TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71

Enterovirus A71(EV-A71) is one of the etiological pathogens leading to hand, foot, and mouth disease(HFMD), which can cause severe neurological complications. The neuropathogenesis of EV-A71 infection is not well understood. The mislocalization and aggregation of TAR DNA-binding protein 43(TDP-43) is the pathological hallmark of amyotrophic lateral sclerosis(ALS). However, whether TDP-43 was impacted by EV-A71 infection is unknown. This study demonstrated that TDP-43 was cleaved during EV-A71 infection. The cleavage of TDP-43 requires EV-A71 replication rather than the activated caspases due to viral infection. TDP-43 is cleaved by viral protease 3 C between the residues 331 Q and332 S, while mutated TDP-43(Q331 A) was not cleaved. In addition, mutated 3 C which lacks the protease activity failed to induce TDP-43 cleavage. We also found that TDP-43 was translocated from the nucleus to the cytoplasm, and the mislocalization of TDP-43 was induced by viral protease 2 A rather than 3 C. Taken together, we demonstrated that TDP-43 was cleaved by viral protease and translocated to the cytoplasm during EV-A71 infection, implicating the possible involvement of TDP-43 in the pathogenesis of EV-A71 infection.     

Cleavage of poly(A)-binding protein by poliovirus 3C protease inhibits host cell translation: a novel mechanism for host translation shutoff

Cleavage of eukaryotic translation initiation factor 4GI (eIF4GI) by viral 2A protease (2Apro) has been proposed to cause severe translation inhibition in poliovirus-infected cells. However, infections containing 1 mM guanidine-HCl result in eIF4GI cleavage but only partial translation shutoff, indicating eIF4GI cleavage is insufficient for drastic translation inhibition. Viral 3C protease (3Cpro) cleaves poly(A)-binding protein (PABP) and removes the C-terminal domain (CTD) that interacts with several translation factors. In HeLa cell translation extracts that exhibit cap-poly(A) synergy, partial cleavage of PABP by 3Cpro inhibited translation of endogenous mRNAs and reporter RNA as effectively as complete cleavage of eIF4GI and eIF4GII by 2Apro. 3Cpro-mediated translation inhibition was poly(A) dependent, and addition of PABP to extracts restored translation. Expression of 3Cpro in HeLa cells resulted in partial PABP cleavage and similar inhibition of translation. PABP cleavage did not affect eIF4GI-PABP interactions, and the results of kinetics experiments suggest that 3Cpro might inhibit late steps in translation or ribosome recycling. The data illustrate the importance of the CTD of PABP in poly(A)-dependent translation in mammalian cells. We propose that enteroviruses use a dual strategy for host translation shutoff, requiring cleavage of PABP by 3Cpro and of eIF4G by 2Apro.     

Degradation of poly(A)-binding protein in apoptotic cells and linkage to translation regulation

We have recently shown that poly(A)-binding protein (PABP) is cleaved during poliovirus and Coxsackievirus infection by viral 3Cprotease and that 3Cprotease modification of a subset of PABP can result in significant translation inhibition. During apoptosis, translation undergoes significant down-regulation that correlates with caspase-3 mediated cleavage of several translation factors, including eIF4G, 4EBP1 and eIF2alpha. The fate of PABP in apoptotic cells has not yet been examined. Here we show that PABP levels decline significantly via proteolytic degradation in apoptotic HeLa, Jurkat and MCF7 cells. The degradation of PABP correlated with translation inhibition but lagged behind cleavage of eIF4GI. In apoptotic MCF7 cells translation inhibition occurred without modification of most translation factors and correlated with PABP degradation. PABP was not cleaved during incubation with several caspases, yet caspase 3 induced weak PABP degradative activity in cells lysates. Both the caspase inhibitor zVAD and calpain inhibitors blocked PABP cleavage in vivo, while the proteosome inhibitor MG132 induced PABP degradation. Protease(s) activated during apoptosis preferentially degraded PABP associated with ribosomes and translation factors, but not PABP in other cellular compartments. The data suggest that targeted degradation of PABP contributes to translation inhibition in apoptotic cells.     

STUB1 regulates antiviral RNAi through inducing ubiquitination and degradation of Dicer and AGO2 in mammals

RNA interference (RNAi) is an intrinsic antiviral immune mechanism conserved in diverse eukaryotic organisms. However, the mechanism by which antiviral RNAi in mammals is regulated is poorly understood. In this study, we uncovered that the E3 ubiquitin ligase STIP1 homology and U-box-containing protein 1 (STUB1) was a new regulator of the RNAi machinery in mammals. We found that STUB1 interacted with and ubiquitinated AGO2, and targeted it for degradation in a chaperon-dependent manner. STUB1 promoted the formation of Lys48 (K48)-linked polyubiquitin chains on AGO2, and facilitated AGO2 degradation through ubiquitin-proteasome system. In addition to AGO2, STUB1 also induced the protein degradation of AGO1, AGO3 and AGO4. Further investigation revealed that STUB1 also regulated Dicer's ubiquitination via K48-linked polyubiquitin and induced the degradation of Dicer as well as its specialized form, termed antiviral Dicer (aviDicer) that expresses in mammalian stem cells. Moreover, we found that STUB1 deficiency up-regulated Dicer and AGO2, thereby enhancing the RNAi response and efficiently inhibiting viral replication in mammalian cells. Using the newborn mouse model of Enterovirus A71 (EV-A71), we confirmed that STUB1 deficiency enhanced the virus-derived siRNAs production and antiviral RNAi, which elicited a potent antiviral effect against EV-A71 infection in vivo. In summary, our findings uncovered that the E3 ubiquitin ligase STUB1 was a general regulator of the RNAi machinery by targeting Dicer, aviDicer and AGO1–4. Moreover, STUB1 regulated the RNAi response through mediating the abundance of Dicer and AGO2 during viral infection, thereby providing novel insights into the regulation of antiviral RNAi in mammals.     

Mitochondria Redistribution in Enterovirus A71 Infected Cells and Its Effect on Virus Replication

Enterovirus A71(EV-A71) is one of the main causative agents of hand, foot and mouth disease(HFMD) and it also causes severe neurologic complications in infected children. The interactions between some viruses and the host mitochondria are crucial for virus replication and pathogenicity. In this study, it was observed that EV-A71 infection resulted in a perinuclear redistribution of the mitochondria. The mitochondria rearrangement was found to require the microtubule network, the dynein complex and a low cytosolic calcium concentration. Subsequently, the EV-A71 non-structural protein 2 BC was identified as the viral protein capable of inducing mitochondria clustering. The protein was found localized on mitochondria and interacted with the mitochondrial Rho GTPase 1(RHOT1) that is a key protein required for attachment between the mitochondria and the motor proteins, which are responsible for the control of mitochondria movement.Additionally, suppressing mitochondria clustering by treating cells with nocodazole, EHNA, thapsigargin or A23187 consistently inhibited EV-A71 replication, indicating that mitochondria recruitment played a crucial role in the EV-A71 life cycle. This study identified a novel function of the EV-A71 2 BC protein and provided a potential model for the regulation of mitochondrial motility in EV-A71 infection.     

肠道病毒A71型致人扁桃体上皮细胞系UT-SCC-60B凋亡和S期阻滞的研究

肠道病毒A71型(Enterovirus A71,EV-A71)是手足口病的重要病原体,为研究EV-A71感染人扁桃体上皮细胞后对细胞凋亡和细胞周期的影响,确定ERK1/2、JNK1/2、PI3K/Akt和含半胱氨酸的天冬氨酸蛋白水解酶(Cysteinyl aspartate specific proteinase,Caspase)的作用,本文以人扁桃体上皮细胞系UT-SCC-60B为细胞模型,CCK-8试剂盒检测EV-A71对UT-SCC-60B的抑制率、流式细胞仪检测EV-A71感染组和抑制剂处理组的凋亡和细胞周期、Caspase活力检测试剂盒测定Caspase-3,Caspase-8,Caspase-9活力。EV-A71以感染剂量和感染时间依赖方式抑制UT-SCC-60B增殖;EV-A71感染致UT-SCC-60B发生细胞凋亡,抑制ERK1/2、JNK1/2和PI3K/Akt能够降低UT-SCC-60B细胞凋亡比例;EV-A71感染UT-SCC-60B后发生S期阻滞,抑制ERK1/2、JNK1/2、PI3K/Akt和Caspase阻止UT-SCC-60B发生S期阻滞;EV-A71感染UT-SCC-60B能够活化Caspase-3,Caspase-8,Caspase-9且ERK1/2、JNK1/2和PI3K/Akt调控Caspase-3,Caspase-8,Caspase-9活力。因此,EV-A71能够导致人扁桃体上皮细胞UT-SCC-60B发生凋亡和S期阻滞,并且ERK1/2、JNK1/2、PI3K/Akt和Caspase参与凋亡和S期阻滞的调控。     

Visualizing and quantifying the differential cleavages of the eukaryotic translation initiation factors eIF4GI and eIF4GII in the enterovirus‐infected cell

Enterovirus (EV) infection has been shown to cause a marked shutoff of host protein synthesis, an event mainly achieved through the cleavages of eukaryotic translation initiation factors eIF4GI and eIF4GII that are mediated by viral 2A protease (2A^pro). Using fluorescence resonance energy transfer (FRET), we developed genetically encoded and FRET‐based biosensors to visualize and quantify the specific proteolytic process in intact cells. This was accomplished by stable expression of a fusion substrate construct composed of the green fluorescent protein 2 (GFP²) and red fluorescent protein 2 (DsRed2), with a cleavage motif on eIF4GI or eIF4GII connected in between. The FRET biosensor showed a real‐time and quantifiable impairment of FRET upon EV infection. Levels of the reduced FRET closely correlated with the cleavage kinetics of the endogenous eIF4Gs isoforms. The FRET impairments were solely attributed to 2A^pro catalytic activity, irrespective of other viral‐encoded protease, the activated caspases or general inhibition of protein synthesis in the EV‐infected cells. The FRET biosensors appeared to be a universal platform for several related EVs. The spatiotemporal and quantitative imaging enabled by FRET can shed light on the protease–substrate behaviors in their normal milieu, permitting investigation into the molecular mechanism underlying virus‐induced host translation inhibition. Biotechnol. Bioeng. 2009; 104: 1142–1152. © 2009 Wiley Periodicals, Inc.     

An antiviral peptide inhibitor that is active against picornavirus 2A proteinases but not cellular caspases

The replication of many viruses is absolutely dependent on proteolytic cleavage. Infected cells also use this biological mechanism to induce programmed cell death in response to viral infection. Specific inhibitors for both viral and cellular proteases are therefore of vital importance. We have recently shown that the general caspase inhibitor zVAD.fmk inhibits not only caspases, but also the 2Apro of human rhinoviruses (HRVs) (L. Deszcz, J. Seipelt, E. Vassilieva, A. Roetzer, and E. Kuechler, FEBS Lett. 560:51-55, 2004). Here, we describe a derivative of zVAD.fmk that inhibits HRV2 2Apro but that has no effect on caspase 9. This gain in specificity was achieved by replacing the aspartic acid of zVAD.fmk with methionine to generate zVAM.fmk. Methionine was chosen because an oligopeptide with methionine at the P1 position was a much better substrate than an oligopeptide with an alanine residue, which is found at the P1 position of the wild-type HRV2 2Apro cleavage site. zVAM.fmk inhibits the replication of HRV type 2 (HRV2), HRV14, and HRV16. In contrast to zVAD.fmk, however, zVAM.fmk did not inhibit apoptosis induced by puromycin in HeLa cells. zVAM.fmk inhibited in vitro the intermolecular cleavage of eukaryotic initiation factor 4GI (eIF4GI) by HRV2 2Apro at nanomolar concentrations. However, much higher concentrations of zVAM.fmk were required to inhibit HRV14 2Apro cleavage of eIF4GI. In contrast, intramolecular self-processing of HRV14 2Apro was much more susceptible to inhibition by zVAM.fmk than that of HRV2 2Apro, suggesting that zVAM.fmk inhibits HRV2 and HRV14 replication by targeting different reactions of the same proteinase.     

细胞膜转运分子ABCC3和SLC7A7在肠道病毒A71型感染中作用的初步研究

肠道病毒A71型(enterovirus A71,EV-A71)是导致手足口病(hand-foot-mouth disease,HFMD)的主要病原体之一,目前对其治疗尚无特异高效的抗病毒药物.研究表明,细胞膜转运相关分子参与病毒的入侵、复制以及感染性子代病毒颗粒的释放.为寻找宿主中可有效抑制EV-A71感染的细胞膜转...     

Eukaryotic initiation factor 4GII (eIF4GII), but not eIF4GI, cleavage correlates with inhibition of host cell protein synthesis after human rhinovirus infection

For many members of the Picornaviridae family, infection of cells results in a shutoff of host protein synthesis. For rhinoviruses and enteroviruses, the shutoff has been explained in part by the cleavage of eukaryotic initiation factor 4GI (eIF4GI), a component of the cap-binding protein complex eIF4F. The cleavage of eIF4GI is mediated by the virus-specific proteinase 2Apro and results in inhibition of cap-dependent, but not cap-independent, translation. The inhibition of host protein synthesis after infection with human rhinovirus 14 (HRV-14) lags behind the cleavage of eIF4GI. Recently, we discovered a functional homolog of eIF4GI, termed eIF4GII, and showed that cleavage of eIF4GII coincides with the shutoff of host cell protein synthesis after poliovirus infection (Gradi et al., Proc. Natl. Acad. Sci. USA 95:11089-11094, 1998). We wished to determine whether eIF4GII cleavage kinetics could also explain the lack of correlation between the kinetics of eIF4GI cleavage and the shutoff of host protein synthesis after rhinovirus infection. In this study, we examined the correlation between human rhinovirus-induced shutoff of host protein synthesis and cleavage of eIF4GI and eIF4GII. In HRV-14-infected HeLa cells, almost no intact eIF4GI could be detected by 4 h postinfection, while only 4% of eIF4GII was cleaved at this time. By 6 h, however, 67% of eIF4GII was cleaved, and this cleavage coincided with a significant (60%) decline of host translation. These results suggest that cleavage of both eIF4GI and eIF4GII is required for HRV-mediated inhibition of host cell protein synthesis and that the cleavage of eIF4GII is the rate-limiting step in the shutoff of host cell protein synthesis after rhinovirus infection.     

Human rhinovirus 2 2Apro recognition of eukaryotic initiation factor 4GI. Involvement of an exosite

The 2A proteinase (2Apro) of human rhinovirus 2 is a cysteine proteinase with a unique chymotrypsin-like fold. During viral replication, 2Apro performs self-processing by cleaving between its own N terminus and the C terminus of the preceding protein, VP1. Subsequently, 2Apro cleaves the two isoforms of the cellular protein, eukaryotic initiation factor (eIF) 4G. We have previously shown that HRV2 2Apro can directly bind to eIF4G isoforms. Here we demonstrate using deletion mutants of eIF4GI that HRV2 2Apro requires eIF4GI amino acids 600-674 for binding; however, the amino acids at the cleavage site, Arg681 downward arrow Gly, are not required. The HRV2 2Apro binding domain for eIF4GI was identified by site-directed mutagenesis. Specifically, mutations Leu17 --> Arg and Asp35 --> Glu severely impaired HRV2 2Apro binding and thus processing of eIF4GI in rabbit reticulocyte lysates; self-processing, however, was not affected. Alanine scanning analysis further identified the loop containing residues Tyr32, Ser33, and Ser34 as important for eIF4GI binding. Although Asp35 is part of the catalytic triad, most of the eIF4GI binding domain lies in a unique exosite structure absent from other chymotrypsin-like enzymes and is distinct from the substrate binding cleft. The exosite represents a novel virulence determinant that may allow the development of specific inhibitors for HRV2 2Apro.     

Differential requirements for caspase-8 activity in the mechanism of phosphorylation of eIF2alpha, cleavage of eIF4GI and signaling events associated with the inhibition of protein synthesis in apoptotic Jurkat T cells

Previously we have reported that induction of apoptosis in Jurkat cells results in an inhibition of overall protein synthesis with the selective and rapid cleavage of eukaryotic initiation factor (eIF) 4GI. For the cleavage of eIF4GI, caspase-3 activity is both necessary and sufficient in vivo, in a process which does not require signaling through the p38 MAP kinase pathway. We now show that activation of the Fas/CD95 receptor promotes an early, transient increase in the level of eIF2alpha phosphorylation, which is temporally correlated with the onset of the inhibition of translation. This is associated with a modest increase in the autophosphorylation of the protein kinase activated by double-stranded RNA. Using a Jurkat cell line that is deficient in caspase-8 and resistant to anti-Fas-induced apoptosis, we show that whilst the cleavage of eIF4GI is caspase-8-dependent, the enhancement of eIF2alpha phosphorylation does not require caspase-8 activity and occurs prior to the cleavage of eIF4GI. In addition, activation of the Fas/CD95 receptor results in the caspase-8-dependent dephosphorylation and degradation of p70(S6K), the enhanced binding of 4E-BP1 to eIF4E, and, at later times, the cleavage of eIF2alpha. These data suggest that apoptosis impinges upon the activity of several polypeptides which are central to the regulation of protein synthesis and that multiple signaling pathways are involved in vivo.     

The eIF4G-eIF4E complex is the target for direct cleavage by the rhinovirus 2A proteinase.

The 2A proteinases (2Apro) of certain picornaviruses induce the cleavage of the eIF4G subunit of the cap-binding protein complex, eIF4F. Several reports have demonstrated that 2Apro of rhinovirus and coxsackievirus B4 cleave eIF4G directly. However, it was suggested that in poliovirus infection, the 2Apro induces the activation of a cellular proteinase which in turn cleaves eIF4G. Furthermore, it is not clear whether eIF4G is cleaved as part of the eIF4F complex or as an individual polypeptide. To address these issues, recombinant eIF4G was purified from Sf9 insect cells and tested for cleavage by purified rhinovirus 2Apro. Here we report that eIF4G alone is a relatively poor substrate for cleavage by the rhinovirus 2Apro. However, an eIF4G-eIF4E complex is cleaved efficiently by the 2Apro, suggesting that eIF4F is a preferred substrate for cleavage by rhinovirus 2Apro. Furthermore, 2Apr drastically reduced the translation of a capped mRNA. An eIF4G-eIF4E complex, but not eIF4G alone, was required to restore translation.     

Differential cleavage of eIF4GI and eIF4GII in mammalian cells. Effects on translation

Two isoforms of the translation initiation factor eIF4G, eIF4GI and eIF4GII, have been described in eukaryotic cells. The exact function of each isoform during the initiation of protein synthesis is still under investigation. We have developed an efficient and reliable method of expressing poliovirus 2Apro, which differentially proteolyzes eIF4GI and eIF4GII in a time- and dose-dependent manner. This system is based on the electroporation of an in vitro transcribed mRNA that contains the encephalomyocarditis virus internal ribosome entry site followed by the sequence of poliovirus 2Apro. In contrast to HeLa cells, expression of this protease in BHK-21 cells induces delayed hydrolysis kinetics of eIF4GI with respect to eIF4GII. Moreover, under these conditions the polyadenylate binding protein is not cleaved. Interestingly, translation of de novo synthesized luciferase mRNA is highly dependent on eIF4GI integrity, whereas ongoing translation is inhibited at the same time as eIF4GII cleavage. Moreover, reinitiation of a preexisting mRNA translation after polysome run-off is dependent on the integrity of eIF4GII. Notably, de novo translation of heat shock protein 70 mRNA depends little on eIF4GI integrity but is more susceptible to eIF4GII hydrolysis. Finally, translation of an mRNA containing encephalomyocarditis virus internal ribosome entry site when the two isoforms of eIF4G are differentially hydrolyzed has been examined.     

Granzyme B inhibits vaccinia virus production through proteolytic cleavage of eukaryotic initiation factor 4 gamma 3

Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD(1408)S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery.     

Reverse Genetic Analysis of Adaptive Mutations within the Capsid Proteins of Enterovirus 71 (EV-A71) Strains Necessary for Infection of CHO-K1 Cells

正Dear Editor,Previous studies had described the adaptation of enterovirus 71 (EV-A71) strains that enabled entry and viral replication in Chinese Hamster Ovary (CHO) cell line(Zaini and Mc Minn 2012; Zaini et al. 2012). These adapted     

Antiviral effects of two Ganoderma lucidum triterpenoids against enterovirus 71 infection

Enterovirus 71 (EV71) is a major causative agent for hand, foot and mouth disease (HFMD), and fatal neurological and systemic complications in children. However, there is currently no clinical approved antiviral drug available for the prevention and treatment of the viral infection. Here, we evaluated the antiviral activities of two Ganoderma lucidum triterpenoids (GLTs), Lanosta-7,9(11),24-trien-3-one,15;26-dihydroxy (GLTA) and Ganoderic acid Y (GLTB), against EV71 infection. The results showed that the two natural compounds display significant anti-EV71 activities without cytotoxicity in human rhabdomyosarcoma (RD) cells as evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. The mechanisms by which the two compounds affect EV71 infection were further elucidated by three action modes using Ribavirin, a common antiviral drug, as a positive control. The results suggested that GLTA and GLTB prevent EV71 infection through interacting with the viral particle to block the adsorption of virus to the cells. In addition, the interactions between EV71 virion and the compounds were predicated by computer molecular docking, which illustrated that GLTA and GLTB may bind to the viral capsid protein at a hydrophobic pocket (F site), and thus may block uncoating of EV71. Moreover, we demonstrated that GLTA and GLTB significantly inhibit the replication of the viral RNA (vRNA) of EV71 replication through blocking EV71 uncoating. Thus, GLTA and GLTB may represent two potential therapeutic agents to control and treat EV71 infection.