Dysregulation of the ubiquitin-proteasome system by curcumin suppresses coxsackievirus B3 replication

Curcumin (diferuloylmethane), a natural polyphenolic compound extracted from the spice turmeric, has been reported to have anti-inflammatory, antioxidant, and antiproliferative properties by modulating multiple cellular machineries. It inhibits several intracellular signaling pathways, including the mitogen-activated protein kinases (MAPKs), casein kinase II (CKII), and the COP9 signalosome (CSN), in various cell types. It has also been recently demonstrated that exposure to curcumin leads to the dysregulation of the ubiquitin-proteasome system (UPS). Coxsackievirus infection is associated with various diseases, including myocarditis and dilated cardiomyopathy. In searching for new antiviral agents against coxsackievirus, we found that treatment with curcumin significantly reduced viral RNA expression, protein synthesis, and virus titer and protected cells from virus-induced cytopathic effect and apoptosis. We further demonstrated that reduction of viral infection by curcumin was unlikely due to inhibition of CVB3 binding to its receptors or CVB3-induced activation of MAPKs. Moreover, gene silencing of CKII and Jab1, a component of CSN, by small interfering RNAs did not inhibit the replication of coxsackievirus, suggesting that the antiviral action of curcumin is independent of these pathways. Finally, we showed that curcumin treatment reduced both the 20S proteasome proteolytic activities and the cellular deubiquitinating activities, leading to increased accumulation of ubiquitinated proteins and decreased protein levels of free ubiquitin. We have recently demonstrated that the UPS-mediated protein degradation and/or modification plays a critical role in the regulation of coxsackievirus replication. Thus, our results suggest an important antiviral effect of curcumin wherein it potently inhibits coxsackievirus replication through dysregulation of the UPS.     

Overexpression of interferon-gamma-inducible GTPase inhibits coxsackievirus B3-induced apoptosis through the activation of the phosphatidylinositol 3-kinase/Akt pathway and inhibition of viral replication

Our previous studies using differential mRNA display have shown that interferon-gamma-inducible GTPase (IGTP), was up-regulated in coxsackievirus B3 (CVB3)-infected mouse hearts. In order to explore the effect of IGTP expression on CVB3-induced pathogenesis, we have established a doxycycline-inducible Tet-On HeLa cell line overexpressing IGTP and have analyzed activation of several signaling molecules that are involved in cell survival and death pathways. We found that following IGTP overexpression, protein kinase B/Akt was strongly activated through phosphorylation, which leads to phosphorylation of glycogen synthase kinase-3 (GSK-3). Furthermore, in the presence of CVB3 infection, the intensity of the phosphorylation of Akt was further enhanced and associated with a delayed activation of caspase-9 and caspase-3. These data indicate that IGTP expression appears to confer cell survival in CVB3-infected cells, which was confirmed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cell viability assay. However, the ability of IGTP to induce phosphorylation of Akt and to promote cell survival was attenuated by the phosphotidylinositol-3 kinase (PI3-K) inhibitor LY294002. Transient transfection of the cells with a dominant negative Akt construct followed by doxycycline induction and CVB3 infection reversed Akt phosphorylation to basal levels and returned caspase-3 activity to levels similar to those when the PI3-K inhibitor LY294002 was added. Moreover, IGTP expression inhibited viral replication and delayed CVB3-induced cleavage of eukaryotic translation initiation factor 4G, indicating that IGTP-mediated cell survival relies on not only the activation of PI3-K/Akt, inactivation of GSK-3 and suppression of caspase-9 and caspase-3 but also the inhibition of viral replication.     

Amiloride derivatives inhibit coxsackievirus B3 RNA replication

Amiloride derivatives are known blockers of the cellular Na⁺/H⁺ exchanger and the epithelial Na⁺ channel. More recent studies demonstrate that they also inhibit ion channels formed by a number of viral proteins. We previously reported that 5-(N-ethyl-N-isopropyl)amiloride (EIPA) modestly inhibits intracellular replication and, to a larger extent, release of human rhinovirus 2 (HRV2) (E. V. Gazina, D. N. Harrison, M. Jefferies, H. Tan, D. Williams, D. A. Anderson and S. Petrou, Antiviral Res. 67:98-106, 2005). Here, we demonstrate that amiloride and EIPA strongly inhibit coxsackievirus B3 (CVB3) RNA replication and do not inhibit CVB3 release, in contrast to our previous findings on HRV2. Passaging of plasmid-derived CVB3 in the presence of amiloride generated mutant viruses with amino acid substitutions in position 299 or 372 of the CVB3 polymerase. Introduction of either of these mutations into the CVB3 plasmid produced resistance to amiloride and EIPA, suggesting that they act as inhibitors of CVB3 polymerase, a novel mechanism of antiviral activity for these compounds.     

Autophagosome supports coxsackievirus B3 replication in host cells

Recent studies suggest a possible takeover of host antimicrobial autophagy machinery by positive-stranded RNA viruses to facilitate their own replication. In the present study, we investigated the role of autophagy in coxsackievirus replication. Coxsackievirus B3 (CVB3), a picornavirus associated with viral myocarditis, causes pronounced intracellular membrane reorganization after infection. We demonstrate that CVB3 infection induces an increased number of double-membrane vesicles, accompanied by an increase of the LC3-II/LC3-I ratio and an accumulation of punctate GFP-LC3-expressing cells, two hallmarks of cellular autophagosome formation. However, protein expression analysis of p62, a marker for autophagy-mediated protein degradation, showed no apparent changes after CVB3 infection. These results suggest that CVB3 infection triggers autophagosome formation without promoting protein degradation by the lysosome. We further examined the role of the autophagosome in CVB3 replication. We demonstrated that inhibition of autophagosome formation by 3-methyladenine or small interfering RNAs targeting the genes critical for autophagosome formation (ATG7, Beclin-1, and VPS34 genes) significantly reduced viral replication. Conversely, induction of autophagy by rapamycin or nutrient deprivation resulted in increased viral replication. Finally, we examined the role of autophagosome-lysosome fusion in viral replication. We showed that blockage of the fusion by gene silencing of the lysosomal protein LAMP2 significantly promoted viral replication. Taken together, our results suggest that the host's autophagy machinery is activated during CVB3 infection to enhance the efficiency of viral replication.     

Murine natural killer cells limit coxsackievirus B3 replication

Previous indirect evidence suggested that natural killer (NK) cells play a role in coxsackie virus B3 serotype 3, myocarditic variant (CVB3m)-induced myocarditis by limiting virus replication. In this study, we present direct evidence that NK cells can limit CVB3m replication both in vitro and in vivo. Virus titers are lowered in primary murine neonatal skin fibroblast (MNSF) cultures incubated with activated splenic large granular lymphocytes (LGL) taken from mice 3 days postinoculation of CVB3m, a time of maximal NK cell activity. The antiviral effect of this cell population is diminished by complement-mediated lysis with the use of anti-asialo GM1 antiserum but not with anti-Lyt-2 monoclonal antibody. Neither interferon nor anti-CVB3m-neutralizing antibody was detected in these cultures. Although activated LGL initiate lysis within CVB3m-infected MNSF in vitro within 3 hr of addition, they do not lyse uninfected MNSF cultures. CVB3m replication is required for expression of surface changes on MNSF that result in lysis by NK cells because cell cultures treated with compounds that prevent CVB3m replication are not killed by LGL. LGL also do not lyse MNSF cultures inoculated with UV-inactivated virus. Mice inoculated with activated LGL and subsequently challenged with CVB3m had reduced titers of virus in heart tissues in comparison to titers of CVB3m in heart tissues of mice not given LGL. The antiviral activity of the LGL preparation was abolished by prior treatment with anti-asialo GM1 antiserum plus complement but not by prior treatment with anti-Lyt-2 monoclonal antibody and complement. These data suggest that NK cells can specifically limit a nonenveloped virus infection by killing virus-infected cells.     

Tyrosine phosphorylation events during coxsackievirus B3 replication.

In order to study cellular and viral determinants of pathogenicity, interactions between coxsackievirus B3 (CVB3) replication and cellular protein tyrosine phosphorylation were investigated. During CVB3 infection of HeLa cells, distinct proteins become phosphorylated on tyrosine residues, as detected by the use of antiphosphotyrosine Western blotting. Two proteins of 48 and 200 kDa showed enhanced tyrosine phosphorylation 4 to 5 h postinfection (p.i.), although virus-induced inhibition of cellular protein synthesis had already occurred 3 to 4 h p.i. Subcellular fractionation experiments revealed distinct localization of tyrosine-phosphorylated proteins of 48 and 200 kDa in the cytosol and membrane fractions of infected cells, respectively. In addition, in Vero cells infected with CVB3, echovirus (EV)11, or EV12, increased tyrosine phosphorylation of a 200-kDa protein was detected 6 h p.i. Herbimycin A, a specific inhibitor of Src-like protein tyrosine kinases, was shown to inhibit virus-induced tyrosine phosphorylations and to reduce the production of progeny virions. In contrast, in cells treated with the inhibitors staurosporine and calphostin C, the synthesis of progeny virions was not affected. Immunoprecipitation experiments suggested that the tyrosine-phosphorylated 200-kDa protein in CVB3-infected cells is of cellular origin. In summary, these investigations have begun to unravel the effect of CVB3 as well as EV11 and EV12 replication on cellular tyrosine phosphorylation and support the importance of tyrosine phosphorylation events for effective virus replication. Such cellular phosphorylation events triggered in the course of enterovirus infection may enhance virus replication.     

Protein kinase B/Akt regulates coxsackievirus B3 replication through a mechanism which is not caspase dependent

The role of signaling pathways including the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) during viral infection has gained much recent attention. Our laboratory reported on an important regulatory role for extracellular signal-regulated kinases (ERK1/2), subfamily members of the MAPKs, during coxsackievirus B3 (CVB3) infection. However, the role of the PI3K pathway in CVB3 infection has not been well characterized. CVB3 is the most common known viral infectant of heart muscle that directly injures and kills infected cardiac myocytes during the myocarditic process. In the present study, we investigated the role of protein kinase B (PKB) (also known as Akt), a general downstream mediator of survival signals through the PI3K cascade, in regulating CVB3 replication and virus-induced apoptosis in a well-established HeLa cell model. We have demonstrated that CVB3 infection leads to phosphorylation of PKB/Akt on both Ser-473 and Thr-308 residues through a PI3K-dependent mechanism. Transfection of HeLa cells with a dominant negative mutant of Akt1 or pretreatment of wild-type HeLa cells with the specific PI3K inhibitor LY294002 significantly suppresses viral RNA expression, as reflected in diminished viral capsid protein expression and viral release. Dominant negative Akt1 and LY294002 also increase apoptosis in infected cells, which can be reversed by addition of the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk). Interestingly, blocking of apoptosis by zVAD.fmk does not reverse the viral RNA translation blockade, indicating that the inhibitory effect of dominant negative Akt1 on viral protein expression is not caspase dependent. In addition, we showed that the attachment of virus to its receptor-coreceptor complex is not sufficient for PKB/Akt activation and that postentry viral replication is required for Akt phosphorylation. Taken together, these data illustrate a new and imperative role for Akt in CVB3 infection in HeLa cells and show that the PI3K/Akt signaling is beneficial to CVB3 replication.     

毛壳素显著抑制柯萨奇病毒 B3的体外复制

柯萨奇病毒B3（CVB3）是一种常见的人类病原体,与多种疾病有关。本研究旨在探讨球毛壳菌代谢产物毛壳素对CVB3在体外培养细胞系中复制的影响。结果显示,毛壳素显著抑制CVB3在体外培养细胞系中的复制。250 nmol/L毛壳素可使 CVB3感染的 HeLa细胞相对存活率从未加毛壳素时的（21．9±1．8）％提高至（70．1±4．3）％。同时,毛壳素处理的HeLa细胞中病毒产量仅为对照组的（5．3±0．8）％,而病毒RNA水平也仅为对照组的（13．0±8．3）％。毛壳素也可使CVB3感染的Vero细胞相对存活率从（64．6±1．7）％提高至（87．2±4．8）％。毛壳素的这种抑制作用可被抗氧化剂 N-乙酰半胱氨酸部分抑制。研究毛壳素抑制病毒复制的具体作用机制将有助于新型抗病毒药物的研制。     

Antiviral effects of pan-caspase inhibitors on the replication of coxsackievirus B3

The induction of apoptosis during coxsackievirus B3 (CVB3) infection is well documented. In order to study whether the inhibition of apoptosis has an impact on CVB3 replication, the pan-caspase inhibitor Z-VAD-FMK was used. The decreased CVB3 replication is based on reduced accumulation of both viral RNA and viral proteins. These effects are due to an inhibitory influence of Z-VAD-FMK on the proteolytic activity of the CVB3 proteases 2A and 3C, which was demonstrated by using the target protein poly(A)-binding protein (PABP). The antiviral effect of the structurally different pan-caspase inhibitor Q-VD-OPH was independently of the viral protease inhibition and resulted in suppression of virus progeny production and impaired release of newly produced CVB3 from infected cells. A delayed release of cytochrome c into the cytoplasm was detected in Q-VD-OPH-treated CVB3-infected cells pointing to an involvement of caspases in the initial steps of mitochondrial membrane-permeabilization.     

Pyrrolidine dithiocarbamate induces cyclooxygenase-2 expression in NIH 3T3 fibroblast cells

Pyrrolidine dithiocarbamate (PDTC) is a metal chelating compound that can exert either pro-oxidant or antioxidant effects in different situations. Several studies demonstrate that it can inhibit cyclooxygenase-2 (COX-2) expression, which may be due to its antioxidant activity. Here, we found that PDTC rather increased COX-2 expression in NIH 3T3. The increase of COX-2 expression was inhibited by adding bathocuproline disulfonic acid, a non-permeable specific copper chelator, in the incubation medium. This result suggests that PDTC exerts its effect by transporting redox-active copper ions into the cells. In support of this observation, PDTC did not induce COX-2 expression in a serum-free environment. When PDTC was added with copper in the serum-free medium, it acted as the inducer of COX-2 expression. In addition, pretreatment of N-acetyl-L-cystein or dithiothreitol, other antioxidants, inhibited the PDTC-induced COX-2 expression. Our data indicate that PDTC induces COX-2 expression in NIH 3T3 cells, which may be due to its activities as a copper chelator and a pro-oxidant.     

QiHong prevents death in coxsackievirus B3 induced murine myocarditis through inhibition of virus attachment and penetration

Viral myocarditis affects about 5% to 20% of the population. So far, there are not many effective antiviral treatments available. QiHong, the combination of the extracts from Astragali (Huangqi), Rhadiola rosea (Hongjingtian), and Sophora flavescens (Kushen), was developed based on laboratory research. The aim of this study was to investigate the effect and mechanism of QiHong on coxsackievirus B3 (CVB3)-induced myocarditis. The antiviral activity of QiHong in vitro was evaluated on HeLa and Vero cells infected by CVB3. Ribavirin was chosen as positive control. Our results showed that QiHong possessed potent antiviral effects on CVB3 by sodium 3'-[1-(phenylamino-carbonyl)-3, 4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid and plaque-forming assay (50% inhibitory concentrations [IC50] were 7.16 +/- 0.8 microg/ml and 2.63 +/- 0.5 microg/ml, respectively). The 50% cytotoxicity concentration (CC50) was 16-fold higher in QiHong-treated cells than in ribavirin-treated cells. Time course studies demonstrated that the antiviral effect of QiHong was mainly found during 0-4 hrs of infection, and it blocked the attachment and penetration of CVB3 into cells. In vivo 4-week-old male Balb/C mice were used and inoculated intraperitoneally with CVB3 suspension or normal saline. At 48 hrs after inoculation, the infected mice were gavaged with QiHong or ribavirin. On Day 6, myocardial virus titers were significantly lower in the QiHong-treated group than in the viral-infected groups. On Day 14, QiHong significantly ameliorated CVB3-induced myocardium necrosis; on Day 28, QiHong treatment increased survival rate 4-fold compared with CVB3-infected controls (64% vs. 16%; P < 0.05). The results showed that QiHong is a very promising potent antiviral agent with a highly significant favorable effect on survival and pathologic changes in CVB3-induced myocarditis with less toxicity than ribavirin. The antiviral activity of QiHong is at least partially due to an inhibitory effect on virus attachment and penetration.     

Gigaxonin interacts with tubulin folding cofactor B and controls its degradation through the ubiquitin-proteasome pathway

Gigaxonin is mutated in human giant axonal neuropathy (GAN), an autosomal recessive neurodegenerative disorder. The presence of generalized cytoskeletal abnormalities , including few microtubules and accumulated intermediate filaments (IFs), in GAN suggests an essential role of gigaxonin in cytoskeletal organization and dynamics. However, the molecular mechanisms underlying the cytoskeletal pathology remain to be elucidated. Over the years, the ubiquitin-proteasome system (UPS) of intracellular protein degradation has been implicated in the control of many fundamental cellular processes. Defects in this system seem to be directly linked to the development of human diseases, including cancers and neurodegenerative diseases . Here, we show that gigaxonin controls protein degradation of tubulin folding cofactor B (TBCB) , a function disrupted by GAN-associated mutations. The substantial TBCB protein accumulation caused by impaired UPS may be a causative factor of cytoskeletal pathology in GAN. Our study provides important insight into pathogenesis of neurodegenerative diseases associated with cytoskeletal abnormalities.     

Glucose deprivation is associated with Chk1 degradation through the ubiquitin-proteasome pathway and effective checkpoint response to replication blocks

Chk1 plays a key role in the DNA replication checkpoint and in preserving genomic integrity. Previous studies have shown that reduced Chk1 function leads to defects in the checkpoint response and is closely associated with tumorigenesis. Here, we report that glucose deprivation caused the degradation of Chk1 protein without perturbing cell cycle progression. The induction of Chk1 degradation in response to glucose deprivation was observed in various cancer cell lines and in normal human fibroblasts. Therefore, it appears to be a universal phenomenon in mammalian cells. A specific proteasome inhibitor blocked glucose deprivation-induced Chk1 degradation. Ubiquitination of Chk1 was detected, indicating that the proteasome-ubiquitin pathway mediates Chk1 degradation upon glucose deprivation. Mechanistic studies have demonstrated that ATR-dependent phosphorylation of Chk1 at the Ser317 and Ser345 sites is not required, suggesting that the molecular mechanism for Chk1 degradation upon glucose deprivation is distinct from genotoxic stress-induced degradation. Under conditions of glucose deprivation, the cells manifested a defective checkpoint response to replication stress, camptothecin or hydroxyurea. The forced expression of Myc-Chk1 partially rescued the defective response to the replication block upon glucose deprivation. Taken together, our results indicate that glucose deprivation induces ubiquitin-mediated Chk1 degradation and defective checkpoint responses, implying its potential role in genomic instability and tumor development.     

Coxsackievirus B3 replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway

Coxsackievirus B3 (CVB3) is the most common human pathogen for viral myocarditis. We have previously shown that the signaling protein p21(ras) GTPase-activating protein (RasGAP) is cleaved and that mitogen-activated protein kinases (MAPKs) ERK1/2 are activated in the late phase of CVB3 infection. However, the role of intracellular signaling pathways in CVB3-mediated myocarditis and the relative advantages of such pathways to host or virus remain largely unclear. In this study we extended our prior studies by examining the interaction between CVB3 replication and intracellular signaling pathways in HeLa cells. We observed that CVB3 infection induced a biphasic activation of ERK1/2, early transient activation versus late sustained activation, which were regulated by different mechanisms. Infection by UV-irradiated, inactivated virus capable of receptor binding and endocytosis triggered early ERK1/2 activation, but was insufficient to trigger late ERK1/2 activation. By using a general caspase inhibitor (zVAD.fmk) we further demonstrated that late ERK1/2 activation was not a result of CVB3-mediated caspase cleavage. Treatment of cells with U0126, a selective inhibitor of MAPK kinase (MEK), significantly inhibited CVB3 progeny release and decreased virus protein production. Furthermore, inhibition of ERK1/2 activation circumvented CVB3-induced apoptosis and viral protease-mediated RasGAP cleavage. Taken together, these data suggest that ERK1/2 activation is important for CVB3 replication and contributes to virus-mediated changes in host cells. Our findings demonstrate coxsackievirus takeover of a particular host signaling mechanism and uncover a prospective approach to stymie virus spread and preserve myocardial integrity.     

Cholesterol shuttling is important for RNA replication of coxsackievirus B3 and encephalomyocarditis virus

Picornaviruses are a family of positive‐strand RNA viruses that includes important human and animal pathogens. Upon infection, picornaviruses induce an extensive remodelling of host cell membranes into replication organelles (ROs), which is critical for replication. Membrane lipids and lipid remodelling processes are at the base of RO formation, yet their involvement remains largely obscure. Recently, phosphatidylinositol‐4‐phosphate was the first lipid discovered to be important for the replication of a number of picornaviruses. Here, we investigate the role of the lipid cholesterol in picornavirus replication. We show that two picornaviruses from distinct genera that rely on different host factors for replication, namely the enterovirus coxsackievirus B3 (CVB3) and the cardiovirus encephalomyocarditis virus (EMCV), both recruited cholesterol to their ROs. Although CVB3 and EMCV both required cholesterol for efficient genome replication, the viruses appeared to rely on different cellular cholesterol pools. Treatments that altered the distribution of endosomal cholesterol inhibited replication of both CVB3 and EMCV, showing the importance of endosomal cholesterol shuttling for the replication of these viruses. Summarizing, we here demonstrate the importance of cholesterol homeostasis for efficient replication of CVB3 and EMCV.     

Group B coxsackievirus diabetogenic phenotype correlates with replication efficiency

Group B coxsackieviruses can initiate rapid onset type 1 diabetes (T1D) in old nonobese diabetic (NOD) mice. Inoculating high doses of poorly pathogenic CVB3/GA per mouse initiated rapid onset T1D. Viral protein was detectable in islets shortly after inoculation in association with beta cells as well as other primary islet cell types. The virulent strain CVB3/28 replicated to higher titers more rapidly than CVB3/GA in the pancreas and in established beta cell cultures. Exchange of 5'-nontranslated regions between the two CVB3 strains demonstrated a variable impact on replication in beta cell cultures and suppression of in vivo replication for both strains. While any CVB strain may be able to induce T1D in prediabetic NOD mice, T1D onset is linked both to the viral replication rate and infectious dose.