Nucleotide sequence of the 5'nontranslated and virion polypeptides regions of coxsackievirus B6.

The nucleotide sequence of coxsackievirus B6 (CVB6) has been determined, and the nucleotides encoding the 5' nontranslated region (5' NTR) and virion polypeptides (VP4, 2, 3 and 1) were compared with other serotype CVBs. An Unweighted Pair-Group Method Analysis (UPGMA) of phylogenetic trees indicated that the 5' NTR of CVB6 locates on an independent branch from the other CVBs. The tree based on the amino acid sequences showed that CVB6 has close correlation with CVB4 in the VP4 and VP2 regions, with CVB1 and CVB5 in the VP3 region, and with CVB5 in the VP1 region. Amino acid sequences of variable regions within the VP2, VP3, and VP1 of CVB6 were unique among CVBs. Thus, by comparison of the nucleotide and amino acid sequences of these variable regions, CVB6 can be easily distinguished from other serotypes. In addition, serine, instead of glycine, was found to locate at the amino-terminus of the VP1 region of CVB6, indicating that CVB6 has a unique cleavage site (i.e., glutamine/serine instead of glutamine/glycine) for proteinase 3C of Picornaviridae.     

Design of LNA-modified siRNAs against the highly structured 5' UTR of coxsackievirus B3

This study describes a strategy to develop LNA-modified small interfering RNA (siRNAs) against the highly structured 5' UTR of coxsackievirus B3 (CVB-3), which is an attractive target site due to its high degree of conservation. Accessible sites were identified based on structural models and RNase H assays with DNA oligonucleotides. Subsequently, LNA gapmers, siRNAs, siLNAs and small internally segmented interfering RNA (sisiLNAs) were designed against sites, which were found to be accessible in the in vitro assays, and tested in reporter assays and experiments with the infectious virus. The best siLNA improved viability of infected cells by 92% and exerted good antiviral activity in plaque reduction assays.     

Purification and partial characterization of coxsackievirus B3 2A protease expressed in Escherichia coli

Reported here is the overexpression, purification and partial characterization of recombinant coxsakievirus B3 2A protease (CVB3 2A(pro)) from bacterial cells transformed with a plasmid containing the CVB3 2A(pro) cDNA sequences. The structural investigation showed that the protein contains mostly beta-strand elements and requires Zn(2+) ions as a structural component which appeared to be inhibitory if added exogenously. The purified enzyme activity was optimal at 4 degrees C and had a short half-life at physiological temperature. This feature can be the result of the presence of a high content of beta-structure and also hydrophobic residues in its structure.     

FTY720 alleviates coxsackievirus B3-induced myocarditis and inhibits viral replication through regulating sphingosine 1-phosphate receptors and AKT/caspase-3 pathways

Fingolimod (FTY720) after phosphorylation, as the ligand of sphingosine 1-phosphate receptors (S1PRs), plays an important role in cell proliferation and differentiation. In this article, FTY720 in the treatment of coxsackievirus B3 (CVB3)-induced viral myocarditis was closely related to apoptosis and AKT/caspase-3 apoptotic pathways. We found that CVB3 inhibited myocardial apoptosis at the early stage with upregulating p-AKT level and downregulating activated caspase-3 level for replication of virus progeny, whereas it promoted apoptosis at a late stage with downregulating p-AKT and upregulating activated caspase-3 for releasing the newly synthesized virus to spread. Interestingly, FTY720 could reverse this trend; it promoted apoptosis at an early stage and inhibited apoptosis at the late stage in vivo and vitro, which proved the antiviral effect. We also found that S1PR1, S1PR4, and S1PR5, rather than S1PR2 and S1PR3, were regulated by FTY720 in this process. The results confirmed that FTY720 alleviates CVB3-induced myocarditis and inhibits viral replication through regulating S1PRs and AKT/caspase-3 pathways with a bidirectional regulation of apoptosis.     

柯萨奇病毒B3型3D蛋白抗体的制备

肠道病毒3D蛋白是其RNA聚合酶。柯萨奇病毒B3型(coxsackievirus B3,CVB3)主要感染心脏,其3D蛋白在心肌表达中的时序和分布尚不清楚。本研究将通过聚合酶链反应(polymerase chain reaction,PCR)获得的CVB 3D片段插入pET28a(＋)的表达框,获得pET28a(＋)-3D重组质粒。异丙基 β-D-硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside,IPTG)诱导pET28a(＋)-3D表达3D-His蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)后,切胶,获得3D-His蛋白。3D-His蛋白加佐剂免疫新西兰大白兔制备3D蛋白多克隆抗体,蛋白免疫印迹法检测抗体效价及特异性。结果显示,本研究获得了高效价且特异性好的抗CVB3 3D蛋白抗体,可用于CVB3 3D蛋白功能的后续研究。     

A small compound targeting the interaction between nonstructural proteins 2B and 3 inhibits dengue virus replication

The non-structural protein NS2B/NS3 serine-protease complex of the dengue virus (DENV) is required for the maturation of the viral polyprotein. Dissociation of the NS2B cofactor from NS3 diminishes the enzymatic activity of the complex. In this study, we identified a small molecule inhibitor that interferes with the interaction between NS2B and NS3 using structure-based screening and a cell-based viral replication assay. A library containing 661,417 small compounds derived from the Molecular Operating Environment lead-like database was docked to the NS2B/NS3 structural model. Thirty-nine compounds with high scores were tested in a secondary screening using a cell-based viral replication assay. SK-12 was found to inhibit replication of all DENV serotypes (EC₅₀ = 0.74–4.92 μM). In silico studies predicted that SK-12 pre-occupies the NS2B-binding site of NS3. Steady-state kinetics using a fluorogenic short peptide substrate demonstrated that SK-12 is a noncompetitive inhibitor against the NS2B/NS3 protease. Inhibition to Japanese encephalitis virus by SK-12 was relatively weak (EC₅₀ = 29.81 μM), and this lower sensitivity was due to difference in amino acid at position 27 of NS3. SK-12 is the promising small-molecule inhibitor that targets the interaction between NS2B and NS3.     

Cleavage and degradation of EDEM1 promotes coxsackievirus B3 replication via ATF6a‐mediated unfolded protein response signalling

Our previous study of coxsackievirus B3 (CVB3)‐induced unfolded protein responses (UPR) found that overexpression of ATF6a enhances CVB3 VP1 capsid protein production and increases viral particle formation. These findings implicate that ATF6a signalling benefits CVB3 replication. However, the mechanism by which ATF6a signalling is transduced to promote virus replication is unclear. In this study, using a Tet‐On inducible ATF6a HeLa cell line, we found that ATF6a signalling downregulated the protein expression of the endoplasmic reticulum (ER) degradation‐enhancing α‐mannosidase‐like protein 1 (EDEM1), resulting in accumulation of CVB3 VP1 protein; in contrast, expression of a dominant negative ATF6a had the opposite effect. Furthermore, we found that EDEM1 was cleaved by both CVB3 protease 3C and virus‐activated caspase and subsequently degraded via the ubiquitin‐proteasome pathway. However, overexpression of EDEM1 caused VP1 degradation, likely via a glycosylation‐independent and ubiquitin‐lysosome pathway. Finally, we demonstrated that CRISPR/Cas9‐mediated knockout of EDEM1 increased VP1 accumulation and thus CVB3 replication. This is the first study to report the ER protein quality control of non‐enveloped RNA virus and reveals a novel mechanism by which CVB3 evades host ER quality control pathways through cleavage and degradation of the UPR target gene EDEM1, to ultimately benefit its own replication.     

Carvedilol has stronger anti-inflammation and anti-virus effects than metoprolol in murine model with coxsackievirus B3-induced viral myocarditis

Aims

This study aims to compare the effects of carvedilol and metoprolol in alleviating viral myocarditis (VMC) induced by coxsackievirus B3 (CVB3) in mice.

Methods

A total of 116 Balb/c mice were included in this study. Ninety-six mice were inoculated intraperitoneally with CVB3 to induce VMC. The CVB3 inoculated mice were evenly divided into myocarditis group (n = 32), carvedilol group (n = 32) and metoprolol group (n = 32). Twenty mice (control group) were inoculated intraperitoneally with normal saline. Hematoxylin and eosin staining and histopathologic scoring were used to investigate the effects of carvedilol and metoprolol on myocardial histopathologic changes on days 3 and 5. In addition, serum cTn-I levels, cytokine levels and virus titers were determined using chemiluminescence immunoassay, enzyme-linked immunosorbent assay and plaque assay, respectively, on days 3 and 5. Finally, the levels of phosphorylated p38MAPK were studied using immunohistochemical staining and Western blotting on day 5.

Results

Carvedilol had a stronger effect than metoprolol in reducing the pathological scores of VMC induced by CVB3. Both carvedilol and metoprolol reduced the levels of cTn-I, but the effect of carvedilol was stronger. Carvedilol and metoprolol decreased the levels of myocardial pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokine, with the effects of carvedilol being stronger than those of metoprolol. Carvedilol had a stronger effect in reducing myocardial virus concentration compared with metoprolol. Carvedilol was stronger than metoprolol in decreasing the levels of myocardial phosphorylated p38MAPK.

Conclusions

In conclusion, carvedilol was more potent than metoprolol in ameliorating myocardial lesions in VMC, probably due to its stronger modulation of the balance between pro- and anti-inflammatory cytokines by inhibiting the activation of p38MAPK pathway through β1- and β2-adrenoreceptors.     

Homology modeling, docking, molecular dynamics simulation, and structural analyses of coxsakievirus B3 2A protease: an enzyme involved in the pathogenesis of inflammatory myocarditis

2A protease of the pathogenic coxsackievirus B3 is key to the pathogenesis of inflammatory myocarditis and, therefore, an attractive drug target. However lack of a crystal structure impedes design of inhibitors. Here we predict 3D structure of CVB3 2A^pro based on sequence comparison and homology modeling with human rhinovirus 2A^pro. The two enzymes are remarkably similar in their core regions. However they have different conformations at the N-terminal. A large number of N-terminal hydrophobic residues reduce the thermal stability of CVB3 2A^pro, as we confirmed by fluorescence, western blot and turbidity measurement. Molecular dynamic simulation revealed that elevated temperature induces protein motion that results in frequent movement of the N-terminal coil. This may therefore induce successive active site changes and thus play an important role in destabilization of CVB3 2A^pro structure.     

Influence of pan-caspase inhibitors on coxsackievirus B3-infected CD19<Superscript>+</Superscript> B lymphocytes

Coxsackievirus B3 (CVB3), together with other enteroviruses of the picornavirus family, is associated with a wide variety of acute and chronic forms of human diseases. Using the murine model of CVB3-caused myocarditis, this pathogen can be detected not only in solid organs but also in different types of immune cells, preferentially in B lymphocytes. Therefore, these cells could represent a non-cardiac virus reservoir and may play an important role with regard to viral dissemination in the infected host. In addition, the infection of specific immune cells might modulate the severity of tissue injury and the pattern of virus-caused pathology in susceptible or resistant individuals. In the present study it could be demonstrated that CVB3 was capable to infect productively a certain percentage of murine CD19⁺ B cells. In vivo studies revealed that CVB3 invaded murine CD19⁺ B cells during an acute infection. Three days p. i. approximately 0.5–1.0% of these cells were productively infected. This proportion could be decreased up to 45%, if 3 days p. i. mice were intravenously treated with the pan-caspase inhibitors Z-VAD-FMK or Q-VD-OPH. These data were compared with results obtained from CVB3-infected human Raji cells.     

Rate-limiting pyrophosphate release by hepatitis C virus polymerase NS5B improves fidelity

The hepatitis C virus RNA-dependent RNA polymerase NS5B is responsible for the replication of the viral genome. Previous studies have uncovered NTP-mediated excision mechanisms that may be responsible for aiding in maintaining fidelity (the frequency of incorrect incorporation events relative to correct), but little is known about the fidelity of NS5B. In this study, we used transient-state kinetics to examine the mechanistic basis for polymerase fidelity. We observe a wide range of efficiency for incorporation of various mismatched base pairs and have uncovered a mechanism in which the rate constant for pyrophosphate release is slowed for certain misincorporation events. This results in an increase in fidelity against these specific misincorporations. Furthermore, we discover that some mismatches are highly unfavorable and cannot be observed under the conditions used here. The calculated fidelity of NS5B ranges between 10⁻⁴–10⁻⁹ for different mismatches.     

miR-155-5p regulates mesenchymal stem cell osteogenesis and proliferation by targeting GSK3B in steroid-associated osteonecrosis

microRNAs (miRNAs) have recently been recognized as playing an important role in bone-associated diseases. This study investigated whether the reduced miR-155-5p in steroid-associated osteonecrosis of the femoral head (ONFH) attenuated osteogenic differentiation and cell proliferation by targeting GSK3B. Bone marrow was collected from the proximal femurs of patients with steroid-associated ONFH (n = 10) and patients with new femoral neck fracture (n = 10) and mesenchymal stem cells (MSCs) were isolated. The expression profile, the biological function of miR-155-5p, and the interaction between miR-155-5p and GSK3B were investigated by cell viability measurement, western blot, real-time polymerase chain reaction, luciferase reporter assay, and Alizarin Red S (ARS) staining of MSCs. The MSCs that were obtained from the femoral neck fracture group and from the steroid-associated ONFH group were transfected with or without miR-155-5p. We found that, in ONFH samples, the level of mature miR-155-5p was significantly lower than that of control samples. By inhibiting GSK3B, miR-155-5p promoted the nuclear translocation of β-catenin, increased the expression of osteogenesis-related genes, and facilitated the proliferation and differentiation of MSCs. Restoring the expression of GSK3B in MSCs partially reversed the effect of miR-155-5p. These findings suggest that reduced miR-155-5p in steroid-associated ONFH attenuates osteogenic differentiation and cell proliferation by increased levels of GSK3B and inhibition of Wnt signaling.     

A quantum mechanics/molecular mechanics study of the reaction mechanism of the hepatitis C virus NS3 protease with the NS5A/5B substrate

Combined quantum mechanics and molecular mechanics (QM/MM) calculations were carried out to characterize the reaction mechanism of the NS3 protease with its preferred substrate (NS5A/5B). The main purpose of this study was to locate the barrier states and intermediates along the distinguished coordinate path (DCP) involved in this process. These structures, and in particular the one corresponding to the first barrier state and intermediate (B1 and I1), could be a starting point for the synthesis of inhibitors of this protease, which could be used to treat hepatitis C. The two first steps of the reaction mechanism were studied, i.e., the acylation step and the breaking of the peptide bond. The first step takes place through a tetracoordinated intermediate, as suggested from previous works on other Serine proteases. The importance of the different amino acid residues was also considered (perturbation study where the MM charges of each residue were set to zero independently). The residues of the oxyanion hole were confirmed as the most important for the electrostatic stabilization of the tetracoordinate intermediate. Moreover, the role of other residues, e.g., Arg-155 and Asp-79, was also explained.     

Antiviral effects of sophoridine against coxsackievirus B3 and its pharmacokinetics in rats

Coxsackievirus B3 (CVB3) is a major pathogen for acute and chronic viral myocarditis. The aim of this study was to investigate the antiviral effects of sophoridine, an alkaloid extracted from Chinese medicinal herb, Sophora flavescens, against CVB3, and the underlying pharmacokinetics. First, we determined the antiviral effects of sophoridine against CVB3 in in vitro (primarily cultured myocardial cells), in vivo (BALB/c mice) and serum pharmacological experiments. Then, we determined the pharmacokinetic behavior in serum samples of SD rats after oral administration by HPLC. Finally, we determined the effects of sophoridine on the production of cytokines in a murine viral myocarditis model by measuring mRNA expression of some important cytokines in hearts of infected BALB/c mice by RT-PCR. We found that sophoridine exhibited obvious antiviral effects both in vitro and in vivo, and serum samples obtained from rats with oral administration of sophoridine reduced the virus titers in infected myocardial cells. The serum concentration profile correlated closely with antiviral activity profile. Moreover, sophoridine significantly enhanced mRNA expression of IL-10 and IFN-gamma, but decreased TNF-alpha mRNA expression. In conclusion, sophoridine possesses antiviral activities against CVB3, by regulating cytokine expression, and it is likely that sophoridine itself, not its metabolites, is mainly responsible for the antiviral activities. Therefore, sophoridine may represent a potential therapeutic agent for viral myocarditis.     

Suppression of miR-221 inhibits glioma cells proliferation and invasion via targeting SEMA3B

Background

Gliomas are the most common primary tumors in the central nervous system. Due to complicated signaling pathways involved in glioma progression, effective targets for treatment and biomarkers for prognosis prediction are still scant.

Results

In this study we revealed that a new microRNA (miR), the miR-221, was highly expressed in the glioma cells, and suppression of miR-221 resulted in decreased cellular proliferation, migration, and invasion in glioma cells. Mechanistic experiments validated that miR-221 participates in regulating glioma cells proliferation and invasion via suppression of a direct target gene, the Semaphorin 3B (SEMA3B). The rescue experiment with miR-221 and SEMA3B both knockdown results in significant reversion of miR-221 induced phenotypes.

Conclusion

Taken together, our findings highlight an unappreciated role for miR-221 and SEMA3B in glioma.     

Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: a 2.2 A resolution structure in a hexagonal crystal form.

The crystal structure of the NS3 protease of the hepatitis C virus (BK strain) has been determined in the space group P6(3)22 to a resolution of 2.2 A. This protease is bound with a 14-mer peptide representing the central region of the NS4A protein. There are two molecules of the NS3(1-180)-NS4A(21'-34') complex per asymmetric unit. Each displays a familiar chymotrypsin-like fold that includes two beta-barrel domains and four short alpha-helices. The catalytic triad (Ser-139, His-57, and Asp-81) is located in the crevice between the beta-barrel domains. The NS4A peptide forms an almost completely enclosed peptide surface association with the protease. In contrast to the reported H strain complex of NS3 protease-NS4A peptide in a trigonal crystal form (Kim JL et al., 1996, Cell 87:343-355), the N-terminus of the NS3 protease is well-ordered in both molecules in the asymmetric unit of our hexagonal crystal form. The folding of the N-terminal region of the NS3 protease is due to the formation of a three-helix bundle as a result of crystal packing. When compared with the unbound structure (Love RA et al., 1996, Cell 87:331-342), the binding of the NS4A peptide leads to the ordering of the N-terminal 28 residues of the NS3 protease into a beta-strand and an alpha-helix and also causes local rearrangements important for a catalytically favorable conformation at the active site. Our analysis provides experimental support for the proposal that binding of an NS4A-mimicking peptide, which increases catalytic rates, is necessary but not sufficient for formation of a well-ordered, compact and, hence, highly active protease molecule.     

BH3 mimetics derived from Bim-BH3 domain core region show PTP1B inhibitory activity

A series of our previously described BH3 peptide mimetics derived from Bim-BH3 domain core region were found to exhibit weak to potent PTP1B binding affinity and inhibitory activities via target-based drug screening. Among these compounds, a 12-aa Bim-BH3 core sequence peptide conjugated to palmitic acid (SM-6) displayed good PTP1B binding affinity (K_D?=?8.38?nmol/L), inhibitory activity (IC₅₀?=?1.20?μmol/L) and selectivity against other PTPs (TCPTP, LAR, SHP-1 and SHP-2). Furthermore, SM-6 promoted HepG2 cell glucose uptake and inhibited the expression of PTP1B, indicating that SM-6 could improve the insulin resistance effect in the insulin-resistant HepG2 cell model. These results may indicate a new direction for the application of BH3 peptide mimetics and promising PTP1B peptide inhibitors could be designed and developed based on SM-6.     

Multi-target siRNA based on DNMT3A/B homologous conserved region influences cell cycle and apoptosis of human prostate cancer cell line TSU-PR1

Abnormal genome hypermethylation participates in the tumorigenesis and development of prostate cancer. Prostate cancer cells highly express DNA methyltransferase 3 (DMNT3) family genes, essential for maintaining genome methylation. In the present study, multi-target siRNA, based on the homologous region of the DNMT3 family, was designed for the in vitro investigation of its effects on the proliferation, migration, and invasion of TSU-PR1 prostate cancer cells. The consequential cell-cycle derangement, through DNMT3A/B or only DNMT3B silencing, was partially efficient, without affecting apoptosis. DNMT3A silencing had absolutely no effect on changing TSU-PR1 cell biological behavior. Hence, DNMT3B alone apparently plays a key role in maintaining the unfavorable behavior of prostate-cancer cells, thereby implying its potential significance as a promising therapeutic target, with DNMT3A simply in the role of helper.