Involvement of caspase-2 activation in aurora kinase inhibitor-induced cell death in axin-expressing L929 cells

Axin is a multifunctional protein that participates in many cellular events including Wnt signaling and cell fate determination. Aurora kinase inhibitor (AKI)-induced cell death and cell membrane rupture is facilitated in L929 cells expressing axin (L-axin cells) through the activation of poly ADP-ribose polymerase (PARP). We observed that caspase-2 activity is required for AKI-induced cell death. Inhibition of caspase-2 activity suppressed AKI-induced PARP activation and mitochondrial dysfunction, resulting in a decrease in AKI-induced cell death. When an axin mutant deleted for the glycogen synthase kinase 3β (GSK3β)-binding domain was expressed in L929 cells (L-ΔGSK cells), AKI-induced caspase-2 activation and cell death decreased. AKI treatment reduced the expression of a 32-kDa caspase-2 splicing variant (caspase-2S) in most L-axin cells, but not in L-ΔGSK cells. These results suggest that AKI-induced caspase-2 activation in L-axin cells might be due to a decrease in the expression of caspase-2S, which inhibits caspase-2 activity. In addition, AKI treatment failed to activate caspase-8 and treatment with necrostatin inhibited AKI-induced cell death in L-axin cells, suggesting that the absence of caspase-8 activation might favor necrotic cell death. Axin expression may facilitate AKI-induced caspase-2 activation followed by activation of PARP and initiation of the necrotic cell death pathway.     

EV71诱导人神经细胞SH-SY5Y自噬的分子机制

【背景】EV71感染所致的重症手足口病易导致神经系统并发症,使患儿预后较差,甚至死亡。【目的】从EV71可诱导神经细胞自噬这一现象出发,探索该病毒诱导神经细胞自噬的miRNA机制,探讨EV71损伤神经细胞可能的分子机制。【方法】通过RT-PCR及Westernblot技术,在感染EV71病毒的人神经母细胞瘤细胞SH-SY5Y中检测细胞自噬变化;通过芯片分析细胞感染前后差异表达的miRNA分子,再使用miRNA mimics调节工具明确与EV71诱导神经细胞自噬有关的miRNA分子。【结果】EV71可诱导SH-SY5Y细胞自噬增加,下调细胞内miRNA29b(miR29b)分子的表达水平;当上调细胞内miR29b的表达后,EV71诱导细胞自噬增加的现象可被逆转,病毒复制水平下降。【结论】EV71诱导神经细胞自噬是通过下调miR29b分子的表达水平实现;miR29b不仅与自噬相关,它与EV71病毒复制也存在密切关系。因此,该研究不仅有助于阐明EV71导致神经系统损伤的具体分子机制,还为miR29b成为治疗EV71感染可能的新药物靶点奠定了理论基础。     

Innate resistance to herpes simplex virus infection. Human lymphocyte and monocyte inhibition of viral replication

Peripheral blood monocytes and lymphocytes isolated from most humans are resistant to HSV infection in vitro. Viral replication is inhibited very early in the cycle, prior to the onset of alpha-protein synthesis; no viral protein or DNA synthesis is detectable even up to 1 week later. The enhanced expression of two 62-kDa and 57-kDa cellular proteins, however, is induced in the lymphocyte population within 3 to 5 h after infection. A 30-kDa protein is induced in the monocyte population immediately after infection. The induced expression of 62-kDa and 57-kDa lymphocyte proteins appears to be virus-mediated because: a) HSV and pseudorabies virus (although not vaccinia virus) induce the expression of 62-kDa and 57-kDa proteins, b) heat shock or exposure of lymphocytes to uninfected cell extracts does not induce expression of either protein, c) 62-kDa protein is not induced in lymphocytes stimulated with a mitogenic concentration of PHA. UV-inactivated HSV induces expression of 62-kDa and 57-kDa proteins in a manner similar to that observed with untreated virus. In contrast, expression of 30-kDa monocyte protein is induced nonspecifically by either uninfected cell extracts or cell extracts containing virus. Sixty-two-kilodalton and 57-kDa protein induction appears to be a marker for human lymphocytes that express profound intracellular resistance to infection with HSV. Induced expression of these proteins occurs only in lymphocytes that inhibit viral replication very early in the growth cycle, prior to the onset of alpha-protein synthesis. Expression of 62-kDa and 57-kDa proteins is not induced in lymphocytes that are permissive or partially permissive to infection with HSV.     

Inhibition of Interferons by Ectromelia Virus

Ectromelia virus (EV) is an orthopoxvirus (OPV) that causes mousepox, a severe disease of laboratory mice. Mousepox is a useful model of OPV infection because EV is likely to be a natural mouse pathogen, unlike its close relatives vaccinia virus (VV) and variola virus. Several studies have highlighted the importance of mouse interferons (IFNs) in resistance to and recovery from EV infection, but little is known of the anti-IFN strategies encoded by the virus itself. We have determined that 12 distinct strains and isolates of EV encode soluble, secreted receptors for IFN-gamma (vIFN-gammaR) and IFN-alpha/beta (vIFN-alpha/betaR) that are homologous to those identified in other OPVs. We demonstrate for the first time that the EV vIFN-gammaR has the unique ability to inhibit the biological activity of mouse IFN-gamma. The EV vIFN-alpha/betaR was a potent inhibitor of human and mouse IFN-alpha and human IFN-beta but, surprisingly, was unable to inhibit mouse IFN-beta. The replication of all of the EVs included in our study and of cowpox virus was more resistant than VV to the antiviral effects induced in mouse L-929 cells by IFN-alpha/beta and IFN-gamma. Sequencing studies showed that this EV resistance is likely to be partly mediated by the double-stranded-RNA-binding protein encoded by an intact EV homolog of the VV E3L gene. The absence of a functional K3L gene, which encodes a viral eIF-2alpha homolog, in EV suggests that the virus encodes a novel mechanism to counteract the IFN response. These findings will facilitate future studies of the role of viral anti-IFN strategies in mousepox pathogenesis. Their significance in the light of earlier data on the role of IFNs in mousepox is discussed.     

Flavivirus NS4A-induced autophagy protects cells against death and enhances virus replication

Flaviviruses include the most prevalent and medically challenging viruses. Persistent infection with flaviviruses of epithelial cells and hepatocytes that do not undergo cell death is common. Here, we report that, in epithelial cells, up-regulation of autophagy following flavivirus infection markedly enhances virus replication and that one flavivirus gene, NS4A, uniquely determines the up-regulation of autophagy. Dengue-2 and Modoc (a murine flavivirus) kill primary murine macrophages but protect epithelial cells and fibroblasts against death provoked by several insults. The flavivirus-induced protection derives from the up-regulation of autophagy, as up-regulation of autophagy by starvation or inactivation of mammalian target of rapamycin also protects the cells against insult, whereas inhibition of autophagy via inactivation of PI3K nullifies the protection conferred by flavivirus. Inhibition of autophagy also limits replication of both Dengue-2 and Modoc virus in epithelial cells. Expression of flavivirus NS4A is sufficient to induce PI3K-dependent autophagy and to protect cells against death; expression of other viral genes, including NS2A and NS4B, fails to protect cells against several stressors. Flavivirus NS4A protein induces autophagy in epithelial cells and thus protects them from death during infection. As autophagy is vital to flavivirus replication in these cells, NS4A is therefore also identified as a critical determinant of flavivirus replication.     

Endoplasmic reticulum stress is induced and modulated by enterovirus 71

Picornavirus infection alters the endoplasmic reticulum (ER) membrane but it is unclear whether this induces ER stress. Infection of rhabdomyosarcoma cells with enterovirus 71 (EV71), a picornavirus, caused overexpression of the ER‐resident chaperone proteins, BiP and calreticulin, and phosphorylation of eIF2α, but infection with UV‐inactivated virus did not, indicating that ER stress was induced by viral replication and not by viral attachment or entry. Silencing (si)RNA knockdown demonstrated that phosphorylation of eIF2α was dependent on PKR: eIF2α phosphorylation was reduced by siPKR but not by siPERK. We provided evidence showing that PERK is upstream of PKR and is thus able to negatively regulate the PKR‐eIF2α pathway. Pulse‐chase experiments revealed that EV71 infection inhibited translation and activation of ATF6. Expression of BiP at the protein level was activated by a virus‐dependent, ATF6‐independent mechanism. EV71 upregulated XBP1 mRNA level, but neither IRE1‐mediated XBP1 splicing nor its active spliced protein was detected, and its downstream gene, EDEM, was not activated. Epigenetic BiP overexpression alleviated EV71‐induced ER stress and reduced viral protein expression and replication. Our results suggest that EV71 infection induces ER stress but modifies the outcome to assist viral replication.     

Bovine herpesvirus type 4 infection modulates autophagy in a permissive cell line

Bovine herpesvirus type 4 (BoHV‐4), like other herpesviruses, induces a series of alterations in the host cell that modify the intracellular environment in favor of viral replication, survival and spread. This research examined the impact of BoHV‐4 infection on autophagy in BoHV‐4 infected Madin Darby bovine kidney (MDBK) cells. Protein extracts of BoHV‐4 infected and control MDBK cells were subjected to Western blot. The concentrations of the autophagy and apoptosis‐related proteins Beclin 1, p21, PI3 kinase, Akt1/2, mTOR, phospho mTOR, p62 and the light chain three (LC3) were normalized to the actin level and expressed as the densitometric ratio. Western blot analysis of virus‐infected cells revealed that autophagic degradation pathway was induced in the late phase of BoHV‐4 infection. After 48 h post‐infection the protein LC3II, which is essential for autophagy was found to be markedly increased, while infection of MDBK cells with BoHV‐4 resulted in a depletion of p62 levels. Becline 1, PI3 kinase, Akt1/2 and p21 expression increased between 24 and 48 h post‐infection. Surprisingly, mTOR and its phosphorylated form, which are negative regulators of autophagy, also increased after 24 h post‐infection. In conclusion, our findings suggest that BoHV‐4 has developed mechanisms for modulation of autophagy that are probably part of a strategy designed to enhance viral replication and to evade the immune system. Additional studies on the relationship between autophagy and BoHV‐4 replication and survival, in both lytic and latent replication phases, are needed to understand the role of autophagy in BoHV‐4 pathogenesis. J. Cell. Biochem. 114: 1529–1535, 2013. © 2013 Wiley Periodicals, Inc.     

Axin1 expression facilitates cell death induced by aurora kinase inhibition through PARP activation

Axin, a negative regulator of Wnt signaling, participates in apoptosis, and Axin1 localizes to centrosomes and mitotic spindles, which requires Aurora kinase activity. In this study, Aurora inhibition of Axin1-expressing cells (L-Axin) produced polyploid cells, which died within 48 h posttreatment, whereas Axin2-expressing cells (L-Axin2) survived the same period. These cell death events showed apoptotic signs, such as chromatin condensation and increased sub-G1 populations, as well as cell membrane rupture. Further analysis showed that Aurora kinase inhibitor (AKI) treatment of L-Axin cells induced poly(ADP-ribose) polymerase (PARP) activation, which increased the poly(ADP-ribosyl)ation of cellular proteins and reduced cellular ATP content. PARP inhibition reduced a proportion of dead cells, suggesting PARP involvement in AKI-induced cell death. Also, AKI treatment of L-Axin cells induced mitochondrial apoptosis-inducing factor (AIF) release, but not mitochondrial cytochrome c release or caspase-3 activation. Knockdown of AIF attenuated AKI-induced cell death in L-Axin cells. Thus, our results suggest that Axin1 expression renders L929 cells sensitive to Aurora inhibition-induced cell death in a PARP- and AIF-dependent manner.     

Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model

Background

We previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo. In the present study we further investigated whether EV71 infection of neuronal SK-N-SH cells induces an autophagic flux. Furthermore, the effects of autophagy on EV71-related pathogenesis and viral load were evaluated after intracranial inoculation of mouse-adapted EV71 (MP4 strain) into 6-day-old ICR suckling mice.

Results

We demonstrated that in EV71-infected SK-N-SH cells, EV71 structural protein VP1 and nonstructural protein 2C co-localized with LC3 and mannose-6-phosphate receptor (MPR, endosome marker) proteins by immunofluorescence staining, indicating amphisome formation. Together with amphisome formation, EV71 induced an autophagic flux, which could be blocked by NH₄Cl (inhibitor of acidification) and vinblastine (inhibitor of fusion), as demonstrated by Western blotting. Suckling mice intracranially inoculated with EV71 showed EV71 VP1 protein expression (representing EV71 infection) in the cerebellum, medulla, and pons by immunohistochemical staining. Accompanied with these infected brain tissues, increased expression of LC3-II protein as well as formation of LC3 aggregates, autophagosomes and amphisomes were detected. Amphisome formation, which was confirmed by colocalization of EV71-VP1 protein or LC3 puncta and the endosome marker protein MPR. Thus, EV71-infected suckling mice (similar to EV71-infected SK-N-SH cells) also show an autophagic flux. The physiopathological parameters of EV71-MP4 infected mice, including body weight loss, disease symptoms, and mortality were increased compared to those of the uninfected mice. We further blocked EV71-induced autophagy with the inhibitor 3-methyladenine (3-MA), which attenuated the disease symptoms and decreased the viral load in the brain tissues of the infected mice.

Conclusions

In this study, we reveal that EV71 infection of suckling mice induces an amphisome formation accompanied with the autophagic flux in the brain tissues. Autophagy induced by EV71 promotes viral replication and EV71-related pathogenesis.     

Sumoylation-promoted enterovirus 71 3C degradation correlates with a reduction in viral replication and cell apoptosis

Enterovirus 71 (EV71), a member of the Picornaviridae family, may cause serious clinical manifestations associated with the central nervous system. Enterovirus 3C protease is required for virus replication and can trigger host cell apoptosis via cleaving viral polyprotein precursor and cellular proteins, respectively. Although the role of the 3C protease in processing viral and cellular proteins has been established, very little is known about the modulation of EV71 3C function by host cellular factors. Here, we show that sumoylation promotes EV71 3C protein ubiquitination for degradation, correlating with a decrease of EV71 in virus replication and cell apoptosis. SUMO E2-conjugating enzyme Ubc9 was identified as an EV71 3C-interacting protein. Further studies revealed that EV71 3C can be SUMO (small ubiquitin-like modifier)-modified at residue Lys-52. Sumoylation down-regulated 3C protease activity in vitro and also 3C protein stability in cells, in agreement with data suggesting 3C K52R protein induced greater substrate cleavage and apoptosis in cells. More importantly, the recombinant EV71 3C K52R virus infection conferred more apoptotic phenotype and increased virus levels in culture cells, which also correlated with a mouse model showing increased levels of viral VP1 protein in intestine and neuron loss in the spinal cord with EV71 3C K52R recombinant viral infection. Finally, we show that EV71 3C amino acid residues 45-52 involved in Ubc9 interaction determined the extent of 3C sumoylation and protein stability. Our results uncover a previously undescribed cellular regulatory event against EV71 virus replication and host cell apoptosis by sumoylation at 3C protease.     

Identification of a human SCARB2 region that is important for enterovirus 71 binding and infection

We previously identified human scavenger receptor class B, member 2 (SCARB2), as a cellular receptor for enterovirus 71 (EV71). Expression of human SCARB2 (hSCARB2) permitted mouse L929 cells to efficiently bind to virions and to produce both viral proteins and progeny viruses upon EV71 infection. Mouse Scarb2 (mScarb2) exhibited 85.8% amino acid identity and 99.9% similarity to hSCARB2. The expression of mScarb2 in L929 cells conferred partial susceptibility. Very few virions bound to mScarb2-expressing cells. The viral titer in L929 cells expressing mScarb2 was approximately 40- to 100-fold lower than that in L929 cells expressing hSCARB2. Using hSCARB2-mScarb2 chimeric mutants, we attempted to map the region that was important for efficient EV71 infection. L929 cells expressing chimeras that carried amino acids 142 to 204 from the human sequence were susceptible to EV71, while chimeras that carried the mouse sequence in this region were not. Moreover, this region was also critical for binding to virions. The determination of this region in hSCARB2 that is important for EV71 binding and infection greatly contributes to the understanding of virus-receptor interactions. Further studies will clarify the early steps of EV71 infection.     

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.     

Proteomic analysis of cells in the early stages of herpes simplex virus type‐1 infection reveals widespread changes in the host cell proteome

During infection by herpes simplex virus type‐1 (HSV‐1) the host cell undergoes widespread changes in gene expression and morphology in response to viral replication and release. However, relatively little is known about the specific proteome changes that occur during the early stages of HSV‐1 replication prior to the global damaging effects of virion maturation and egress. To investigate pathways that may be activated or utilised during the early stages of HSV‐1 replication, 2‐DE and LC‐MS/MS were used to identify cellular proteome changes at 6 h post infection. Comparative analysis of multiple gels representing whole cell extracts from mock‐ and HSV‐1‐infected HEp‐2 cells revealed a total of 103 protein spot changes. Of these, 63 were up‐regulated and 40 down‐regulated in response to infection. Changes in selected candidate proteins were verified by Western blot analysis and their respective cellular localisations analysed by confocal microscopy. We have identified differential regulation and modification of proteins with key roles in diverse cellular pathways, including DNA replication, chromatin remodelling, mRNA stability and the ER stress response. This work represents the first global comparative analysis of HSV‐1 infected cells and provides an important insight into host cell proteome changes during the early stages of HSV‐1 infection.     

Persistence of mumps virus in mouse L929 cells

The characteristics of a persistent infection of L929 cells with mumps virus (MuV) is presented. The persistent infection (L-MuV cells) was regulated by interferon (IFN) produced endogenously and almost all the properties showed that the carrier culture was maintained by horizontal transmission of the virus. Small-plaque mutants, but not temperature-sensitive variants, were selected during the persistent infection. MuV released from L-MuV cells (MuV-pi) replicated efficiently in L929 cells, while infection of L929 cells with the original MuV-o resulted in an abortive infection. The efficient replication of MuV-pi in L929 cells can be explained by the findings that MuV-pi induced IFN more slowly and had lower susceptibility to IFN in L929 cells than MuV-o did. M protein was synthesized to a considerable degree in MuV-pi-infected cells, while it could not be detected in MuV-o-infected cells. By contrast, MuV-pi formed small plaques in Vero cell monolayers and the yield of MuV-pi in Vero cells was lower than that of MuV-o. M protein induced by MuV-pi decayed easily in Vero cells. M protein was considered to be a limiting factor for MuV replication in both cell lines.     

Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes

Autophagy is a lysosomal degradative pathway that has diverse physiological functions and plays crucial roles in several viral infections. Here we examine the role of autophagy in the life cycle of JEV, a neurotropic flavivirus. JEV infection leads to induction of autophagy in several cell types. JEV replication was significantly enhanced in neuronal cells where autophagy was rendered dysfunctional by ATG7 depletion, and in Atg5-deficient mouse embryonic fibroblasts (MEFs), resulting in higher viral titers. Autophagy was functional during early stages of infection however it becomes dysfunctional as infection progressed resulting in accumulation of misfolded proteins. Autophagy-deficient cells were highly susceptible to virus-induced cell death. We also observed JEV replication complexes that are marked by nonstructural protein 1 (NS1) and dsRNA colocalized with endogenous LC3 but not with GFP-LC3. Colocalization of NS1 and LC3 was also observed in Atg5 deficient MEFs, which contain only the nonlipidated form of LC3. Viral replication complexes furthermore show association with a marker of the ER-associated degradation (ERAD) pathway, EDEM1 (ER degradation enhancer, mannosidase α-like 1). Our data suggest that virus replication occurs on ERAD-derived EDEM1 and LC3-I-positive structures referred to as EDEMosomes. While silencing of ERAD regulators EDEM1 and SEL1L suppressed JEV replication, LC3 depletion exerted a profound inhibition with significantly reduced RNA levels and virus titers. Our study suggests that while autophagy is primarily antiviral for JEV and might have implications for disease progression and pathogenesis of JEV, nonlipidated LC3 plays an important autophagy independent function in the virus life cycle.     

HtrA2/Omi terminates cytomegalovirus infection and is controlled by the viral mitochondrial inhibitor of apoptosis (vMIA)

Viruses encode suppressors of cell death to block intrinsic and extrinsic host-initiated death pathways that reduce viral yield as well as control the termination of infection. Cytomegalovirus (CMV) infection terminates by a caspase-independent cell fragmentation process after an extended period of continuous virus production. The viral mitochondria-localized inhibitor of apoptosis (vMIA; a product of the UL37x1 gene) controls this fragmentation process. UL37x1 mutant virus-infected cells fragment three to four days earlier than cells infected with wt virus. Here, we demonstrate that infected cell death is dependent on serine proteases. We identify mitochondrial serine protease HtrA2/Omi as the initiator of this caspase-independent death pathway. Infected fibroblasts develop susceptibility to death as levels of mitochondria-resident HtrA2/Omi protease increase. Cell death is suppressed by the serine protease inhibitor TLCK as well as by the HtrA2-specific inhibitor UCF-101. Experimental overexpression of HtrA2/Omi, but not a catalytic site mutant of the enzyme, sensitizes infected cells to death that can be blocked by vMIA or protease inhibitors. Uninfected cells are completely resistant to HtrA2/Omi induced death. Thus, in addition to suppression of apoptosis and autophagy, vMIA naturally controls a novel serine protease-dependent CMV-infected cell-specific programmed cell death (cmvPCD) pathway that terminates the CMV replication cycle.     

Interactome analysis of the EV71 5′ untranslated region in differentiated neuronal cells SH‐SY5Y and regulatory role of FBP3 in viral replication

Enterovirus 71 (EV71), a single‐stranded RNA virus, is one of the most serious neurotropic pathogens in the Asia‐Pacific region. Through interactions with host proteins, the 5′ untranslated region (5′UTR) of EV71 is important for viral replication. To gain a protein profile that interact with the EV71 5′UTR in neuronal cells, we performed a biotinylated RNA‐protein pull‐down assay in conjunction with LC–MS/MS analysis. A total of 109 proteins were detected and subjected to Database for Annotation, Visualization and Integrated Discovery (DAVID) analyses. These proteins were found to be highly correlated with biological processes including RNA processing/splicing, epidermal cell differentiation, and protein folding. A protein–protein interaction network was constructed using the STRING online database to illustrate the interactions of those proteins that are mainly involved in RNA processing/splicing or protein folding. Moreover, we confirmed that the far‐upstream element binding protein 3 (FBP3) was able to bind to the EV71 5′UTR. The redistribution of FBP3 in subcellular compartments was observed after EV71 infection, and the decreased expression of FBP3 in host neuronal cells markedly inhibited viral replication. Our results reveal various host proteins that potentially interact with the EV71 5′UTR in neuronal cells, and we found that FBP3 could serve as a positive regulator in host cells.