Autophagy in coxsackievirus-infected neurons

Autophagy is a process to engulf aberrant organelles or protein aggregates into double-membrane vesicles for lysosomal breakdown. Autophagy is a protective process against some intracellular bacteria and viruses, however, it is also used for replication by some viruses, such as poliovirus. We recently found that coxsackievirus B4 (CVB4) also induces the autophagy pathway and activates the calpain system for replication in neurons. Notably, the inhibition of autophagy with 3-methyladenine (3MA) reduced calpain activation and virus replication. Calpain inhibitors also reduced autophagosome formation and virus replication. This finding indicates that calpain and the autophagy pathway are closely connected with each other during the infection. Interestingly, we also found that 3MA and calpain inhibitors enhanced the caspase-3 specific cleavage of spectrin during CVB4 infection, suggesting that autophagy inhibition by these drugs triggered apoptosis. Thus, autophagy and apoptosis may balance each other in CVB4-infected neurons. Here, we show that inhibition of caspase with zVAD increased autophagosome formation, further proposing the cross-talk between autophagy and apoptosis in CVB4-infected neurons.     

Calpain regulates CVB3 induced viral myocarditis by promoting autophagic flux upon infection

Calpains are calcium-activated neutral cysteine proteases. The dysregulation of calpain activity has been found to be related to cardiovascular diseases, for which calpain inhibition is used as a treatment. Viral myocarditis (VMC) is primarily caused by Coxsackievirus group B3 virus infection (CVB3). CVB3 virus infection induces autophagy and hijacks this process to facilitate its replication. In this study, we found that calpain was significantly activated in hearts affected by VMC. However, pharmacologically inhibiting calpain aggravated VMC symptoms in mice due to myocardial inflammation and cardiac dysfunction. The inhibition of calpain activity in vitro led to the accumulation of LC3-II and increased levels of p62/SQSTM1 protein expression, suggesting that autophagic flux was impaired by calpain inhibition. These effects of calpain inhibition were also observed in capn4-specific myocardial knockout mice in vivo. Furthermore, our results provided evidence that calpain inhibition in VMC, unlike other cardiovascular diseases, exacerbated the disease symptom by impairing CVB3-induced autophagic flux, which may subsequently reduce virus autolysosome degradation. Our findings indicated that calpain inhibition may not be a good treatment for VMC disease in a clinical setting.     

Coxsackievirus can exploit LC3 in both autophagy-dependent and -independent manners in vivo

RNA viruses modify intracellular membranes to produce replication scaffolds. In pancreatic cells, coxsackievirus B3 (CVB3) hijacks membranes from the autophagy pathway, and in vivo disruption of acinar cell autophagy dramatically delays CVB3 replication. This is reversed by expression of GFP-LC3, indicating that CVB3 may acquire membranes from an alternative, autophagy-independent, source(s). Herein, using 3 recombinant CVB3s (rCVB3s) encoding different proteins (proLC3, proLC3^G120A, or ATG4B^C74A), we show that CVB3 is, indeed, flexible in its utilization of cellular membranes. When compared with a control rCVB3, all 3 viruses replicated to high titers in vivo, and caused severe pancreatitis. Most importantly, each virus appeared to subvert membranes in a unique manner. The proLC3 virus produced a large quantity of LC3-I which binds to phosphatidylethanolamine (PE), affording access to the autophagy pathway. The proLC3^G120A protein cannot attach to PE, and instead binds to the ER-resident protein SEL1L, potentially providing an autophagy-independent source of membranes. Finally, the ATG4B^C74A protein sequestered host cell LC3-I, causing accumulation of immature phagophores, and massive membrane rearrangement. Taken together, our data indicate that some RNA viruses can exploit a variety of different intracellular membranes, potentially maximizing their replication in each of the diverse cell types that they infect in vivo.     

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.     

细胞自噬促进柯萨奇病毒B3复制的研究

本研究探索柯萨奇病毒B3(Coxsackievirus B3,CVB3)感染引起的自噬与病毒复制之间的关系。CVB3感染HeLa细胞,并在病毒感染后6 h、8 h和10 h时检测LC3-Ⅰ蛋白、LC3-Ⅱ蛋白和p62蛋白的表达水平。结果显示CVB3病毒感染促使LC3-Ⅱ/LC3-Ⅰ比值升高,同时降低p62蛋白的表达。分别将自噬诱导剂雷帕霉素(Rapamy-cin)、自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3MA)或溶酶体抑制剂阿洛司他丁(Aloxistatin,E46D)预处理HeLa细胞2 h,CVB3感染药物处理细胞并在病毒感染6 h后收集细胞、检测CVB3病毒VP1蛋白的表达。结果显示雷帕霉素和E64D促使CVB3病毒VP1蛋白表达增加,而3MA降低CVB3病毒VP1蛋白的表达。本研究得出结论 CVB3病毒感染诱导自噬进而促进病毒复制。     

The role of autophagy during coxsackievirus infection of neural progenitor and stem cells

Coxsackievirus B3 (CVB3) has previously been shown to utilize autophagy in an advantageous manner during the course of infection of the host cell. However, few studies have determined whether stem cells induce autophagy in a similar fashion, and whether virus-induced autophagy occurs following infection of stem cells. Therefore, we compared the induction of autophagy following CVB3 infection of neural progenitor and stem cells (NPSCs), which we have recently shown to be highly susceptible to CVB3 infection, to HL-1 cells, a transformed cardiomyocyte cell line. As previously demonstrated for other susceptible host cells, HL-1 cells showed an increase in the activity of autophagic signaling following infection with a CVB3 expressing dsRed protein (dsRed-CVB3). Furthermore, viral titers in HL-1 cells increased in the presence of an inducer of autophagy (CCPA), while viral titers decreased in the presence of an inhibitor of autophagy (3-MA). In contrast, no change in autophagic signaling was seen in NPSCs following infection with dsRed-CVB3. Also, basal levels of autophagy in NPSCs were found to be highly elevated in comparison to HL-1 cells. Autophagy could be induced in NPSCs in the presence of rapamycin without altering levels of dsRed-CVB3 replication. In differentiated NPSC precursors, autophagy was activated during the differentiation process, and a decrease in autophagic signaling was observed within all three CNS lineages following dsRed-CVB3 infection. Hence, we conclude that the role of autophagy in modulating CVB3 replication appears cell type-specific, and stem cells may uniquely regulate autophagy in response to infection.     

The role of autophagy during coxsackievirus infection of neural progenitor and stem cells

Coxsackievirus B3 (CVB3) has previously been shown to utilize autophagy in an advantageous manner during the course of infection of the host cell. However, few studies have determined whether stem cells induce autophagy in a similar fashion, and whether virus-induced autophagy occurs following infection of stem cells. Therefore, we compared the induction of autophagy following CVB3 infection of neural progenitor and stem cells (NPSCs), which we have recently shown to be highly susceptible to CVB3 infection, to HL-1 cells, a transformed cardiomyocyte cell line. As previously demonstrated for other susceptible host cells, HL-1 cells showed an increase in the activity of autophagic signaling following infection with a CVB3 expressing dsRed protein (dsRed-CVB3). Furthermore, viral titers in HL-1 cells increased in the presence of an inducer of autophagy (CCPA), while viral titers decreased in the presence of an inhibitor of autophagy (3-MA). In contrast, no change in autophagic signaling was seen in NPSCs following infection with dsRed-CVB3. Also, basal levels of autophagy in NPSCs were found to be highly elevated in comparison to HL-1 cells. Autophagy could be induced in NPSCs in the presence of rapamycin without altering levels of dsRed-CVB3 replication. In differentiated NPSC precursors, autophagy was activated during the differentiation process, and a decrease in autophagic signaling was observed within all three CNS lineages following dsRed-CVB3 infection. Hence, we conclude that the role of autophagy in modulating CVB3 replication appears cell type-specific, and stem cells may uniquely regulate autophagy in response to infection.     

Pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo

Autophagy protects against many infections by inducing the lysosomal-mediated degradation of invading pathogens. However, previous in?vitro studies suggest that some enteroviruses not only evade these protective effects but also exploit autophagy to facilitate their replication. We generated Atg5(f/f)/Cre(+) mice, in which the essential autophagy gene Atg5 is specifically deleted in pancreatic acinar cells, and show that coxsackievirus B3 (CVB3) requires autophagy for optimal infection and pathogenesis. Compared to Cre(-) littermates, Atg5(f/f)/Cre(+) mice had an ～2,000-fold lower CVB3 titer in the pancreas, and pancreatic pathology was greatly diminished. Both in?vivo and in?vitro, Atg5(f/f)/Cre(+) acinar cells had reduced intracellular viral RNA and?proteins. Furthermore, intracellular structural elements induced upon CVB3 infection, such as compound membrane vesicles and highly geometric paracrystalline arrays, which may represent viral replication platforms, were infrequently observed in?infected Atg5(f/f)/Cre(+) cells. Thus, CVB3-induced subversion of autophagy not only benefits the virus but also exacerbates pancreatic pathology.     

Calpain‐2 plays a pivotal role in the inhibitory effects of propofol against TNF‐α‐induced autophagy in mouse hippocampal neurons

Calpains are calcium‐dependent proteases and play critical roles in neuronal autophagy induced by inflammation. Propofol has been reported to exert anti‐inflammatory effects in neurons. We aimed to identify whether and how propofol‐modulated calpain activity and neuron autophagy in response to tumour necrosis factor‐α (TNF‐α). Mouse hippocampal neurons were pre‐treated with propofol and exposed to TNF‐α. Autophagy was evaluated by fluorescent autophagy assay and by measuring LC3I and LC3II expression. Intracellular calcium concentration was measured by fluorescent assay. Calpain activation was measured by calpain activity assay. The protein expression of intracellular signalling molecules was detected by Western blot analysis. Compared with untreated control neurons, 40 ng/mL TNF‐α treatment for 2 hours induced neuron autophagy, which was attenuated by 25 μmol/L propofol. TNF‐α induced intracellular calcium accumulation, the phosphorylation of calcium/calmodulin‐dependent protein kinase II (CAMK II) and calpain‐2, calpain activation and lysosomal cathepsin B release as well as tyrosine kinase receptor B (TrkB) truncation. These effects were alleviated by propofol, calcium chelator, CAMK II inhibitor, calpain‐2 inhibitor, calpain‐2 siRNA transfection and N‐Methyl‐d ‐aspartic acid (NMDA) receptor antagonist. Propofol, via NMDA receptor, inhibited TNF‐α‐mediated hippocampal neuron autophagy. The mechanism may involve calcium and calcium‐dependent signalling pathway, especially CAMK II and calpain‐2.     

RNase L and double-stranded RNA-dependent protein kinase exert complementary roles in islet cell defense during coxsackievirus infection

Coxsackievirus (CV) is an important human pathogen that has been linked to the development of autoimmunity. An intact pancreatic beta cell IFN response is critical for islet cell survival and protection from type 1 diabetes following CV infection. In this study, we show that IFNs trigger an antiviral state in beta cells by inducing the expression of proteins involved in intracellular antiviral defense. Specifically, we demonstrate that 2',5'-oligoadenylate synthetases (2-5AS), RNase L, and dsRNA-dependent protein kinase (PKR) are expressed by pancreatic islet cells and that IFNs (IFN-alpha and IFN-gamma) increase the expression of 2-5AS and PKR, but not RNase L. Moreover, our in vitro studies uncovered that these pathways play important roles in providing unique and complementary antiviral activities that critically regulate the outcome of CV infection. The 2-5AS/RNase L pathway was critical for IFN-alpha-mediated islet cell resistance from CV serotype B4 (CVB4) infection and replication, whereas an intact PKR pathway was required for efficient IFN-gamma-mediated repression of CVB4 infection and replication. Finally, we show that the 2-5AS/RNase L and the PKR pathways play important roles for host survival during a challenge with CVB4. In conclusion, this study has dissected the pathways used by distinct antiviral signals and linked their expression to defense against CVB4.     

Inhibition of HIV-1 replication with stable RNAi-mediated knockdown of autophagy factors

Autophagy is a cellular process leading to the degradation of cytoplasmic components such as organelles and intracellular pathogens. It has been shown that HIV-1 relies on several components of the autophagy pathway for its replication, but the virus also blocks late steps of autophagy to prevent its degradation. We generated stable knockdown T cell lines for 12 autophagy factors and analyzed the impact on HIV-1 replication. RNAi-mediated knockdown of 5 autophagy factors resulted in inhibition of HIV-1 replication. Autophagy analysis confirmed a specific defect in the autophagy pathway for 4 of these 5 factors. We also scored the impact on cell viability, but no gross effects were observed. Upon simultaneous knockdown of 2 autophagy factors (Atg16 and Atg5), an additive inhibitory effect was scored on HIV-1 replication. Stable knockdown of several autophagy factors inhibit HIV-1 replication without any apparent cytotoxicity. We therefore propose that targeting of the autophagy pathway can be a novel therapeutic approach against HIV-1.     

Interactions between enteroviruses and autophagy in vivo

Autophagy plays a protective role during many viral and bacterial infections. Predictably, evolution has led to several viruses developing mechanisms by which to evade the inhibitory effects of the pathway. However, one family of viruses, the picornaviruses, has gone one step further, by actively exploiting autophagy. Using mice in which Atg5 has been conditionally deleted in pancreatic acinar cells, we have studied the outcome of infection by coxsackievirus B3 (CVB3), a member of the enterovirus genus and picornavirus family. Two key findings emerged: disruption of autophagy (1) dramatically compromised virus replication in vivo, and (2) significantly limited pancreatic disease.     

Interactions between enteroviruses and autophagy in vivo

Autophagy plays a protective role during many viral and bacterial infections. Predictably, evolution has led to several viruses developing mechanisms by which to evade the inhibitory effects of the pathway. However, one family of viruses, the picornaviruses, has gone one step further, by actively exploiting autophagy. Using mice in which Atg5 has been conditionally deleted in pancreatic acinar cells, we have studied the outcome of infection by coxsackievirus B3 (CVB3), a member of the enterovirus genus and picornavirus family. Two key findings emerged: disruption of autophagy (1) dramatically compromised virus replication in vivo, and (2) significantly limited pancreatic disease.     

IL-12 protects against coxsackievirus B3-induced myocarditis by increasing IFN-gamma and macrophage and neutrophil populations in the heart

Th1-type immune responses, mediated by IL-12-induced IFN-gamma, are believed to exacerbate certain autoimmune diseases. We recently found that signaling via IL-12Rbeta1 increases coxsackievirus B3 (CVB3)-induced myocarditis. In this study, we examined the role of IL-12 on the development of CVB3-induced myocarditis using mice deficient in IL-12p35 that lack IL-12p70. We found that IL-12 deficiency did not prevent myocarditis, but viral replication was significantly increased. Although there were no changes in the total percentage of inflammatory cells in IL-12-deficient hearts compared with wild-type BALB/c controls by FACS analysis, macrophage and neutrophil populations were decreased. This decrease corresponded to reduced TNF-alpha and IFN-gamma levels in the heart, suggesting that macrophage and/or neutrophil populations may be a primary source of TNF-alpha and IFN-gamma during acute CVB3 myocarditis. Increased viral replication in IL-12-deficient mice was not mediated by reduced TNFRp55 signaling, because viral replication was unaltered in TNFRp55-deficient mice. However, STAT4 or IFN-gamma deficiency resulted in significantly increased viral replication and significantly reduced TNF-alpha and IFN-gamma levels in the heart, similar to IL-12 deficiency, indicating that the IL-12/STAT4 pathway of IFN-gamma production is important in limiting CVB3 replication. Furthermore, STAT4 or IFN-gamma deficiency also increased chronic CVB3 myocarditis, indicating that therapeutic strategies aimed at reducing Th1-mediated autoimmune diseases may exacerbate common viral infections such as CVB3 and increase chronic inflammatory heart disease.     

Calpain裂解Atg5决定中性粒细胞发生自噬还是凋亡，以及C5a在其中的可能作用

通过阐明C5a、calpain和Atg5相互作用,为开展新的研究寻找方向．中性粒细胞凋亡控制炎症反应及其强度,多种疾病和中性粒细胞凋亡失调有关,但其发生机制尚未阐明．C5a为补体片段,有多种功能,如诱导中性粒细胞趋化、呼吸爆发、增强吞噬、颗粒酶释放和延迟凋亡．已知calpain涉及中性粒细胞功能及凋亡调节并对该凋亡发生具有特异性．不同刺激因素可通过不同路径调节不同calpain亚型的活性． 已有报道C5a可以通过调节calpain亚型活性而调节中性粒细胞的趋化反应．另外,自噬是真核细胞中广泛存在的生物过程,具有细胞保护作用,Atg5对于自噬体形成必不可少．Calpain可裂解Atg5为24 ku tAtg5,使其失去形成自噬体的功能并介导凋亡．Atg5参与了自噬和凋亡的转换．     

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.     

West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

Autophagy is a homeostatic process responsible for recycling cytosolic proteins and organelles. Moreover, this pathway contributes to the cell’s intrinsic innate defenses. While many viruses have evolved mechanisms to antagonize the antiviral effects of the autophagy pathway, others subvert autophagy to facilitate replication. Here, we have investigated the role of autophagy in West Nile virus (WNV) replication. Experiments in cell lines derived from a variety of sources, including the kidney, liver, skin, and brain, indicated that WNV replication does not upregulate the autophagy pathway. Furthermore, WNV infection did not inhibit rapamycin-induced autophagy, suggesting that WNV does not disrupt the authophagy signaling cascade. Perturbation of the autophagy pathway by depletion of the major autophagy factors Atg5 or Atg7 had no effect on WNV infectious particle production, indicating that WNV does not require a functional autophagy pathway for replication. Taken together, the results of our study provide evidence that WNV, unlike several other viruses of the family Flaviviridae, does not significantly interact with the conventional autophagy pathway in mammalian cells.     

Apoptosis-suppressing and autophagy-promoting effects of calpain on oridonin-induced L929 cell death

Calpain, calcium-dependent cysteine protease, is reported here to impose the crucial influence on oridonin-induced L929 cell apoptosis and autophagy. We found that inhibition of calpain increased oridonin-induced Bax activation, cytochrome c release and PARP cleavage, indicating that calpain plays an anti-apoptotic role in oridonin-induced L929 cell apoptosis. To explore this potential anti-apoptotic mechanism, we inhibited calpain and proteasome activity in oridonin-induced L929 cell apoptosis, and discovered that the inducible IκBα proteolysis was partially blocked by the inhibition of either calpain or proteasome, but completely blocked by the inhibition of both. It demonstrated that calpain and proteasome were two distinct pathways participating in IκBα degradation. To further study the role of calpain in oridonin-induced L929 cell autophagy, we discovered that calpain inhibitor decreased oridonin-induced autophagy, as well as Beclin 1 activation and the conversion from LC3-I to LC3-II. Moreover, Inhibition of autophagy by 3-MA increased oridonin-induced apoptosis. In conclusion, besides suppressing apoptosis, calpain promotes autophagy in oridonin-induced L929 cell death, and inhibition of autophagy might contribute to up-regulation of apoptosis.     

Interaction between coxsackievirus B3 infection and α-synuclein in models of Parkinson’s disease

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. PD is pathologically characterized by the death of midbrain dopaminergic neurons and the accumulation of intracellular protein inclusions called Lewy bodies or Lewy neurites. The major component of Lewy bodies is α-synuclein (α-syn). Prion-like propagation of α-syn has emerged as a novel mechanism in the progression of PD. This mechanism has been investigated to reveal factors that initiate Lewy pathology with the aim of preventing further progression of PD. Here, we demonstrate that coxsackievirus B3 (CVB3) infection can induce α-syn-associated inclusion body formation in neurons which might act as a trigger for PD. The inclusion bodies contained clustered organelles, including damaged mitochondria with α-syn fibrils. α-Syn overexpression accelerated inclusion body formation and induced more concentric inclusion bodies. In CVB3-infected mice brains, α-syn aggregates were observed in the cell body of midbrain neurons. Additionally, α-syn overexpression favored CVB3 replication and related cytotoxicity. α-Syn transgenic mice had a low survival rate, enhanced CVB3 replication, and exhibited neuronal cell death, including that of dopaminergic neurons in the substantia nigra. These results may be attributed to distinct autophagy-related pathways engaged by CVB3 and α-syn. This study elucidated the mechanism of Lewy body formation and the pathogenesis of PD associated with CVB3 infection.