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Two new ursane triterpenoids along with twelve known compounds were isolated from 80% ethanol extract of Agastache rugosa (Fisch. et. Mey.) O. Kuntze by using silica gel column, MCI column, ODS column and HPLC. The structures of the new compounds were identified as 2α,3α-dihydroxy-24-nor-urs-4(23),12(13)-dien-28-oic acid (1) and 2α,3α-dihydroxy-24-nor-urs-4(23),12(13),20(30) -trien-28-oic acid (2) by HR-ESI-MS, NMR and ECD spectral data, named agasursacid A and agasursacid B. In addition, compounds 3, 4, 6, 8 showed anti-coxsackievirus B3 (CVB3) activities with a IC50 as 4.77, 1.59, 11.11 and 25.87 μmol·L-1, resepectively.
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Objective@#This study aimed to identify internal ribosome entry sites (IRESs) in the open reading frame (ORF) of the Coxsackievirus B3 (CVB3) genome.@*Methods@#The sequences of P1, P2, or P3 of the CVB3 genome or the truncated sequences from each antithymocyte globulin (ATG) to the end of the P1, P2, or P3 gene were inserted into the pEGFP-N1 vector. After transfection, possible IRES-dependent green fluorescent protein (GFP)-fused proteins were detected by anti-GFP western blotting. The sequences of possible IRESs were inserted into specific Fluc/Rluc bicistronic vectors, in which the potential IRESs were determined according to the Fluc/Rluc activity ratio. Expression of Fluc and Rluc mRNA of the bicistronic vector was detected by RT-qPCR.@*Results@#After transfection of full length or truncated sequences of the P1, P2, or P3 plasmids, six GFP-fused protein bands in P1, six bands in P2 and nine bands in P3 were detected through western blotting. Two IRESs in VP2 (1461-1646 nt) and VP1 (2784-2983 nt) of P1; one IRES in 2C (4119-4564 nt) of P2; and two IRESs in 3C (5634-5834 nt) and 3D (6870-7087 nt) of P3 were identified according to Fluc/Rluc activity ratio. The cryptic promoter was also excluded by RT-qPCR.@*Conclusion@#Five IRESs are present in the CVB3 coding region.
Subject(s)
Internal Ribosome Entry Sites/genetics , Open Reading Frames , RNA, Messenger/geneticsABSTRACT
Micro RNAs (microRNAs,miRNAs) are non-coding,endogenous,small RNA molecules that regulate gene expression.MiRNAs are involved in the development of heart and blood vessels.The abnormal expression of miRNAs is associated with atherosclerosis,coronary heart disease,heart failure and hypertension.In recent years,a series of advances have been made in the study of coronary heart disease,hypertension,arrhythmia,heart failure,cardiovascular remodeling,heart development,and the functional control of myocardial cells,endothelial cells,smooth muscle cells.Viral myocarditis (VMC)is a common cardiovascular disease.The pathogenesis of VMC is not fully elucidated.VMC is mainly caused by coxsackie virus B3 (CVB3)infection.The research confirms that miRNAs are closely related to CVB3.MiRNAs are involved in the pathogenesis of VMC,which play an important role in the development of VMC.The research of miRNAs in the role in viral myocarditis will contribute to further understanding for the pathogenesis of myocarditis and highlighting new direction for the treatment of VMC.This paper reviews the relationship between miRNAs and CVB3,and the role of miRNAs in the pathogenesis of VMC.
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OBJECTIVE@#Autophagy is a highly conserved intracellular degradation pathway. Many picornaviruses induce autophagy to benefit viral replication, but an understanding of how autophagy occurs remains incomplete. In this study, we explored whether coxsackievirus B3 (CVB3) infection induced autophagy through endoplasmic reticulum (ER) stress.@*METHODS@#In CVB3-infected HeLa cells, the specific molecules of ER stress and autophagy were detected using Western blotting, reverse transcription polymerase chain reaction (RT-PCR), and confocal microscopy. Then PKR-like ER protein kinase (PERK) inhibitor, inositol-requiring protein-1 (IRE1) inhibitor, or activating transcription factor-6 (ATF6) inhibitor worked on CVB3-infected cells, their effect on autophagy was assessed by Western blotting for detecting microtubule-associated protein light chain 3 (LC3).@*RESULTS@#CVB3 infection induced ER stress, and ER stress sensors PERK/eIF2α, IRE1/XBP1, and ATF6 were activated. CVB3 infection increased the accumulation of green fluorescent protein (GFP)-LC3 punctuation and induced the conversion from LC3-I to phosphatidylethanolamine-conjugated LC3-1 (LC3-II). CVB3 infection still decreased the expression of mammalian target of rapamycin (mTOR) and p-mTOR. Inhibition of PERK, IRE1, or ATF6 significantly decreased the ratio of LC3-II to LC3-I in CVB3-infected HeLa cells.@*CONCLUSION@#CVB3 infection induced autophagy through ER stress in HeLa cells, and PERK, IRE1, and ATF6a pathways participated in the regulation of autophagy. Our data suggested that ER stress may inhibit mTOR signaling pathway to induce autophagy during CVB3 infection.
Subject(s)
Humans , Activating Transcription Factor 6 , Metabolism , Autophagy , Coxsackievirus Infections , Metabolism , Endoplasmic Reticulum Stress , Endoribonucleases , Metabolism , Enterovirus B, Human , HeLa Cells , Protein Serine-Threonine Kinases , Metabolism , Signal Transduction , eIF-2 Kinase , MetabolismABSTRACT
12-N-Benzenesulfonyl-11-matrinic acid derivatives are a new class of anti-CVB3 compounds, but the mechanism of action is still unknown. Therein, two kinds of molecule probes were designed and constructed in this study, including matrinic amines that might be applied to the BIAcore fishing technique and biotin-tagged matrinic derivatives, which could be applied in the biotin affinity chromatography. Moreover, their anti-CVB3 activities were evaluated. Among them, 10a displayed a good activity with an IC50 value of 0.8 μmol·L-1. This active molecule probe provides a key chemical tool for exploration of the anti-CVB3 mechanism of this type of compounds.
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To screen interaction proteins of CVB3 VP3 from cDNA library of human heart ,yeast two hybridization was conducted in this study .The bait plasmid pGBKT7-VP3 was constructed ,VP3 fusion protein and its self-activation in AH109 yeast cells was then detected .The positive clones were confirmed by PCR amplification of cDNA inserts ,Alu I digesting ,DNA sequencing ,and Blasting were used to sort positive colonies to eliminate duplicates .Positive clones were confirmed by one-to-one yeast two hybridization ,and them were sequenced and analyzed for homology .Theα-galactosidase assay was performed to detect the interaction strength .Totally ,10 positive proteins interacting with VP3 of CVB3 were obtained by homology analy-sis,namely,EIF4A2,HADHB,GAPDH,ASPG,ACTA1,TNNI3,CKM,LMOD3,ERGIC1,and ALDH2.The strength of interactions between VP3 and 10 candidate proteins were proved byα-galactosidase assay .This study will contribute to explore the CVB3 VP3 function on molecular level and provides some new clues to explain the pathogenic mechanism of myo-carditis and cardiomyopathy .
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Coxsackievirus B type 3 (CVB3) is one of the major causative pathogens associated with viral meningitis and myocarditis, which are widespread in the human population and especially prevalent in neonates and children. These infections can result in dilated cardiomyopathy (DCM) and other severe clinical complications. There are no vaccines or drugs approved for the prevention or therapy of CVB3-induced diseases. During screening for anti-CVB3 candidates in our previous studies, we found that jiadifenoic acids C exhibited strong antiviral activities against CVB3 as well as other strains of Coxsackie B viruses (CVBs). The present studies were carried out to evaluate the antiviral activities of jiadifenoic acids C. Results showed that jiadifenoic acids C could reduce CVB3 RNA and proteins synthesis in a dose-dependent manner. Jiadifenoic acids C also had a similar antiviral effect on the pleconaril-resistant variant of CVB3. We further examined the impact of jiadifenoic acids C on the synthesis of viral structural and non-structural proteins, finding that jiadifenoic acids C could reduce VP1 and 3D protein production. A time-course study with Vero cells showed that jiadifenoic acids C displayed significant antiviral activities at 0-6 h after CVB3 inoculation, indicating that jiadifenoic acids C functioned at an early step of CVB3 replication. However, jiadifenoic acids C had no prophylactic effect against CVB3. Taken together, we show that jiadifenoic acids C exhibit strong antiviral activities against all strains of CVB, including the pleconaril-resistant variant. Our study could provide a significant lead for anti-CVB3 drug development.
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Guanylate binding protein-1(GBP-1) is an interferon-induced protein. To observe its antiviral effect against Hepatitis B virus (HBV) and Coxsackie virus B3 (CVB3), we constructed an eukaryotic expression vector of human GBP-1(hGBP-1). Full-length encoding sequence of hGBP-1 was amplified by long chain RT-PCR and inserted into a pCR2.1 vector, then subcloned into a pCDNA3.1(-) vector. Recombinant hGBP-1 plasmids and pHBV1.3 carrying 1.3-fold genome of HBV were contransfected into HepG2 cells, and inhibition effect of hGBP-1 against HBV replication was observed. Hela cells transfected with recombinant hGBP-1 plasmids were challenged with CVB3, and viral yield in cultures were detected. The results indicated that recombinant eukaryotic expression plasmid of hGBP-1 was constructed successfully and the hGBP-1 gene carried in this plasmid could be efficiently expressed in HepG2 cells and Hela cells. hGBP-1 inhibit CVB3 but not HBV replication in vitro. These results demonstrate that hGBP-1 mediates an antiviral effect against CVB3 but not HBV and perhaps plays an important role in the interferon-mediated antiviral response against CVB3.
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Objective To investigate the effect of the prescription of Yiqiyangyin Huoxuehuayu on the mice model with viral dilated cardiomyopathy by examining the levels of serum ?-IFN.Methods Replicated the animal models of early viral dilated cardiomyopathy by injecting the CVB3m virus to the peritoneal cavity of the Balb/c mice.Two hundred and ten Balb/c mice were divided into 4 groups at random:blank group,model group,high and low dose traditional Chinese medicine group.The blank group has 30 mice.The other three group has 60 mice each.The mice of high dose traditional Chinese medicine group and low dose traditional Chinese medicine group were treated with the prescription of Yiqiyangyin Huoxuehuayu.The change of pathomorphology in the cardiac muscle tissue,the rate of death,the weight change and the cardiac weight index were observed after 4 weeks of the treatment.At the same time,ELISA method was used to exam the levels of serum ?-IFN.Results The prescription of Yiqiyangyin Huoxuehuayu can induce the expression of serum ?-IFN in the mice with viral dilated cardiomyopathy.The mean level of serum ?-IFN of the high dose traditional Chinese medicine group and low dose traditional Chinese medicine group were significantly higher than that of the model group.Conclusions The prescription of Yiqiyangyin Huoxuehuayu can inhibit the viral replication and regulate the immunologic function.
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Objective:To study the expression of coxsackievirus group B3 gene fragment encoding VP1 in procaryon and to explore its application.Methods:Stablie expression of VP1 gene of CVB3 in E.coli was obtained.The expressed protein was purified by NAT chromatography and its immunoactivity was identified by indirect ELISA.Results:The expressed product of VP1,similar to native protein antigen of CVB3,could strong bind with the mouse's antibody serum against CVB3(polyclonal antibody).Irrelevant monoclonal antibody as contrast presents negative activity. Using the expressed VP1 product,we have had a special IgM ELISA for the patient's serum of the clinical acute viral myocarditis.The result was same with the cellular protein antigens of the tissue-cultivated CVB3.Conclusion:The protein antigen CVB3-VP1 which is obtained by the method of gene engineering has character of high product, and its immunoactivity after being purified was basically unchanged. At present, this kind of antigen is difficult to be obtained from the viral cullture medium and a potent hazard for being infected by this virus may take place in such manipulateion. By the method of gene engineering we can obtain antigenic VP1 of CVB3 and use as immuneogen for the detection of serum antibody,by which to provide reliable test reference for the early-stage diagnosis and clinical therapy of the acute myocarditis.
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OBJECTIVE:To investigate the antiviral action of emodin in combination with other Chinese active medicine components on Coxsackie virus B3(CVB3) and explore the possible antiviral mechanism.METHODS:Hep-2 cells infected by CVB3 were cultured with different concentration and proportion of the emodin and other Chinese medicine components for 72 hours and their inhibitory effects on CVB3 replication were evaluated with cell survival rates by MTT assay.RESULTS:In the Hep-2 cell system,all the combinations between emodin and other medicine components could inhibit cytopathic effect(CPE) of CVB3-infected cells,increase cells' survival rates and decrease the cytotoxicity.The drug combination which showed the best effect of killing viruses directly was emodin:curcumine:banlangen(2:1:2),and the one showing the highest efficacy in inhibiting the replication of CVB3 in cells was emodin:curcumine:banlangen(2:0:1).CONCLUSION:The toxicity of emodin was reduced and its anti-CVB3 activity was enhanced by its combined use with other Chinese active medicine components.
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Objective:Infection with CVB3 may cause severe cardiovascular disease. The mechanisms are not clear,cytokines may have immportant functions,but which kind of cells are they come from is not known well, myocardial fibroklasts is the main cells.The cytokines were determined after infection with CVB3.Methods:IL 6, IL 8, IL 1?, IL 1? and TNF ? in the supernate were measured with ELISA. Results:IL 6 and IL 8 increased significantly after CVB3 infection( P