Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines

Different rabies virus (RABV) strains have their own biological characteristics, but little is known about their respective impact on autophagy. Therefore, we evaluated whether attenuated RABV HEP-Flury and wild-type RABV GD-SH-01 strains triggered autophagy. We found that GD-SH-01 infection significantly increased the number of autophagy-like vesicles, the accumulation of enhanced green fluorescent protein (EGFP)-LC3 fluorescence puncta and the conversion of LC3-I to LC3-II, while HEP-Flury was not able to induce this phenomenon. When evaluating autophagic flux, we found that GD-SH-01 infection triggers a complete autophagic response in the human neuroblastoma cell line (SK), while autophagosome fusion with lysosomes was inhibited in a mouse neuroblastoma cell line (NA). In these cells, GD-SH-01 led to apoptosis and mitochondrial dysfunction while triggering autophagy, and apoptosis could be decreased by enhancing autophagy. To further identify the virus constituent causing autophagy, 5 chimeric recombinant viruses carrying single genes of HEP-Flury instead of those of GD-SH-01 were rescued. While the HEP-Flury virus carrying the wild-type matrix protein (M) gene of RABV triggered LC3-I to LC3-II conversion in SK and NA cells, replacement of genes of nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) produced only minor autophagy. But no one single structural protein of GD-SH-01 induced autophagy. Moreover, the AMPK signaling pathway was activated by GD-SH-01 in SK. Therefore, our data provide strong evidence that autophagy is induced by GD-SH-01 and can decrease apoptosis in vitro. Furthermore, the M gene of GD-SH-01 may cooperatively induce autophagy.     

Autophagy is activated for cell survival after endoplasmic reticulum stress

Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 “dots”), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.     

Free fatty acids stimulate autophagy in pancreatic β-cells via JNK pathway

Recent studies have suggested that free fatty acids stimulate autophagy of pancreatic beta cells. The aim of this study was to verify the free fatty acids (FFA)-induced autophagy and investigate its molecular mechanism. As reported previously, palmitate strongly enhanced the conversion of light chain (LC)3-I to LC3-II, a marker of activation of autophagy in INS-1 beta cells. Palmitate-induced conversion of LC3-I to LC3-II was also observed in neuron-, muscle-, and liver-derived cells. In addition, palmitate induced the formation of typical autophagosomes and autolysosomes and enhanced the degradation rate of long-lived proteins. These results confirmed that palmitate activates autophagic flux in most of the cells. While FFAs reportedly activate several signal transduction pathways in beta cells, palmitate-induced autophagy was blocked by a JNK inhibitor. Although enhanced oxidative stress and endoplasmic reticulum (ER) stress are suspected to mediate FFA-induced activation of JNK1, the induction of autophagy was not associated with changes in molecular markers related to oxidative and endoplasmic reticulum stresses. On the other hand, phosphorylation of double stranded RNA-dependent protein kinase (PKR) paralleled JNK1 activation. Considered together, our study suggested that FFA stimulated functional autophagy possibly through the PKR-JNK1 pathway independent of ER or oxidative stress.     

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.     

Autophagy Benefits the Replication of Newcastle Disease Virus in Chicken Cells and Tissues

Newcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chicken-derived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double- or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.     

Efficiency of Nitrous Acid as An Inactivating and Mutagenic Agent of Intact Tobacco Mosaic Virus and Its Isolated Nucleic Acid

When tobacco mosaic virus (TMV) and its isolated nucleic acid (TMV-RNA) were treated with nitrous acid, the nucleic acid was inactivated six times faster than the intact virus. Inactivation of both the infectious entities was exponential with treatment time to 0.1% level of survival. Eight different mutant phenotypes were scored after inactivation of TMV and TMV-RNA to 50, 10, 1.0, and 0.1% survival levels. Significantly more mutants in relation to unaltered isolates were induced at all levels of survival upon nitrous acid treatment of TMV than of TMV-RNA. Furthermore, the proportion of two specific mutant phenotypes was significantly greater in treated TMV than in treated TMV-RNA. No qualitative differences, however, were observed between the mutational spectra of nitrous acid-treated TMV and TMV-RNA. These results indicate that, in the intact virus, the viral capsid protects some of the sites involved in lethality; thus, proportionately more mutants are induced on nitrous acid treatment of TMV versus TMV-RNA.     

Are mitochondrial reactive oxygen species required for autophagy?

Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H₂O₂ was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ρ° HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.     

Evidence for Multiplicity Reactivation of Ultraviolet Light Irradiated Ribonucleic Acid Isolated from Tobacco Mosaic Virus

Multiplicity reactivation (MR) seems to take place in leaves of Nicotiana glutinosa inoculated with ultraviolet (UV) light irradiated RNA from tobacco mosaic virus (TMV-RNA). A similar phenomenon was not observed with UV-irradiated TMV particles. Considering MR as resulting from genetic recombination between viral genomes, a recombination mechanism, which has been difficult to prove with plant viruses, is proposed as being operative during multiplication of TMV. From the pattern of MR of TMV-RNA, the location of the gene for the RNA replicase within a TMV-RNA strand is discussed.     

VEGF-A promotes angiogenesis after acute myocardial infarction through increasing ROS production and enhancing ER stress-mediated autophagy

Proangiogenesis is generally regarded as an effective approach for treating ischemic heart disease. Vascular endothelial growth factor (VEGF)-A is a strong and essential proangiogenic factor. Reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy are implicated in the process of angiogenesis. This study is designed to clarify the regulatory mechanisms underlying VEGF-A, ROS, ER stress, autophagy, and angiogenesis in acute myocardial infarction (AMI). A mouse model of AMI was successfully established by occluding the left anterior descending coronary artery. Compared with the sham-operated mice, the microvessel density, VEGF-A content, ROS production, expression of vascular endothelial cadherin, positive expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/Bip), and LC3 puncta in CD31-positive endothelial cells of the ischemic myocardium were overtly elevated. Moreover, VEGF-A exposure predominantly increased the expression of beclin-1, autophagy-related gene (ATG) 4, ATG5, inositol-requiring enzyme-1 (IRE-1), GRP78/Bip, and LC3-II/LC3-I as well as ROS production in the human umbilical vein endothelial cells (HUVECs) in a dose and time-dependent manner. Both beclin-1 small interfering RNA and 3-methyladenine treatment predominantly mitigated VEGF-A-induced tube formation and migration of HUVECs, but they failed to elicit any notable effect on VEGF-A-increased expression of GRP78/Bip. Tauroursodeoxycholic acid not only obviously abolished VEGF-A-induced increase of IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I, but also negated VEGF-A-induced tube formation and migration of HUVECs. Furthermore, N-acetyl- l -cysteine markedly abrogated VEGF-A-increased ROS production, IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I in the HUVECs. Taken together, our data demonstrated that increased spontaneous production of VEGF-A may induce angiogenesis after AMI through initiating ROS–ER stress-autophagy axis in the vascular endothelial cells.     

Initiation of apoptosis and autophagy by photodynamic therapy

This study was designed to examine modes of cell death after photodynamic therapy (PDT). Murine leukemia L1210 cells and human prostate Bax-deficient DU-145 cells were examined after PDT-induced photodamage to the endoplasmic reticulum (ER). Previous studies indicated that this treatment resulted in a substantial loss of Bcl-2 function. Both apoptosis and autophagy occurred in L1210 cells after ER photodamage with the latter predominating after 24 hr. These processes were characterized by altered cellular morphology, chromatin condensation, loss of mitochondrial membrane potential and formation of vacuoles containing cytosolic components. Western blots demonstrated processing of LC3-I to LC3-II, a marker for autophagy. In DU145 cells, PDT initiated only autophagy. Phosphatidylinositol (PI) 3-kinase inhibitors suppressed autophagy in both cell lines as indicated by inhibition of vacuolization and LC3 processing. Inhibitors of apoptosis and/or autophagy were then used to delineate the contributions of the two pathways to the effects of PDT. Given the ability of autophagy to upregulate MHC-11 peptide presentation, autophagy may play a role in the ability of photodynamic therapy to stimulate immunologic recognition of target cells.     

Blocking autophagy enhances the apoptosis effect of bufalin on human hepatocellular carcinoma cells through endoplasmic reticulum stress and JNK activation

Bufalin extracts are a part of traditional Chinese medicine, Chansu. In the current study, we investigated the effect of bufalin on the proliferation of the human hepatocellular carcinoma (HCC) cell lines, Huh-7 and HepG-2, and explored the therapeutic potential of the drug. Our results demonstrated that bufalin markedly inhibited cell proliferation and promoted apoptosis in the Huh-7 and HepG-2 cells in vitro. The underlying mechanism of the bufalin-induced apoptosis was the induction of endoplasmic reticulum (ER) stress via the IRE1–JNK pathway. In addition, during the ER stress response, the autophagy pathway, characterized by the conversion of LC3-I to LC3-II, was activated, resulting in increased Beclin-1 protein levels, decreased p62 expression and stimulation of autophagic flux. Our data supported the pro-survival role of bufalin-induced autophagy when the autophagy pathway was blocked with specific chemical inhibitors; the involvement of the IRE1 pathway in the ER stress-induced autophagy was also demonstrated when the expression of IRE1 and CHOP was silenced using siRNA. These data indicate that combining bufalin with a specific autophagy inhibitor could be a promising therapeutic approach for the treatment of HCC.     

TMEM166, a novel transmembrane protein,regulates cell autophagy and apoptosis

Programmed cell death can be divided into apoptosis and autophagic cell death. We describe the biological activities of TMEM166 (transmembrane protein 166, also known as FLJ13391), which is a novel lysosome and endoplasmic reticulum-associated membrane protein containing a putative TM domain. Overexpression of TMEM166 markedly inhibited colony formation in HeLa cells. Simultaneously, typical morphological characteristics consistent with autophagy were observed by transmission electron microscopy, including extensive autophagic vacuolization and enclosure of cell organelles by double-membrane structures. Further experiments confirmed that the overexpression of TMEM166 increased the punctate distribution of MDC staining and GFP-LC3 in HeLa cells, as well as the LC3-II/LC3-I proportion. On the other hand, TMEM166-transfected HeLa and 293T cells succumbed to cell death with hallmarks of apoptosis including phosphatidylserine externalization, loss of mitochondrial transmembrane potential, caspase activation and chromatin condensation. Kinetic analysis revealed that the appearance of autophagy-related biochemical parameters preceded the nuclear changes typical of apoptosis in TMEM166-transfected HeLa cells. Suppression of TMEM166 expression by small interference RNA inhibited starvation-induced autophagy in HeLa cells. These findings show for the first time that TMEM166 is a novel regulator involved in both autophagy and apoptosis.     

LRP6 regulates Rab7-mediated autophagy through the Wnt/β-catenin pathway to modulate trophoblast cell migration and invasion

Pre-eclampsia is a common complication during pregnancy; however, the underlying mechanisms of the crosstalk between low-density lipoprotein receptor-related protein 6 (LRP6) and autophagy in trophoblast cells are still not fully explored. Messenger RNA (mRNA) and protein levels of LRP6, beclin 1, Unc-51-like autophagy activating kinase 1 (ULK1), p62, vimentin, matrix metallopeptidase-9 (MMP-9), β-catenin, c-Myc, and Rab7, as well as the ratio of LC3-II/LC3-I, were analysed by quantitative real-time polymerase chain reaction or Western blot analysis, respectively. An MTT assay was used to measure cell growth, and transwell and wound healing assays were carried out to evaluate the invasion and migration abilities of the trophoblasts used. An immunofluorescence assay was used to measure LC3. The mRFP-GFP-LC3 tandem fluorescence assay was applied to detect autophagic flow. LRP6 overexpression was achieved by constructing pcDNA3.1-LRP6 vectors. LRP6 was expressed at low levels in HTR-8/SVneo cells under hypoxia/reoxygenation (H/R) conditions. H/R inhibited the activation of autophagy. LRP6 overexpression promoted cell proliferation and activated autophagy, which led to the upregulation of beclin 1 and ULK1, as well as the ratio of LC3-II/LC3-I and the downregulation of p62. Furthermore, LRP6 overexpression elevated the migration and invasion abilities of the indicated cells and increased vimentin and MMP-9 expression levels. Furthermore, LRP6 upregulated Rab7 and activated autophagy through the Wnt/β-catenin pathway. The late autophagy inhibitor bafilomycin A1 (Baf-A1) and the Wnt/β-catenin pathway inhibitor PKF115-584 reversed the effects of LRP6 on trophoblast autophagy, migration and invasion. LRP6 promotes Rab7-mediated autophagy by activating the Wnt/β-catenin pathway, which leads to increasing migration and invasion of trophoblast cells. Our study paves a new avenue for clinical treatment, and LRP6 may serve as an essential target in pre-eclampsia.     

Autophagy promotes the replication of encephalomyocarditis virus in host cells

A growing number of studies have demonstrated that autophagy has a diverse role in the infection process of different pathogens. However, to date, it is unknown whether autophagy is activated in encephalomyocarditis virus (EMCV)-infected host cells, and if so, what its role is in this process. In the present study, we first demonstrated that EMCV infection significantly increases the number of double- and single-membrane vesicles in the cytoplasm of host cells. It was then confirmed that these observed vesicles are indeed related to autophagy, and that EMCV replication is required for the induction of autophagy by examining LC3-I/-II conversion and p62/SQSTM1 degradation using immunoblotting. Next, we performed confocal immunofluorescence analysis and discovered that, during EMCV replication, both the nonstructural protein 3A and capsid protein VP1 colocalized with LC3. The colocalizations of both 3A and VP1 protein with autophagosome-like vesicles were further confirmed using immunoelectron microscopy, indicating that EMCV undergoes RNA replication on the membranes of these vesicles. Finally, we used pharmacological regulators and siRNAs to examine the role of autophagy in EMCV replication. Our results suggest that autophagy not only promotes the replication of EMCV in host cells, but it also provides a topological mechanism for releasing cytoplasmic viruses in a nonlytic manner. Noticeably, the autophagic pharmaceuticals we used had no significant effect on virus entry or cell viability, both of which may affect viral replication. To our knowledge, ours is the first strong evidence indicating that autophagy is involved in EMCV infection in host cells.     

Autophagy promotes the replication of encephalomyocarditis virus in host cells

A growing number of studies have demonstrated that autophagy has a diverse role in the infection process of different pathogens. However, to date, it is unknown whether autophagy is activated in encephalomyocarditis virus (EMCV)-infected host cells, and if so, what its role is in this process. In the present study, we first demonstrated that EMCV infection significantly increases the number of double- and single-membrane vesicles in the cytoplasm of host cells. It was then confirmed that these observed vesicles are indeed related to autophagy, and that EMCV replication is required for the induction of autophagy by examining LC3-I/-II conversion and p62/SQSTM1 degradation using immunoblotting. Next, we performed confocal immunofluorescence analysis and discovered that, during EMCV replication, both the nonstructural protein 3A and capsid protein VP1 colocalized with LC3. The colocalizations of both 3A and VP1 protein with autophagosome-like vesicles were further confirmed using immunoelectron microscopy, indicating that EMCV undergoes RNA replication on the membranes of these vesicles. Finally, we used pharmacological regulators and siRNAs to examine the role of autophagy in EMCV replication. Our results suggest that autophagy not only promotes the replication of EMCV in host cells, but it also provides a topological mechanism for releasing cytoplasmic viruses in a nonlytic manner. Noticeably, the autophagic pharmaceuticals we used had no significant effect on virus entry or cell viability, both of which may affect viral replication. To our knowledge, ours is the first strong evidence indicating that autophagy is involved in EMCV infection in host cells.     

Transient Receptor Potential Cation Channel V1 (TRPV1) Is Degraded by Starvation- and Glucocorticoid-Mediated Autophagy

A mammalian cell renovates itself by autophagy, a process through which cellular components are recycled to produce energy and maintain homeostasis. Recently, the abundance of gap junction proteins was shown to be regulated by autophagy during starvation conditions, suggesting that transmembrane proteins are also regulated by autophagy. Transient receptor potential vanilloid type 1 (TRPV1), an ion channel localized to the plasma membrane and endoplasmic reticulum (ER), is a sensory transducer that is activated by a wide variety of exogenous and endogenous physical and chemical stimuli. Intriguingly, the abundance of cellular TRPV1 can change dynamically under pathological conditions. However, the mechanisms by which the protein levels of TRPV1 are regulated have not yet been explored. Therefore, we investigated the mechanisms of TRPV1 recycling using HeLa cells constitutively expressing TRPV1. Endogenous TRPV1 was degraded in starvation conditions; this degradation was blocked by chloroquine (CLQ), 3MA, or downregulation of Atg7. Interestingly, a glucocorticoid (cortisol) was capable of inducing autophagy in HeLa cells. Cortisol increased cellular conversion of LC3-I to LC-3II, leading autophagy and resulting in TRPV1 degradation, which was similarly inhibited by treatment with CLQ, 3MA, or downregulation of Atg7. Furthermore, cortisol treatment induced the colocalization of GFP-LC3 with endogenous TRPV1. Cumulatively, these observations provide evidence that degradation of TRPV1 is mediated by autophagy, and that this pathway can be enhanced by cortisol.     

Protein kinase Ctheta is required for autophagy in response to stress in the endoplasmic reticulum

Autophagy is an evolutionally conserved process for the bulk degradation of cytoplasmic proteins and organelles. Recent observations indicate that autophagy is induced in response to cellular insults that result in the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER). However, the signaling mechanisms that activate autophagy under these conditions are not understood. Here, we report that ER stress-induced autophagy requires the activation of protein kinase C (PKC), a member of the novel-type PKC family. Induction of ER stress by treatment with either thapsigargin or tunicamycin activated autophagy in immortalized hepatocytes as monitored by the conversion LC3-I to LC3-II, clustering of LC3 into dot-like cytoplasmic structures, and electron microscopic detection of autophagosomes. Pharmacological inhibition of PKC or small interfering RNA-mediated knockdown of PKC prevented the autophagic response to ER stress. Treatment with ER stressors induced PKC phosphorylation within the activation loop and localization of phospho-PKC to LC3-containing dot structures in the cytoplasm. However, signaling through the known unfolded protein response sensors was not required for PKC activation. PKC activation and stress-induced autophagy were blocked by chelation of intracellular Ca(2+) with BAPTA-AM. PKC was not activated or required for autophagy in response to amino acid starvation. These observations indicate that Ca(2+)-dependent PKC activation is specifically required for autophagy in response to ER stress but not in response to amino acid starvation.     

Porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication

An increasing number of studies demonstrate that autophagy, an intrinsic mechanism that can degrade cytoplasmic components, is involved in the infection processes of a variety of pathogens. It can be hijacked by various viruses to facilitate their replication. In this study, we found that PRRSV infection significantly increases the number of double- or single-membrane vesicles in the cytoplasm of host cells in ultrastructural analysis. Our results showed the LC3-I was converted into LC3-II after virus infection, suggesting the autophagy machinery was activated. We further used pharmacological agents and shRNAs to confirm that autophagy promoted the replication of PRRSV in host cells. Confocal microscopy analysis showed that PRRSV inhibited the fusion between autophagosomes and lysosomes, suggesting that PRRSV induced incomplete autophagy. This suppression caused the accumulation of autophagosomes which may serve as replication site to enhance PRRSV replication. It has been shown that NSP2 and NSP3 of arterivirus are two components of virus replication complex. We also found in our studies that NSP2 colocalized with LC3 in MARC-145 cells by performing confocal microscopy analysis and continuous density gradient centrifugation. Our studies presented here indicated that autophagy was activated during PRRSV infection and enhanced PRRSV replication in host cells by preventing autophagosome and lysosome fusion.     

The Interplays between Autophagy and Apoptosis Induced by Enterovirus 71

Background

Enterovirus 71 (EV71) is the causative agent of human diseases with distinct severity, from mild hand, foot and mouth disease to severe neurological syndromes, such as encephalitis and meningitis. The lack of understanding of viral pathogenesis as well as lack of efficient vaccine and drugs against this virus impedes the control of EV71 infection. EV71 virus induces autophagy and apoptosis; however, the relationship between EV71-induced autophagy and apoptosis as well as the influence of autophagy and apoptosis on virus virulence remains unclear.

Methodology/Principal Findings

In this study, it was observed that the Anhui strain of EV71 induced autophagy and apoptosis in human rhabdomyosarcoma (RD-A) cells. Additionally, by either applying chemical inhibitors or knocking down single essential autophagic or apoptotic genes, inhibition of EV71 induced autophagy inhibited the apoptosis both at the autophagosome formation stage and autophagy execution stage. However, inhibition of autophagy at the stage of autophagosome and lysosome fusion promoted apoptosis. In reverse, the inhibition of EV71-induced apoptosis contributed to the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to LC3-II and degradation of sequestosome 1 (SQSTM1/P62). Furthermore, the inhibition of autophagy in the autophagsome formation stage or apoptosis decreased the release of EV71 viral particles.

Conclusions/Significance

In conclusion, the results of this study not only revealed novel aspect of the interplay between autophagy and apoptosis in EV71 infection, but also provided a new insight to control EV71 infection.