlighting the importance of a HA-NA functional balance.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza A Virus, H7N9 Subtype/metabolism , Influenza in Birds/transmission , Neuraminidase/metabolism , Orthomyxoviridae Infections/transmission , Animals , Birds , Female , Glycosylation , Guinea Pigs , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Host-Pathogen Interactions , Humans , Influenza A Virus, H7N9 Subtype/genetics , Influenza A Virus, H7N9 Subtype/physiology , Influenza in Birds/virology , Influenza, Human/transmission , Influenza, Human/virology , Mutation , Neuraminidase/genetics , Orthomyxoviridae Infections/virology , Time Factors
5.
Virus Res ; 199: 1-8, 2015 Mar 02.
Article in English | MEDLINE | ID: mdl-25485472

ABSTRACT

BACKGROUND: Coxsackievirus B3 (CVB3) is a common cause of myocarditis and dilated cardiomyopathy. CVB3 3C protease (3CP) cleaves the viral polyprotein during replication. We tested whether a water soluble 3CP inhibitor (3CPI) had antiviral effects in a chronic myocarditis model. METHODS: Chronic myocarditis was established using DBA/2 strain mice. Starting on post-infection (p.i) day 3, CVB3-infected mice (n=41) were treated with 3CPI by daily intraperitoneal (i.p.) injection at a concentration of 50 µM (1.7 mg/kg/day) per day for 3 consecutive days. Additional mice (n=49) were injected with PBS as a control. RESULTS: The 5-week survival rate was significantly higher with 3CPI treatment (82.3% versus 47.9%; P<0.05). Organ virus titers at day 3 and 7 and myocardial damage were significantly lower in 3CPI-treated mice. Echocardiography at day 31 indicated strong protection of heart function by 3CPI (FS, 51.2±1.5 versus 26.1±1.5%; P<0.001). Hemodynamic measurements indicated that 3CPI treatment markedly reduced CVB3-induced LV dysfunction on day 31 (dP/dTmax, 5302±352 versus 4103±408 mmHg/s, P<0.05; dP/dTmin, -3798±212 versus -2814±206 mmHg/s, P<0.01). CONCLUSIONS: Water soluble 3CPI was delivered through i.p. injection after CVB3 infection. This agent preserved heart function and decreased organ viral titers and myocardial damage. Soluble 3CPI may be beneficial in the treatment of cardiomyopathy associated with enterovirus infection.


Subject(s)
Antiviral Agents/administration & dosage , Cardiomyopathies/prevention & control , Coxsackievirus Infections/prevention & control , Enterovirus B, Human/enzymology , Protease Inhibitors/administration & dosage , Viral Proteins/antagonists & inhibitors , 3C Viral Proteases , Animals , Cysteine Endopeptidases , Disease Models, Animal , Enterovirus B, Human/drug effects , Heart/virology , Heart Function Tests , Male , Mice, Inbred DBA , Survival Analysis , Treatment Outcome , Viral Load
6.
Microbiol Immunol ; 57(4): 281-7, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23586632

ABSTRACT

The coxsackieviruses type B3 (CVB3) are members of the genus Enterovirus of the family Picornaviridae. They are the commonest cause of chronic myocarditis and dilated cardiomyopathy. However, there is still no effective method for diagnosing CVB3 infection in humans. Here, a fast and accurate system that uses a capsid-protein-specific peptide sequence to detect CVB3 in the sera of patients with viral myocarditis was established. The peptide sequence was selected from the whole CVB3 capsid protein sequence by computationally predicting fragments with high antigenicity and low hydrophobicity. Two of eight possible peptide sequences were selected and commercially synthesized. The synthesized peptides encoded either the VP2 or VP1 capsid protein and induced immunoglobulin G antibody expression in immunized rabbits. Anti-VP2 and anti-VP1 sera detected the viral proteins extracted from CVB3-infected HeLa cells. The newly synthesized peptides successfully induced antibody production. These peptides, applied in an ELISA system, detected anti-CVB3 antibodies in virus-infected mouse serum. Moreover, an ELISA system based on the VP2 peptide detected CVB3 infection in patients with positively identified CVB3-induced fulminant myocarditis. These results indicate that these new peptides specifically interact with anti-CVB3 IgG antibodies in mouse and human sera. This ELISA system should be useful for the clinical diagnosis of enterovirus-induced myocarditis.


Subject(s)
Coxsackievirus Infections/diagnosis , Enterovirus B, Human/immunology , Myocarditis/diagnosis , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , Capsid Proteins/chemistry , Capsid Proteins/immunology , Coxsackievirus Infections/immunology , Coxsackievirus Infections/virology , Enzyme-Linked Immunosorbent Assay/methods , HeLa Cells , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Mice , Myocarditis/immunology , Myocarditis/virology , Peptides/chemical synthesis , Peptides/immunology , Rabbits
7.
Bioorg Med Chem Lett ; 22(22): 6952-6, 2012 Nov 15.
Article in English | MEDLINE | ID: mdl-23062551

ABSTRACT

Peptidomimetic anti-viral agents against Coxsackievirus B3 (CVB3) were developed using a strategy involving the inhibition of 3C protease (CVB3 3C(pro)), a target for CVB3-mediated myocarditis or pericarditis. In an attempt to improve the inhibitory activity against CVB3, a variety of hetero-aromatic groups were incorporated into the α,ß-unsaturated ester as Michael acceptor moiety, which is the position of interaction with the cysteine moiety in the P1' active site of CVB3 3C(pro). Among these hetero-aromatic groups, the quinoline analogs 9c and 9e, with IC(50) values of 250 and 130 nM as determined from an enzyme assay, significantly inhibited the CVB3-mediated cell cytotoxicity, indicating parallel anti-viral activities. A comparison of the binding modes of the potent inhibitor 9e and the relatively weak inhibitor 9n was explored in a molecular docking study, which revealed that compound 9n lacked hydrogen bonds in its interactions with Gly129, 128, and 145.


Subject(s)
Antiviral Agents/chemistry , Enterovirus/enzymology , Oligopeptides/chemistry , Protease Inhibitors/chemistry , Quinolines/chemistry , Viral Proteins/antagonists & inhibitors , 3C Viral Proteases , Antiviral Agents/chemical synthesis , Antiviral Agents/toxicity , Binding Sites , Catalytic Domain , Cell Survival/drug effects , Cysteine Endopeptidases/metabolism , HeLa Cells , Human Umbilical Vein Endothelial Cells , Humans , Hydrogen Bonding , Molecular Docking Simulation , Oligopeptides/chemical synthesis , Oligopeptides/toxicity , Protease Inhibitors/chemical synthesis , Protease Inhibitors/toxicity , Quinolines/chemical synthesis , Quinolines/toxicity , Viral Proteins/metabolism
8.
Biosci Biotechnol Biochem ; 76(6): 1173-6, 2012.
Article in English | MEDLINE | ID: mdl-22790942

ABSTRACT

We generated a cardiotropic replication-competent chimeric coxsackievirus B3 (CVB3) to express alcohol dehydrogenase (ADH). Although exogenously expressed ADH was found by Western blot analysis, its enzyme function was repressed. To define the factor that inhibits the enzymatic function of ADH, we introduced a site-directed mutation at the second amino acid (MGAQEF···) of the CVB3 VP0 capsid protein, effectively changing glycine to alanine. This glycine is known to be a myristoylation site during viral capsid protein maturation in infected cells. In contrast to the unmodified virus, ADH expression and enzymatic function were readily detectable in the mutated rCVB3-ADH (G2A) virus. While expression of ADH required mutation of the CVB3 VP0 myristoylation site for proper function, another chimeric virus that expresses green fluorescent protein (rCVB3-GFP (G or A)) worked independently of the myristoylation site. Indeed, infected HeLa cells displayed GFP under a fluorescent microscope. These results indicate that the myristoylation site in the VP0 capsid protein inhibited the expression of enzymatically active ADH but not GFP. VP0 myristoylation is dispensable for chimeric CVB3 virus replication.


Subject(s)
Alcohol Dehydrogenase/genetics , Capsid Proteins/genetics , Drosophila Proteins/genetics , Enterovirus B, Human/genetics , Alanine/chemistry , Alanine/metabolism , Alcohol Dehydrogenase/chemistry , Alcohol Dehydrogenase/metabolism , Amino Acid Substitution , Animals , COS Cells , Capsid Proteins/metabolism , Chlorocebus aethiops , Drosophila , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Gene Expression , Genetic Vectors , Glycine/chemistry , Glycine/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Microscopy, Fluorescence , Mutagenesis, Site-Directed , Myristic Acid/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Virus Replication
9.
Intervirology ; 55(3): 201-9, 2012.
Article in English | MEDLINE | ID: mdl-21821992

ABSTRACT

BACKGROUND: Replication-competent coxsackievirus B3 (CVB3) has been used as a gene transfer vector for cultured cardiomyocytes and hearts in vivo. However, CVB3 induces cell lysis when it replicates in infected cells. In this study, we investigated whether a replication-defective rCVB3 vector could be generated and used as a noncytotoxic gene transfer vector for cardiomyocytes. METHODS: We generated a replication-defective luciferase-expressing CVB3 plasmid. This recombinant cDNA and pCMV-P1 plasmids were amplified and cotransfected into Hek293 cells using transfection reagents. Replication-defective rLuCVB3 virus was recovered from the cells and cell culture supernatants for 3 days after transfection. The generated rLuCVB3 viruses were concentrated on a 30% sucrose cushion and semiquantified using a luciferase assay. In addition, foreign gene delivery by the rLuCVB3 was tested in cultured cardiomyocytes and intact mouse hearts after rLuCVB3 infection. RESULTS: Luciferase was expressed in Hek293, HeLa cells and cardiomyocytes after rLuCVB3 infection. In addition, these cells did not show a significant cytopathic effect after 72 h. Luciferase protein expression or activity were detected for 3 days in the myocardium of rLuCVB3-infected mouse hearts without producing cytotoxicity or inflammation. CONCLUSION: As a proof-of-concept, these data indicate that a replication-defective rCVB3 vector can be generated and used as a novel gene transfer system to transfect exogenous genes into cardiomyocytes without generating cytotoxicity.


Subject(s)
Enterovirus B, Human/genetics , Gene Transfer Techniques , Genetic Vectors , Myocytes, Cardiac/virology , Transduction, Genetic , Animals , Cells, Cultured , Enterovirus B, Human/pathogenicity , Genes, Reporter , Humans , Luciferases/analysis , Luciferases/genetics , Mice
10.
J Infect Dis ; 205(3): 491-7, 2012 Feb 01.
Article in English | MEDLINE | ID: mdl-22207647

ABSTRACT

BACKGROUND: We investigated the efficacy of a 3C protease inhibitor (3CPI) in a murine coxsackievirus B3 (CVB3) myocarditis model. CVB3 is a primary cause of viral myocarditis. The CVB3 genome encodes a single polyprotein that undergoes a series of proteolytic events to produce several viral proteins. Most of this proteolysis is catalyzed by the 3C protease (3CP). METHODS AND RESULTS: By way of a micro-osmotic pump, each mouse received 50 mM 3CPI in 100 µL of 100% dimethyl sulfoxide (DMSO) during a 72-hour period. On the day of pump implantation, mice (n = 40) were infected intraperitoneally with 10(6) plaque-forming units of CVB3. For the infected controls (n = 50), the pump was filled with 100% DMSO without 3CPI. The 3-week survival rate of 3CPI-treated mice was significantly higher than that of controls (90% vs 22%; P < .01). Myocardial inflammation, viral titers, and viral RNA levels were also reduced significantly in the 3CPI-treated group compared with these measures in the controls. CONCLUSIONS: The protein-based drug 3CPI inhibited the activity of 3CP of CVB3, significantly inhibited viral proliferation, and attenuated myocardial inflammations, subsequent fibrosis, and CVB3-induced mortality in vivo. Thus, this CVB3 3CPI has the potential to be a novel therapeutic agent for the treatment of acute viral myocarditis during the viremic phase.


Subject(s)
Antiviral Agents/administration & dosage , Coxsackievirus Infections/drug therapy , Enterovirus B, Human/drug effects , Enzyme Inhibitors/administration & dosage , Myocarditis/drug therapy , Viral Proteins/antagonists & inhibitors , 3C Viral Proteases , Animals , Coxsackievirus Infections/mortality , Coxsackievirus Infections/virology , Cysteine Endopeptidases , Disease Models, Animal , Heart/virology , Histocytochemistry , Male , Mice , Mice, Inbred BALB C , Microscopy , Myocarditis/mortality , Myocarditis/virology , Myocardium/pathology , Pancreas/virology , Survival Analysis , Viral Load
11.
Heart ; 97(13): 1067-73, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21558477

ABSTRACT

BACKGROUND: In an animal model of viral myocarditis, plasma levels of thioredoxin and adiponectin have been reported to be associated with the severity of inflammation and recovery of ventricular dysfunction, respectively. However, there have been few reports about the clinical significance of these cytokine levels in human myocarditis. OBJECTIVES: To examine the hypothesis that cytokine levels correlate with clinical courses of patients with acute fulminant myocarditis (FM). METHODS: A total of 33 consecutive patients with biopsy-proven acute myocarditis were evaluated. Twenty patients were ascribed to an FM group and the other 13 patients were grouped as a non-fulminant group (NFM). Plasma cytokine levels at the time of admission and after 2 weeks were evaluated and correlated with the duration of mechanical circulatory support application. RESULTS: Plasma thioredoxin level at admission was raised in the FM group (3.08±2.15 ng/ml) compared with the NFM group (1.63±0.45 ng/ml, p=0.011) and reduced after an initial unstable period. However there was no significant difference in plasma adiponectin level between the two groups. In a multivariable regression model, increased plasma thioredoxin level (OR=5.79, 95% CI 1.67 to 20.1, p=0.006) and reduced plasma adiponectin level (OR=0.16, 95% CI 0.055 to 0.49, p=0.001) were associated with longer duration of mechanical circulatory support application in the patients with FM, which in turn was significantly related to death or cardiac transplantation. CONCLUSION: In patients with acute myocarditis, the plasma thioredoxin level was increased in the more severe form, and a reduced level of adiponectin was closely correlated with worse short-term outcome in patients with FM.


Subject(s)
Adiponectin/blood , Myocarditis/diagnosis , Thioredoxins/blood , Acute Disease , Adolescent , Adult , Aged , Biomarkers/blood , Cytokines/blood , Epidemiologic Methods , Female , Humans , Male , Middle Aged , Myocarditis/blood , Myocarditis/virology , Prognosis , Virus Diseases/blood , Virus Diseases/diagnosis , Young Adult
12.
J Microbiol Biotechnol ; 19(3): 307-13, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19349757

ABSTRACT

In order to systemically investigate the possibility of using coxsackievirus B3 (CVB3) to deliver foreign genes in vivo, a recombinant strain of CVB3 encoding the renilla gene (CVB3- renilla) was constructed. The recombinant CVB3 resulted in extensive and transient expression of the renilla protein within mouse organs, especially the pancreas. The level of expression was generally dependent upon the viral titer present. Moreover, the CVB3-renilla strain was completely attenuated. Interestingly, the recombinant CVB3 vector was expressed much more strongly in mouse organs than was a comparable adenoviral vector. The CVB3-renilla strain did not express the renilla gene in mice with pre-existing coxsackievirus-specific neutralizing antibodies, but direct organ-specific administration of the virus during openperitoneum surgery was able to circumvent this immunity. This coxsackievirus vector may represent a useful means for delivering and expressing foreign genes in mouse models in an acute and extensive fashion.


Subject(s)
Enterovirus B, Human/physiology , Gene Transfer Techniques , Adenoviridae/genetics , Animals , Antibodies, Viral/metabolism , Genes, Reporter , Genetic Vectors/administration & dosage , Genetic Vectors/biosynthesis , HeLa Cells , Humans , Injections, Intraperitoneal , Luciferases, Renilla/administration & dosage , Luciferases, Renilla/biosynthesis , Luminescent Agents/administration & dosage , Luminescent Agents/metabolism , Mice , Pancreas/drug effects , Virus Replication
13.
Circ Res ; 102(6): 711-9, 2008 Mar 28.
Article in English | MEDLINE | ID: mdl-18258855

ABSTRACT

PICOT (protein kinase C-interacting cousin of thioredoxin) was previously shown to inhibit pressure overload-induced cardiac hypertrophy, concomitant with an increase in ventricular function and cardiomyocyte contractility. The combined analyses of glutathione S-transferase pull-down experiments and mass spectrometry enabled us to determine that PICOT directly interacts with muscle LIM protein (MLP) via its carboxyl-terminal half (PICOT-C). It was also shown that PICOT colocalizes with MLP in the Z-disc. MLP is known to play a role in anchoring calcineurin to the Z-disc in the sarcomere, which is critical for calcineurin-NFAT (nuclear factor of activated T cells) signaling. We, therefore, suggested that PICOT may affect calcineurin-NFAT signaling through its interaction with MLP. Consistent with this hypothesis, PICOT, or more specifically PICOT-C, abrogated phenylephrine-induced increases in calcineurin phosphatase activity, NFAT dephosphorylation/nuclear translocation, and NFAT-dependent transcriptional activation in neonatal cardiomyocytes. In addition, pressure overload-induced upregulation of NFAT target genes was significantly diminished in the hearts of PICOT-overexpressing transgenic mice. PICOT interfered with MLP-calcineurin interactions in a dose-dependent manner. Moreover, calcineurin was displaced from the Z-disc, concomitant with an abrogated interaction between calcineurin and MLP, in the hearts of PICOT transgenic mice. Replenishment of MLP restored the hypertrophic responses and the increase in calcineurin phosphatase activity that was inhibited by PICOT in phenylephrine-treated cardiomyocytes. Finally, PICOT-C inhibited cardiac hypertrophy to an extent that was comparable to that of full-length PICOT. Taken together, these data suggest that PICOT inhibits cardiac hypertrophy largely by negatively regulating calcineurin-NFAT signaling via disruption of the MLP-calcineurin interaction.


Subject(s)
Calcineurin/metabolism , Cardiomegaly/prevention & control , Myocytes, Cardiac/metabolism , NFATC Transcription Factors/metabolism , Signal Transduction , Thioredoxins/metabolism , Active Transport, Cell Nucleus , Animals , Animals, Newborn , Binding, Competitive , Cardiomegaly/metabolism , Cardiomegaly/pathology , Cardiotonic Agents/pharmacology , Cell Size , Cells, Cultured , LIM Domain Proteins , Mechanotransduction, Cellular , Mice , Mice, Transgenic , Muscle Proteins/genetics , Muscle Proteins/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/enzymology , Myocytes, Cardiac/pathology , NIH 3T3 Cells , Nerve Tissue Proteins/metabolism , Phenylephrine/pharmacology , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins/metabolism , Signal Transduction/drug effects , Thioredoxins/chemistry , Thioredoxins/genetics , Transduction, Genetic
14.
Virus Genes ; 36(1): 141-6, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18172750

ABSTRACT

Coxsackieviruses are important human pathogens that induce myocarditis and pancreatitis. However, there are no vaccines or therapeutic reagents for their clinical treatment. Although RNA interference (RNAi)-based approaches to the prevention of viral production have been developed recently, limitations to the in vivo delivery systems and variations in the viral target sequences still hamper the strategy. In this study, to overcome these limitations, we have constructed recombinant lentivirus-delivered short hairpin RNAs (shRNAs) against sequences in highly conserved cis-acting replication element (CRE) within the 2C protein of coxsackievirus B3 (CVB3), designated MET-2C. A recombinant lentivirus, designated Met-2C lenti, was constructed that contains the MET-2C sequence, which acts as a shRNA. Met-2C lenti clearly reduced viral production in CVB3-infected cells in vitro. Moreover, the mice injected intraperitoneally with Met-2C lenti had significant reductions in viral titers, viral myocarditis, and proinflammatory cytokines after challenge with CVB3, compared with those in GFP lenti infected control mice. Moreover, Met-2C lenti improved survival rate compared with that of the GFP lenti infected control group. Therefore, Met-2C lenti is potentially a clinical therapeutic agent for the treatment of viral myocarditis.


Subject(s)
Carrier Proteins/genetics , Coxsackievirus Infections/therapy , Enterovirus B, Human/genetics , Myocarditis/therapy , RNA Interference , Viral Nonstructural Proteins/genetics , Animals , Carrier Proteins/metabolism , Cell Line , Coxsackievirus Infections/immunology , Coxsackievirus Infections/prevention & control , Coxsackievirus Infections/virology , Disease Models, Animal , Genetic Vectors , Humans , Interleukin-6/metabolism , Lentivirus/genetics , Male , Mice , Mice, Inbred BALB C , Myocarditis/immunology , Myocarditis/prevention & control , Myocarditis/virology , RNA, Small Interfering/genetics , Survival Rate , Tumor Necrosis Factor-alpha/metabolism , Viral Nonstructural Proteins/metabolism
15.
Biochem Biophys Res Commun ; 358(1): 7-11, 2007 Jun 22.
Article in English | MEDLINE | ID: mdl-17485072

ABSTRACT

Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was modified by substitution of the ethyl group at the P2' position with various hydrophobic aromatic rings that are predicted to interact preferentially with the Tyr residue in the S2' pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro.


Subject(s)
Antiviral Agents/chemical synthesis , Enterovirus/drug effects , Isoxazoles/chemical synthesis , Models, Molecular , Pyrrolidinones/chemical synthesis , Viral Proteins/antagonists & inhibitors , 3C Viral Proteases , Amino Acid Substitution , Amino Acids/metabolism , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites , Cell Survival/drug effects , Cysteine Endopeptidases , Enterovirus/enzymology , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions , Isoxazoles/chemistry , Isoxazoles/pharmacology , Phenylalanine/analogs & derivatives , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , Rhinovirus/enzymology , Valine/analogs & derivatives , Virus Replication/drug effects
16.
Virus Res ; 126(1-2): 62-8, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17350707

ABSTRACT

Cardiac fibrosis and inflammation are major pathologic conditions that result from viral myocarditis. Connective tissue growth factor (CTGF) stimulates fibroblast proliferation and induces production of extracellular matrix molecules. We studied the correlation between CTGF and cardiac fibrosis in an acute Coxsackievirus B3 (CVB3) myocarditis animal model. Eight-week-old BALB/c mice were infected intraperitoneally with 10(4) plaque forming units (PFU) of CVB3. Myocardial inflammation peaked on day 7 and decreased markedly by day 14 post-infection (pi); cardiac fibrosis was noted from day 7 and peaked on day 14. By contrast, CTGF was weakly expressed by the interstitial cells in uninfected control hearts and also in the hearts of day 3 pi. CTGF expression measured by real-time PCR was elevated on day 3 and peaked on day 7 pi. TGF-beta expression peaked at day 7 pi. The cell type of CTGF expression changed from interstitial cells to myocytes after virus infection. On day 7, CTGF was strongly expressed by myocytes and inflammatory cells surrounding calcified necrotic areas. In addition, cardiac myocytes expressed CTGF on day 14. Our results, based on an acute CVB3 model of myocarditis, provide evidence that CTGF may mediate the development of fibrosis after viral myocarditis, and that the cells expressed CTGF changes during the course of viral myocarditis.


Subject(s)
Coxsackievirus Infections/genetics , Enterovirus B, Human/pathogenicity , Immediate-Early Proteins/genetics , Intercellular Signaling Peptides and Proteins/genetics , Myocarditis/genetics , Acute Disease , Animals , Base Sequence , Collagen Type I/genetics , Connective Tissue Growth Factor , Coxsackievirus Infections/metabolism , Coxsackievirus Infections/pathology , DNA Primers/genetics , Female , Fibrosis , Gene Expression , Immediate-Early Proteins/metabolism , Immunohistochemistry , In Situ Hybridization , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred BALB C , Myocarditis/metabolism , Myocarditis/pathology , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tissue Distribution , Transforming Growth Factor beta/genetics
17.
Cardiovasc Res ; 71(3): 517-26, 2006 Aug 01.
Article in English | MEDLINE | ID: mdl-16806133

ABSTRACT

OBJECTIVE: The coxsackie and adenovirus receptor (CAR) and the decay-accelerating factor (DAF) are receptors for coxsackievirus B3 (CVB3), which is known as the major cause of human viral myocarditis. We investigated the potential for therapeutic use of soluble virus receptor fusion proteins. METHODS: We designed and generated a novel virus receptor trap (hCAR-hDAF:Fc) consisting of both CVB3 receptors and the Fc portion of human IgG1 and evaluated its antiviral effects in experimental CVB3 myocarditis. RESULTS: Among four soluble virus receptor fusion proteins (hCAR:Fc, hDAF:Fc, hCAR-hDAF:Fc and hDAF-hCAR:Fc), hCAR:Fc and hCAR-hDAF:Fc in the supernatant of transfected cells neutralized echovirus, adenovirus, and various serotypes of CVB in a dose-dependent manner. Both soluble viral receptor proteins bound to the VP0 and VP1 capsid proteins of CVB3. The in vivo efficacy of viral receptor proteins was evaluated by intramuscular injection of plasmid (hCAR:Fc or hCAR-hDAF:Fc) followed by electroporation in a murine model of CVB3 myocarditis. Serum levels of the virus receptor proteins increased relative to baseline values from day 3 and peaked on day 14 at 12.9-fold for hCAR:Fc and 7.1-fold for hCAR-hDAF:Fc. The 3-week survival rate was significantly higher in hCAR-hDAF:Fc-treated mice (61%) than in hCAR:Fc-treated mice (29%) and in controls (15%; p<0.05). Myocardial inflammation, fibrosis, and myocardial virus titers were all significantly reduced in the hCAR:Fc and hCAR-hDAF:Fc groups compared to the controls. CONCLUSION: Our soluble virus receptor trap, hCAR-hDAF:Fc, attenuated viral infection, myocardial inflammation, and fibrosis, resulting in higher survival rates in mice with coxsackieviral myocarditis. Furthermore, it consists exclusively of human components, and we demonstrated that this soluble virus receptor trap may be used as a potential candidate for a novel therapeutic agent for the treatment of acute viral myocarditis during the viremic phase.


Subject(s)
Coxsackievirus Infections/therapy , Genetic Therapy/methods , Myocarditis/therapy , Receptors, Virus/metabolism , Acute Disease , Animals , Capsid Proteins/metabolism , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Coxsackievirus Infections/metabolism , Coxsackievirus Infections/pathology , Disease Models, Animal , Electroporation/methods , HeLa Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Myocarditis/metabolism , Myocarditis/pathology , Myocarditis/virology , Receptors, Virus/genetics , Recombinant Fusion Proteins/therapeutic use , Survival Analysis
18.
Biochem Biophys Res Commun ; 344(3): 765-71, 2006 Jun 09.
Article in English | MEDLINE | ID: mdl-16631119

ABSTRACT

Tumor necrosis factor-alpha (TNF-alpha) is one of the major cytokines that modulate the immune response in viral myocarditis, but its role has not yet been thoroughly evaluated. We antagonized TNF-alpha using the expressed soluble p75 TNF receptor linked to the Fc portion of the human IgG1 gene (sTNFR:Fc) by in vivo electroporation, and evaluated its effects on experimental coxsackieviral B3 (CVB3) myocarditis. A plasmid DNA encoding sTNFR:Fc (15microg/mouse) was injected into the gastrocnemius muscles of Balb/C male mice followed by electroporation (day -1). Control mice were injected with an empty vector. One day after electroporation, mice were infected with CVB3 (day 0). Serum levels of sTNFR:Fc increased from day 2 and peaked at day 5 following electroporation. The heart virus titers of sTNFR:Fc mice were higher than those of controls at day 3. However, subsequent to day 12, the survival rates of the sTNFR:Fc mice were significantly higher than those of the controls (36% versus 0% at day 27, P<0.01). Histopathological examination indicated that inflammation and myocardial fibrosis were significantly decreased in sTNFR:Fc mice at day 12. The expressed sTNFR:Fc could modulate the inflammatory process during the post-viremic phase of viral myocarditis.


Subject(s)
Coxsackievirus Infections/pathology , Coxsackievirus Infections/therapy , Immunoglobulin G/administration & dosage , Myocarditis/pathology , Myocarditis/therapy , Receptors, Tumor Necrosis Factor/administration & dosage , Receptors, Tumor Necrosis Factor/genetics , Animals , Coxsackievirus Infections/metabolism , Electroporation/methods , Genetic Therapy/methods , Immunoglobulin G/genetics , Male , Mice , Mice, Inbred BALB C , Myocarditis/metabolism , Myocarditis/virology , Recombinant Fusion Proteins/administration & dosage , Survival Analysis , Survival Rate , Transfection/methods , Treatment Outcome
19.
Virus Res ; 113(2): 153-7, 2005 Nov.
Article in English | MEDLINE | ID: mdl-15993506

ABSTRACT

MAP kinase signaling has been implicated in coxsackievirus B3 (CVB3) pathogenesis and as necessary in the virus lifecycle. We studied the correlation with extracellular signal-regulated kinase 1/2 (ERK1/2) signaling and virus replication in the presence of coxsackievirus and adenovirus receptor (CAR). In CHO cells that do not expressed CAR, specific ERK1/2 phosphorylation (pERK1/2) was not detected, and progeny virus was not produced after infection. By contrast, in HeLa and CHO-CAR cells, which expressed CAR, the specific early and late pERK1/2 at 0.5 and 8 h were induced, and progeny viruses were produced progressively through 24 h after infection. However, when CHO-CAR cells were infected with replication-defective CVB3, specific pERK1/2 was not detected. In addition, when late pERK1/2 is inhibited by the MEK1 inhibitor PD98059, at 4 h after infection, virus replication significantly decreased. Therefore, our findings suggest that early pERK1/2 is a response to virus binding to CAR, whereas late pERK1/2 is related to the viral replication.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Enterovirus B, Human/physiology , Mitogen-Activated Protein Kinase 3/metabolism , Animals , CHO Cells , Calcium-Calmodulin-Dependent Protein Kinases/pharmacology , Cricetinae , Enterovirus B, Human/genetics , Flavonoids/pharmacology , Gene Deletion , Gene Expression Regulation, Viral , HeLa Cells , Humans , Signal Transduction , Virus Replication
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