Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei 112, Taiwan, Republic of China1
Faculty of Medical Technology and Institute of Biotechnology in Medicine, School of Medical Technology and Engineering, National Yang-Ming University, 155 Li-Nong St Sec. 2, Shih-Pai, Taipei 112, Taiwan, Republic of China2
Division of Clinical Virology, Department of Pathology and Laboratory Medicine, Veterans General Hospital-Taipei, Taipei 112, Taiwan, Republic of China3
Author for correspondence: Wu-Tse Liu at Faculty of Medical Technology. Fax +886 2 2826 4092. e-mail wtliu{at}ym.edu.tw
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Abstract |
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Introduction |
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EV71 is a small, nonenveloped, icosahedral RNA virus that possesses a single-stranded RNA genome of approximately 7400 nucleotides of positive polarity (Brown & Pallansch, 1995 ). The genome is predicted to comprise a 5' untranslated region (UTR), a long open reading frame that encodes a protein of approximately 2100 amino acids, a short 3' UTR and a polyadenylated tail (Brown & Pallansch, 1995
). Although the replication mechanism of EV71 is largely unknown, studies from members in the Picornaviridae family revealed that the plus-strand RNA genome is translated into a single large polyprotein. Maturation cleavage of the polyprotein to generate functional viral proteins is mainly mediated by virus-encoded proteases, designated 2A (2Apro) and 3C (3Cpro). Most of the proteolytic reactions are accomplished by 3Cpro, whereas 2Apro catalyses only two cleavages on the polyprotein, one between the capsid protein precursor and itself and another on 3CD to generate 3C' and 3D'.
Apoptosis, or programmed cell death, is a genetically determined cell death programme and provides a natural mechanism to remove damaged cells from tissue. The most typical signs involve a series of cellular events that include the nucleolytic internucleosome degradation of chromosomal DNA, compaction and fragmentation of chromatin, membrane blebbing and cellular shrinkage (Martin et al., 1994 ; Green, 2000
). Recently, several reports focused on the ability of picornaviruses to affect the apoptosis-inducing activities within infected cells, most evidently exemplified by Theiler's murine encephalomyelitis virus (TMEV) (Jelachich & Lipton, 1999
), coxsackievirus B3 (CVB3) (Carthy et al., 1998
) and poliovirus (PV) (Tolskaya et al., 1995
; Agol et al., 1998
, 2000
; López-Gurrero et al., 2000
). Further investigation revealed that expression of 2Apro as the only PV component results in apoptotic cell death (Goldstaub et al., 2000
). Moreover, infection of mice with TMEV or PV induces apoptosis in the central nervous system (CNS) and is associated with the neurovirulent effect or fatal outcome in infected mice (Tsunoda et al., 1997
; Girard et al., 1999
).
Infection with picornaviruses can cause dramatic inhibition of host protein synthesis (Haller & Semler, 1995 ; Sachs et al., 1997
), which is followed by a selective and efficient translation of the viral mRNA. Cellular mRNAs contain a 5'-terminal cap structure that is responsible for initiation of translation (Sonenberg & Gingras, 1998
). In contrast, picornavirus RNAs have in their 5' UTRs a complex structure known as an internal ribosome entry site (IRES) element, which directs a cap-independent mechanism of translation (reviewed by Jackson et al., 1994
; Belsham & Sonenberg, 1996
). An early event occurring during many picornavirus infections is cleavage of the eukaryotic translation initiation factor 4G (eIF4G), either directly or indirectly, by viral 2Apro (Etchison et al., 1982
). There are two related eIF4G species, eIF4GI and eIF4GII, which appear to be functional homologues of each other but share only 46% identity (Gradi et al., 1998
). IRES-directed initiation of virus protein synthesis is maintained following cleavage of the eIF4G species, with concomitant inhibition of capped cellular mRNA translation (Ohlmann et al., 1996
; Borman et al., 1997
; Gradi et al., 1998
; Roberts et al., 1998
).
In this study, we showed that infection with EV71 induces apoptosis, as shown by the data from morphological and biochemical studies. Despite a moderate level of homology to poliovirus 2Apro, EV71 2Apro expression is sufficient to trigger cleavage of eIF4GI and apoptotic cell death.
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Methods |
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Infection with EV71.
Cells were seeded on culture Petri dishes a day before infection with EV71 at an m.o.i. of 0·1. After a 2 h adsorption, the cell layer was rinsed with PBS and MEM containing 2% FBS was added. Cells were then incubated at 37 °C throughout the infection.
Plasmid construction.
Total RNA was extracted from EV71 virus stock with 200 µl RNAzol B solution (TEL-TEST). The cDNA was first synthesized using 100 units of Moloney murine leukaemia virus reverse transcriptase (Promega) at 37 °C for 60 min. One-fifth volume of the product from the reverse transcription reaction was used as a template for PCR for the EV71 (BrCr strain) 2A-coding region (nt 33303779 relative to the predicted transcription initiation site; Brown & Pallansch, 1995 ), with the primers forward, 5' CGGATCCATGGGGAAATTCGGTCAGCAGTC 3', and reverse, 5' GCTCGAATTCCTGCTCCATCGCTTCCTCAT 3'. The 5' and 3' primers were engineered to contain BglII and EcoRI restriction sites for cloning. The conditions for PCR were as follows: 94 °C for 3 min, then 35 cycles of a denaturation step at 94 °C for 1 min, an annealing step at 58 °C for 1 min 30 s and an elongation step at 72 °C for 3 min, and 1 cycle of an elongation step at 72 °C for 10 min. The RTPCR product was subsequently digested and directionally cloned into the BglII and EcoRI sites of plasmid pEGFP-N2 (Clontech). This construct contains the 2A-coding sequences fused in frame to the N terminus of the green fluorescent protein (GFP) gene and is referred to as p2A-GFP. Plasmid p2A-IRES-GFP was constructed by inserting the 2A cDNA fragment into pIRES2-EGFP (Clontech), a plasmid harbouring the GFP reporter gene controlled by the IRES from encephalomyocarditis virus (EMCV). This permits both 2A and GFP genes to be translated from a single bicistronic mRNA. The authenticity of each construct was confirmed by automated DNA sequencing.
Immunofluorescence assay and Hoechst staining.
Infected cells were fixed with 3% paraformaldehyde for 30 min and 0·2% Triton X-100 for 5 min and then incubated with a mouse anti-EV71 monoclonal antibody (MAb) (Chemicon, 3324) at 37 °C for 30 min. Following two washes in PBS, cells were incubated with the secondary antibody, a goat anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC) (ICN), at 37 °C for 30 min. Nuclei were counterstained with a DNA-binding dye, Hoechst 33258 (0·5 µg/ml, Sigma). Cell morphology was visualized under a fluorescence microscope equipped with both FITC and UV filters.
DNA analysis.
Cells were incubated in versene and then suspended in the buffer containing 20 mM EDTA and 10 mM TrisHCl (pH 7·4). Next, cells were treated with 0·5% Triton X-100 at 0 °C for 20 min. After centrifugation (12000 r.p.m. for 15 min at 4 °C), the supernatant was first treated with SDS (at a final concentration of 1%) and then with 10 µg RNase A at 37 °C for 30 min. DNA was then isolated by phenol extraction and ethanol precipitation. DNA was fractionated by electrophoresis in a 1·5% agarose gel, stained with ethidium bromide and visualized under UV light.
DNA transfection.
Approximately 70% confluent monolayer of Vero or HeLa-229 cells grown on 100 mm diameter dishes were transfected with 30 µg of plasmid DNA using the calcium phosphate precipitation method (Sambrook et al., 1989 ). After incubation at 37 °C in 5% CO2 for the appropriate amount of time, the cells were analysed as indicated for each experiment.
Western blot analysis.
Vero, RD and HeLa-229 cells were harvested at various time-points following the infection of EV71 or transfection of 2A-expressing plasmids. Cell extracts were prepared by washing with cold PBS and scraped into the lysis buffer (50 mM TrisHCl, pH 7·5, 150 mM NaCl, 0·5 mM EDTA and 0·5% NP-40). Cell extracts were then mixed with an equal volume of 2x sample buffer (125 mM TrisHCl, pH 6·8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol and 0·2% bromophenol blue), separated by SDSPAGE and transferred onto nitrocellulose membranes. Blots were incubated with mouse anti-PARP [poly(ADP-ribose) polymerase] MAb (1:500 dilution; BD Transduction Laboratories) or mouse anti-eIF4GI antibody (1:250 dilution; BD Transduction Laboratories). Blots were then incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (Sigma), the secondary antibody. Proteins were detected using the Enhanced Chemiluminescence Western Blotting kit (Amersham).
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Results |
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Discussion |
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Cell death occurs by either necrotic or apoptotic pathways. Necrosis is accidental death characterized by rupture of the plasma membrane and release of cytoplasmic constituents. Apoptosis is an active and energy-dependent process of cell death in response to a wide variety of stimuli (Martin et al., 1994 ; Green, 2000
). In vivo, apoptotic cell death is reported to be noninflammatory, while necrotic death is typically accompanied by inflammation (Melcher et al., 1999
). The ability of numerous viruses to elicit apoptosis either directly or indirectly upon infection has been demonstrated (Teodoro & Branton, 1997
; O'Brien, 1998
). This process may represent an important step in the spread of progeny to neighbouring cells, while limiting the inflammatory and immune responses (O'Brien, 1998
), and protecting progeny virus from action of host antibodies. In some cases, specific viral proteins have been identified that are potent apoptosis inducers by themselves, such as E1A of human adenovirus and Tat and Nef of human immunodeficiency virus (Roulston et al., 1999
; Teodoro & Branton, 1997
). In other cases, the presence of viral RNA, rather than viral proteins, is implicated as a trigger. Examples of these are the apoptotic action of the RNA-dependent 2-5A synthetase/RNase L system (Castelli et al., 1997
) and the RNA-dependent protein kinase PKR (Lee & Esteban, 1994
; Lee et al., 1997
). Among human EV, PV was first described as being capable of evoking an apoptotic reaction in several cultured cell lines (Ammendolia et al., 1999
; López-Guerrero et al., 2000
; Tolskaya et al., 1995
), with its 2Apro being implicated in the apoptosis-inducing function (Goldstaub et al., 2000
). In addition, CVB3 was shown to induce apoptosis of HeLa cells (Carthy et al., 1998
). Its capsid protein VP2 was identified to interact with the cellular pro-apoptotic protein Siva and may contribute to the induction of apoptotic events (Henke et al., 2000
). We addressed herein that EV71 2Apro is capable of triggering apoptosis, adding one more to the growing list of pro-apoptotic genes from enteroviruses.
Elucidation of the exact molecular mechanism used by EV71 2Apro to provoke apoptosis must take into account that this protease may cleave a variety of host proteins, including eIF4GI and PARP shown in this report. eIF4GI is a component of the cap-binding complex (eIF4F), which plays a pivotal role in the interaction between capped mRNA and the 40S ribosomal subunit, leading to the initiation of translation (Morley et al., 1997 ). Cleavage of eIF4GI results in the disruption of the eIF4F complex and, hence, inhibition of cap-dependent translation. In addition, it was demonstrated that recombinant 2Apro of EV and HRV can cleave eIF4G directly in vitro, albeit to a much lesser extent (Bovee et al., 1998
; Haghighat et al., 1996
; Lamphear et al., 1993
). This could be explained by the finding that eIF4G alone is a relatively poor substrate, as opposed to the eIF4F complex, which is cleaved efficiently by the HRV 2Apro (Haghighat et al., 1996
). It has been proposed that the shut-off of cap-dependent translation is a major mechanism of the execution phase of apoptosis, which leads to rapid cell death (Clemens et al., 1998
; Marissen & Lloyd, 1998
). Furthermore, it has been noted that eIF4GI also serves as a substrate for activated caspases when cells undergo apoptosis (Clemens et al., 1998
; Marissen & Lloyd, 1998
). However, the cleavage products (ca. 150 and 80 kDa) are distinct from those generated in EV71-infected or EV71 2Apro-transfected cells (Fig. 7
), consistent with the data documented previously for picornavirus-infected cells (Roberts et al., 2000
). Also, it was shown that caspase inhibitors did not inhibit the cleavage of eIF4GI during PV infection (Roberts et al., 2000
). These data suggested that apoptosis could be induced as a consequence of cleavage of eIF4GI in a caspases-independent manner.
With regard to PARP, another host protein susceptible to cleavage by 2Apro reported herein, it is proteolytically cleaved by family members of the ICE-like cysteine protease family, in particular by caspase-3 during the process of apoptosis (Lazebnik et al., 1994 ). More interestingly, the caspase-3 cleavage products have a molecular mass of 85 and 25 kDa, reminiscent of those seen from EV71-infected (Fig. 3
) or 2Apro-transfected cells (Fig. 6
). Moreover, recent reports showed that caspase-3 is activated after CVB3 infection of HeLa cells (Carthy et al., 1998
) or PV infection of U937 cells (López-Guerrero et al., 2000
). Thus, the mechanism by which EV71 2Apro-induced apoptosis may be a caspase-3-dependent event. In fact, our preliminary study indicated that PARP remained intact in the EV71-infected or EV71 2Apro-transfected MCF-7 cells deficient in caspase-3 (unpublished data). Taken together, it is tempting to assume that the cleavage of PARP and eIF4G isoforms occurs at distinct stages during the apoptotic process. In this case, eIF4G cleavage may be directed toward blocking de novo cap-dependent translation. It may initiate an apoptotic event without the action of caspases, while PARP cleavage is the consequence of caspase-3 activation at the later phase of apoptosis. Nonetheless, it is conceivable that EV71 2Apro induces apoptosis indirectly by activation of other still unidentified cellular substrates pertinent to an endogenous cell suicide program. Further experiments are needed to better delineate the mechanism(s) by which EV71 2Apro induces apoptosis.
Induction of apoptosis by virus infection is emerging as an important aspect of pathogenesis. Cell damage in the CNS that is caused by its response to a virus infection can involve apoptosis (Oberhaus et al., 1997 ; Tsunoda et al., 1997
). Virus-induced apoptosis has been shown to be an important component of PV-induced cell death and tissue injury in the CNS of infected mice (Girard et al., 1999
). EV71, like PV, invades the CNS to give rise to several syndromes, notably aseptic meningitis, encephalomyelitis and flaccid paralysis. Therefore, this work represents an initial attempt to unveil the relevance of EV71-induced apoptosis for human diseases. Moreover, discerning the trigger that initiates apoptosis in a virus-infected cell, in this case the EV71 2Apro, should be of benefit to understanding virus pathology as well as the development of effective inhibitors targeted to this viral protein.
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Acknowledgments |
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Received 23 November 2001;
accepted 30 January 2002.