Nicolas Hili
email: hili@cs.queensu.ca

A Metamodeling Framework for Promoting Flexibility and Creativity Over Strict Model Conformance

This paper defines FlexiMeta, a metamodeling framework intended to promote more flexibility and creativity while not compromising validation through model conformance. It advocates less coupling between models and metamodels in order to make the creation of models and user-defined metamodels possible in an arbitrary order. It comes along with a generic process structured into several phases. For each phase, a proper balance between flexibility and validation is found in order to bridge the gap between creativity and strict model conformance.

Introduction and Motivation

Metamodeling techniques [START_REF] Fondement | Defining model driven engineering processes[END_REF] increase validation through model conformance. However, they drastically decrease flexibility as user-defined metamodels have to be created first and each change to the metamodels questions the validity of existing models [START_REF] Gomez | An approach to the cocreation of models and metamodels in Enterprise Architecture Projects[END_REF]. Consequently, traditional Model-Driven Engineering (MDE) approaches fail to address some issues, such as the availability, evolution, and multiplicity of metamodels as well as the prototyping of models.

Whether introducing flexibility in MDE processes is efficiently addressed or not will ultimately depend on how they are realized by modeling frameworks. Consequently, considerations have to be made about the implementation of such frameworks. Particularly, design considerations of the internal representations of a model, given a programming language and a data serialization format, and their impact at both object-and meta-levels, should be cautiously made.

This paper presents FlexiMeta, a metamodeling framework for promoting flexibility and creativity during the model creation process over a strict model conformance according to a metamodel. This framework derives from previous lessons learned during an inter-organizational project involving both industrial and academic partners from the nuclear-plant system field [START_REF] Hili | Innovative Key Features for Mastering Model Complexity: Flexilab, a Multimodel Editor Illustrated on Task Modeling[END_REF]. In opposition to traditional MDE frameworks that promote a strict coupling between models and metamodels, less coupling between models and metamodels is advocated in FlexiMeta, in order to address the problem of conception of models and userdefined metamodels in an arbitrary order, i.e., without assuming the existence of one before the other, and without assuming any relationship (one-to-one or one-to-many) between them.

This paper is structured as follows: Section 2 details FlexiMeta; Section 3 sketches a first implementation in which FlexiMeta is used; Related work is discussed in section 4; Section 5 concludes.

FlexiMeta

This section introduces FlexiMeta, a metamodeling framework which promotes more flexibility and creativity over a strict model conformance. Throughout this section, FlexiMeta is illustrated over the Families case study, a popular example to illustrate model-to-model transformations 3 .

Fig. 1 shows the architecture of FlexiMeta, highlighting the internal representations (horizontally) of a model at object-and meta-levels (vertically). Dashed nodes and edges depict components that can be used at different times, depending on the intent of the modeling. Therefore, during the first phases of a project, relying on the metamodel is not required to create models. It prevents from validating the created models but it increases creativity and flexibility. On the other side, the metamodel definition is required if one wants to ensure model conformance. In other words, the proposed framework allows for balancing flexibility and validity during time and with respect to the intent of the modeling. In the following, we will describe each part of the framework. The instanciated objects are not distinguishable by genre, as the model does not conform to a metamodel. Consequently, both the Simpson and the Homer objects are instanciated the same way. In FlexiMeta, each object is uniquely idenfitied by a Universal Unique IDentifier (UUID) which is generated during its instanciation by the Base meta-object. Object attributes and references are created using the get and set functions defined by the Base meta-object.

Code Generation. The JavaScript Base meta-object fosters creativity and flexibility by providing a minimal structure to extend. However, objects cannot be validated according to a metamodel definition. FlexiMeta provides a code generation process to generate meta-objects from a metamodel. The code generation process is similar to how Eclipse Modeling Framework (EMF) generates Java classes from an Ecore metamodel. However, the main difference is that this step is optional, and one can simply inherit from the prototype of the Base meta-object, as described above.

Fig. 3 gives a glimpse of the artifact created during the code generation process. For each concept of the metamodel, a corresponding meta-object is created. The code generator allows for the generation of different artifacts, such as: [START_REF] Fondement | Defining model driven engineering processes[END_REF] getter and setter functions for each attribute of the concept; (2) a validate function to ensure that the model object is well constructed with respect to the meta-object definition; (3) import and export functions to ensure the interoperability with Ecore models. It decreases flexibility and creativity but improves validation and model conformance.

Fig. 3: FlexiMeta Code Generator

The code generator has been written using Acceleo and takes an Ecore metamodel as an input to generate JavaScript meta-objects that inherit from the Base meta-object (cf. Fig. 1). This choice was made to ensure interoperability between Ecore and FlexiMeta. The generator creates the required import and export functions to be able to import models from and export them to Eclipse.

This step is optional. When a metamodel is created, meta-objects can be generated from it. Existing objects that were previously typed with the Base meta-object can then be retyped using the newly generated meta-objects. Fig. 3 gives an example of the created meta-objects. Two meta-objects are created: Family and Individual. Each of them inherits from the Base meta-object, and getters and setters are generated from the metamodel. Two additional functions have been generated to validate the model and to export it into Ecore.

The validate function is recursive and detects different constraint violations. So far, it can check multiplicity violations, non-existence of required attributes, malformed value for enumerations, and existence of unexpected attributes. For example, in Fig. 3, the validator can detect that the catchphrase attribute of Homer Simpson is unexpected as it does not exist in the metamodel, and that the city attribute does not exist in the Simpson family object while it was defined as a required attribute by the metamodel. Once all these constraints are verified for one model object, the validate function hands over to the validate function of each composed element. It is worth noting that references and compositions are not distinguishable in JavaScript. Instead, this distinction exists in the metamodel. Consequently, the order of calls of the validate sub-function is inferred during the code generation process.

The toEcore function is generated from the Ecore metamodel and is specific to this metamodel definition. Listing 1.1 illustrates the export of the Simpson family model into Ecore. As the export feature is specific to the metamodel definition, compositions and references are observed to fit with the metamodel structure. In addition, unexpected object attributes are simply ignored and not exported. Finally, it is worth noting that, given the same set of attributes during the JSON export and the eXtensible Markup Language (XML) export, the size of the serialized model in JSON is reduced up to 40% compared to the size of the serialized model in XML. This result can be explained by the lightweight notation of JSON and shows how FlexiMeta addresses scalability. Serialization. FlexiMeta can serialize models to and deserialize models from JSON. JSON is a standardized, platform-independent data serialization format that is natively supported by several languages without loading extra libraries. It has a very lighweight notation which makes it efficient to process. For example, it does not distinguish nested nodes from attributes4 as XML does. Therefore, it may be less human-readable5 , however, it deters the temptation to replicate the metamodel structure to the serialized data.

For serializing models to and deserializing models from JSON, an opportunist serialization engine has been implemented. The term "opportunist" designates that the serialization engine serializes data "as it arrives". It brings several benefits. It is not specific to a metamodel, and is therefore generic. Consequently, it can be used to serialize and deserialize models even if the metamodel is implicit or not formalized. If the metamodel does not exist, the deserialization engine will instanciate objects using the Base meta-object that has been defined. If the metamodel is defined, then the deserialization process deserializes the model by using the generated meta-objects defined during the code generation process. Listing 1.2 shows how the model is serialized in JSON. An object ({}) contains several key-value pairs separated with commas. E.g., the Simpson object has four pairs: uuid, lastname, address, and members. The value of the members key is an array ([]) of objects. Listing 1.2 illustrates how data is serialized as it arrives. When the serializer has to process the spouse attribute of the Homer object, the Marge object has not been serialized yet. Consequently, the Marge object is serialized inside the spouse attribute. When it comes to serialize the Marge object in the Simpson's members attribute, only its UUID is serialized.

Listing 1.2: Serialization in JSON

Implementation

A preliminary implementation of a web-based modeling environment has been developed to allow everyone to exercise FlexiMeta6 (cf. Fig. 4). It allows for the creation of the Simpson family model. It is composed of several areas.

The main area 1 depicts a graphical editor to edit the Simpson Family model. The model consists of the family composed of several members. Model Fig. 4: Case Study: The Simpson Family editing can be done using the contextual menu by right-clicking on the different graphical elements. Below the graphical editor, two views 2 and 3 allow for model serialization in both JSON and XML. JSON serialization is available every time while XML serialization can only be done when the metamodel is known (i.e., after the meta-objects are generated from the metamodel). On the left-hand side of the graphical editor, a model explorer 4 offers a tree view representation of the model. It is updated every time the model changes. Below the model explorer, a validation view 5 displays the list of conformance errors that occur on the model. As for the XML exporter view, validation can only be processed after the code is generated.

FlexiMeta comes along with a generic process structured into three phases exploration, consolidation, and finalization -, which address specific intents. The exploration phase allows the user to create models without relying on userdefined metamodels. It promotes creativity but prevents from validating models as no metamodel has been defined yet. In the consolidation phase, the metamodel is known and this phase is used to align the created models to fit with the metamodel definition. Therefore, validation is possible, but it is still possible to create models which do not conform with the metamodel. Finally, the finalization phase is intended to create only valid models regarding the metamodel definition. This generic process has been proposed to offer a trade-off between flexibility and validation at specific times during the creation of models. Due to the limited space, the generic process is not further detailed.

To go through the generic process, a view 6 reminds the user in which phase he or she is. A radar chart displays level and comment for each challenge Flexi-Meta intends to address during the current phase. In addition, a button allows the user to move forward to the next phase. Finally, a last view 7 illustrates the metamodel used during the consolidation and the finalization phases.

A significant body of literature addresses the problem of flexibility in existing MDE approaches. Model / metamodel co-evolution techniques were proposed to withstand metamodel evolution and to automatically or semi-automatically adapt models [START_REF] Wachsmuth | Metamodel Adaptation and Model Co-adaptation[END_REF][START_REF] Cicchetti | Automating Coevolution in Model-Driven Engineering[END_REF]. These techniques usually rely on the identification of some transformation patterns (e.g., creation of new concepts, deletion of existing concepts, addition to some attributes, etc.). Unfortunately, they cannot be fully automated as some model evolutions cannot be inferred. At best, a variation could be semi-assisted by a human intervention. At worst, a variation could require to design a specific model transformation to migrate existing models.

Bottom-up metamodeling techniques consist in inferring what the metamodel should be regarding a set of existing models [START_REF] Gomez | An approach to the cocreation of models and metamodels in Enterprise Architecture Projects[END_REF][START_REF] Sánchez-Cuadrado | Bottom-up meta-modelling: An interactive approach[END_REF][START_REF] Cho | Creating Visual Domain-specific Modeling Languages from End-user Demonstration[END_REF]. It allows for the creation of a model independent of the metamodel definition. By analogy, we can compare this approach with NoSQL databases from which schemas do not have to be defined. In that case, the metamodel exists, implicitly behind the inference mechanism. As of the first technique, some automations could not be fully automated. For instance, it is not possible to infer the multiplicity of a relation.

Metamodel extension techniques leverage the use of general-purpose modeling languages, such as Unified Modeling Language (UML), instead of defining new metamodels from scratch [START_REF] Weisemöller | A Comparison of Standard Compliant Ways to Define Domain Specific Languages[END_REF][START_REF] Selic | A systematic approach to domain-specific language design using uml[END_REF][START_REF] Langer | Emf profiles: A lightweight extension approach for emf models[END_REF]. Several mechanisms exist. For example, UML can be extended using the UML profile mechanism. Metamodel extension answers to specific challenges, such as the availability of user-defined metamodels, and model prototyping, as the existing metamodel to extend already exists. Then, even if the extension has not been defined yet, it is still possible to quickly prototype the system to define.

As for Multi-level modeling techniques [START_REF] Atkinson | The Essence of Multilevel Metamodeling[END_REF] and tools (e.g., MetaDepth [START_REF] Lara | Deep meta-modelling with metadepth[END_REF]), the same distinction is made between linguistic (i.e., the language) and ontological type. With respect to the nature of the prototype relation in JavaScript, objects in FlexiMeta can be created with no ontological type first, and then be further retyped given a user-defined metamodel. In addition, the framework precisely defines how and when (i.e., during which phase) objects are retyped, in order to align the model definition with the user-defined metamodel.

Existing techniques usually address specific and localized issues, such as metamodel evolution or model co-adaptation. Our work intends to address all the aforementioned challenges at once. An attempt was done to address them in our previous work [START_REF] Hili | Innovative Key Features for Mastering Model Complexity: Flexilab, a Multimodel Editor Illustrated on Task Modeling[END_REF], where a metamodel-tolerant approach and a loose-model conformity control were defined, but the problem space and the methodology to address it were both not formalized. In FlexiMeta, a model can be created before the metamodel (model prototyping). Once the metamodel is defined, model conformance can be checked to help designers migrate both the model and the metamodel (metamodel evolution and co-adaptation).

Several modeling frameworks were proposed over time. EMF [START_REF] Steinberg | EMF Eclipse Modeling Framework, ser. The Eclipse Series[END_REF] and Visualization and Modeling SDK (VMSDK) [START_REF] Cook | Domain-specific Development with Visual Studio Dsl Tools[END_REF] rely on a code generation approach. In Eclipse, Java classes are generated for each concept of the metamodel. Models are serialized in XML using a specific serializer generated alongside the Java implementation of the metamodel concepts. The mapping between the modeling language, data serialization format, and programming language appears at the object-level and the meta-level. Unfortunately, the combination of the class-based programming language with a schema-dependent data serialization format severely affects the flexibility during the modeling activities. Some attempts were done to bring capabilities of EMF into web-based environments. EMF-Rest [START_REF] Costa | Towards a View-Based Process for Designing and Documenting RESTful Service Architectures[END_REF][START_REF] Ed-Douibi | Emf-rest: Generation of restful apis from models[END_REF] is a framework that intends to bring EMF capabilities through a REST API. Another interesting framework is Ecore.js7 , which is developed in JavaScript and available through NodeJS.

Moddle8 is a utility library for creating user-defined metamodels using Java-Script and JSON. A metamodel can be defined at run-time and models can be created using it. However, models cannot be created without defining the metamodel in JSON first. Besides, models cannot be validated and Moddle provides a limited model coverage. For example, it is not possible to define enumerations.

MoDiGen [START_REF] Gerhart | Approach to define highly scalable metamodels based on json[END_REF] is an interesting work to address the scalability challenge. Models and metamodels are defined using JSON. However, it seems that there is no implementation to use it as no programming language is mentioned. Moreover, there is no mention about how models are concretely "instanciated" from the metamodel definition in JSON.

Compared to other frameworks, FlexiMeta relies on a code generation process to generate JavaScript implementation of concepts from external metamodels (Ecore metamodels, so far). This part is not mandatory though, and one can use the minimal implementation using the Base JavaScript meta-object (cf. Section 2). Flexibility and validation challenges are not addressed simultaneously and can be balanced over time, which allows one to benefit from both.

Conclusion

This paper introduces a new metamodeling framework for promoting more flexibility when creating models and metamodels. Unlike existing approaches, it balances flexibility and strict model conformance throughout the development life-cycle. To do so, less coupling between a model and a metamodel is advocated to give more freedom during the modeling activities. A preliminary tool has been sketched to exercise the new metamodeling framework.

The prototype-based programming style of JavaScript, combined to the use of a schema-free data serialization format opens up some interesting horizons for the development of new modeling frameworks. For instance, JavaScript supports the dynamic creation of meta-objects at run-time and at different levels of modeling (deep instanciation). FlexiMeta could take advantage of it to support the binding of models to several metamodels or several versions of the same metamodel at the same time, in order to adress issues such as the multiplicity of metamodels and metamodel evolution.

Listing 1 . 1 :

 11 Export into Ecore 1 < family:Family xmi:version = " 2.0 " 2 xmlns:xmi = " http: // www . omg . org / XMI " 3 xmlns:xsi = " http: // www . w3 . org /2001/ XMLSchema -instance " 4 xmlns:family = " http: // family /0.1 " x s i : s c h e m a L o c a t i o n = " http: // family /0.1 family . ecore " lastname = " Simpson " uuid = " d229e52c -4 e18 -4261 -9144 -... " > 5 < f a m i l y : I n d i v i d u a l uuid = " 6720 a6c4 -eedc -4 b0c -... " age = " 43 " > </ f a m i l y : I n d i v i d u a l > 6 < f a m i l y : I n d i v i d u a l uuid = " cdb9eb06 -4 c13 -492 b -9262 -... " age = " 49 " spouse = " 6720 a6c4 -eedc -4 b0c -... " > </ f a m i l y : I n d i v i d u a l > 7 </ family:Family >

http://www.eclipse.org/atl/documentation/old/ATLUseCase_ Families2Persons.pdf.

The concept of attributes does not exist in JSON.

It is controversial though, as the learning curve to understand JSON is smoother thanks to its light notation.

This environment is available at http://fleximeta.net/demo-models2016.

Available here: https://github.com/emfjson/ecore.js.

Available here: https://github.com/bpmn-io/moddle.


Dr Sandra Martin-Latil

Laurence Mousson

Arnaud Autret

Florence Colbère-Garapin

Bruno Blondel

Bax is activated during rotavirus-induced apoptosis through the mitochondrial pathway

Rotaviruses are the leading cause of infantile viral gastroenteritis worldwide. Mature enterocytes of the small intestine infected by rotavirus undergo apoptosis and their replacement by less differentiated dividing cells probably leads to defective absorptive function of the intestinal epithelium, and this contributes to osmotic diarrhea and rotavirus pathogenesis.

Here, we show that the infection of MA104 cells by the simian rhesus rotavirus strain RRV induced caspase-3 activation, DNA fragmentation and cleavage of poly(ADP-ribose) polymerase (PARP); all three phenomena are features of apoptosis. RRV induced the release of cytochrome c from mitochondria to the cytosol indicating that the mitochondrial apoptotic pathway was activated. RRV infection of MA104 cells activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change. Most importantly, Bax-specific small interfering RNAs partially inhibited the cytochrome c release in RRV-infected cells.

Thus, mitochondrial dysfunction induced by rotavirus is Bax-dependent. Apoptosis presumably leads to impaired intestinal functions, so our findings contribute to improve our understanding of rotavirus pathogenesis at the cellular level.

INTRODUCTION

Rotavirus is a nonenvelopped, double-stranded RNA virus belonging to the Reoviridae family. It is the major etiologic agent of severe gastroenteritis in children under 5 years of age and causes 600,000 deaths per year [START_REF] Parashar | Rotavirus and severe childhood diarrhea[END_REF]. Rotavirus infection is mainly restricted to the small intestinal villus epithelium, resulting in villus atrophy. Diarrhea associated with rotavirus is a multifactorial process: it involves dysfunctions of both nutrient digestion and enterocyte absorption at the top of villi, and the secretion of water and electrolytes [START_REF] Lundgren | Pathogenesis of rotavirus diarrhea[END_REF][START_REF] Morris | Microbes and microbial toxins: paradigms for microbial-mucosal interactions. VIII. Pathological consequences of rotavirus infection and its enterotoxin[END_REF][START_REF] Ramig | Pathogenesis of intestinal and systemic rotavirus infection[END_REF]. The defective absorptive function and the increased intestinal permeability may be a consequence of the replacement of rotavirus-infected mature enterocytes by less differentiated cells and this could contribute to rotavirus pathogenesis. In a murine model, rotavirus-infected enterocytes undergo apoptosis and are therefore lost [START_REF] Boshuizen | Changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice[END_REF]. It is therefore likely that apoptosis makes a major contribution to diarrhea associated with rotavirus infection.

Cell death by apoptosis is part of the normal development and maintenance of homeostasis [START_REF] Vaux | Cell death in development[END_REF] but is also involved in pathological situations associated with infections [START_REF] Hofman | Roles and mechanisms of apoptosis in infectious diseases[END_REF][START_REF] Roulston | Viruses and apoptosis[END_REF] and other (3) causes. Apoptosis is characterized by chromatin condensation, cell shrinkage, membrane bledding, and DNA fragmentation. It can be generally divided into two nonexclusive pathways, the death receptor pathway (extrinsic) and the mitochondrial pathway (intrinsic) [START_REF] Danial | Cell death: critical control points[END_REF][START_REF] Green | The pathophysiology of mitochondrial cell death[END_REF][START_REF] Jin | Overview of cell death signaling pathways[END_REF]. In the extrinsic pathway, stimulation of death receptors, such as Fas and TNF receptor 1, leads to formation of the death-inducing signaling complex (DISC) which allows the activation of caspase-8 and/or caspase-10, and then to that of downstream effector caspases, particularly caspase-3 [START_REF] Ashkenazi | Death receptors: signaling and modulation[END_REF][START_REF] Muzio | FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex[END_REF]. The intrinsic pathway is initiated in response to diverse apoptotic stimuli and leads to the loss of mitochondrial transmembrane potential and the release of several pro-apoptotic proteins, including cytochrome c and Smac/Diablo, from the mitochondrial intermembrane space to the cytosol. Once released, cytochrome c forms a complex with Apaf-1 and pro-caspase 9, resulting in activation of this initiator caspase, and then of effector caspases [START_REF] Wang | The expanding role of mitochondria in apoptosis[END_REF]. Smac/Diablo promotes caspase activation by directly binding to and inhibiting the caspase inhibitors of the IAP family [START_REF] Van Loo | The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet[END_REF][START_REF] Wang | The expanding role of mitochondria in apoptosis[END_REF]. This mitochondrial apoptotic pathway is tightly controlled by protein members of the Bcl-2 family. Some, including Bcl-2 and Bcl-X L , inhibit apoptosis, whereas others, including Bax and Bid, induce apoptosis [START_REF] Cory | The Bcl2 family: regulators of the cellular life-ordeath switch[END_REF].

The extrinsic and intrinsic pathways may cross-talk through the pro-apoptotic protein Bid.

Indeed, caspase-8 can cleave Bid to generate a truncated form, tBid that targets mitochondria and activates the pro-apoptotic protein Bax [START_REF] Li | Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis[END_REF][START_REF] Li | Relief of extrinsic pathway inhibition by the Bid-dependent mitochondrial release of Smac in Fas-mediated hepatocyte apoptosis[END_REF][START_REF] Luo | Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors[END_REF].

There have been very few studies of rotavirus-induced apoptosis in primate cells in vitro. An early study in human carcinoma HT29 cells indicated that rotavirus induced peripheral condensation of the chromatin and fragmentation of the nuclei, suggesting that apoptosis was induced in infected cells [START_REF] Superti | Induction of apoptosis in HT-29 cells infected with SA-11 rotavirus[END_REF]. A more recent study in fully differentiated Caco-2 cells indicated that rotavirus induced apoptosis in these cells, and did so through the mitochondrial pathway [START_REF] Chaibi | Rotavirus induces apoptosis in fully differentiated human intestinal Caco-2 cells[END_REF]. However, the precise signaling pathways leading to mitochondrial dysfunction following rotavirus infection have not been investigated.

Here, we studied apoptosis induced by the Rhesus Rotavirus (RRV) strain in the monkey kidney MA104 cells, the cellular model in which the rotavirus cycle has been best characterized. We first confirmed that RRV-induced apoptosis in this model occurs through the mitochondrial pathway as observed in Caco-2 cells. We then investigated the cascade of events related to mitochondrial dysfunction. We report here that mitochondrial apoptotic pathway in RRV-infected MA104 cells is Bax-dependent. It is, to our knowledge, the first demonstration of Bax-dependent apoptosis in rotavirus-infected cells.

MATERIALS AND METHODS

Materials -Chemicals

Protein G (P3296), Staurosporine (S4400) and mouse anti-tubulin antibody (T5168) were obtained from Sigma-Aldricht. CompleteTM protease inhibitor mixture was obtained from Roche Applied Science. z-VAD-fmk ( 627610

Cells and Virus

The monkey kidney MA104 cell line (MA104 cells) was cultured in minimum Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine and 0.1 mM non-essential amino acids, in a 5% CO2 incubator. The rotavirus RRV strain was kindly provided by Didier Poncet (Gif-sur-Yvette, France). Virus stocks were generated in MA104 cells in a serum-free culture medium supplemented with trypsin (0.5 µg/ml). Viruses were activated by treatment with trypsin at 37°C for 30 min, and MA104 cell monolayers were infected at a multiplicity of infection (m.o.i.) of 0.002 plaque-forming units/cell. After 1 h of adsorption at 37°C, the inoculum was removed and the infected cells were incubated in serum-free medium containing trypsin. When cytopathic effects were complete, the cultures were frozen and then thawed, and the cell debris was removed by centrifugation.

To infect MA104 cells, RRV was activated in serum-free medium with 0.5 µg/ml trypsin.

Cells were washed twice in serum-free medium and then infected at an m.o.i of 10. The inoculum was removed 1 h later and replaced with medium containing 10% FBS. This time was defined as 0 h post-infection for all experiments.

Assessment of apoptosis by flow cytometry

The percentages of cells that were apoptotic was determined by flow cytofluorimetric analysis of aliquots of 2 x 10 6 cells incubated with acridine orange (AO), a metachromatic nuclear dye (λEx max : 500 nm, λEm max : 526 nm), for 15 min at 37°C. AO fluorescence was measured with a FACScan machine (Becton Dickinson). Two populations of cells were separated, one consisting of the living cells characterized by a bright fluorescence labeling, and the second being apoptotic cells with a characteristic distinct pattern of reduced fluorescence intensity [START_REF] Estaquier | Programmed cell death and AIDS: significance of T-cell apoptosis in pathogenic and nonpathogenic primate lentiviral infections[END_REF]. We analyzed at least 10,000 cells for each sample. Data were analyzed with Cellquest software (Becton Dickinson).

Quantification of oligonucleosomal DNA fragmentation

DNA fragmentation was assayed using a cell death detection ELISA kit (Roche Applied Science) with RRV-infected MA104 cells plated in 6-well culture dishes according to the manufacturer's instructions; the test measures the cytosolic histone-associated mono-and oligonucleosome fragments. The concentration of nucleosomal fragments in cytosolic fractions was determined by a sandwich enzyme-linked immunosorbent assay, using histone-specific antibodies preadsorbed onto microtiter plates and peroxidase-conjugated antibodies against DNA. Peroxidase activity was measured photometrically at 405 nm. The experiments were run in triplicate.

Detection of active caspase-8

Caspase-8 activation was detected using a carboxyfluorescein FLICA apoptosis detection kit (Immunochemistry Technologies, LLC) according to manufacturer's protocol. This assay is based on a fluorescein-labeled inhibitor (FAM-LETD-fmk) that binds specifically to active caspase-8. Apoptotic cells containing active caspase-8 were detected with a FACScan (Becton Dickinson) machine. We analyzed at least 10,000 cells for each sample. Data were analyzed with Cellquest software (Becton Dickinson).

Transfection of small interfering RNA (siRNA)

RNA interference was used to silence Bax gene expression in MA104 cells. Cells were plated in 6-well culture dishes with complete medium and allowed to grow for 24 h, so as to reach 50-80% confluence. The cells were then transfected with siRNA (6321, Cell Signaling) targing Bax mRNA. An irrelevant siRNA (6201, Cell Signaling) that does not lead to the specific degradation of any cellular mRNA was used as a negative control. A mixture of OptiMEM medium and LipofectAMINE was incubated for 10 min at room temperature and was then incubated with siRNA (25 nM) for 20 min at room temperature to allow complex formation. This siRNA mixture was then added to each well following the manufacturer's suggested protocol. Twenty-four hours after transfection, the medium was changed and cells were infected with the virus 24 h later. Gene silencing was verified by testing for proteins by immunoblot analysis after transient transfection of MA104 cells with siRNA.

Whole cell extracts

Approximately 5.10 6 cells were collected and washed with PBS, then resuspended in lysis buffer (20 mM Tris pH 7.5, 135 mM NaCl, 2 mM EDTA, 1% Triton-X100, 10% glycerol) supplemented with a protease inhibitor mixture (Roche). The cells were homogenized on ice using dounce homogenizer and incubated for 10 min at 4°C in the lysis buffer. The lysates were clarified by centrifugation for 10 min at 1200 g. The supernatant was collected as the whole cell extract.

Subcellular fractionation

A subcellular proteome extraction kit (Calbiochem) was used to isolate cytosol and heavy membrane fractions of MA104 cells according to the manufacturer's instructions. Cells (5.10 6 ) were harvested, pelleted, washed twice, resuspended in the ice-cold Extraction I buffer containing a protease inhibitor mixture and incubated for 10 min at 4°C with gentle agitation.

The suspension was centrifuged at 1000 g at 4°C for 10 min. The supernatant was used as the cytosol fraction. The pellet was resuspended in the ice-cold Extraction II buffer containing a protease inhibitor mixture and incubated for 30 min at 4°C with gentle agitation. It was then centrifuged at 6000 g for 10 min at 4°C, and the supernatant was used as the heavy membrane fraction.

Detection of Bax conformation change by immunoprecipitation

Cells were harvested, washed in PBS and suspended in lysis buffer (10 mM hepes pH 7.4, 150 mM NaCl, 1% Chaps) containing a mixture of protease inhibitors. The zwitterionic detergent Chaps was used because it does not affect Bax conformation [START_REF] Hsu | Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations[END_REF]. Cells were homogenized on ice with dounce homogenizer, incubated for 2 h in the lysis buffer and centrifuged for 30 min at 14000 g. The resulting supernatant was incubated overnight at 4°C with 20 µl of protein G and 2 µg of the anti-Bax antibody (6A7). The immunoprecipitates were collected by centrifugation at 14000 g (4°C, 5 min). The pellets were washed with immunoprecipitation buffer and suspended in 50 µl of Laemmli's buffer containing reducing agent (Invitrogen).

Western-blot analysis

Protein concentrations were determined by using a Bio-Rad protein assay kit (Bio-Rad, Richmond, CA). Samples of equal protein content were resuspended in Laemmli's electrophoresis sample buffer containing reducing agent, denatured by boiling for 5 min, subjected to SDS-PAGE (10-20% Tricine gels; Novex) and transferred to nitrocellulose membranes (Amersham Biosciences). Nonspecific sites were blocked by incubating the membranes for 1 h at room temperature with 5% nonfat milk and 0.1% Tween 20 in Phosphate-Buffered Saline (PBST, pH 7.4), and the membranes were incubated overnight at 4°C or 2 h at room temperature with the primary antibody. Membranes were then washed in PBST and treated with appropriate horse-radish peroxidase-conjugated secondary antibody for 1 h at room temperature. The immunoblots were washed in PBST and developed using an enhanced chemiluminescence detection kit (Amersham Biosciences). Anti-tubulin and Cox IV antibodies were used to verify equal protein loading.

Statistical Analysis

Data are expressed as means ± SEM of three independent experiments. Student's test was used to compare experimental conditions and controls. A value of P<0.05 was considered significant.

RESULTS

RRV infection induces apoptosis in MA104 cells through the mitochondrial pathway.

Apoptosis induced by RRV in MA104 cells was investigated by following the kinetics of DNA fragmentation with the Cell Death ELISA kit (Roche) (Fig. 1A). Early p.i. (until 8h p.i.), DNA fragmentation was not significantly different in infected and uninfected cells.

Enrichment of nucleosomal DNA fragments started by 12 h p.i. and increased until 20 h p.i. DNA fragmentation 20 h p.i. was 3-to 4-fold that of mock-infected cells and similar to that observed after 14 h of treatment with staurosporine, used as positive control for DNA fragmentation. These results confirm that rotavirus induced apoptosis in MA104 cells.

We then looked for activation of caspase-3, the central executioner in the apoptotic program, in RRV-infected MA104 cells. Cleavage of 32 kDa procaspase-3 revealed by the detection, in a whole cell lysate, of its 19 kDa cleavage product was substantial at about 14 h p.i. (Fig. 1B).

To confirm the activation of caspase-3 in RRV-infected MA104 cells, we investigated the processing of one of its characteristic substrates, the poly(ADP-ribose) polymerase (PARP) (Fig. 1B). The amount of full-length PARP in a whole lysate of RRV-infected MA104 cells declined from 14 to 22 h p.i., thus paralleling the pattern of caspase-3 activation. These results show that rotavirus induced caspase executioner activation in MA104 cells. Furthermore, the peak of caspase-3 activation coincided with that of virus production during a single-cycle infection in the same experimental conditions (data not shown).

To assess the role of caspases in cell death resulting from RRV infection, infected cells were treated with a broad-spectrum caspase inhibitor, z-VAD-fmk (10 µM, Calbiochem). The roles of caspase-9 and caspase-3 were investigated with specific inhibitors, z-LEHD-fmk (10 µM, Calbiochem) and z-DEVD-fmk (10 µM, Calbiochem), respectively. Apoptosis was analyzed 14 h p.i., by measuring chromatin condensation and fragmentation by flow cytometry after Acridine Orange (AO) staining (Fig. 1C). The three caspase inhibitors significantly reduced the percentage of apoptotic cells following RRV infection with respect to that in their untreated counterparts. (The same level of apoptosis inhibition was observed with z-VADfmk at a concentration of 100 µM, data not shown). These results indicate that caspases are, at least partly, involved in RRV-induced apoptosis. Furthermore, the decrease in apoptosis in cells treated with the caspase 9 inhibitor seems to indicate that the mitochondrial pathway is activated in RRV-infected cells.

To confirm that the mitochondrial pathway is activated during RRV infection in MA104 cells, we investigated the kinetics of cytochrome c and Smac/Diablo efflux from mitochondria into the cytosol. Infected cells were fractionated to separate the cytosolic fraction from the heavy membrane fraction containing mitochondria, at various times after infection, and the fractions were studied by western blotting (Fig. 2). Cytochrome c and Smac/Diablo were detected in the cytoplasm of infected MA104 cells from 9 h p.i. and 12 h p.i., respectively: they increased in amount in the the cytosol thereafter until the last time point investigated (18 h p.i.) whereas they decreased in amount in the heavy membrane fraction. These findings indicate that rotavirus causes the release of cytochrome c and Smac/Diablo into the cytosol in MA104 cells, consistent with apoptotic mitochondrial dysfunction.

RRV-induced mitochondrial apoptotic pathway is caspases-and Bid-independent.

We then looked for upstream signals that could trigger the activation of the mitochondrial apoptotic pathway following RRV infection. This pathway can be activated by the BH3-only protein Bid following its cleavage by caspase-8 [START_REF] Li | Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis[END_REF][START_REF] Luo | Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors[END_REF]. First, the kinetics of the processing of procaspase-8 was analyzed in RRV-infected MA104 cells, by immunoblotting (Fig. 3A).

The amounts of procaspase-8 decreased from 14 h p.i., relative to those in mock-infected cells, suggesting caspase-8 activation. Caspase-8 activation was confirmed at 14 h p.i. by flow cytometry with a FLICA apoptosis detection kit, based on the specific binding of a fluorescein-labeled inhibitor (FAM-LETD-fmk, Immunochemistry Technologies, LLC) to active caspase (Fig 3B). The kinetics of Bid processing was then analyzed in RRV-infected MA104 cells, by immunoblotting (Fig. 3A). The amounts of Bid and procaspase-8, decreased with respect to those in mock-infected cells, suggesting a cleavage of Bid mediated by activated caspase-8.

To determine whether caspase-8 and Bid were required for engaging mitochondrial pathway of apoptosis, we assessed cytochrome c release in the presence of the broad-spectrum caspase inhibitor, z-VAD-fmk. The release of cytochrome c from the mitochondria into the cytosol in RV-infected MA104 cells (14 h p.i.) was analyzed by western blotting following subcellular fractionation (Fig. 4). As expected, cleavage of both Bid and PARP were inhibited in the presence of the caspase inhibitor (Fig. 4A). In contrast, z-VAD-fmk was unable to prevent cytochrome c release (Fig. 4B), suggesting that mitochondrial dysfunction in RRV-infected MA104 cells is induced by a caspase-and Bid-independent mechanism. Thus, caspases seem to be involved in RRV-induced apoptosis at a position in the pathway downstream from the mitochondrial dysfunction.

Bax is activated during RRV infection by a caspase independent pathway.

The proapoptotic Bcl-2 family protein Bax is a pivotal regulator of cytochrome c release from mitochondria to the cytosol [START_REF] Adams | Life-or-death decisions by the Bcl-2 protein family[END_REF][START_REF] Oltvai | Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death[END_REF][START_REF] Wei | Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death[END_REF]. In healthy cells, most Bax is in the cytoplasm and in an inactive form. Bax-mediated cell death occurs through a conformational change and its translocation to the mitochondrial outer membrane, resulting in the loss of mitochondrial membrane potential and the release of proapoptogenic factors including cytochrome c [START_REF] Hsu | Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations[END_REF][START_REF] Murphy | Bcl-2 inhibits a Fas-induced conformational change in the Bax N terminus and Bax mitochondrial translocation[END_REF][START_REF] Wolter | Movement of Bax from the cytosol to mitochondria during apoptosis[END_REF]. We investigated kinetics of the conformational change of Bax in RRV-infected cells.

Total cell lysates were prepared at the indicated times post-infection (p.i) (Fig. 5A) in a lysis buffer (containing Chaps) that did not affect Bax conformation. Bax was then immunoprecipitated with an anti-Bax antibody (6A7) specific for the active conformation of Bax [START_REF] Hsu | Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations[END_REF][START_REF] Hsu | Nonionic detergents induce dimerization among members of the Bcl-2 family[END_REF] and visualized by western blotting (Fig. 5A). In mock-infected cells, no activated Bax was detected in the immunoprecipitates. From 12 h p.i, Bax was precipitated by antibody 6A7, indicating that RRV infection induced the conformational change of Bax as observed in STS-treated cells used as positive control (data not shown). Thus, rotavirus infection triggers a conformational change in Bax in MA104 cells. Furthermore, we showed that Bax activation still occured in the presence of the broad-spectrum caspase inhibitor, z-VAD-fmk (100 µM, Calbiochem) (Fig. 5B), indicating that Bax activation during RRV infection is caspase independent.

MA104 cell infection by RRV increases the Bax/Bcl-2 ratio.

The Bcl-2-family, obviously, includes the antiapoptotic protein Bcl-2, which converges on mitochondria and competes with proapoptotic Bax to regulate the release of cytochrome c in response to apoptotic signal [START_REF] Adams | Life-or-death decisions by the Bcl-2 protein family[END_REF][START_REF] Gross | BCL-2 family members and the mitochondria in apoptosis[END_REF][START_REF] Reed | Bcl-2 family proteins[END_REF]. As the ratio of pro-and anti-apoptotic proteins determines, at least in part, the susceptibility of cells to the death signal [START_REF] Oltvai | Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death[END_REF], we studied the time-dependent effects of RRV infection on Bax and Bcl-2 abundance in MA104 cells (Fig 5C). The amount of Bcl-2 decreased as the viral infection proceeded, whereas the total amount of Bax was unaffected. Thus rotavirus infection increased the Bax/Bcl-2 ratio and this could contribute to the mitochondrial apoptotic pathway by increasing the amount of free Bax thereby allowing its activation.

RRV-induced mitochondrial apoptotic pathway is Bax-dependent.

These various findings seem to indicate that RRV induces Bax-mediated apoptosis in MA104

cells. To assess the requirement for Bax in RRV-induced mitochondrial dysfunction, MA104 cells were transiently transfected with specific small interfering RNA (siRNA) to knock down Bax expression. Western blot analysis of MA104 cells transfected with the Bax siRNA (Cell Signaling) and with the irrelevant control siRNA (Cell Signaling) confirmed that the specific siRNA reduced significantly the abundance of Bax (50 %) (Fig 6A). We analyzed cytochrome c release 14 h after infection of MA104 cells previously transfected with siRNA. Bax siRNA partially inhibited cytochrome c release (52 %) in RRV-infected cells whereas the irrelevant control siRNA had no effect. Furthermore, a much larger proportion of cytochrome c remained in the membrane fraction containing mitochondria from RRV-infected cells treated with Bax siRNA than from those treated with the irrelevant control (Fig 6B). We also investigated RRV-induced apoptosis in MA104 cells, previously transfected with siRNA. The fraction of apoptotic cells analyzed at 14 h p.i., by flow cytometry after Acridine Orange (AO) staining, decreased in cells with reduced Bax expression (Fig. 6C). These results demonstrate that Bax plays a key role in rotavirus-induced mitochondrial pathway of apoptosis.

DISCUSSION

The pathophysiological mechanisms behind rotavirus-induced diarrhea have not been completely described. In mice, apoptosis may be associated with functional changes during rotavirus infection, particularly the modification of digestion and absorption functions.

Evidence for apoptosis has been reported in intestinal cell lines following rotavirus infection

in vitro [START_REF] Chaibi | Rotavirus induces apoptosis in fully differentiated human intestinal Caco-2 cells[END_REF][START_REF] Superti | Induction of apoptosis in HT-29 cells infected with SA-11 rotavirus[END_REF]. However, the precise cellular and molecular mechanisms underlying rotavirus-induced apoptosis have not been defined. Here, we investigated the apoptosis induced by the RRV strain in MA104 cells, a monkey kidney epithelial cell line in which the rotavirus cycle has been best characterized.

We showed that several hallmarks of apoptosis, notably DNA fragmentation, caspase-3 activation and PARP cleavage, were detected following RRV infection of MA104 cells. DNA fragmentation was not observed in a previous study [START_REF] Castilho | The in vitro cytopathology of a porcine and the simian (SA-11) strains of rotavirus[END_REF] in the same RRV-infected cells, probably because of differences in the sensitivity of the tests used. Moreover, we report the release of cytochrome c and Smac/DIABLO from mitochondria into the cytosol, implicating mitochondrial dysfunction in the apoptotic pathway. Cytochrome c release is known to be the pro-apoptotic signal causing the autocatalytic activation of procaspase-9 that triggers caspase-3 cleavage. We showed that specific inhibition of either caspase-3 or caspase-9 reduced the percentage of apoptotic cells following RRV infection. Thus, RRV-induced apoptosis is, at least partly, dependent on caspase activation. It is also possible that Smac/Diablo is associated with RRV-induced apoptosis through its action on cellular inhibitors of apoptosis proteins (IAPs), as observed notably during reovirus-induced apoptosis [START_REF] Kominsky | Reovirus-induced apoptosis requires mitochondrial release of Smac/DIABLO and involves reduction of cellular inhibitor of apoptosis protein levels[END_REF].

We then investigated events upstream from RRV-induced mitochondrial dysfunction. We focused on the Bcl-2 family proteins because they are central regulators of the mitochondrial apoptotic pathway [START_REF] Adams | Life-or-death decisions by the Bcl-2 protein family[END_REF][START_REF] Gross | BCL-2 family members and the mitochondria in apoptosis[END_REF][START_REF] Narita | Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria[END_REF][START_REF] Reed | Bcl-2 family proteins[END_REF] and have been implicated in various models of virusinduced apoptosis [START_REF] Liu | Role of the mitochondrial signaling pathway in murine coronavirus-induced oligodendrocyte apoptosis[END_REF][START_REF] Natoni | The mitochondrial pathway of apoptosis is triggered during feline calicivirus infection[END_REF]. Bax, one of the pro-apoptotic Bcl-2 family proteins, is the most downstream activator molecule known of cytochrome c release machinery [START_REF] Puthalakath | Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins[END_REF]. We showed that Bax is activated following RRV infection in MA104 cells: we detected its conformational modification involving the exposure of its N-terminal extremity required for insertion into mitochondrial membranes [START_REF] Hsu | Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations[END_REF][START_REF] Murphy | Bcl-2 inhibits a Fas-induced conformational change in the Bax N terminus and Bax mitochondrial translocation[END_REF]. Once integrated into the outer mitochondrial membrane, Bax can trigger cytochrome c release leading to the activation of the effector caspase-3 and apoptotic cell death [START_REF] Desagher | Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis[END_REF][START_REF] Gross | Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis[END_REF][START_REF] Korsmeyer | Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c[END_REF][START_REF] Wei | Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death[END_REF]. Our analysis of RRV-infected MA104 cells by using siRNA-mediated gene silencing indicates, for the first time, that the apoptotic mitochondrial pathway is activated through a Bax-dependent mechanism.

The inhibition of the prosurvival function of Bcl-2 is essential for the activation of Bax because Bcl-2 can compete with Bax to regulate the release of cytochrome c [START_REF] Adams | Life-or-death decisions by the Bcl-2 protein family[END_REF][START_REF] Gross | BCL-2 family members and the mitochondria in apoptosis[END_REF][START_REF] Reed | Bcl-2 family proteins[END_REF]. The amount of antiapoptotic Bcl-2 protein decreased in RRV-infected cells without any change in the amount of Bax. This results in an increase in the Bax/Bcl-2 ratio in RRV-infected cells that could favor an increase in free Bax. Thus, one mechanism by which rotavirus may contribute to Bax activation is by decreasing the number of Bax/Bcl-2 complexes at the mitochondrial membrane. Activated BH3-only proteins such as Bid can suppress the capacity of Bcl-2 to inhibit apoptosis by interacting with it to displace and subsequently activate Bax [START_REF] Kuwana | BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly[END_REF][START_REF] Letai | Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics[END_REF]. Bid can be activated by caspase-8, resulting in Bax activation and release of cytochrome c [START_REF] Korsmeyer | Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c[END_REF]. We showed that Bid processing was inhibited by the broad caspase inhibitor z-VAD-fmk in RRVinfected cells; therefore rotavirus infection causes caspase-8 activation leading to Bid activation. However, the inhibitor did not inhibit Bax activation and cytochrome c release, suggesting that caspases and Bid do not play a crucial role in the Bax-dependent cytochrome c release pathway during RRV infection. Thus, caspases seem to be involved in RRV-induced apoptosis at a position in the pathway downstream from the mitochondrial dysfunction.

Several mechanisms for promoting Bax activation and mitochondrial dysfunction have been described. These mechanisms include a signal transduction cascade involving mitogenactivated protein kinases (MAPK) such as c-jun N-terminal protein kinase (JNK) and p38, as described in other models [START_REF] Kim | JNK-and p38 kinase-mediated phosphorylation of Bax leads to its activation and mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells[END_REF][START_REF] Tsuruta | JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins[END_REF]. The regulation of mitochondrial dysfunction by JNK following virus infection was first demonstrated in reovirus-infected cells [START_REF] Clarke | JNK regulates the release of proapoptotic mitochondrial factors in reovirus-infected cells[END_REF]. Coulson et al. [START_REF] Holloway | Rotavirus Activates JNK and p38 Signaling Pathways in Intestinal Cells, Leading to AP-1-Driven Transcriptional Responses and Enhanced Virus Replication[END_REF] recently showed that JNK and p38, are activated by rotavirus infection in MA104 cells.

However the involvement of MAPK activation in Bax-dependent mitochondrial dysfunction

has not yet been demonstrated in rotavirus-infected cells.

Rotavirus infection of cultured cells induces a progressive rise in the cytosolic Ca 2+ concentration [START_REF] Brunet | Rotavirus infection induces an increase in intracellular calcium concentration in human intestinal epithelial cells: role in microvillar actin alteration[END_REF][START_REF] Michelangeli | Effect of rotavirus infection on intracellular calcium homeostasis in cultured cells[END_REF][START_REF] Ruiz | Role of Ca2+in the replication and pathogenesis of rotavirus and other viral infections[END_REF][START_REF] Tian | The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum[END_REF]. This increase may be involved in rotavirus-induced apoptosis, and indeed, the intracellular calcium ion chelator BAPTA-AM partially inhibits apoptosis [START_REF] Chaibi | Rotavirus induces apoptosis in fully differentiated human intestinal Caco-2 cells[END_REF].

The rise in cytosolic Ca 2+ concentration in rotavirus-infected cells results from a progressive increase in plasma membrane permeability to Ca 2+ and a depletion of endoplasmic reticulum (ER) pools. Xu et al. showed that rotavirus infection leads to ER stress [START_REF] Xu | BiP (GRP78) and endoplasmin (GRP94) are induced following rotavirus infection and bind transiently to an endoplasmic reticulum-localized virion component[END_REF]. This stress and ER Ca 2+ depletion may lead to Bax activation [START_REF] Pan | Synergistic movements of Ca(2+) and Bax in cells undergoing apoptosis[END_REF]. It would be valuable to determine in future studies whether rotavirus induces apoptosis by inducing both ER stress and ER Ca 2+ depletion leading to mitochondrial dysfunction. Several anti-and pro-apoptotic members of the Bcl-2 family have been found, in addition to their mitochondrial localization, to be associated to the ER and have been implicated in controlling apoptosis by affecting cellular Ca 2+ homeostasis [START_REF] Nutt | Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores[END_REF][START_REF] Pan | Synergistic movements of Ca(2+) and Bax in cells undergoing apoptosis[END_REF][START_REF] Pinton | Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum[END_REF]. As demonstrated in this study, Bax is involved in mitochondrial dysfunction in RRV infected-cells, but it could also be involved in ER Ca 2+ release and thus amplify the apoptotic signal in RRV-infected cells. It would therefore be interesting to investigate whether Bax plays a role in ER Ca 2+ release.

Apoptosis of mature rotavirus-infected enterocytes induces the replacement of these cells by less differentiated dividing cells. This event may well be the cause of the defective absorptive functions of the intestinal epithelial and consequently diarrhea associated with rotavirus pathogenesis (4). Thus, the identification of the apoptotic signaling pathways in RRV-infected cells would improve our understanding of the mechanisms by which rotavirus infection causes functional alterations. Our demonstration of the activation of Bax during rotavirus infection and its involvement in mitochondrial apoptotic pathway provide new insights concerning rotavirus-induced pathogenesis.

FIGURE LEGENDS

  ), z-DEVD-fmk (264155) and z-LEHD-fmk (218761) were purchased from Calbiochem. Mouse anti-CoxIV (A21347) antibody was purchased from Molecular Probes. Mouse anti-Bcl-2 (sc-509) and anti-Bax (clone 6A7, sc-23959) antibodies were purchased from Santa Cruz Biotechnology. Rabbit anti-Bax antibody (NT 06-499) and mouse anti-cytochrome c antibody (556433) were obtained from Upstate and BD Pharmingen, respectively. Rabbit anti-Smac/Diablo (2409) was purchased from ProSci incorporated. Anti-mouse (NA9310V) and anti-rabbit (NA9340V) HRP-conjugated secondary antibodies were obtained from Amersham Biosciences. Goat anti-Bid antibody (AF860) and donkey anti-goat (HAF109) HRP-conjugated antibody were purchased from Research & Diagnostic. Mouse anti-PARP antibody (clone C-2-10) was obtained from Biomol. Mouse anti-Caspase-3 (9668) and anti-caspase-8 (9746) antibodies, and Bax (6321) and non-targeted control (6201) siRNAs were purchased from Cell Signaling.

Fig. 1 .

 1 Fig. 1. RRV induces apoptosis in MA104 cells. A. DNA-fragmentation in RRV-infected MA104 cells. At the indicated times post-infection, DNA fragmentation was analyzed using the Cell Death Detection ELISA PLUS assay which detects the appearance of histone-associated low molecular weight DNA in the cytoplasm of cells. Mock-infected and staurosporine (STS)-treated MA104 cells were used as negative and positive controls, respectively. Enrichment Factor was calculated as the absorbance at 405/492 nm for treated cells divided by that of the corresponding untreated cells. Data are means ± S.D. of three experiments. *P<0.05 by Student t-test comparing RRV-infected MA104 cells to mock-infected MA104 cells. B. RRV infection triggered caspase-3 activation and PARP processing in MA104 cells. At the indicated times post-infection, whole cell extracts were subjected to western blot analysis with anti-caspase-3 and anti-PARP antibodies. Mock-infected MA104 cells were used as negative controls. An asterisk (*) indicates a nonspecific protein band which was used as a protein loading control. Positions of markers of molecular weight are indicated at the left of panel. C. RRV-induced apoptosis is dependent on caspases activation in MA104 cells. MA104 cells were treated or non-treated with the broad caspase inhibitor z-VAD-fmk (10 µM), the specific caspase-9 inhibitor z-LEHD-fmk (10 µM) or the specific caspase-3 inhibitor z-DEVD-fmk (10 µM) 2 h before RRV infection and maintained throughout the infection. Mock-and RRVinfected MA104 cells were analyzed 14 h post-infection (p.i) by flow cytometry after acridine orange nuclear dye staining and the fold increase of apoptosis was calculated as the ratio of the apoptotic cell percentages for RRV-infected MA104 cells to that for mock-infected MA104 cells. Data are expressed as means of three independent experiments. Error bars

Fig. 2 .

 2 Fig. 2. Mitochondrial apoptotic pathway is activated in RRV-infected MA104 cells. Time course of cytochrome c and Smac/Diablo release in RRV-infected MA104 cells. At the indicated times post-infection, cells were collected and subjected to subcellular fractionation as described in Material and Methods. The cytosolic (C) and heavy membrane (HM) fraction proteins were assayed by western blot analysis for cytochrome c and Smac/Diablo. Mockinfected MA104 cells were used as negative controls. Cox IV and tubulin were used as protein loading controls for heavy membrane and cytosolic fractions, respectively. Positions of markers of molecular weight are indicated at the left of panel. Cytochrome c and Smac/Diablo protein levels of heavy membrane and cytosolic fractions were determined by densitometry and plotted as ratios relative to the levels of Cox IV and tubulin, respectively (lower panel).

Fig. 3 .

 3 Fig. 3. RRV induces caspase-8 activation and Bid processing in MA104 cells. A. Time course of caspase-8 activation and Bid processing in RRV-infected MA104 cells. At the indicated times post-infection, whole cell extracts were subjected to immunoblot analysis with anti-caspase-8 and anti-Bid antibodies. Mock-infected MA104 cells were used as negative controls. Tubulin was used as a control for protein loading. Positions of markers of molecular weight are indicated at the left of panel. Bid protein levels were determined by densitometry and plotted as ratios relative to the levels of tubulin (lower panel). B. Caspase-8 activation was determined in mock-and RRV-infected MA104 cells (14 h p.i.) by flow cytometry using cell-permeable, fluorescein-labeled inhibitor (FAM-LETD-fmk), that binds specifically on active caspase-8, as described in Material and Methods. Histogram

Fig. 4 .

 4 Fig. 4. Cytochrome c release in RRV-infected MA104 cells is caspase-independent. A. The broad-spectrum caspase inhibitor z-VAD-fmk inhibits Bid and PARP processing in RRV-infected MA104 cells. Mock-and RRV-infected MA104 cells were pretreated or not pretreated with 100 µM z-VAD-fmk for 2 h before and during RRV infection. Whole cell extracts were prepared 14 h post-infection and Bid and PARP were assayed by western blot analysis. Mock-infected MA104 cells were used as negative controls. Tubulin was used as a control for protein loading. Positions of markers of molecular weight are indicated at the left of panel.B. z-VAD-fmk did not inhibit cytochrome c release in RRV-infected MA104 cells. Mockand RRV-infected MA104 cells were pretreated or not pretreated with 100 µM z-VAD-fmk for 2 h before and during infection. Fourteen hours after RRV infection, the cytosolic and heavy membrane fractions were assayed for cytochrome c by western blotting. Mock-infected MA104 cells were used as negative controls, Cox IV and tubulin were used as protein loading controls for heavy membrane and cytosolic fractions, respectively. Positions of markers of molecular weight are indicated at the left of panel.

Fig. 5 .

 5 Fig. 5. RRV induces Bax protein activation in MA104 cells. A. RRV induced Bax conformational change in MA104 cells. At the indicated time postinfection (p.i), MA104 cells were lysed in immunoprecipitation buffer containing the zwitterionic detergent Chaps (which maintains Bax in its activated conformation). Anti-Bax 6A7 antibody was used to immunoprecipitate conformationally active Bax protein. The immunoprecipitates were analyzed by immunoblotting with anti-Bax antibody.

  irrelevant siRNA-transfected to Bax siRNA-transfected MA104 cells (lower panel). B. siRNA-induced silencing of the Bax gene results in reduction of RRV-induced cytochrome c release in MA104 cells. Cells were infected with RRV 48 h post-transfection and cytochrome c was analyzed in cytosolic (C) and heavy membrane fractions (M) by western blotting 14h post-infection. Mock-infected MA104 cells were used as negative controls. Tubulin and Cox IV were used as controls for protein loading of cytosolic (C) and heavy membrane (M) fractions, respectively. Positions of markers of molecular weight are indicated at the left of panel. Cytochrome c protein levels of heavy membrane and cytosolic fractions were determined by densitometry and plotted as ratios relative to the levels of Cox IV and tubulin, respectively (lower panel). C. siRNA-induced silencing of the Bax gene results in reduction of RRV-induced apoptosis in MA104 cells. Cells were infected with RRV 48 h post-transfection and the percentage of apoptosis was determined by flow cytometry after Acridine Orange (AO) staining. MA104 cells transfected with irrelevant siRNA was used as negative control.

ACKNOWLEDGEMENTS

We thank Didier Poncet (Gif-sur-Yvette, France) for the gift of MA104 cells and the rotavirus RRV strain. We thank Isabelle Pelletier and Aure Saulnier for their help. This work was supported by the Institut Pasteur, Danone Research Centre Daniel Carasso and the Ministère de l'Education Nationale, de la Recherche et de la Technologie.


Karine Labadie

Aure Saulnier

Sandra Martin-Latil

Florence Colbère-Garapin

Reduced apoptosis in human intestinal cells cured of persistent poliovirus infection

Cells cured of persistent virus infection can be used to investigate cellular pathways of resistance to viral cytopathic effects. Persistent poliovirus (PV) infections were established in human intestinal Caco-2 cells and spontaneously cured cell cultures were obtained. Two cell clones, cl6 and b13, cured of type 3 PV mutant infection, and their parental Caco-2 cells were compared for susceptibility to PV infection, PV receptor CD155 expression, capacity to differentiate into polarized enterocytes, and PV-, staurosporine-and actinomycin D-induced apoptosis. Our results strongly suggest that cells partially resistant to apoptosis can be selected during persistent virus infection.

Cells cured of persistent virus infection can be used to investigate cellular pathways of resistance to virus infection [START_REF] Borzakian | Persistent poliovirus infection: establishment and maintenance involve distinct mechanisms[END_REF][START_REF] Calvez | Cell clones cured of persistent poliovirus infection display selective permissivity to the wild-type poliovirus strain Mahoney and partial resistance to the attenuated Sabin 1 strain and Mahoney mutants[END_REF][START_REF] Ebert | Cathepsin B Is Inhibited in Mutant Cells Selected during Persistent Reovirus Infection[END_REF][START_REF] Sanchez | RNA interferencemediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus[END_REF][START_REF] Saulnier | Complete cure of persistent virus infections by antiviral siRNAs[END_REF][START_REF] Vallbracht | Persistent infection of human fibroblasts by hepatitis A virus[END_REF]. The spontaneous cure of persistent virus infection in vitro has been described only rarely [START_REF] Borzakian | Persistent poliovirus infection: establishment and maintenance involve distinct mechanisms[END_REF][START_REF] De La Torre | Establishment of cell lines persistently infected with foot-and-mouth disease virus[END_REF][START_REF] Labadie | Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells[END_REF][START_REF] Ron | Spontaneous curing of a minute virus of mice carrier state by selection of cells with an intracellular block of viral replication[END_REF]. Poliovirus (PV), the prototype member of the enterovirus genus [START_REF] Stanway | Molecular and biological basis of picornavirus taxonomy[END_REF], is composed of a single-stranded positive-sense RNA enclosed in an icosaedral capsid. We have developed several models of persistent PV infection to elucidate the mechanisms of persistent infections and investigate cellular resistance to viral cytopathic effects [START_REF] Colbère-Garapin | Persistent infections by picornaviruses[END_REF]. Mutated forms of the PV receptor, CD155, have been found in persistently PV-infected IMR-32 cells [START_REF] Blondel | Poliovirus, pathogenesis of poliomyelitis, and apoptosis[END_REF][START_REF] Pavio | Expression of mutated poliovirus receptors in human neuroblastoma cells persistently infected with poliovirus[END_REF]. The allelic form with a threonine in position 67 (Thr67) of CD155 conferred on murine L cells a partial resistance to PV-induced apoptosis, with reference to the other form (Ala67) expressed in the parental IMR-32 cell line [START_REF] Gosselin | Poliovirusinduced apoptosis is reduced in cells expressing a mutant CD155 selected during persistent poliovirus infection in neuroblastoma cells[END_REF]. HEp-2 cells cured of PV infection have a variety of phenotypes, often involving PV-CD155 interactions [START_REF] Borzakian | Persistent poliovirus infection: establishment and maintenance involve distinct mechanisms[END_REF][START_REF] Calvez | Cell clones cured of persistent poliovirus infection display selective permissivity to the wild-type poliovirus strain Mahoney and partial resistance to the attenuated Sabin 1 strain and Mahoney mutants[END_REF]. Persistent infections with PV mutants have also been established in human intestinal Caco-2 cells [START_REF] Labadie | Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells[END_REF]. We used PV type 3 mutant L2-2 [START_REF] Duncan | Two amino acid substitutions in the type 3 poliovirus capsid contribute to the establishment of persistent infection in HEp-2c cells by modifying virus-receptor interactions[END_REF], and Sabin 3 (S3) mutants H136-11, H442-11 and H637-11, isolated from a chronically infected hypogammaglobulinemic patient [START_REF] Martin | Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637day period of virus excretion[END_REF]. Mutations differentiating these PV mutants and S3 have been published [START_REF] Labadie | Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells[END_REF][START_REF] Martin | Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637day period of virus excretion[END_REF]. The mechanisms of the persistent infection in human intestinal cells involve viral and cellular determinants [START_REF] Labadie | Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells[END_REF]. Caco-2 cultures (20-80%) stopped excreting infectious virus between 1 month and 1 year post-infection (pi) [START_REF] Labadie | Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells[END_REF]. Clonal cell populations were grown from well-isolated colonies of cured cells, as described [START_REF] Calvez | Cell clones cured of persistent poliovirus infection display selective permissivity to the wild-type poliovirus strain Mahoney and partial resistance to the attenuated Sabin 1 strain and Mahoney mutants[END_REF], and their resistance to de novo PV type 3 infection was tested. The absence of PV RNA in the cured clones was first verified by RT-nested PCR, as described (24) (not shown). All cell clones were permissive to PV infection, but 2 of 5 were partially resistant to virus-induced cell lysis. These clones, cl6 and b13, cured of L2-2 and H442-11 infection respectively, were studied in comparison with parental Caco-2 cells. Cells (10 6 ) were seeded in duplicate in wells of 24-well plates in DMEM medium containing 10% fetal bovine serum, and incubated overnight at 37 °C. Cells were then mock-infected or infected with a type 3 PV strain: S3, L2-2 or H442-11, at a multiplicity of infection (moi) of 10 -2 infectious doses 50 (ID 50 ) per cell. The percentage of cells surviving infection was determined 1, 2 and 3 days pi by staining cells with trypan blue. The resistance of the clones to lysis was highly significant 3 days pi: with all type 3 PV strains tested, more than 80% of parental Caco-2 cells were lysed whereas 60-100% of cl6 and 40-80% of b13 cells survived infection (Fig. 1a-1c).

Similar results were obtained with infections with Sabin 1 and Sabin 2 PV strains (not shown), indicating that the resistance of cl6 and b13 cells to PV infection was not typespecific. As S3 multiplication was slightly delayed in the clones (Fig. 1d), the resistance of the clones was tested at a higher moi. At an moi of 10 ID 50 per cell, S3 multiplication in cl6 was delayed by 1-2 h during the exponential phase. However, 24 h pi, viral yields were similar for the three cell types and cl6 cells were much more resistant than Caco-2 cells to S3induced lysis (Fig. 1e and1f). At this moi, b13 was more susceptible to infection than cl6, in agreement with data at low moi.

Low levels of CD155 expression may contribute to resistance to virus-induced cell lysis [START_REF] Colston | Poliovirus variants selected on mutant receptor-expressing cells identify capsid residues that expand receptor recognition[END_REF]. We quantified the expression of CD155 at the surface of Caco-2, cl6 and b13 cells, over a period of 6 days between two cell passages. We used flow cytometry after indirect immunofluorescence labeling with the anti-CD155 monoclonal antibody 404 [START_REF] Lopez | Identification of a new class of Ig superfamily antigens expressed in hemopoiesis[END_REF], as described [START_REF] Gosselin | Poliovirusinduced apoptosis is reduced in cells expressing a mutant CD155 selected during persistent poliovirus infection in neuroblastoma cells[END_REF][START_REF] Labadie | Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells[END_REF]. More than 90% of cells of each cell type were brightly stained and the level of CD155 expression was slightly higher on the cured cells than on Caco-2 cells (Fig. 2a). This was confirmed normalizing the results for ICAM-1, detected with the control antibody 8.4A6 (Sigma) (Fig. 2b). Thus, the delay before the cytopathic effects in the clones was not due to a low level of CD155 expression.

We tested whether mutated forms of CD155 had been selected in cl6 and b13, because it has been shown that PV multiplication could occur without cell lysis in cells expressing mutated CD155 [START_REF] Morrison | Homolog-scanning mutagenesis reveals poliovirus receptor residues important for virus binding and replication[END_REF]. We determined the sequence of CD155 mRNA from these cells and from Caco-2 cells, in the region corresponding to the domain of CD155 that includes the PVbinding site [START_REF] Koike | The poliovirus receptor protein is produced both as membrane-bound and secreted forms[END_REF][START_REF] Mendelsohn | Cellular receptor for poliovirus: molecular cloning, nucleotide sequence and expression of a new member of the immunoglobulin superfamily[END_REF], as described [START_REF] Pavio | Expression of mutated poliovirus receptors in human neuroblastoma cells persistently infected with poliovirus[END_REF][START_REF] Saulnier | Complete cure of persistent virus infections by antiviral siRNAs[END_REF]. Nucleotide positions that were mutated in CD155 mRNA from persistently PV-infected IMR-32 cells were not mutated in Caco-2, cl6 or b13 cells: they were Gly 39 , Ala 67 and Arg 104 . Also, no other mutation was detected, suggesting that the partial resistance of cl6 and b13 cells to PV-induced lysis was not due to the expression of CD155 forms mutated in the PV-binding domain. To exclude the role of other mutations in CD155, cells were infected with coxsackievirus B3 (CVB3), an enterovirus that uses the coxsackievirus and adenovirus receptor (3). Results were similar to those obtained with PV (Fig. 2c), confirming that the resistance of clones to virus-induced lysis did not depend on CD155.

We looked for a correlation between the partial resistance of clones to PV-induced lysis and their differentiation into polarized enterocytes, because enterocytes are more resistant to PV-induced lysis than undifferentiated Caco-2 cells [START_REF] Labadie | Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells[END_REF][START_REF] Tucker | Vectorial release of poliovirus from polarized human intestinal epithelial cells[END_REF]. No correlation between these phenotypes could be established (not shown).

PV infection induces apoptosis in several cell types, including Caco-2 cells (1, 2, 12, 21, 26). We therefore tested whether the resistance of clones to PV-induced lysis correlated with partial resistance to PV-induced apoptosis, by comparing the percentage of the three cell types with DNA fragmentation 3 days after infection with S3 at a moi of 10 -2 ID 50 /cell: terminal deoxynucleotidyltransferase-mediated dUTP-rhodamine nick end labeling (TUNEL) was used as described [START_REF] Girard | Poliovirus induces apoptosis in the mouse central nervous system[END_REF]. In mock-infected cultures, less than 1 % of cells were TUNELpositive (not shown). In infected cultures, viral antigens were detected by indirect immunofluorescence with a rabbit anti-PV type 3 antiserum [START_REF] Borzakian | Persistent poliovirus infection: establishment and maintenance involve distinct mechanisms[END_REF]. The percentages of cells that were viral antigen-positive did not differ significantly between the cell types (Fig. 3a).

Interestingly, there were 3-fold fewer viral antigen-and TUNEL-double positive cells in cultures of cl6 and b13 than in Caco-2 cultures, suggesting that the clones were more resistant to PV-induced apoptosis than Caco-2 cells (Fig. 3a).

To test whether cells cured of persistent infection were also partially resistant to apoptosis induced by non-viral inducers, we treated confluent cultures of each cell type with either actinomycin D (AMD) (8 µM for 22 h) or staurosporine (2 µM for 18 h). Like PV, both drugs activate the mitochondrial pathway of apoptosis, but staurosporine has a much more specific effect than AMD. DNA fragmentation was evaluated with a Cell Death Detection ELISA plus kit (Roche). Cells of cl6 and b13 appeared to be about 2-and 3-fold more resistant to AMD-and staurosporine-induced apoptosis, respectively, than Caco-2 cells (Fig 3b and3c). These results indicate that the partial resistance of the cured clones to apoptosis is not exclusively virus-specific.

In conclusion, our results strongly suggest that cells partially resistant to apoptosis can be selected during persistent virus infection. This may contribute to the mechanism of persistent infection.

We are grateful to I. Pelletier for her help and to J. Estaquier, A. Martin

Legend to Figure 1 .Legend to Figure 2 .

 12 , C. Prehaud, S. Lay, S. Guillot and N. Escriou for helpful suggestions. We thank B. Blondel for critical reading of the manuscript and Marc Lopez for the gift of anti-CD155 monoclonal antibody. KL and AS were supported by fellowships from the Ministry (MENESR) and this work by grants from the Institut Pasteur (PTR 120) and Danone Research Centre Daniel Carasso. Partial resistance of Caco-2-cell-derived clones cl6 and b13 to PV-induced cell lysis after de novo infection. PV type 3 strains Sabin 3 (S3), L2-2 and H442-11 were used at an moi of 10 -2 (a-d), or 10 ID 50 /cell (e, f). Caco-2 (dark gray), cl6 (light gray) and b13 (white) cells were infected with the indicated virus strains and the number of surviving cells was determined by trypan blue exclusion, 1, 2 and 3 days pi at the moi of 10 -2 (a-c) and 1 day pi at the moi of 10 (e). The results were obtained by calculating surviving cells in infected cultures as a percentage of those in mock-infected homologous cultures, for the same day. S3 growth curves in Caco-2 (circles), cl6 (squares) and b13 (triangles) cells infected at moi of 10 -2 (d) and 10 ID 50 /cell (f) are shown. The results are means ± SEM from at least four cultures from two independent experiments. The resistance of clones to virus-induced lysis did not depend on CD155 expression. The level of CD155 expression at the surface of Caco-2 (circles), cl6 (squares) and b13 (triangles) cells was determined by indirect immunofluorescence and the mean fluorescence per positive cell was quantified by flow cytometry in arbitrary units, 1 to 6 days after cell passage by trypsinization (a). The ratio of CD155 to ICAM-1 expression, used as a reference, is shown for the three cell types (b). Partial resistance of Caco-2-cell-derived clones cl6 and b13 to cell lysis after infection with CVB3 (c). Caco-2 (dark gray), cl6 (light gray) and b13 (white) cells were infected at an moi of 10 -2 ID 50 /cell, and the number of surviving cells was determined by trypan blue exclusion 2 days pi. The results are means ± SEM from at least four cultures from two independent experiments. Legend to Figure 3.

Cell clones cured of persistent PV infection were partially resistant to apoptosis.

Percentage of viral antigen-and TUNEL-positive cells in Caco-2, cl6 and b13 cultures 3 days pi by PV Sabin 3 (moi of 10 -2 ID 50 /cell); about 300-400 cells were analyzed under the microscope for each culture (a). Specific enrichment in mono-and oligo-nucleosomes released into the cytoplasm of confluent Caco-2, cl6 and b13 cultures following treatment with actinomycin D (AMD, 8 µM for 22 h) (b) or staurosporine (ST, 2 µM for 18 h) (c). The enrichment factor was determined with a Cell Death Detection ELISA plus kit (Roche). The results are means ± SEM from at least two independent experiments.