Characterization of Epstein-Barr BALF0/BALF1 proteins Zhouwulin Shao

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Zhouwulin Shao. Characterization of Epstein-Barr virus BALF0/BALF1 proteins. Human health and pathology. Sorbonne Université, 2019. English. ￿NNT : 2019SORUS358￿. ￿tel-03139823￿

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Sorbonne Université

Ecole doctorale Complexité du Vivant Centre de Recherche Saint-Antoine Equipe “Biologie et thérapeutiques du cancer”

Characterization of Epstein-Barr virus

BALF0/BALF1 proteins

Par Zhouwulin SHAO

Thèse de doctorat de Virologie

Dirigée par Vincent MARECHAL

Présentée et soutenue publiquement le 16 octobre 2019

Devant un jury composé de :

M. Henri GRUFFAT Chargé de Recherche Rapporteur M. Pierre Emmanuel CECCALDI Professeur Rapporteur M. Guennadi SEZONOV Professeur Président M. Jean-Pierre VARTANIAN Directeur de Recherche Examinateur Mme. Joëlle WIELS Directrice de Recherche Examinatrice M. Vincent MARECHAL Professeur Directeur de thèse

Résumé de thèse

Etat de la question

Le virus Epstein-Barr (EBV) est gammaherpesvirus humain oncogène qui infecte de

façon persistante plus de 95% des adultes dans le monde. La primo-infection à EBV est

asymptomatique chez les enfants alors qu’elle peut être responsable de la

mononucléose infectieuse chez les jeunes adultes. L'EBV se transmet principalement

par la salive et établit une infection à vie après la primo-infection. Cette infection

persistante alterne infection latente, principalement dans les cellules B mémoire, et

réactivation sporadique menant éventuellement à la production de virus et à sa

transmission ultérieure. L’infection à EBV est associée un certain nombre de tumeurs

malignes d’origine lymphoïde et épithéliale, notamment le lymphome de Burkitt (BL),

le lymphome de Hodgkin (HL) et le carcinome du nasopharynx (NPC). L'association

étroite entre EBV et la forme non différenciée du NPC est illustrée par la présence du

virus dans 100% des tumeurs et un profil de réponse sérologique anti-viral spécifique.

BALF0/1 est un gène viral dont les produits – qui n’ont jamais été observés au cours de l’infection naturelle – ont été décrits comme des modulateurs de l'apoptose. Deux codons initiateurs potentiels sont présents au début du cadre de lecture ouvert (ORF)

BALF0/1, suggérant que deux protéines pourraient être codées avec différentes extrémités N-terminales. BALF1 serait codée par l'ORF le plus court. La protéine codée

à partir de la première méthionine est appelée BALF0. Seule la seconde méthionine est conservée chez les orthologues BALF0/1 des gammaherpesviridae. Des travaux antérieurs ont montré que les patients atteints de NPC pouvaient produire des anticorps reconnaissant une protéine de 31 kDa dans des cellules NIH3T3 transfectées par plasmide codant BALF0/1, ce qui est compatible avec la taille attendue de BALF0.

Néanmoins, l'existence de BALF1 n'a pas pu être confirmée dans le même contexte.

Jusqu'à présent, l'existence des protéines BALF0 et BALF1 dans des cellules

naturellement infectées n’a pu être démontrée en raison de l'absence de réactifs immunologiques spécifiques. Une condition préalable essentielle à la production de tels réactifs est la production de formes solubles purifiées de BALF0/1 pouvant

éventuellement être utilisées pour l’immunisation.

Résultats

1) Detection d’anticorps anti-BALF0/1 chez les patients atteints de NPC

Nous avons tout d’abord essayé d’exprimer BALF0/1 à partir de deux vecteurs d’expression procaryotes (pET-22b et pGEX-2T) avec une étiquette poly-histidine C- terminale ou une étiquette GST (Gluthation S tranferase) N-terminale. De nombreuses conditions d’expression ont été évaluées, sans succès. Nous avons supposé que des domaines hydrophobes internes pouvaient altérer l'expression de BALF0/1 dans E. coli.

L'analyse structurelle de la protéine indique en effet la présence de 2 hélices α avec une hydrophobie dans la région C-terminale. Un premier projet publié décrit l'expression et la purification d'une forme tronquée de BALF0/1 (tBALF0, acides aminés 1 à 140), un mutant avec délétion du domaine transmembranaire C-terminal, en utilisant un plasmide d'expression bactérien hétérologue (pET-22b-tBALF0). tBALF0 a été purifiée à homogénéité en conditions dénaturantes par chromatographie d'affinité sur colonne de nickel. Après analyse SDS-PAGE, une seule bande a été observée à la masse moléculaire attendue (MW) dans des conditions réductrices. Inversement, au moins sept bandes ont été observées dans des conditions non réductrices, suggérant que les conditions Red-Ox d’expression/purification pourraient favoriser la multimérisation du tBALF0, que ce soit pendant l'expression de la protéine, sa purification ou pendant l'analyse. L'identité de tBALF0 a été confirmée par spectrométrie de masse. tBALF0 a

également été utilisé comme antigène pour le développement d’un test sérologique

ELISA. Ce test a permis de détecter la présence d'IgG de faible titre contre BALF0/1 au cours de certaines infections primaires (10.0%) et passée (13.3%). Inversement, des

IgG anti- BALF0/1 à titre élevé ont été détectés chez 33.3% des patients NPC, ce qui suggère que BALF0/1 est exprimé dans cette situation clinique, et qu’elle stimule une réponse humorale spécifique. La présence d'anticorps dirigés contre BALF0/1 chez des patients infectés par le virus EBV pourrait donc être considérée comme une preuve indirecte importante de l'existence de BALF0 et/ou BALF1 in vivo.

(Shao, Z.; Borde, C.; Marchand, C.H.; Lemaire, S.D.; Busson, P.; Gozlan, J.-M.;

Escargueil, A.; Maréchal, V. Detection of IgG directed against a recombinant form of

Epstein-Barr virus BALF0/1 protein in patients with nasopharyngeal carcinoma.

Protein Expression and Purification 2019, 162, 44–50.)

2) Modulation de l’autophagie par les protéines BALF0/1

L'autophagie est un processus catabolique essentiel qui dégrade les composants cytoplasmiques pour assurer la survie des cellules notamment. En plus de sa contribution au contrôle de la qualité et de la quantité de la biomasse intracellulaire et des organelles, l'autophagie agit également comme un mécanisme d'élimination des microbes pour protéger les cellules eucaryotes contre les agents pathogènes intracellulaires et notamment des virus. Cependant, certains virus ont mis au point des stratégies efficaces pour échapper au contrôle immunitaire ou pour promouvoir leur propre réplication en manipulant l'autophagie à leur avantage. Des études récentes indiquent que l'EBV peut moduler l'autophagie à la fois pendant la latence et la réactivation. Pendant la latence, LMP1 induit l'autophagie pour contrôler sa propre dégradation, LMP2A peut induire l'autophagie pour favoriser la formation d'un acinus anormal et EBNA3C, qui est requise à l'inhibition de l'apoptose et au maintien de la proliferation des cellules infectées, module également l'autophagie. La contribution des gènes viraux à l’autophagie durant le cycle lytique constitue un axe de recherche important, mais encore peu documenté. Au cours du cycle lytique, Rta stimule l'expression de gènes liés à l'autophagie en suivant une voie dépendante du facteur ERK, un processus qui serait favorable à la production de particules virales. Il a également

été rapporté que l'autophagie est bloquée aux dernières étapes de la dégradation pour éventuellement favoriser l'acquisition de l'enveloppe virale par EBV. Cependant, les protéines virales responsables de l’altération des voies de l'autophagie sont encore inconnues.

Bcl-2 et certains homologues de Bcl-2 codés par les , à savoir M11

(MHV68) et Ks-Bcl-2 (KSHV) inhibent l'autophagie via une interaction avec Beclin 1, une protéine cellulaire essentielle à l’initiation de l’autophagie. Au cours du cycle lytique, EBV code deux trois protéines orthologues à Bc-2, à savoir BHRF1 et

BALF0/1. Nous avons supposé, en raison de cette homologie que BHRF1 et BALF0/1 pourraient également moduler l’autophagie.

Alors que BHRF1 a été largement caractérisée en tant que protéine anti-apoptotique, la fonction de BALF0 et/ou BALF1 est encore équivoque. Il a été proposé que BALF0/1 jouerait un rôle dans l'inhibition de l'apoptose par association avec Bax et Bak. A l'inverse, BALF1 ne protège pas contre l'apoptose induite par le virus Sindbis ou par

Bax et antagonise l'activité anti-apoptotique de BHRF1. BALF0 peut également antagoniser l'activité anti-apoptotique de BHRF1 mais ne co-immunoprécipite pas avec

BHRF1. BALF0/1 est transcrit à la fois au stade lytique et à la latence dans les lignées cellulaires de lymphomes de Burkitt positives pour EBV et les biopsies de NPC. Jusqu'à présent, l'existence de BALF0 et/ou BALF1 dans des cellules naturellement infectées par le virus EBV n'a jamais été confirmée en raison de l'absence de réactifs immunologiques dédiés.

Nous démontrons ici que l’ORF BALF0/1 code effectivement deux protéines dans une lignées B EBV+ dérivée de Burkitt, et que leur expression est augmentée au cours de la réactivation. BALF0 et BALF1 sont à peine détectables dans les cellules non réactivées mais s’accumulent pendant la phase précoce du cycle lytique, comme le montre une immuno-empreinte utilisant l'antisérum polyclonal spécifique dirigé contre tBALF0. De manière surprenante, l’accumulation de BALF1 précède celle de BALF0, alors que l’accumulation de BALF0 est associée à une baisse marquée du niveau de BALF1. En utilisant des plasmides exprimant séparément BALF0 et BALF1, nous

avons pu confirmer que la surexpression de BALF1 favorisait l’accumulation de

BALF0 qui, à son tour, inhibait les BALF1 de de façon dose-dépendante, expliquant

ainsi la cinétique déséquilibrée des deux protéines lors de la réactivation du EBV.

Puisque BALF1 et BALF0 sont exprimées de manière séquentielle lors de la

réactivation d'EBV, l'autophagie a été analysée dans des cellules HeLa exprimant

BALF1 seul ou co-exprimant BALF0 et BALF1. En raison de son niveau d'expression

très faible, l'impact de BALF0 sur l'autophagie n'a pas pu être évalué. Dans les cellules

HeLa qui exprimaient de manière stable la GFP-LC3, BALF1 induit une augmentation

significative du nombre de vesiclues de GFP-LC3 (autophagosomes) qui

s’accumulaient en présence d’inhibiteur lysosomal (CQ). Cette observation suggère

que BALF1 stimule le flux autophagique. De plus, l'expression de BALF1 induit une

augmentation significative de LC3-II, une forme lipidée de LC3 étroitement associée

aux membranes des autophagosomes. Cette accumulation est plus marquée encore en

présence de CQ. Cette expérience a donc confirmé que BALF1 stimule la formation

d'autophagosomes dans les cellules HeLa. Pour étudier une phase ultérieure de

l'autophagie, c'est-à-dire la fusion entre autophagosomes et lysosomes, nous avons

utilisé des cellules HeLa exprimant de manière stable une sonde tandem mRFP-GFP-

LC3. Dans ces cellules, les autophagosomes sont doublement marqués avec GFP et

mRFP alors que les autolysosomes acides, résultant de la fusion entre autophagosomes

(neutres) et lysosomes (acides), ne sont marqués qu'avec les mRFP en raison de

l’inactivation de la GFP qui se produit à pH bas. Dans ces cellules, BALF1 induit

l'accumulation concomitante de vésicules à double marquage (autophagosomes) et

rouge uniquement (autolysosomes), confirmant ainsi une augmentation significative du

flux autophagique jusqu'à la formation d'autolysosomes. Comme démontré

précédemment, BALF0 réduit l'accumulation de BALF1. Contrairement à l'effet pro- autophagique de BALF1, la co-expression simultanée de BALF0 et BALF1 à partir du même plasmide d'expression entraine une réduction du flux autophagique. Pris ensemble, ces résultats permettent de conclure que BALF1 stimule le flux autophagique, lequel est à son tour limité en présence de BALF0.

Les analyses en microscopie confocale montrent que BALF1 colocalise avec des vésicules GFP-LC3 positives. Cela nous a conduit à supposer que BALF1 pourrait être associé à des vésicules contenant GFP-LC3, éventuellement par le biais d'un motif LIR.

Cette hypothèse est corroborée par la présence d'un motif LIR putatif entre les acides aminés 146 à 149 (146-WSRL-149). Deux mutants BALF1 ponctuels ont été générés pour évaluer la contribution du domaine supposé du domaine LIR (1) à la localisation de BALF1 dans les vésicules LC3-positives et (2) à la capacité de BALF1 à promouvoir l'autophagie. Ces mutations ont eu un effet spectaculaire à la fois sur la localisation subcellulaire de BALF1 et ont partiellement ou totalement annulé la capacité de BALF1

à stimuler la formation d'autophagosomes. Alors que nous n’avons pas été en mesure de fournir des preuves supplémentaires de l’interaction directe entre les protéines de la famille ATG8 et BALF1, nous avons pu démontrer ici que ce domaine était nécessaire

à la fois pour le ciblage efficace de BALF1 vers les autophagosomes ainsi que pour la stimulation de l’autophagie par BALF1.

(Shao, Z.; Borde, C.; Quignon, F.; Escargueil, A.; Maréchal, V. Epstein-Barr virus

BALF0 and BALF1 modulate . 2019, under review)

3) BHRF1 induit la mitophagie, ce qui inhibe la réponse innée

BHRF1, l'autre orthologue viraal de Bcl-2, a été décrit comme modulateur anti- apoptotique dans différents systèmes cellulaires expérimentaux. Elle a d’abord été considérée comme une protéine précoce bien qu’elle ait également été détectée au cours de certains programmes de latence. On pense que le mécanisme par lequel BHRF1 exerce sa fonction anti-apoptotique, comme son équivalent chez les mammifères, passe par la liaison et la séquestration d'un sous-ensemble de protéines cellulaires pro- apoptotiques de la famille Bcl-2. Cela rappelle fortement ce qui a été décrit pour la

protéine de survie Bcl-xL de mammifère dans laquelle les interactions impliquaient le peptide BH3 de protéines pro-apoptotiques dans un sillon de surface formé par des hélices a de Bcl-xL. De plus, BHRF1, comme Bcl-2, réside principalement dans la membrane mitochondriale.

Les mitochondries sont impliquées dans de nombreuses fonctions cellulaires telles que la production d'énergie, le maintien de l'homéostasie du calcium, la génération d'espèces réactives de l'oxygène (ROS) et l'initiation de l'apoptose. Les mitochondries jouent également un rôle central dans l'immunité innée contre les virus. Ceci est notamment dû à leur rôle dans l'activation des voies de signalisation de l'interféron (IFN) par le biais des protéines de signalisation antivirale mitochondriales (MAVS) présentes

à la surface de la mitochondrie. Les fonctions des mitochondries sont étroitement liées

à la morphologie et au nombre de mitochondries. En effet, les mitochondries sont des organites dynamiques et mobiles qui subissent en permanence un remodelage de la membrane au cours de cycles répétés de fusion et de fission et dont la longueur est déterminée par l’équilibre entre les taux de fission / fusion. Le maintien de l'homéostasie mitochondriale comprend également le contrôle du nombre de mitochondries par la stimulation de leur biogenèse et l'élimination des mitochondries endommagées par autophagie sélective (mitophagie).

La capacité de BHRF1 à bloquer l’apoptose est bien documentée, alors qu’aucune information n’est disponible à ce jour concernant son rôle présumé sur la dynamique mitochondriale et l’autophagie. Nous avons démontré que l'expression ectopique de

BHRF1 conduit à une fission mitochondriale dépendante de la protéine 1 (Drp1) liée à la dynamine. Par ailleurs, BHRF1 induit l'accumulation d'autophagosomes très probablement en interagissant avec Beclin 1, une protéine de la machinerie autophagique qui peut être inhibée par Bcl-2. Ces modifications cellulaires conduisent

à la formation d'un mito-aggresome, un regroupement mitochondrial périnucléaire qui précède la mitophagie. Etant donné le rôle central des mitochondries dans l'immunité innée, la contribution de BHRF1 dans le contrôle de l'immunité innée a été explorée,

ce qui a permis de démontrer que la dégradation mitochondriale induite par BHRF1

entraînait l'inhibition de l'induction de l'IFN de type I en réponse à divers stimuli.

(Vilment, G.; Glon, D.; Siracusano, G.; Lussignol, M.; Shao, Z.; Hernandez, E.; Perdiz,

D.; Quignon, F.; Mouna, L.; Poüs, C.; Maréchal, V.; Esclatine, A. BHRF1, a Bcl-2 viral homolog, disturbs mitochondrial dynamics and stimulates mitophagy to dampen type I

IFN induction. Autophagy 2019, under review)

4) Perspectives du projet : comprendre les interactions fonctionnelles en

BALF0/BALF1 et BHRF1

Des travaux antérieurs ont indiqué que BALF1 et BHRF1 pouvaient interagir, modulant ainsi la capacité de BHRF1 à inhiber l'apoptose. Cela nous a amenés à nous demander si ces deux protéines pouvaient agir de concert pour moduler l'autophagie. La co- expression de BALF1 et BHRF1 entraîne une accumulation majorée de LC3-II par rapport à BALF1 et BHRF1 seuls. Cependant, l'ajout de CQ ne modifie pas le niveau de LC3-II, démontrant que la dégradation de LC3-II induite par l'autophagie est inhibée lorsque les deux protéines sont co-exprimées. De plus, nous avons démontré que

l'accumulation de BHRF1, BALF0 et BALF1 résultait d'une interaction complexe dans

laquelle BHRF1 favorisait l'accumulation de BALF0 et BALF1, tandis que BALF0 et

BALF1 étaient tous deux capables de réduire considérablement l'expression de BHRF1.

Ces travaux, encore préliminaires, ont démontré pour la première fois que BHRF1

pourrait moduler l’activité du protéasome.

Acknowledgments

I wish to thank my supervisor Prof. Vincent Maréchal for his excellent guidance and

endless patience throughout my studies. I also wish to extend gratitude to Dr. Henri

Gruffat and Prof. Pierre Emmanuel Ceccaldi for sharing their expertise on my work to

review this thesis. I also would like to thank members of the thesis jury for evaluating

my work.

Gratitude also goes to Prof. Alexandre Escargueil, Dr. Michèle Sabbah and Dr. Chloé

Borde for their advice and assistance. I also wish to thank Dr. Frédérique Quignon, Dr.

Nathalie Ferrand and members of team “Biologie et thérapeutiques du cancer” for all their help and for making the team such a nice place to work.

I also wish to acknowledge Prof. Audrey Esclatine for her expertise on autophagy and

the provision of experimental materials. Thanks also go to Dr. Pierre Busson and Dr.

Joël-Meyer Gozlan for the provision of patient samples; Dr. Christophe Marchand for

the technical assistance of mass spectrometry; Mr. Christophe Piesse for the help of

antibody production and Dr. Grégoire Stym-Popper for the assistance of IMARIS

operation.

Finally, I wish to thank my parents and friends for their love and support.

 I Abstract

Autophagy is an essential catabolic process that degrades cytoplasmic components

within the autolysosome therefore ensuring cell survival and homeostasis. A growing

number of viruses including members of the Herpesviridae family have been shown to

manipulate autophagy to facilitate their persistency or to optimize their replication.

Previous works have shown that Epstein-Barr virus (EBV), a human transforming γ-

herpesvirus, hijacked autophagy during the lytic cycle possibly to favor the formation

of viral particles. However, the viral proteins that are responsible for EBV-mediated

subversion of the autophagy pathways are still to be characterized. Here we provide

first evidences that EBV BALF0/1 open reading frame encodes for two proteins,

namely BALF0 and BALF1, that are expressed during the early phase of the lytic cycle.

BALF1 stimulates the autophagic flux which, in turn, was limited in the presence of

BALF0. A putative LC3-interacting region (LIR) was identified that is required both for BALF1 to colocalize with autophagosomes as well as to stimulate autophagy.

BHRF1, one of the well-characterized Bcl-2 homologs of EBV, has been described as an anti-apoptotic modulator in different experimental cell systems. In this thesis, it also shown that BHRF1 stimulates mitophagy, a process that prevents the initiation of the innate immune response mediated by mitochondrial pathways. Co-expression of both

BHRF1 and BALF1 resulted in a slight blockage in the degradative step of autophagy.

Finally, we demonstrated that the accumulation of BHRF1, BALF0 and BALF1 resulted from a complex interplay in which BHRF1 promoted the accumulation of

BALF0 and BALF1 whereas BALF0 and BALF1 were both able to dramatically reduce

BHRF1 expression. Additionally, BALF1 was required for BALF0 accumulation which, in turn, repressed BALF1 expression. Thus, BHRF1 is proposed to have new functions on proteasome-dependent pathways in addition to its activity as an anti- apoptotic and a pro-autophagic protein.

Keywords

Epstein-Barr virus; BALF1; BALF0; BHRF1; autophagy; vBcl-2

 II Résumé

L'autophagie est un processus catabolique essentiel qui dégrade les composants

cytoplasmiques assurant ainsi la survie des cellules et l'homéostasie. Un nombre

croissant de virus comprenant des membres de la famille des Herpesviridae se sont

avérés capable de manipuler l'autophagie pour faciliter leur persistance ou optimiser

leur réplication. Des travaux antérieurs ont montré que le virus d'Epstein-Barr (EBV),

un γ-herpesvirus oncogène humain, détournait l'autophagie au cours de la phase lytique

de son cycle pour favoriser la formation de particules virales. Cependant, les protéines

virales responsables de la manipulation des voies autophagiques restent à caractériser.

Nous montrons ici que le cadre ouvert de lecture BALF0/1 code deux protéines, à savoir

BALF0 et BALF1, qui sont exprimées au cours de la phase précoce du cycle lytique.

BALF1 stimule le flux autophagique, une activité partiellement limitée par BALF0.

Une région supposée d'interaction avec LC3 (LIR) a été identifiée, qui est nécessaire à

la fois pour que BALF1 puisse se localiser avec les autophagosomes et pour stimuler

l'autophagie. Nous avons aussi contribué à démontrer que BHRF1, un orthologue viral

de Bcl-2 bien connu pour ses fonctions anti-apoptotiques, stimule la mitophagie, un

processus qui empêche l'initiation de la réponse immunitaire innée médiée par les voies

mitochondriales. Enfin, nous montrons que les protéines BALF0, BALF1 et BHRF1

sont au cœur d’un réseau de régulation complexe: BHRF1 favorise l'accumulation de

BALF0 et BALF1, alors que BALF0 et BALF1 sont toutes deux capables de limiter

l’accumulation de BHRF1. Par ailleurs, BALF1 est nécessaire à l’accumulation de

BALF0, qui limite en retour celle de BALF1. Nous démontrons ainsi, pour la première

fois, qu’outre ses fonctions pro-autophagiques et anti-apoptotiques, BHRF1 serait

également capable de moduler les voies de dégradations médiées par le protéasome.

Mots clés

Virus Epstein-Barr; BALF1; BALF0; BHRF1; autophagie; vBcl-2

 III Table of Contents

Acknowledgments...... Ⅰ Abstract...... Ⅱ Résumé...... Ⅲ Table of Contents...... Ⅳ Abbreviations...... Ⅶ List of Illustrations...... Ⅻ 1. Introduction...... 1 1.1 Epstein-Barr virus...... 1 1.1.1 Discovery of Epstein-Barr virus...... 1 1.1.2 Classification of EBV...... 2 1.1.3 Structure of EBV virion and genome...... 2 1.2 EBV infection...... 5 1.2.1 Transmission...... 5 1.2.2 Primary infection and viral persistence...... 5 1.2.3 Viral entry...... 8 1.2.4 Immune response...... 10 1.3 Life cycle of EBV...... 11 1.3.1 Latency...... 11 1.3.2 Lytic cycle...... 15 1.3.3 Reactivation...... 20 1.4 EBV-associated human diseases...... 20 1.4.1 Infectious mononucleosis...... 21 1.4.2 X‑linked lymphoproliferative disease...... 21 1.4.3 Post-transplant lymphoproliferative disease...... 21 1.4.4 Burkitt’s lymphoma...... 22 1.4.5 Hodgkin’s lymphoma...... 23 1.4.6 NK/T-cell lymphoma...... 24 1.4.7 Nasopharyngeal carcinoma...... 24 1.4.8 Gastric carcinoma...... 26 1.5 EBV viral Bcl-2 homologs...... 26 1.5.1 Bcl-2 family proteins and viral Bcl-2 homologs...... 26

 IV 1.5.2 Herpesvirus-encoded Bcl-2 homologs...... 30 1.5.3 EBV vBcl-2s...... 30 1.6 BHRF1...... 32 1.6.1 Expression...... 32 1.6.2 Subcellular localization...... 33 1.6.3 Function...... 34 1.7 BAL0/1...... 35 1.7.1 Expression...... 35 1.7.2 Subcellular localization...... 37 1.7.3 Function...... 37 1.8 Autophagy...... 40 1.8.1 Different types of autophagy...... 40 1.8.2 A multistage process...... 42 1.8.3 The autophagic machinery and its regulation pathways...... 44 1.8.4 Physiological and pathological roles of autophagy...... 52 1.8.5 Selective autophagy...... 53 1.9 EBV and autophagy...... 57 1.9.1 Autophagy and EBV latency...... 57 1.9.2 Autophagy modulation during EBV lytic cycle...... 59 1.10 Herpesvirus and autophagy: a lesson to explore the contribution of autophagy to EBV infection...... 62 1.10.1 Subversion of autophagy by Herpesviruses...... 62 1.10.2 Herpesvirus-encoded vBcl-2s inhibit autophagy...... 63 1.11 Aims and objectives...... 65 2. Results...... 67 2.1 Detection of IgG directed against a recombinant form of Epstein-Barr virus BALF0/1 protein in patients with nasopharyngeal carcinoma...... 67 2.2 Epstein-Barr virus BALF0 and BALF1 modulate autophagy...... 76 2.3 BHRF1, a Bcl-2 viral homolog, disturbs mitochondrial dynamics and stimulates mitophagy to dampen type I IFN induction...... 102 3. Discussion...... 142 3.1 The existence of EBV BAFL0 and BALF1 in vivo...... 142 3.2 EBV vBcl-2s modulate autophagy...... 145

 V 3.3 The interplay between vBcl-2s of EBV...... 150 4. Take home message...... 154 5. References...... 156

 VI Abbreviations

AMPK 5’-AMP-activated protein kinase AIDS acquired immune deficiency syndrome ATF activating transcription factor AP-1 activator protein-1 AIM ATG8-interacting motif ATG autophagy-related BL Burkitt’s lymphoma BAC bacterial artificial BALF1 BamH1 A fragment leftward reading frame 1 BHRF1 BamH1 H fragment rightward reading frame 1 BARF1 BamHI A fragment rightward reading frame 1 BART miRNA BamHI A rightward transcript microRNA b-Zip basic leucine zipper BCR B-cell receptor Bcl-2 B cell lymphoma 2 BBD Beclin-binding domain CREB cyclic AMP-response element-binding proteins CMA chaperone-mediated autophagy CQ chloroquine DC dendritic cell E early EA-D early antigen diffuse ENKL extranodal NK/T-cell lymphoma, nasal type EBV Epstein-Barr virus EBNA EBV nuclear antigens EBVaGC EBV-associated GC EBER EBV-encoded RNAs eBL endemic Burkitt’s lymphoma ER endoplasmic reticulum

 VII ELISA -linked Immunosorbent Assay EGFR epidermal growth factor receptor E. coli Escherichia coli ERK extracellular signal-regulated kinase GFP-LC3 green fluorescent protein (GFP)-tagged LC3 GABARAP gamma-aminobutyric receptor-associated protein GC gastric carcinoma HIV human immunodeficiency virus HVT herpesvirus of turkeys HVS herpesvirus saimiri HDACs histone deacetylases HL Hodgkin’s lymphoma HRS Hodgkin–Reed–Sternberg HOPS homotypic fusion and protein sorting HCMV human FL human follicular lymphoma IPTG isopropyl β-D-1-thiogalactopyranoside IE immediate early IRS1 insulin receptor substrate 1 IRS2 insulin receptor substrate 2 IR internal repeats ICTV International Committee on Taxonomy of Viruses IRGM immunity related GTPase M IM Infectious mononucleosis IAV Influenza A virus JNK1 Jun N-terminal protein kinase 1 KSHV Kaposi’s sarcoma herpesvirus Kbp kilobase pairs kDa kilodaltons LCLs lymphoblastoid cell lines L late

 VIII LMP latent membrane protein LKB1 liver kinase B1 LOH loss of heterozygosity MEF2 myocyte enhancer factor 2 mAb monoclonal antibody MHC major histocompatibility complex mTOR mammalian target of rapamycin MEK1/2 MAPK/ERK kinases 1/2 M2 Matrix 2 MAP1LC3 microtubule-associated protein 1 light chain 3 MAMs mitochondria-associated membranes MOM mitochondrial outer membrane MAPK mitogen-activated protein kinase MW molecular weight MOMP MOM permeabilization MHV-68 murine γ-herpesvirus 68 NMR nuclear magnetic resonance NK natural killer cell NCBI National Center for Biotechnology Information NGS next-generation sequencing NLPHL nodular lymphocyte-predominant HL ORF open reading frame orf16 open reading frame 16 PKR protein kinase R PE phosphatidylethanolamine PI3P phosphatidylinositol 3-phosphate PI3K phosphoinositide 3-kinase PDK1 phosphoinositide-dependent protein kinase 1 PI3KC3 phosphoinositol 3-kinase C3 PLCγ phospholipase C gamma pDCs plasmacytoid DCs

 IX PTLD post-transplant lymphoproliferative disorder PKB protein kinase B Rubicon RUN domain and cysteine-rich domain containing, Beclin 1- interacting protein RHEB Ras homolog enriched in brain Rag Ras-related small ROS reactive oxygen species RFP-GFP-LC3 red fluorescent protein (RFP)-GFP-LC3 SNARE soluble N-ethylmaleimide–sensitive factor attachment protein receptor SH2D1A SH2 domain containing 1A SLAM signaling lymphocyte activation molecule shRNA small hairpin RNA siRNA small interfering RNA sBL sporadic Burkitt’s lymphoma SAP SLAM-associated protein TM transmembrane domain TNF tumor necrosis factor TSC2 tuberous sclerosis complex 2 tBid truncated Bid TR terminal repeats TRAIL TNFα-related apoptosis-inducing ligand TP53 tumor protein p53 TGF-β transforming growth factor β TGN trans-Golgi network TPA/PMA 12-O-tetradecanoylphorbol-13-acetate UVRAG ultraviolet radiation resistance‑associated gene protein ULK1 Unc-51-like kinase 1 vPIC viral preinitiation complex vBcl-2 viral Bcl-2 homolog vFLIP viral homolog of cellular FLICE-like inhibitor protein VCA viral capsid antigen

 X Vif viral infectivity factor XBP-1 X-box binding protein 1 XLP X‑linked lymphoproliferative disease Y2H yeast two-hybrid 3-MA 3-methyladenine

 XI List of Illustrations

Figure 1. Electron microscopy observation of thin sectioned EBV particles Figure 2. Classification of EBV Figure 3. Structure of EBV virion Figure 4. Schematic diagram of the linear EBV genome and part of the viral gene products. Figure 5. The model of EBV infection and persistence in vivo Figure 6. Model of EBV entry into major target cells Figure 7. Pre-latency of EBV infection Figure 8. EBV episome and latent Figure 9. Patterns of EBV latent gene expression Figure 10. Replication cycle of EBV. Figure 11. Schematic representation of mammalian Bcl-2 family members Figure 12. Model of apoptotic regulation by cellular and viral Bcl-2 proteins Figure 13. Alignment of amino acid sequences of the indicated cellular and viral Bcl- 2 family proteins Figure 14. The colocalization between BHRF1 and mitochondria Figure 15. The binding between BH3 peptides and BHRF1 Figure 16. The BALF1 ORF potentially encodes two isoforms Figure 17. Subcellular localization of BALF0 and BALF1 Figure 18. BALF1 does not colocalize with BHRF1 in CHO cells Figure 19. Different types of autophagy Figure 20. The process of autophagy Figure 21. The origin and source of the autophagosome membranes Figure 22. The autophagic machinery in mammalian cells Figure 23. Two ubiquitin-like conjugation systems in autophagic machinery Figure 24. The autophagosome–lysosome fusion Figure 25. Regulation pathways of autophagy Figure 26. Pharmacological and genetic modulators of autophagy Figure 27. Methods for monitoring autophagy Figure 28. The roles of autophagy in human Figure 29. Different types of selective autophagy

 XII Figure 30. The overview of selective autophagy Figure 31. LIR-containing proteins Figure 32. EBV subverts autophagy and hijacks the autophagic vesicles for transportation towards the plasma membrane Figure 33. Herpesviruses subvert autophagy Figure 34. BHRF1 stabilization effect and cooperation with BALF1 to modulate autophagy Figure 35. Autophagic modulation by BALF0 Figure 36. Viral proteins encoded by EBV involved in the regulation of autophagy Figure 37. Subcellular localization of BALF1 in HeLa cells Figure 38. Complex interplay between BALF0, BALF1 and BHRF1

Table 1. Function of EBV-encoded latent genes

 XIII 1. Introduction

1.1 Epstein-Barr virus

1.1.1 Discovery of Epstein-Barr virus

The encounter between Anthony Epstein, a British pathologist, and Denis Burkitt, a

surgeon working in Uganda, led to the discovery of Epstein-Barr virus (EBV). In 1958,

Burkitt’s lymphoma (BL) was first described as a lymphosarcoma of young children

with a prevalence distributed along central Africa [1]. Burkitt suggested that this

lymphoma may be vector-transmitted and therefore be virus-induced [2,3]. In 1961,

Epstein attended a lecture about BL which was given by Burkitt, and attempted to

identify the putative virus resident within BL cells [4]. After 3 years of unsuccessful

work, the first cell line derived from BL biopsies was established through a

collaboration with Bert Achong and Yvonne Barr, that led to the detection of herpes-

like particles in 1964 [5,6]. Observation by electron microscopy revealed the presence

of unequivocal viral particles with a characteristic morphology of herpesvirus (Figure

1). This virus was later confirmed as a new human herpesvirus through collaboration

with Walter and Gertrude Henle, who worked in United States, and named Epstein-

Barr virus [4,7].

Figure 1. Electron microscopy observation of thin sectioned EBV particles. In an infected cell, immature virions cut in various planes and a mature enveloped particle shown in the inset. (Ref. 4)

 1 1.1.2 Classification of EBV

EBV, formally named Human herpesvirus 4 (HHV4), belongs to the family

Herpesviridae within the order [8]. Herpesviridae consists of three subfamilies including , and

Gammaherpesvirinae, and these subfamilies are divided into different genera. In human, eight viruses have been described including type 1 (HSV-1/HHV-

1), Herpes simplex virus type 2 (HSV-2/HHV-2), Varicella-zoster virus (VZV/HHV-

3), Human cytomegalovirus (HCMV/HHV-5), (two variants,

HHV-6 A and B), Human herpesvirus 7 (HHV-7), EBV (HHV-4) and Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) (Figure 2). EBV belongs to the

Lymphocryptovirus genus, a subdivision of , which characterized by its predominantly B cell lymphotropic and oncogenic properties.

Figure 2. Classification of EBV. Adapted from the Virus Taxonomy: 2018b Release of International Committee on Taxonomy of Viruses (ICTV) 9th Report (2011).

1.1.3 Structure of EBV virion and genome

EBV consists of a linear, double-stranded DNA genome encased within an icosahedral nucleocapsid, surrounded by a proteinaceous matrix dubbed the tegument and then wrapped by a glycoprotein-embedded lipid envelope [8] (Figure 1 and 3).

 2

Figure 3. Structure of EBV virion. The linear, double-stranded DNA genome is encased within capsid which assembly as nucleocapsid. Nucleocapsid is wrapped by tegument layer under the envelope containing glycoproteins. (Adapted from ViralZone, Swiss Institute of Bioinformatics)

The EBV genome is approximately 170 kilobase pairs (kbp) in length, and the sequence has been annotated with reference to fragments generated by BamHI restriction endonuclease digestion and labelled according to the size (from large to small: A-Z, a- e) (Figure 4). Based on the direction of transcription, the letters L and R standing for

"leftward reading frame" and "rightward reading frame", respectively, have also been used for annotation (e.g. BHRF1: BamH1 H fragment rightward reading frame 1). The

EBV genome has various repetitive sequences scattered throughout the viral genome either within the coding regions of viral latent proteins or near the viral replication origins, including internal repeats (IR) and terminal repeats (TR) [9]. In comparison to most other viruses, the relatively large genome of EBV has the potential to encode for more than 80 proteins [9–11] (Figure 4).

EBV strains have been generally classified into 2 major subtypes, i.e. type 1 and type

2 (also known as type A and B, respectively) mainly based on polymorphisms within the EBV nuclear antigens EBNA2, EBNA3A, EBNA3B and EBNA3C [12]. Type 1

EBV strains are prevalent worldwide and able to transform human B lymphocytes into lymphoblastoid cell lines (LCLs) more efficiently compared to type 2 strains, which is due to differences in the EBNA2 gene [13,14]. The prototypical EBV B95-8 strain was

 3 first fully cloned and sequenced in 1984 by using conventional procedure (GenBank accession number V01555) [15]. In 2003, the wild-type EBV sequence was constructed by replacing a deletion of approximately 12 kb in B95-8 strain with corresponding sequence of Raji strain isolated from BL [16], generating a reference sequence

(GenBank accession number NC_007605). Until 2014, accessible EBV whole genome sequences from GenBank were limited to less than 15 strains (B95‑8/Raji, GD1, AG876,

GD2, HKNPC1, Akata, Mutu, M81, K4123‑Mi, K4413‑Mi, HKNPC2–HKNPC9,

NA19114, NA19315 and NA19384) [16–25]. Recently, a new experimental strategy was developed to enable high-throughput EBV genome sequencing by using a genome capture method analogous to human exome sequencing [24,26], and resulted in an explosive increase in EBV whole genome sequences [27,28].

Figure 4. Schematic diagram of the linear EBV genome and part of the viral gene products. The scale of DNA size is shown at the top. The BamHI restriction map is generated based on the sequence of the SNU-719 strain (middle). EBV latent and lytic gene products (selected, green) are illustrated at the bottom. EBV latent gene products comprise 6 EBV nuclear antigens (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C and EBNA-LP), 3 latent membrane proteins (LMP1,

LMP2A and LMP2B). A series of non-coding RNAs are also expressed during latent infection, including the BamHI A rightward transcript microRNAs (BART miRNAs), BHRF1 miRNAs and EBV-encoded RNAs (EBER1 and EBER2). EBV lytic genes have not been extensively characterized, and previous reports have shown that lytic genes encode for viral transcription factors

(e.g. BZLF1), a viral DNA polymerase (BALF5) and viral glycoproteins (e.g. gp350/220 and gB). Repetitive sequences are shaded in purple. FR, family of repeats; IR, internal repeats; TR, terminal repeats. (Ref. 9)

 4 1.2 EBV infection

EBV is a ubiquitous human γ-herpesvirus which is mainly transmitted through saliva and persistently infects more than 95% of adults worldwide [5,9,29]. B lymphocytes and epithelial cells are the two major targets of EBV, and it can also infect T lymphocytes, natural killer (NK) cells, dendritic cells (DCs) and their precursors under different circumstances [30].

1.2.1 Transmission

EBV is primarily transmitted through saliva. It would notably be acquired by deep kissing among adolescents and young adults [31,32]. Sexual intercourse has been reported to enhance the EBV transmission [33]. Blood transfusion and allograft transplantation also involve in the EBV transmission from donors to recipients [34–37].

The exposure and acquisition of EBV in preadolescent children have been proposed to be associated with close contact with household members or caregivers who carry EBV and shed the virus periodically into their oral secretions [38,39].

1.2.2 Primary infection and viral persistence

Orally transmitted virus from the EBV-positive to EBV-negative individual occurs mainly through the epithelium of the oropharynx, establishing a lytic replication, eliciting the release of active virions and shedding into the throat. However, it has reported that the mucosal apical surface of the intact oral epithelium is resistant to cell- free virus infection [40,41]. Subsequently, the same group reported that initial EBV entry into mucosal epithelium might occur by rapid viral transcytosis from apical to basolateral membranes [42]. B cells in the underlying lymphoid tissues can also become infected but the virus switches to a growth-transforming latent infection resulting in the

EBV-positive lymphoblasts, which are typically controlled by T-cell responses directed against viral latent proteins. However, the switch between different latent expression programs eventually leads to the silencing of immunogenic viral proteins, which favors

 5 immune escape. This allows the formation of a stable reservoir of resting memory B cells containing multicopy viral genomes with a very limited viral gene expression (so called latency 0) (Figure 5A). The memory B cells harboring EBV genomes can recirculate between blood and lymphoid tissue within the oropharynx region, and periodical reactivation of the virus into lytic cycle results in the seeding of new viral particles in the oropharynx therefore reestablishing and replenishing the viral life cycle.

In immunocompetent hosts, EBV can therefore persist under the tight control of the immune system and establish a lifelong infection.

During primary infection, several models have been proposed for how EBV latently infect B cells, including infection of naïve B cells which migrate through the germinal center and become EBV-positive memory B cells [43], or, alternatively, selective infection of pre-existing memory B cells [44]. In the first scenario, EBV infects naïve

B cells and drives them into lymphoblasts which allows the cells to migrate into the follicle and initiate a germinal center reaction, and then EBV-infected cells exit the germinal centre as resting memory B cells in which all viral protein expression is turned off (Figure 5A). Alternatively, EBV infection of pre-existing memory B cells as a direct route into the reservoir of resting memory cells (Figure 5A). During persistent infection, memory B cells harboring the EBV might be recruited into germinal center reactions and either replenish the memory B cell reservoir or commit to plasma-cell differentiation which triggers viral replication (Figure 5B). The resulting virus might infect permissive epithelial cells and other uninfected B cells as well as being shed into saliva for spread to other hosts.

 6

Figure 5. The model of EBV infection and persistence in vivo. (A) Primary infection. Once transmitted, EBV establishes a lytic replication in the epithelium of the oropharynx releasing active virions so that B cells in the underlying lymphoid tissues can become infected. Distinct models have been proposed for how EBV latently infect B cells, including infection of naïve B cells which migrate through the germinal center and become EBV-positive memory B cells or, alternatively, selective infection of pre-existing memory B cells. (B) Persistent infection. EBV-positive memory

B cells might be recruited into germinal center reactions and either replenish the memory B cell reservoir or commit to plasma-cell differentiation which triggers viral replication. According to the pattern of gene expression, latency has been further categorized into various types and indicated on the top of each figure. Latency 0 is characterized by none of viral gene expression. The rest of latent gene expression patterns will be more discussed later in following sections. (Ref. 42)

 7 1.2.3 Viral entry

Once transmitted, EBV infects target cells by fusion between the viral and cellular lipid bilayer membranes using multiple viral factors and host receptors.

EBV utilizes five glycoproteins (gp) for efficient B cell entry including attachment protein gp350/220, receptor binding protein gp42, core fusion machinery gH/gL and gB. The attachment protein gp350/220 binds to complement receptor type 2

(CR2/CD21) or type 1 (CR1/CD35) which tethering EBV to the host cells through an attachment step that is not essential for the entry whereas increases the infection efficiency [45,46]. Then, the binding of gp42 with its B lymphocyte receptor human leukocyte antigen (HLA) class II results in a widening hydrophobic pocket within gp42 that allows the activation of gH/gL. The interaction between the tripartite complex gH/gL/gp42 and gB triggers the conformational transition of gB from profusion to postfusion leading to the fusion with B cell membranes (Figure 6A). The interaction between gp42 and gH/gL inhibits epithelial cell fusion and entry, which determines the cellular tropism [47,48]. Since epithelial cells lack both HLA class II and CD21, gH binds directly to its epithelial cell receptor integrin ανβ6 giving rise to a conformational change within the large groove of gH/gL that allows the triggering of gH/gL. The conformational transition from profusion to postfusion gB induced by the interaction between gH/gL and gB results in the fusion with plasma membranes (Figure 6B).

The cellular receptors of T cells and NK cells involved in the entry of EBV have not been clearly identified. It has been observed that peripheral blood T cells do not normally express the EBV receptor CD21 [46,49], which suggests that EBV might use alternative viral glycoproteins and cellular receptors to infect T cells. NK cells express

HLA class II but not CD21, and the NK-cell attack of EBV-infected B cells leads to trans-synaptic acquisition and transient expression of functional CD21 which might be used for EBV infection [50].

 8 A

B

Figure 6. Model of EBV entry into major target cells. (A) EBV entry into B lymphocytes. After attachment, gp42 binding to HLA class II allows the activation of gH/gL. The transition of gB from profusion to postfusion leading to the fusion with B cell membranes. (B) EBV entry into epithelial cells. gH binds directly to integrin ανβ6 giving rise to the triggering of gH/gL. The conformational transition from profusion to postfusion gB induced by the interaction between gH/gL and gB results in the fusion with plasma membranes. The residues Q54/K94 (yellow spheres) of gH/gL are supposed to be involved in gB interactions and are directed towards a model of prefusion gB. The structural view of EBV gH/gL shows the disulfide bonds (orange spheres), gp42-binding region (blue spheres) and the KGD-motif (red spheres). TM, transmembrane domain; α, α-helix; β, β-strand. (Ref. 43)

 9 1.2.4 Immune response

Since EBV primary infection is asymptomatic more often, it has been difficult to investigate the dynamics of adaptive immune response to EBV in the general population, whereas accumulating evidences have shown that immunocompetent individuals with infectious mononucleosis (IM) elicit T cell responses to primary EBV infection [51]. IM is associated with a large expansion of activated CD8+ T cells in the circulation. It is shown that these CD8+ T cells are primarily specific for lytic viral proteins, including 2 immediate early genes: BZLF1 and BMLF1 [52], whereas the late proteins elicit minimal CD8+ T cell response. In comparison to lytic proteins, CD8+ T

cells specific for latent proteins, such as EBNA1, EBNA3, LMP1 and LMP2, can be

detected at a much lower frequency [53]. In IM patients, expansions of CD4+ T cells

do not exhibit the similar magnitude as observed in CD8+ T cells [54]. Serologic

evaluation of the humoral response to EBV viral proteins has been used as the gold

standard for assessing the status of exposure to the virus. Antibodies against lytic

proteins including viral capsid antigen (VCA), gp350, early antigen-diffuse (EA-D) as

well as latent protein EBNA1 can be measured for the diagnosis of the presence or

absence of infection and the infection stage (primary versus past infection).

Although the adaptive immune response to EBV has been extensively investigated, the

role of the innate immune system, particularly NK cells, has been recently addressed

[55,56]. There are emerging lines of evidence indicating that NK cells are involved in

the regulation of primary lytic infection [56]. The NK cells expansion has been

observed in individuals with IM and correlated with decreasing level of viral load in

some cases [31,57]. In vivo, EBV infection triggered an increase of NK cells in

humanized mice. Conversely, depletion of NK cells led to increasing viral titers, more

severe symptoms and the development of EBV-induced lymphomas [58]. In vitro,

human NK cells can kill EBV-infected B cells undergoing lytic replication [59].

 10 1.3 Life cycle of EBV

The life cycle of EBV comprises two modalities: the latency and lytic cycle. During latency, EBV genomic DNA persists as an episome in the nucleus of host cell. A limited number of latent genes is expressed, and no viral particles are produced. In the lytic cycle, extensive viral gene expression takes place and progeny viruses are produced.

The switch from latency to lytic cycle is termed reactivation, which can be achieved by diverse stimuli in vivo (B cell differentiation into plasmocytes, B cell receptor linking…) and ex vivo (many biological and chemical inducers have been described).

1.3.1 Latency

After primary infection, it has long been regarded that EBV establishes latent infection immediately. However, it has been reported that a subset of lytic genes expressed whereas no viral DNA replication were detectable [60–62] (Figure 7A) , suggesting the pre-latency might be the initial mode of EBV infection [63] (Figure 7B).

During latency, viral genomic DNA exists in the nucleus as multicopy chromatinized episomes. Latent genomes are replicated from the origin of plasmid replication (OriP) in synchronization with the host genome during the S phase and delivered to daughter cells during mitosis in a chromosome associated form [64]. Only a limited number of latent genes are expressed under the control of viral promoter Cp, Wp and Qp. Latent proteins consist of the 6 EBV nuclear antigens (EBNA1, EBNA2, EBNA3A, EBNA3B,

EBNA3C and EBNA-leader protein (LP)), 3 latent membrane proteins (LMP1, LMP2A and LMP2B), BamHI A fragment rightward reading frame 1 (BARF1) and BHRF1.

Multiple of non-coding RNAs are also expressed during latent infection, including 44

BamHI A rightward transcript microRNAs (BART miRNAs), 3 BHRF1 miRNAs and

2 EBV-encoded RNAs (EBER1 and EBER2) (Figure 8). Latent gene products of EBV play an essential role in the growth, survival and immune escape of EBV-infected cells.

For these reasons, several of them are involved in EBV-associated malignancies

(Table1).

 11 A

B

Figure 7. Pre-latency of EBV infection. (A) A subset of viral genes expressed during the pre-latent phase. The newly infected primary B cells, which also referred to as pre-latent, express a subset of lytic and latent genes. The pre-latent B cells transit to several types of latency with various gene expression modes. (B) Upon primary infection of EBV, the infected cells undergo pre-latent, abortive lytic cycles in which only immediate-early (blue) and early genes (red) are expressed without viral lytic DNA replication. Then, the transient lytic state is silenced and only a limited number of latent genes (green) is expressed, which followed by a transition into the abortive lytic cycle whereas then re-silenced to the latent state again. A part of latent cells enters into the complete lytic cycle after latency with viral lytic DNA replication and production of the progeny virus. (Adapted from Ref. 60,61)

 12

Figure 8. EBV episome and latent genes. Only a limited number of latent genes are expressed from the double-stranded viral DNA episome. OriP is shown in orange. TR refers to the terminal repeats shown in red. The short thick green arrows represent exons encoding latent proteins: six EBNAs, three LMPs, BHRF1 and BARF1. The short blue arrows represent EBV encoded RNAs. The middle long green arrow represents EBV transcription during latency III. The inner red arrow represents the EBNA1 transcript originating from the Qp promoter during latency I and latency II. The outer long blue arrow represents transcription during Wp-restricted latency which is initiated from the

Wp promoter with a deletion of EBNA2. (Ref. 5)

 13

Table1. Function of EBV-encoded latent genes.

 14 According to the pattern of gene expression, latency has been further categorized into

four types (Figure 9), which have significance both in the life cycle of the virus and

EBV-associated malignancies. Latency III pattern is characterized by expression of all latent proteins. It has been associated with LCLs and in proliferating B-cells in patients with post-transplant lymphoproliferative disorder (PTLD). EBNA proteins are expressed from mRNAs generated by alternative splicing of a long primary transcript initiated from promoter Cp or Wp, which is located in the BamHI C or W region, while

LMPs are expressed from separate promoters located in the BamHI N region [65–67].

BHRF1 has recently been described as a latent protein which is also expressed from

Wp-initiated transcript in Latency III [68]. Latency I pattern is characterized by expression of a single latent protein EBNA1 which is transcribed from a promoter located in the BamHI Q region (Qp) and has been observed in the majority of BL (85%)

[69,70]. Wp-restricted latency (Wp Latency) is characterized by expression of BHRF1 and EBNA proteins except EBNA2 as well as the LMPs. It has been observed in a minority of BL (15%) which carry EBV strains with a deletion of the EBNA2 region

[68,71,72]. Latency II pattern is characterized by expression of EBNA1 accompanied by the expression of LMPs. It has been observed in EBV-associated gastric carcinoma

(GC), nasopharyngeal carcinoma (NPC) and Hodgkin’s lymphoma (HL) [73–75].

1.3.2 Lytic cycle

The lytic cycle of EBV is orchestrated by more than 80 genes which are coordinately expressed as a successive cascade divided into three functional stages i.e. immediate early (IE), early (E) and late (L) (Figure 10). The IE genes encode for transcription factors which are in charge of turning on the expression of E genes and are critical for the switch from latency to lytic replication. The E genes encode for proteins that are notably responsible for nucleotide metabolism and viral DNA amplification whereas the L genes encode for viral structural proteins such as capsid proteins, tegument proteins and glycoproteins.

 15

Figure 9. Patterns of EBV latent gene expression. Latency III is characterized by expression of all latent proteins and observed in LCLs and patients with PTLD. Latency I is characterized by expression of a single latent protein EBNA1 and has been observed in the majority of BL (85%).

Wp Latency has been found in a minority (15%) of EBV-positive BLs (termed Wp-BL). Latency II is characterized by expression of EBNA1 and accompanied by expression of LMPs, that has been observed in EBV-associated GC, NPC and HL. Latent proteins are shown in blue. Non-coding

RNAs are shown in red. Latent promoters (Cp,Wp and Qp) are shown in green. In Wp-BL, EBNA-

LP is truncated due to a genomic deletion and therefore is denoted as t-EBNA-LP. (Ref. 74)

 16

Figure 10. Replication cycle of EBV. After transmission, viral particle attaches and enters into host cell either by fusion of its envelope with the plasma membrane or by endocytosis resulting in the release of nucleocapsid and the tegument into the cytoplasm. The nucleocapsid is transported to the nuclear pore by using the microtubule network and the viral genome is released into the nucleus and circularized. The replication cycle begins with the expression of the IE proteins which are the transcription factors required for the expression of the E proteins. Part of the E proteins are involved in the formation of the core viral replication proteins. Following amplification of the viral genome, the L proteins are probably expressed from the newly replicated viral DNA contributing to the formation of the viral particle. At the end of viral multiplication, assembled nucleocapsid are budding through the inner nuclear membrane acquiring the first envelope (primary envelopment). Then, the envelope of nucleocapsids fuses with the outer nuclear membrane to release the unenveloped nucleocapsids into the cytoplasm. During re-budding into a cytoplasmic compartment, probably the trans-Golgi network (TGN), the unenveloped nucleocapsids acquire the tegument proteins, viral glycoproteins and the final envelope (secondary envelopment). Once formed, mature virions are released from cells by using exocytosis. (Adapted from Ref. 75)

 17 IE genes BZLF1 and BRLF1 encode for the proteins ZEBRA (Zta/Z/EB1) and Rta (R), respectively, which are essential transactivators required to induce the switch from latent to lytic stage of EBV infection in most of latently infected cell lines [76,77]. The promoters of these IE genes are initially activated by cellular transcription factors. The promoter of ZEBRA is activated by cellular transcription factors, including myocyte enhancer factor 2 (MEF2), Sp1 and Sp3 [78,79]. Transcription factors of the basic leucine zipper (b-Zip) family , such as the cyclic AMP-response element-binding proteins (CREB), activating transcription factor (ATF), activator protein-1 (AP-1) and a spliced form of the X-box binding protein 1 (XBP-1) also involve in the activation of

ZEBRA promoter [80–84]. Subsequently, ZEBRA and Rta synergistically activate the promoters of E genes that encode for the viral replication proteins [85]. In comparison to Rta, ZEBRA is much more effective in inducing the expression of EBV lytic genes in many cell lines, and only ZEBRA but not Rta can switch from latency to lytic replication in BL cell line Raji as well as some LCLs [86,87]. The origin of lytic viral

DNA replication, termed oriLyt, is distinct from the origin of replication that used for latent genome maintenance [88]. In addition to acting as a transactivator, ZEBRA has been found to play an essential role in EBV DNA replication through direct interactions with oriLyt and the viral replication proteins [89–91]. During primary infection, the transcription of lytic cycle has been found both in B cells and epithelial cells [61,92].

Immediately after infection, a complete lytic cycle cannot be induced because ZEBRA preferentially binds to and activates the methylated viral genome (at that very early step, the viral genome is still free of methylations) which results in a pre-latent phase that has been proposed as the initial mode of EBV primary infection [93,94].

E gene products are mainly involved in the lytic viral replication. EB2 (Mta/SM), gene product encoded by BSLF2 and BMLF1, functions as a mRNA export factor for a subset of early and late viral mRNAs and plays an essential role in the production of infectious virus [95,96]. There are six core viral replication proteins mediating the lytic viral replication including BALF5 (the viral DNA polymerase), BMRF1 (the DNA

 18 polymerase processivity factor), BALF2 (the single-stranded DNA binding protein),

BBLF4 (the helicase), BSLF1 (the primase) and BBLF2/3 (the primase-associated

protein) [97]. There are at least two E genes encoding the transcription factors including

BMRF1 and BRRF1 [98,99]. Notably, BMRF1 is not only a DNA polymerase

processivity factor but also a transactivator inducing the expression of oriLyt promoter,

BHLF1 [98]. BGLF4 (a serine/threonine protein kinase) modulates the function of

nuclear pore complex and promotes the nuclear import of several EBV proteins

including viral DNA replication and the major capsid protein for viral DNA

replication and the nuclear egress of nucleocapsids [100,101]. The early genes of EBV

also encode for two to three homologs of cellular Bcl-2 namely BHRF1, BALF0 and/or

BALF1 [102–105], that will be discussed with more details in following sections. The

viral DNA amplification is followed by expression of L genes, and the early protein

BcRF1 with other viral proteins (BGLF3, BDLF4, BVLF1, BDLF3.5 and BFRF2)

forming a viral preinitiation complex (vPIC) to interact with cellular RNA polymerase

II and activate the L gene transcription [106–108].

L genes primarily encode for structural viral proteins: nucleocapsid proteins including

BcLF1 (major capsid protein) and BFRF3 (minor capsid protein VCAp18) that around the viral genome as well as glycoproteins including BLLF1 (gp350/220), BXLF2 (gH),

BKRF2(gL), BZLF2 (gp42) and BALF4 (gB) that mediate attachment and fusion to host cells [109]. In addition, L genes encode for two immunomodulatory proteins which are transcribed by a mechanism distinct from that used for encoding viral structural proteins [110]. The two immunoevasins, BCRF1 (viral homolog of cellular interleukin-

10) and BPLF1 (deubiquitinase/deneddylase) are transcribed independently of the vPIC and suppress antiviral immune responses during primary infection [110].

At the end of viral replication, assembly of capsid proteins and viral genome takes place in the nucleus (Figure 10). Once assembled, the completed nucleocapsids associate with some of the tegument proteins and bud through the inner nuclear membrane acquiring the first envelope (primary envelopment). Then, the envelope of nucleocapsids fuses

 19 with the outer nuclear membrane to release the unenveloped nucleocapsids into the cytoplasm. During re-budding into a cytoplasmic compartment, probably the trans-

Golgi network (TGN), the unenveloped nucleocapsids acquire the tegument proteins, viral glycoproteins and the final envelope (secondary envelopment). Once formed, mature virions are released from cells by using exocytosis.

1.3.3 Reactivation

EBV reactivation is probably triggered in vivo by stimulation of B-cell receptor (BCR) and differentiation of memory B cells to finite antibody-producing plasma cells

[111,112]. Upon activation of the BCR, a series of signaling pathways are turned on and several transcription factors downstream of BCR initiate transcription from the

BZLF1 promoter [113]. In latently infected B cells, viral reactivation can be mimicked through activation of the BCR-induced phospholipase C gamma (PLCγ) and mitogen- activated protein kinase (MAPK) pathways by using the 12-O-tetradecanoylphorbol-

13-acetate (TPA/PMA) phorbolester [113,114]. In addition, reactivation can be triggered by BCR crosslinking antibodies, histone deacetylases (HDACs), DNA methyltransferase inhibitors, calcium ionophores, transforming growth factor β (TGF-

β) and hypoxia [10,113]. In epithelial cells, cellular transcription factor KLF4, which is required for normal epithelial cell differentiation, induces differentiation-dependent lytic EBV reactivation by binding to and activating transcription from the BZLF1 and

BRLF1 [115].

1.4 EBV-associated human diseases

EBV is a ubiquitous lymphocrytovirus which persistently infects more than 95% of population worldwide. EBV is mainly transmitted by saliva and establishes lifelong infection. Whereas EBV persistent infection is usually symptomless, it has also been associated with several lymphoproliferative diseases and with a number of human

 20 malignancies of lymphoid and epithelial origins. Based on ex vivo data as well as medical and epidemiological evidences, EBV is currently considered as carcinogenic to humans by the International Agency for Research on cancer (IARC) (group 1).

1.4.1 Infectious mononucleosis

Infectious mononucleosis (IM) is an acute and self-limiting infectious disease with

clinical symptoms including fever, marked fatigue, lymphadenopathy and pharyngitis,

that is accompanied by a large number of atypical lymphocytes in the blood [116].

Primary infection by EBV is the major cause of IM. Whereas primary infection with

EBV is asymptomatic when occurring in childhood, it might be responsible for IM in

adolescents or young adults. During acute illness, high viral loads are detectable both

in the oral cavity and blood, that is accompanied by a massive expansion of CD8+ T

cells directed against EBV-infected B cells and the production of immunoglobulin M

antibodies against VCA, whereas the number of CD8+ T cells decreases to normal level

and antibodies develop against EBNA-1 in convalescence [117,118].

1.4.2 X‑linked lymphoproliferative disease

X‑linked lymphoproliferative disease (XLP) was first reported in 1975 as “Duncan’s

disease” of 18 boys in Duncan kindred [119]. XLP is an inherited immunodeficiency,

which in the majority of cases exacerbates following the primary infection with EBV,

resulting in fatal IM, hypogammaglobulinemia, and malignant lymphoma [120,121].

The genetic defect responsible for XLP has been identified as a mutation in the SH2

domain containing 1A (SH2D1A) gene of the X chromosome, which encodes for a

defective signaling lymphocyte activation molecule (SLAM)-associated protein (SAP)

leading to inability to regulate immune responses to control B-cell proliferation caused

by EBV infections [122,123].

1.4.3 Post-transplant lymphoproliferative disease

 21 In immunocompetent individuals, EBV-induced B-cell transformation is controlled by

EBV-specific T-cell response. Conversely impaired T-cell response occurring during

acquired or innate immunodeficiency can lead to the unregulated EBV-driven B-cell

proliferation and transformation. After transplantation of solid organs or hematopoietic

stem cells, latently infected B-cells may proliferate and be the cause of post-transplant

lymphoproliferative diseases (PTLD) [124]. Following solid organ and hematopoietic

stem cell transplantation, PTLD is thought to derive from lymphoid cells of the

recipient or from the donor [125,126]. This severe and life-threatening disease is

characterized by clinical symptoms including fever, lymphadenopathy, fulminant

sepsis, and mass lesions in lymph nodes, spleen, or central nervous system [127], which

is associated with EBV infection displaying a latency III pattern of gene expression.

1.4.4 Burkitt’s lymphoma

EBV has been first discovered in a cell line derived from biopsies of Burkitt’s

lymphoma (BL) in 1964. Subsequently it has been reported that EBV infection of

umbilical cord lymphocytes could give rise to continuously proliferating

lymphoblastoid cell lines (LCLs) [6,128], suggesting a connection between EBV

infection and development of lymphomas.

BL is an aggressive B-cell malignancy which is classified into three distinct subtypes

referred to as endemic (eBL), sporadic (sBL) and immunodeficiency-related BL based

on the geographic distribution and EBV-association [129]. Almost all eBL in equatorial

Africa are EBV-positive. Alternatively, worldwide sBL are rarely associated with EBV

(∼10–15%). The human immunodeficiency virus (HIV)-associated BL exhibit an

intermediate rate of association with EBV (∼40%) suggesting that the role of EBV in

this subtype is less clear than in eBL [130–132]. The eBL is the most common pediatric

malignancy in the equatorial belt of Africa in which Plasmodium falciparum malaria is holoendemic [132]. It has been shown that the intensity of malaria infection correlates with the expression level of activation induced cytidine deaminase, which is necessary

 22 for inducing the characteristic BL‑causing mutations, suggesting that malaria and EBV infection have a synergistic effect on the development of BL [133].

EBV genome has been first identified in biopsies of BL by using DNA hybridization in

1970 [134], and then the expression of EBNA1 has been shown in BL cell lines by using anti-complement immunofluorescence in 1974 [135]. Subsequent analysis demonstrated that EBV-positive BL cells can express other EBV nuclear antigens as well as latent membrane proteins under culture condition [136]. The precise role of

EBV infection in pathogenesis of BL is still discussed and the presence of monoclonal

EBV episomes in EBV-positive BL cells suggests that EBV infection has preceded and might contribute to proliferation of the precursor B-cells [137]. BL are characterized by the reciprocal chromosomal translocation of the MYC gene on chromosome 8 and the immunoglobulin heavy or κ and λ light chain loci on chromosome 14. This genetic event leads to the MYC oncogene under the transcriptional control of an immunoglobulin locus [138]. The resulting overexpression of c-myc not only drives the high proliferation of BL cells but also induces apoptosis [139,140]. In contrast to EBV- positive counterparts, BL cell lines losing the viral episomes in culture are more sensitive to apoptosis suggesting that EBV might contribute to counteract c-myc pro- apoptotic activity [72]. In the majority of BL, latency I pattern has been observed and characterized by expression of a single latent protein (EBNA1) [69,70], which plays an essential role in the maintenance of viral episome and mediates anti-apoptotic effect of

EBV in BL cells [141].

1.4.5 Hodgkin’s lymphoma

Hodgkin’s lymphoma (HL) was first described by Thomas Hodgkin as Hodgkin’s disease in 1832 [142], which was subsequently renamed to HL because of its lymphoid origin and characterized by the mononucleated Hodgkin cells and the multinucleated

Reed–Sternberg cells (known as Hodgkin–Reed–Sternberg (HRS) cells) [143].

According to the differences in the morphology and phenotype of the lymphoma cells

 23 and the composition of the cellular infiltrate, HL is divided into two subtypes including

the classical HL and nodular lymphocyte-predominant HL (NLPHL) [144]. EBV has

been detected in HRS cells in about 40% of classical HL in the Western countries

whereas it is rarely found in NLPHL [145,146], and EBV-infected HRS cells are nearly

universal in HL patients with acquired immune deficiency syndrome (AIDS) [147,148].

1.4.6 NK/T-cell lymphoma

Extranodal NK/T-cell lymphoma is a rare and distinct malignancy of putative NK cell

origin with a minority deriving from the T-cell lineage, which is pathologically categorized into two forms including extranodal NK/T-cell lymphoma, nasal type

(ENKL) and aggressive NK-cell leukemia [149]. It is characterized by an aggressive clinical course, poor prognosis and strong association with EBV infection [150]. ENKL is more common in Asian than in Western countries and exhibits a latency II pattern with variable LMP1 expression [151,152]. Although the role of EBV in ENKL development is unclear, transcriptional defects of the BART miRNAs by deletion and the disruption of host NHEJ1 by integrated EBV fragment have been described [153].

1.4.7 Nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) usually originates in the lateral wall of the nasopharynx with clinical symptoms including trismus, pain, otitis media, nasal regurgitation due to paresis of the soft palate, hearing loss and cranial nerve palsies in the primary tumor. Subsequently the tumor growth may produce nasal obstruction or bleeding and a "nasal twang" [154]. Based on histology, NPC has been classified into three subtypes including type 1 squamous cell carcinoma, type 2 non-keratinizing carcinoma and type 3 undifferentiated carcinoma whose association with EBV infection is well established [154,155]. Undifferentiated NPC is endemic to southern

China and Southeast Asia and affects mostly middle-aged individuals with predominance among males [155–157].

 24 In spite of geographical origin, almost all undifferentiated NPC are found to be EBV positive [158]. EBV strains have been generally classified into 2 subtypes hereby referred to as EBV-1 and EBV-2 [12], which both have been implicated in NPC. The majority of NPC cases are associated with EBV-1 infection in Southern China,

Southeast Asia, Mediterranean, Africa, and the United States, whereas some cases are mostly EBV-2-related among Inuit populations of Alaska but contain polymorphisms with characteristics of Asian EBV-1 [159]. The association between EBV infection and

NPC was first identified in patients with high titers antibodies directed against EBV antigens including VCA and early antigen diffuse (EA-D/BMRF1) [160]. In NPC tumor cells, EBV genome was detected by in situ hybridization and subsequent studies revealed that NPC tumor cells carry monoclonal EBV genomes indicating that EBV infection has preceded the expansion of the malignant cell clone [161–165]. In addition,

EBV exhibits latency II pattern with variable LMP1 expression in undifferentiated NPC

[166]. Previously, the level of VCA-specific immunoglobulin A (IgA) has been used as a potential prognostic indicator for NPC. Current approach for early screening of

NPC has been improved by using the quantitative PCR to detect circulating cell-free

EBV DNA which has become a gold standard biomarker that can be used to stage patients and provide prognostic information [167–169].

Exposure to environmental risk factors, such as smoking and dietary components, results in the loss of heterozygosity (LOH) at 3p and 9p occurring as an early event in NPC pathogenesis, which generates low-grade pre-invasive lesions that become susceptible to EBV infection after additional genetic and epigenetic events

[170–172]. Once infected, upregulated expression of the anti-apoptotic protein Bcl-2 and elevated activity of telomerase in low-grade dysplastic lesions promote the establishment of latent EBV infection. EBV latent gene products might provide growth and survival benefits resulting in the development of NPC [173,174]. Recent analysis of genomic landscape of NPC revealed an enrichment of genetic lesions affecting

 25 several important cellular processes and pathways, including chromatin modification,

ERBB-PI3K signaling and autophagy machinery [175].

1.4.8 Gastric carcinoma

The EBV genome was initially detected in gastric carcinoma (GC) by polymerase chain reaction (PCR) in 1990 [176]. Subsequently, it was shown that nearly 10% of GC are

EBV positive [170]. In contrast to endemic BL and NPC, EBV-associated GC

(EBVaGC) represents a worldwide prevalence in a similar proportion suggesting that it might be the most common EBV-related malignancy [177–179]. EBVaGC is characterized by a monoclonal proliferation of carcinoma cells with latent EBV infection as demonstrated by the detection of EBER through in situ hybridization

[180,181]. As similar to NPC, EBVaGC displays a latency II pattern of latent gene expression [182]. EBVaGC has characteristic clinicopathological features including lymphoepithelioma-like histology, a predominance among males, a proximal location in the stomach and a favorable prognosis [178–180]. The precise role of EBV in the pathogenesis of GC is still unknown, whereas it has been proposed that EBV can only infect neoplastic gastric cells and therefore would be a late event in gastric carcinogenesis [183].

1.5 EBV viral Bcl-2 homologs

1.5.1 Bcl-2 family proteins and viral Bcl-2 homologs

Apoptosis is a process of fundamental importance for cellular and tissue homeostasis,

pathophysiological processes and development. Bcl-2 (B cell lymphoma 2) has been

identified as the first inhibitor of apoptosis [184]. The activation of Bcl-2 resulted from

a t(14;18) chromosomal translocation in follicular lymphoma, a malignant B-cell lymphoma. Following this recombination, Bcl-2 expression was placed under the control of the immunoglobulin transcription enhancer which lead to its over-expression

 26 [185–187]. Subsequent studies have determined that Bcl-2 plays an important role in

the tumorigenesis by inhibiting apoptosis rather than promoting proliferation [188,189].

The Bcl-2 family proteins, including Bcl-2 and its homologs, have been characterized

by the presence of conserved short regions termed as Bcl-2 homology (BH) domains

[190]. Based on their structure and function, the Bcl-2 family proteins can be classified into three groups: the anti-apoptotic proteins, the pro-apoptotic proteins and the BH3- only proteins (Figure 11). Most of the Bcl-2 family proteins contain an α-helix with high hydrophobicity at their C-terminus. This region is considered as a potential transmembrane (TM) domain that may target Bcl-2 related proteins to cellular membranes, such as nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes [191,192]. In mammals, the anti-apoptotic group consists of

Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1/Bfl-1, Boo/Diva/Bcl-2-L-10, Bcl-B and NR-13 whereas pro-apoptotic group contains Bax Bak Bok and Bcl-Xs [193–195]. The

BH3-only proteins only bear the BH3 domains and include Bid, Bad, Noxa, Puma, Bmf,

BimL/Bod, Bik/Nbk, Blk, Hrk/DP5, Bnip3 and Bnip3 L [193,194].

Activation of Bax and Bak in the mitochondrial outer membrane (MOM) results in the

MOM permeabilization (MOMP), which leads to the release of cytochrome c and other pro-apoptotic factors from the mitochondrial intermembrane space to initiate the apoptotic cascades [193,196] (Figure 12). In response to apoptotic stimuli, BH3-only proteins bind to and neutralize the pro-survival proteins, thereby activate Bax/Bak and trigger apoptosis [197]. Alternatively, it has been shown that BH3-only proteins can initiate apoptosis through direct interaction with pro-apoptotic proteins [197]. In addition, the BH3-only protein BID is activated by proteolytic cleavage to form tBid

(truncated Bid). This releases its BH3 domain that becomes available for interaction

[196,198–200].

 27

Figure 11. Schematic representation of mammalian Bcl-2 family members. Bcl-2 homology (BH) domains 1–4 (BH1–4) are indicated. TM, putative transmembrane domain. α, α-helix. (Ref. 194)

 28

Figure 12. Model of apoptotic regulation by cellular and viral Bcl-2 proteins. Activation of Bax and Bak by BH3-only protein through either the removal of inhibition on pro-survival proteins or direct interaction, leads to MOMP, and releases factors such as cytochrome c from the mitochondrial intermembrane space to initiate the caspase cascade. vBcl-2 proteins can act on the BH3-only proteins, or directly block the action of Bax and Bak to prevent the initiation of apoptosis. (Ref. 193)

Apoptosis plays a crucial role in the regulation of innate immune responses against intracellular pathogens. Accordingly, viruses can inhibit apoptosis to promote genome replication, viral proteins production, virus assembly and the spreading to new host.

 29 Conversely viruses are capable of initiating apoptosis to facilitate shedding and hence

dissemination as well as evade from immune responses [201]. Viruses have evolved

mechanisms by using molecular mimicry of host proteins to stimulate or inhibit

apoptosis during their life cycles [201]. There is accumulating evidences demonstrating that some DNA viruses can encode for Bcl-2 orthologs (vBcl-2), which mimic the anti- apoptotic Bcl-2 proteins and hijack the intrinsic apoptotic pathway for their own benefits. So far vBcl-2 have been identified notably in Herpesviridae, Poxviridae,

Asfarviridae and Iridoviridae family [202–204]. vBcl-2 homologs can inhibit apoptosis by interacting with cellular BH3-only or pro-apoptotic proteins (Figure 12).

1.5.2 Herpesvirus-encoded Bcl-2 homologs

A multitude of Herpesviridae members encode for Bcl-2 like proteins, including α- herpesvirinae (herpesvirus of turkeys [HVT]), β-herpesvirinae (HCMV), γ- herpesvirinae (KSHV, EBV, herpesvirus saimiri [HVS] and murine γ-herpesvirus 68

[MHV-68]).

Amongst the α-herpesviruses, vnr-13 encoded by HVT shares 80% homology with Nr-

13, an apoptosis inhibitor in avian cells, and inhibits apoptosis after serum deprivation

[205]. HCMV encodes vMIA, which is targeted to mitochondria and inhibits oligomerization of pro-apoptotic Bcl-2 family members Bax and Bak. vMIA appears to be distinct both in sequence and structure from Bcl-2 proteins [206]. Ks-Bcl-2, a vBcl-2 protein encoded by KSHV, is able to bind Bim, Bid, Bik, Bmf, Hrk, Noxa, and

Puma. Ks-Bcl-2 plays a key role in the completion of the lytic cycle during viral infection [193,207,208]. EBV encodes for two vBcl-2 proteins, namely BHRF1 and

BALF0/1 [102–105] that will be more discussed later in following sections. Oncogenic

HVS also encodes for a Bcl-2 homolog named ORF16, which protects cells from heterologous virus-induced apoptosis [209]. Finally, M11 encoded by MHV-68 has been identified as an inhibitor of Fas- and TNF-induced apoptosis [193,210–212].

1.5.3 EBV vBcl-2s

 30 The members of γ-herpesviruses usually encode for only one vBcl-2, whereas EBV

uniquely encodes for two to three putative vBcl-2 proteins, namely BHRF1, BALF0

and/or BALF1 [103–105,213]. BHRF1, the first identified vBcl-2 of EBV, has been characterized as an anti-apoptotic protein [214]. It has been suggested that BALF0/1 may compensate for the lack of v-FLICE-inhibitory proteins (v-FLIP), another group of anti-apoptotic proteins that has been found in some herpesviruses such as HVS and

KSHV [104].

BALF1 (BamH1 A fragment leftward reading frame 1), was initially identified by a

BLAST search [104]. Two in-frame methionine codons were found near the beginning of BALF1 open reading frame (ORF) suggesting that two proteins with different N- terminus could be encoded from the same ORF. BALF1 protein would be encoded by the shorter ORF, while the protein encoded from the first albeit non-conserved methionine is referred to as BALF0 [105] (Figure 13). The BH1 domain of BALF0 and

BALF1 differs from that in other Bcl-2 family proteins, which contains a serine rather

than a highly conserved glycine that is essential for the anti-apoptotic function in most of Bcl-2 homologs [105]. BALF0 and BALF1 also lack the C-terminal transmembrane domain which is found in other cellular and viral Bcl-2 proteins [105].

EBV vBcl-2s have long been described as early proteins and they have been proposed to protect the host cell from apoptosis during the lytic phase. More recently it has been shown that EBV vBcl-2s are also expressed in the very early stage of infection in primary B cells, since BHRF1 and BALF0/1 mRNA are detectable one day post- infection [215,216]. Importantly recombinant EBV viruses lacking either BHRF1 or

BALF0/1 are still able to transform primary resting B lymphocytes whilst slightly impaired. However, the genetic inactivation of both vBcl-2 genes severely abrogates the ability of EBV to transform primary B cells and to evade apoptosis in infected cells

[216], suggesting that they may protect the cells during the early phase of infection,

before latency is established.

 31

Figure 13. Alignment of amino acid sequences of the indicated cellular and viral Bcl-2 family proteins. By using ClustalW method, alignment of amino acid sequences has been performed in cellular apoptotic proteins including Bcl-xL, Bcl-2, Bax and Bak; vBcl-2 proteins of EBV including

BHRF1, BALF0 and BALF1 as well as BALF1-like proteins from pongine herpesvirus, callitrichine herpesvirus 3, pan herpesvirus and herpesvirus papio. Identical and similar amino acids are indicated by dark shading and light shading, respectively. Bcl-2 homology (BH) domains 1–4

(BH1–4) are indicated. The serine within the BH1 domain is marked by red dot. TM, putative transmembrane domain. (Ref. 105)

1.6 BHRF1

1.6.1 Expression

BHRF1 (191 amino acids), has been previously regarded as a lytic cycle gene product, presumably to promote survival of host cells to ensure efficient virus replication. It has been proposed that BHRF1 is translated from Wp-initiated transcripts in Latency III as well as in Wp-restricted latency. Immunoblot analysis has shown that BHRF1 is

 32 expressed as a latent protein in Wp-restricted BL cells [68], whereas it accumulated in

lytically induced BL cells from 6h post-induction [217]. During infection of primary B cells with B95.8 EBV strain BHRF1 mRNA can be detected within 24h post-infection

[216]. Similarly, BHRF1 protein is also detectable by 24–48 hour following infection with a recombinant EBV strain that has been rendered lytic cycle deficiency, confirming that its expression is not strictly limited to the lytic phase of the viral cycle.

Wp-restricted expression of BHRF1 was therefore proposed to counteract the high apoptotic sensitivity inherent to the c-myc–driven growth program in BL cells [68].

1.6.2 Subcellular localization

It has been initially reported that BHRF1 localized at the periphery of the mitochondria similarly to cellular Bcl-2 [218]. As a matter of fact, overexpression of BHRF1 induces an unusual mitochondrial morphology compared to the traditional filigreed mitochondria (Figure 14). Indeed, BHRF1 colocalized with perinuclear mitochondrial aggregates, a process that is linked to an EBV-mediated degradation of mitochondrial fragments by autophagy (mitophagy). This unexpected discovery will be described later in this work.

Figure 14. The colocalization between BHRF1 and mitochondria. In CHO cells, overexpression of FLAG-tagged BHRF1 exhibits the colocalization with mitochondria which co-stained by mitotracker, observed by indirect immunofluorescence microscopy. (Adapted from Ref. 105)

 33 1.6.3 Function

BHRF1, one of the well-characterized Bcl-2 homologs of EBV, has been described as

an anti-apoptotic modulator in different experimental cell systems. The anti-apoptotic

function of BHRF1 has been initially demonstrated by using EBV-positive BL cell lines stably transfected with BHRF1, which reveals its ability to protect against apoptosis induced by serum depletion or ionomycin [214]. Subsequent studies have also shown

that BHRF1 is capable of protecting various cell types from apoptosis induced by a

broad set of extrinsic and intrinsic apoptotic stimuli, including DNA-damaging reagents

[214], infection with an E1B 19K-deleted adenovirus or Sindbis virus [219,220],

overexpression of BIK or BOK [221,222], gamma radiation [223], deprivation of

growth factors [224], TNF-α [224], anti-Fas antibody [224] and exposure to TNFα-

related apoptosis-inducing ligand (TRAIL) [225].

Furthermore, BHRF1 has also been shown to protect against apoptosis in a mouse

model (Eμ-myc mouse) of human BL [226]. In this model, BHRF1 expression was

demonstrated to confer tumor cells a dramatic resistance against therapeutics such as

DNA damaging agents [227].

The mechanism by which BHRF1 exerts its anti-apoptotic function, as its mammalian

counterparts, is thought to be through the binding and sequestration of a sub-set of

cellular pro-apoptotic Bcl-2 family proteins, including Bid, Bim, PUMA and Bak [227–

229]. The three-dimensional structures show that these interactions closely resemble

what has been described for mammalian pro-survival protein Bcl-xL where interactions

involved the BH3 peptide of pro-apoptotic proteins into a surface groove formed by α-

helices of Bcl-xL [228] (Figure 15).

 34

Figure 15. The binding between BH3 peptides and BHRF1. (A) BHRF1 (blue) in complex with the

Bim BH3 domain (yellow). (B) is rotated by 180° through the vertical axis from (A). (C) BHRF1

(blue) in complex with the Bak BH3 domain (orange). (D) Bcl-xL (cyan) in complex with the Bim

BH3 domain. (Ref. 227)

1.7 BALF0/1

1.7.1 Expression

The characterization of BALF0/1 proteins is still matter to controversy, and the name of both proteins has been changed over years. So far, it is considered that two proteins can be encoded from the BALF0/1 ORF: the shorter form (182 amino acids) would be encoded from the second ATG and is called BALF1 whereas the longer (220 amino acids) encoded from the first ATG and initially named BALF1 was further renamed as

BALF0 (Figure 16A). Importantly only BALF1 would be conserved among lymphocryptoviruses and the extra N-terminus of BALF0 has no obvious homology with known functional domains (Figure 13 and 16B). However, both proteins could be expressed by in vitro translation from this unique ORF [105], confirming that both ATG could be used as translation initiation sites. Previous work has also shown that NPC patients may produce antibodies directed against a 31 kDa protein in BALF0/1- transfected NIH3T3 cells [230], which was compatible with BALF0 expected size.

Nonetheless the existence of BALF1 could not be confirmed in the same context. Due to the poor quality of this immunological characterization, this result is therefore

 35 questionable. As a conclusion, the existence of BALF0/1 in naturally infected cells could not be assessed due to the lack of specific immunological reagents.

Figure 16. The BALF1 ORF potentially encodes two isoforms. (A) Diagram of the EBV genome segment containing putative BALF0 and BALF1 transcripts (drawn in reverse of the usual orientation). Nucleotide sequences for putative TATA boxes (bent arrows), initiation codons, stop codon (underlined), and poly(A) signal (underlined) are shown. Numbers of intervening nucleotides (nt) are shown. Calculated transcript sizes [excluding poly(A)] are shown. (B) Alignment of the

DNA sequences of BALF1 from EBV and other lymphocryptoviruse including pongine herpesvirus 3, pan herpesvirus and herpesvirus papio indicating that only the second methionine codon of EBV is conserved and this internal start codon has a conserved Kozak initiation sequence that is lacking at the first methionine codon. (Ref. 105)

Upon infection of primary B cells with EBV B95.8 strain, the transcription of BALF0/1 was detectable within 24h post-infection [216]. Although BALF0/1 transcript can be detected at low levels in LCLs as well [215], it is not known whether these arose from a small number of lytically infected cells or, as BHRF1, whether BALF0/1 was also transcribed during latency. BALF0/1 is also transcribed both in latency and during the lytic phase in EBV-positive BL cell lines, including latently infected IB4 and Namalwa

 36 as well as B95.8, Akata and P3HR1 in which complete lytic stage induced by different

stimulus [230]. In Akata and P3HR1 cells, the transcription of BALF0/1 increases

gradually after reactivation [230]. Importantly BALF0/1 transcription during

reactivation was not blocked by inhibitors of viral DNA replication demonstrating that

BALF0/1 is not a late viral gene [230]. Altogether, this suggests that BALF0/1 may be

expressed at low level during latency and could be induced during reactivation in B

cells. In addition, almost 80% of NPC biopsies contain BALF0/1 mRNA [230].

1.7.2 Subcellular localization

In COS-7 living cells, the localization of enhanced yellow fluorescent protein (EYFP)-

tagged BALF0/1 is mainly cytoplasmic. In this study BALF0/1 cytoplasmic

localization was mainly diffuse with some local spots (Figure 17A). When ectopically

expressed in CHO cells, HA-tagged BALF0 and BALF1 localize diffusely throughout

the cytoplasm and display an excluded pattern from mitochondria which clearly differs

from BHRF1 (Figure 17B).

1.7.3 Function

Whereas BHRF1 has been mainly characterized as an anti-apoptotic protein [214],

BALF0/1 has not been extensively investigated and its function is still equivocal.

BALF0/1 has been proposed to play a role in inhibiting IFN-γ- or camptothecin-induced

apoptosis through association with Bax and Bak [104]. BALF0/1 also increase cell

survival by suppressing serum starvation-induced apoptosis [230,231]. Conversely,

BALF1 fails to protect against Sindbis virus- or BAX-induced apoptosis. More

surprisingly, BALF1 could antagonize the anti-apoptotic activities of BHRF1 and Ks-

Bcl-2 whereas it was not able to inhibit the function of cellular Bcl-xL [105]. BALF0 may also antagonize the anti-apoptotic activity of BHRF1 but does not co- immunoprecipitated with BHRF1 contrary to BALF1 [105]. No colocalization has been found between BALF1 and BHRF1 (Figure 18), and co-immunoprecipitation experiments do not show direct interaction, which suggested that BHRF1 function

 37 regulated by BALF1 may occur through competition for the same downstream cellular

machinery [105]. An interactome map of EBV proteins was generated by using a

stringent high-throughput yeast two-hybrid (Y2H) system, showing that BALF0/1

might interact with BdRF1 and BORF2 [232]. Subsequently, a more detailed EBV viral

protein interaction network has been established by using genome-wide Y2H screen

displaying interactions between BALF0/1 and EBNA3A, BALF3, BFRF4, BGLF5,

BKRF3, BALF4 and BSRF1. It has been recently confirmed that BSRF1 precipitates

with BALF0/1 but not BHRF1, and BALF0/1 was suggested to be incorporated into

the tegument fraction with BSRF1 [233]. So far, none of these interactions has been

functionally investigated.

Figure 17. Subcellular localization of BALF0 and BALF1. (A) In COS-7 live cells, the localization of EYFP-tagged BALF0/1 was observed by fluorescence microscopy. (B) In CHO cells, overexpression of HA-tagged BALF0 and BALF1 was localized diffusely throughout the cytoplasm and apparently excluded from mitochondria that were co-stained by mitotracker. (Adapted from Ref.

105,230)

 38 BALF0/1 promotes tumor formation and metastasis in nude mice which suggests that

BALF0/1 may play a role during tumorigenesis and may be a new marker for the screen

and diagnosis of EBV-related cancers [231]. It has been reported that BALF0/1 may

also interact with about 135 cellular proteins. Notably both BALF0/1 and BHRF1 might

target proteins of the Notch pathway and interact with oncogene epidermal growth

factor receptor (EGFR) [234]. Bioinformatics analysis has revealed the coordinated

expression pattern between EBV and human GC transcriptome [235]. In this study,

BALF0/1 expression correlated with human hub gene, CNTD2, suggesting a putative role for BALF0/1 in EBV-positive GC pathogenesis. Once again, these large scales genetic or proteomic investigations would deserve experimental investigations.

Figure 18. BALF1 does not colocalize with BHRF1 in CHO cells. Cells were co-transfected with plasmids encoding FLAG-tagged BHRF1 (green) plus either HA-tagged BALF1 (red, top row) or HA-tagged BALF0 (red, bottom row). No any colocalization observed in the merged images (third column). (Ref. 105)

 39 1.8 Autophagy

Autophagy (literally “self-eating”, derived from Greek words auto – self, phagein – to

eat) is an essential catabolic process that degrades cytoplasmic components within the

autolysosome to ensure cell survival and homeostasis [236]. Autophagy was first

observed in the late 1950s, in mammalian cells by electron microscopy. This led to the

observation of characteristic vesicles containing cytoplasmic organelles [237,238]. In

the mid 1990s, the development of biochemical methods and genetic approaches led to

the discovery of autophagy-related gene (ATG) [239,240] in the model yeast

Saccharomyces cerevisiae, which has been a milestone in the understanding of the

importance of this process.

1.8.1 Different types of autophagy

According to the delivery means of intracellular macromolecules and organelles, which collectively referred to as autophagic cargo, to the lysosome, autophagy has been categorized into three major types including macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) (Figure 19). Macroautophagy is characterized by the sequestration of a portion of cytoplasm including organelles through an isolation membrane to form a double-membrane vesicle called autophagosome [241]. The outer membrane of the autophagosome then fuses with the lysosome to form an autolysosome, and the internal materials of autophagosome are eventually degraded within the autolysosome. The resultant metabolites are transported into the cytoplasm and used either for the synthesis of new macromolecules or as a source of energy.

Microautophagy refers to a process by which cytoplasmic contents are directly engulfed by inward invagination of the lysosomal or late endosomal membrane [242]. Unlike macroautophagy and microautophagy, CMA is highly specific to substrate proteins containing a KFERQ-like pentapeptide sequence. This motif allow the targeted proteins to be recognized by cytosolic Hsc70 and cochaperones, then to be translocated into the

 40 lysosome interior after association with lysosome membrane protein LAMP-2A [243].

The present study focuses on macroautophagy, hereafter referred to as autophagy.

Figure 19. Different types of autophagy. Macroautophagy: bulk cytoplasmic components are sequestered in the autophagosome which subsequently trafficks and fuses with the lysosome, therefore resulting in the degradation of its contents. Microautophagy: cargo is directly taken up by the lysosome through invagination of the lysosomal membrane. Chaperone-mediated autophagy: unfolding and translocation of proteins with a specific signal sequence that is recognized by the

LAMP2A receptor on the lysosome. (Ref. 244)

 41 1.8.2 A multistage process

Autophagy can be divided into six sequential steps, including initiation, nucleation,

elongation, maturation, fusion and degradation [244,245] (Figure 20).

Figure 20. The process of autophagy. Autophagy can be divided into several steps: initiation, the formation of isolation membrane; nucleation, the sequestration of cytoplasmic cargos; elongation, phagophore elongates and expands; maturation: completion of phagophore and transportation of the autophagosome to lysosome; fusion, docking and fusion of autophagosome with lysosome; degradation, degradation of the autophagic cargos within the autolysosome. (Adapted from Ref.

246)

Autophagy begins with the formation of a primitive membrane structure named isolation membrane, which originates from intracellular membranous domains. The membrane origin of autophagic vacuoles remains controversial, and varieties of organelles have been proposed to be implicated in this process, including mitochondria

[246], mitochondria-associated membranes (MAMs) [247], endoplasmic reticulum (ER)

[248,249], Golgi apparatus [250], plasma membrane [246,251] and recycling endosomes [252] (Figure 21). The isolation membrane sequesters cytoplasmic cargos and elongates. After completion, the autophagosome fuses with lysosome to form autolysosome, where the cargos are degraded by lysosomal hydrolytic enzymes. In addition, autophagosomes may also fuse with early or late endosomes to form amphisomes, which then fuse with lysosomes to become autolysosomes [253,254]. The

 42 dynamic process of autophagosome formation, delivery of cargo to the lysosome and the degradation of cargo within the autolysosome is called autophagic flux [255]. The difference between the rate of autophagosome formation and autolysosome degradation has been used for describing the autophagic flux [256]. The impaired fusion of autophagosomes with lysosomes, or the inhibition of the acidic environment of the lysosomal compartment prevents the degradation of cargos and thus results in the accumulation of autophagosomes, therefore suppressing the autophagic flux [256].

Figure 21. The origin and source of the autophagosome membranes. Various organelles including mitochondria, mitochondria-associated membranes (MAMs), endoplasmic reticulum (ER), Golgi apparatus, plasma membrane, and recycling endosomes have been proposed to involve in the autophagosome formation. (Ref. 238)

 43 1.8.3 The autophagic machinery and its regulation pathways

The identification of ATG in yeast led to the discovery of their homologs in mammalian cells, whose functions have been extensively studied [239,257]. The ATG proteins play essential roles in the initiation of autophagy and the formation of autophagosome, herein referred to as the autophagic machinery [255]. In mammalian cells, the core autophagic machinery consists of several complexes, including the Unc-51-like kinase

1 (ULK1) initiation complex, the class III phosphoinositide 3-kinase (PI3K) nucleation complex, the phosphatidylinositol 3-phosphate (PI3P)-binding complex, the ATG12 conjugation system and the microtubule-associated protein 1 light chain 3/gamma- aminobutyric receptor-associated protein (MAP1LC3/GABARAP) conjugation system, as well as ATG9 (Figure 22).

Figure 22. The autophagic machinery in mammalian cells. The core autophagic machinery consists of the ATG9, the ULK1 initiation complex, the class III PI3K nucleation complex, the PI3P-binding complex, the ATG12 conjugation system and the MAP1LC3/GABARAP conjugation system

(simply referred to as LC3 in this figure). (Adapted from Ref. 246)

 44 The ULK1 initiation complex contains ULK1, ATG13, ATG101 and FAK family

kinase interacting protein of 200 kDa (FIP200), which is crucial for the initiation of

autophagy by mediating mammalian target of rapamycin (mTOR) signaling to the

autophagy machinery [258–261]. The class III PI3K nucleation complex is required for

the assembly of primitive isolation membrane. It is also important for initiating

autophagosome formation, which comprises Beclin 1, ATG14, vacuolar protein sorting

34 (VPS34) and VPS15 [238,262,263]. ULK1 has been shown to phosphorylate VPS34

resulting in an enhanced activity of the PI3K complex, which drives the nucleation of

the isolation membrane and the recruitment of additional ATG proteins, as well as the

PI3P-binding complex, including WD-repeat domain phosphoinositide-interacting

proteins (WIPIs) and double FYVE-containing protein 1 (DFCP1) [238,264]. ATG9

has been identified as a transmembrane ATG protein and the ATG9-containing vesicles

are an important membrane source during the early stages of autophagosome formation

[265].

Following nucleation, the elongation of phagophore is mediated by two sequentially

acting ubiquitin-like conjugation systems (Figure 22). In the ATG12 conjugation

system, ATG12 is irreversibly conjugated to ATG5 by ATG7 and ATG10, which

functions as an activating E1-like enzyme and a conjugating E2-like enzyme, respectively (Figure 23A). The ATG12–ATG5 conjugate binds non-covalently to

ATG16L1 through ATG5 to form the ATG12-ATG5-ATG16L1 complex, which

dimerizes and interacts with the PI3P-binding complex resulting in the association with

the phagophore membrane for subsequent LC3 conjugation [266–270] (Figure 22 and

23A). In mammalian cells, the seven ATG8 homologs have been divided into two

subfamilies, including the MAP1LC3 subfamily (shortly named LC3A, LC3B, LC3B2

and LC3C) and the GABARAP subfamily (GABARAP, GABARAP-L1 and

GABARAP-L2) [271–275], all of which are hereafter referred to as LC3. In LC3

conjugation system, LC3 is cleaved by ATG4 immediately after synthesis to generate

a cytosolic LC3-I. ATG7 activates LC3-I and transfers it to ATG3 (E2-like enzyme).

 45 The ATG12-ATG5-ATG16L1 complex functions as an E3 in the conjugation of phosphatidylethanolamine (PE) to LC3-I to form lipidated LC3-II, which associates both with the inner and outer membranes of phagophore [273,276] (Figure 22 and 23B).

Unlike the irreversible ATG12-ATG5-ATG16L1 complex, LC3-II can be deconjugated from the outer membranes by ATG4 during autophagosome maturation, which is necessary for autophagosome biogenesis [273]. LC3B is the most widely used and well- established autophagosome marker to monitor autophagy [256,271].

A B

Figure 23. Two ubiquitin-like conjugation systems in autophagic machinery. (A) ATG12 conjugation system. (B) LC3 conjugation system. The lipidated LC3-II associates with the inner and outer membranes of phagophore. LC3-II can be deconjugated from the outer membranes by

ATG4 (Deconjugation). PE, phosphatidylethanolamine. (Ref. 241)

 46 Membrane fusion is generally regulated by Rabs, soluble N-ethylmaleimide–sensitive

factor attachment protein receptors (SNAREs) and tethering complexes [277]. During

the fusion of autophagosome with lysosome, the attachment of a hairpin-type tail-

anchored SNARE, syntaxin 17, to the autophagosome membranes leads to the fusion

with lysosomes [278]. Homotypic fusion and protein sorting (HOPS) promotes

autophagosome–lysosome fusion through the interaction with syntaxin 17 [279]. At

this stage, Beclin 1-VPS34 complex is associated with the ultraviolet radiation

resistance‑associated gene protein (UVRAG), which is required for the autophagosome

and endosome maturation. Conversely, this process is impaired when UVRAG is

combined with Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-

interacting protein) [262,280]. (Figure 24)

Figure 24. The autophagosome–lysosome fusion. During this step, autophagic machinery and additional proteins are involved, including the Beclin 1-VPS34-UVRAG complex, syntaxin 17 and

HOPS. (Adapted from Ref. 236)

Autophagy is regulated by a multitude of signaling pathways under various circumstances. The mTOR kinase is a key autophagy inhibitor of the insulin/growth

 47 factor pathway, whose downstream targets are ULK1 and ULK2 [281] (Figure 25).

Growth factor pathway is activated upon the binding of insulin to its receptor, resulting

in the recruitment and phosphorylation of insulin receptor substrate 1 and 2 (IRS1 and

IRS2), which creates a docking scaffold for the binding of class I PI3K. Following the

enhanced membrane recruitment, protein kinase B (PKB)/Akt is activated by

phosphoinositide-dependent protein kinase 1 (PDK1) [282]. The tumor suppressor

PTEN also positively regulates autophagy by inhibiting the PKB/Akt signaling [283].

Phosphorylation of tuberous sclerosis complex 2 (TSC2) by activated Akt/PKB inhibits

the interaction of TSC2 with TSC1 therefore preventing the formation of the TSC1/2

complex [284], which activates Ras homolog enriched in brain (RHEB) (GTP-bound

form) [285,286] and allows it to directly bind and activate mTOR [287]. Ras signaling

is also involved in the regulation of autophagy by growth factors [288]. Under a reduced

energy level, the energy sensor 5’-AMP-activated protein kinase (AMPK) is activated through the upstream liver kinase B1 (LKB1) by an increased AMP/ATP ratio, which leads to the phosphorylation and activation of the TSC1/2 complex and subsequent suppression of the mTOR activity through RHEB [289] (Figure 25). Under high amino acid levels, the mTOR is activated via the class III PI3K and the Ras-related small

GTPases (Rag) [290–292] whereas the Ras effector Raf-1, an amino acid sensor, is inhibited leading to the downregulation of the downstream mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinases 1/2 (MEK1/2) and

ERK1/2 [293] (Figure 25). In addition, autophagy is induced by ER stress, a process regulated through the RNA-dependent protein kinase-like ER kinase (PERK)- eukaryotic initiation factor 2α (eIF2α) pathway and the inositol-requiring kinase 1

(IRE1)-c-Jun N-terminal kinase (JNK1) pathway [288]. Autophagy has also been shown to be upregulated upon viral infection through the antiviral eIF2α kinase signaling pathway, including eIF2α and the IFN-inducible double-stranded RNA- dependent protein kinase R (PKR) [294,295].

 48

Figure 25. Regulation pathways of autophagy. Several signaling pathways involve in the regulation of autophagy including the growth factors, insulin, nutrients and energy. (Ref. 289)

In order to explore in detail, the mechanism of autophagy, several pharmacological modulators and genetic manipulation techniques can be used (Figure 26). Rapamycin induces autophagy both in vitro and in vivo through the inhibition of autophagy suppressor mTOR [296]. 3-methyladenine (3-MA) inhibits autophagy by repressing the activity of class III PI3-kinase which is required for the autophagosome formation [297].

Bafilomycin A1 has been previously reported to inhibit the fusion of autophagosome with lysosome; subsequently it has been shown that the primary effect of bafilomycin

A1 was to impair the acidification of lysosomal compartment, therefore inhibiting the degradation of autophagic cargos [298,299]. Chloroquine (CQ) prevents the acidification of lysosomes as well [300,301]. The techniques for genetic inhibition of autophagy consist of knockout or knockdown of various ATG genes and the use of dominant-negative mutant of autophagy proteins [255].

 49

Figure 26. Pharmacological and genetic modulators of autophagy. A full process of autophagy with partial list of autophagic inducers and inhibitors are shown. (Ref. 256)

These tools can be used in combination to explore the different steps of autophagy

[255,302]. The measurement of the total number of autophagosomes represents the steady state at a specific time point, including the identification of autophagosomes and autolysosomes by electron microscopy (Figure 27A), immunoblot analysis of the total levels of lipidated LC3 II and quantification of the number of LC3 containing puncta in cells expressing green fluorescent protein (GFP)-tagged LC3 (GFP-LC3) (once autophagy is induced, cytosolic LC3 I is converted into lipidated LC3 II forming puncta which mark autophagosome) [303] (Figure 27B). Since autophagy is a dynamic process, increased levels of autophagosomes result either from autophagy induction or alternatively from an impaired fusion of autophagosomes with lysosomes. Thus, the measurements of autophagic flux might represent a reliable and direct analysis of the autophagic activity. In the presence of lysosomal inhibitors, increased accumulation of

LC3 II or GFP-LC3 puncta indicates the induction of autophagy (Figure 27C and 27D).

Additionally, tandem red fluorescent protein (RFP)-GFP-LC3 (RFP-GFP-LC3) has

also been utilized for monitoring autophagic flux: in cells expressing this probe,

autophagosomes are dually labelled with RFP and GFP whereas autolysosomes are

only labelled with RFP (Figure 27E). Indeed, the different stability in response to a low

pH of GFP and RFP proteins leads to the quenching of GFP but not RFP at acidic pH

 50 (autolysosomes) [304]. Therefore, autophagosomes are identified as yellow puncta

(green+red) while autolysosomes are labelled by red fluorescence. An increase in both signals indicates autophagy induction whereas the increase in yellow puncta with a decrease in red puncta indicates a blockage in maturation (Figure 27E).

Figure 27. Methods for monitoring autophagy. (A) Identification of autophagosomes and autolysosomes by electron microscopy. (B) Measurements of the number of autophagosomes by immunoblot analysis and quantification of the GFP-LC3 puncta. (C) and (D) Measurements of the autophagic flux in the presence lysosomal inhibitors. (E) In cells expressing RFP-GFP-LC3, autophagosomes are identified as yellow puncta and autolysosomes as red puncta. AP, autophagosome; AL, autolysosome; Y, yellow; R, red. (Adapted from Ref. 306)

 51 1.8.4 Physiological and pathological roles of autophagy

The genetic studies in yeast have identified a set of ATG genes, and most of them have

highly conserved functional homologs in the mammalian systems [305]. The analysis

of autophagy-defective organisms revealed various physiological roles and

pathological effects of autophagy at both the cellular and whole-organism levels [306]

(Figure 28).

Figure 28. The roles of autophagy in human. XMEA: X-linked myopathy with excess autophagy;

UCMD: Ullrich congenital muscular disorder. (Ref. 244)

Autophagy occurs constitutively at basal level under normal conditions to maintain

homeostasis by recycling intracellular long-lived proteins, lipids, and damaged

organelles. It is stimulated under starvation for replenishing energy stores as well as by

various stress such as hypoxia, ER stress, the presence of free radicals, and genotoxic

stress [244,307]. A reduced autophagic activity is associated with normal and

pathological aging whereas pharmacological or genetic methods stimulating autophagy

give rise to the extended lifespan of model organisms from flies to mice [308,309].

Autophagy also plays an important role in the preimplantation development, survival

 52 during neonatal starvation, and cell differentiation during erythropoiesis, lymphopoiesis and adipogenesis [310]. Additionally, defects in autophagy may be involved in the pathogenesis of multiple diseases, including Crohn’s disease [311,312], type 2 diabetes [313,314], Huntington’s disease [296], Alzheimer’s disease [315] and

Parkinson’s disease [316]. The implication of autophagy in tumorigenesis is complex and likely dependent on the stage of tumor development [317]. At early tumor stages, autophagy rather suppress tumorigenesis. Conversely, once the tumor is formed, autophagy might promote tumor progression and metastasis [318].

It is to note that the autophagic pathways play an essential role in innate and adaptive immunity. Autophagic vesicles allow the direct elimination of intracellular microbes by engulfing and targeting them to lysosomal degradation [319,320]. Virus can be eliminated by autophagy, a process called virophagy [321]. In addition, peptides generated from the autophagic pathways can be processed and matured for major histocompatibility complex (MHC) class I and class II presentation [236,322].

1.8.5 Selective autophagy

Whereas autophagy has first been regarded as a non-specific and bulk degradation process, emerging evidences reveal that autophagy can degrade targets in a selective manner which refers to as selective autophagy [257,323]. Multiple subcellular structures are degraded by selective autophagy, which has been named by various terms reflecting the specificity of exquisite target. This includes pexophagy (peroxisomes)

[324], lipophagy (lipids) [325], nucleophagy (nucleus) [326], ER-phagy (ER) [327], xenophagy (microbes) [328], and mitophagy (mitochondria) [329] (Figure 29). This process is mediated by selective autophagy receptors which sequester specific cargos into the forming phagophores through the interaction with LC3 or other ATG8 family proteins that are anchored on the autophagic membranes. The binding to membrane- coupled LC3 requires short amino acid sequences named LC3-interacting region (LIR) motif or ATG8-interacting motif (AIM) [330,331] (Figure 30).

 53

Figure 29. Different types of selective autophagy. Various terms reflecting the specificity of exquisite target have been used for naming different types of selective autophagy. Established (black) and putative (red) selective autophagy receptors for the respective processes are listed. Unidentified receptors are indicated by question marks. (Ref. 333)

p62 is well known as a scaffold protein of the NF-κB signaling pathway [332]. p62 was

the first identified selective autophagy receptor. It is involved in the autophagic

degradation of ubiquitylated protein aggregates as well as other selective autophagy

substrate [330,333]. The LIR motif of p62 was elucidated by detailed deletion mapping

and point mutation analysis, together with X-ray crystallography and nuclear magnetic

resonance (NMR) [334,335]. An increasing number of verified LIR motifs reveals a

core consensus sequence [W/F/Y]xx[L/I/V], which consists of four key amino acids

including an aromatic residue (W/F/Y) at the first position and a hydrophobic residue

(L/I/V) at the fourth position, while x may be any amino acid [331,336] (Figure 31).

However, atypical LIR motifs have also been found that did not match with the

canonical consensus [337]. In addition, LIR-containing proteins are involved not only

 54 in the cargo targeting for selective autophagy but also in the autophagosome formation

and maturation [331].

Figure 30. The overview of selective autophagy. After induction, the cargos for selective autophagy

are recruited to the inner membrane of the growing phagophore by autophagy receptors that mediate interaction between the cargo and lipidated ATG8/LC3. Following maturation and fusion, the enclosed cargos are degraded within the autolysosomes. (Adapted from Ref. 332)

Autophagy has been adapted for host defense of invading pathogens. Thus, it is not surprising to observe that several viruses deploy subversion strategies to counteract and/or benefit from host autophagic machinery to improve their replication and promote their survival. In most cases, the molecular mechanisms are still not known

[338–340]. To date, only two viral proteins have been reported to interact directly with

ATG8 family proteins: the viral infectivity factor (Vif) of HIV-1 and Matrix 2 (M2) of influenza A virus (IAV) [341,342]. Of these, M2 is the only one that contains an experimentally validated LIR motif [342]. M2 is responsible for the inhibition of autophagic degradation of IAV by blocking the fusion of autophagosome with lysosome [343]. Subsequently, it was reported that the cytoplasmic tail of M2 harbors

 55 a LIR motif mediating the interaction with LC3, which is also required for the re- distribution of LC3 to plasma membrane in IAV-infected cell [342].

Figure 31. LIR-containing proteins. LIR-containig proteins are grouped according to the aromatic residue (W, F, or Y) of the consensus sequence [W/F/Y]xx[L/I/V]. The conserved aromatic and hydrophobic residues are underlined and in bold. The pictograms represent the domain architectures of LIR-containing proteins: LIR motifs (magenta: characterized, yellow: putative); autophagy- related Ub-binding domains (yellow cylinder); characterized functional domains (blue cylinder); any other elements (green connection). H. sapiens, Homo sapiens; S. cerevisiae, Saccharomyces cerevisiae; P. pastoris, Pichia pastoris; aa, amino acids. (Ref. 333)

 56 1.9 EBV and autophagy

EBV establishes lifelong infection after primary infection. This persistent infection alternates latent infection and sporadic reactivations that eventually leads to virus production and subsequent transmission. During EBV life cycle, autophagy modulation has been shown to be mediated either directly by EBV-encoded proteins or indirectly through the activation and inhibition of the cellular pathways involved in the autophagic regulation.

1.9.1 Autophagy and EBV latency

During latency, EBV genomic DNA persists as an episome in the nucleus of host cell.

Only a few latent genes are expressed, and no viral particles produced. According to the pattern of gene expression, latency has been further categorized into four types including latency I, II, III and Wp-restricted (as described in 1.3.1), which are important both for the life cycle of the virus and for EBV-associated malignancies.

In comparison to EBV-negative BL cell lines, a constitutively high level of autophagy has been observed in latency III EBV-positive B cells. These cells express high levels of the pro-autophagic protein Beclin 1 and exhibit a marked resistance to apoptosis

[344]. Following treatment with nutlin-3, a TP53 pathway activator inducing apoptosis, several autophagy-stimulating genes were upregulated both in EBV-negative and latency III EBV-positive B cells. The autophagic flux was stimulated only in the EBV- positive cells and was associated with an upregulation of sestrin 1 and an inhibition of mTOR that was more rapid than in EBV-negative cells [344]. Inhibition of autophagy with CQ potentiates the pro-apoptotic effect of nutlin-3 in EBV-positive cells demonstrating autophagy is involved in cell survival [344]. By using immunohistochemical staining, clinical samples comprising latency II or III tumor cells exhibit a constitutively active autophagy [344]. Notably, basal autophagy was not activated in EBV-positive latency I BL cell lines [344]. EBNA1 is the single and characteristic latent protein expressed during latency I. EBNA1 does not promote the

 57 autophagic process but can be presented on MHC class II molecules when autophagy

is induced [345].

During latency III, all latent proteins are expressed. Of these, LMP1 is an oncogene

which is essential for transformation of B lymphocyte and can enhance its survival

[346,347]. LMP1 is an integral membrane protein consisting of six membrane-spanning domains and a C-terminal signaling domain [348]. LMP-1 induces autophagy through the 3-6 transmembrane domains in a dose-dependent manner [349]. The low expression levels of LMP1 induce early stage of autophagy as evidenced by an accumulation of autophagosomes. Conversely high levels of LMP1 expression induce an accumulation of autolysosomes corresponding to the late stage of autophagy [349]. In addition, inhibition of autophagy in EBV-transformed B cells leads to the accumulation of LMP1 resulting in an inhibition of the colony formation indicating that induction of autophagy by LMP1 contributes to its own regulation which in turn controls the transformation of the host cells [349]. The mechanism of LMP1 induces autophagy is controversial.

Previous study had shown that only transmembrane domains 3–6 are required for this induction rather than the C-terminal signaling domain which can activate the NF-κB,

AP-1 and Stat-1 signaling pathways [349], whereas another group recently reported that the induction of autophagy by LMP1 is NF-κB-dependent [344]. This difference between two reports might be due to the distinct cell systems have been utilized.

The EBV infection is associated with several human malignancies of epithelial origins including NPC and GC, which exhibit latency II expression pattern. The expression of

LMP2A leads to resistance to anoikis, a cell detachment-induced apoptosis, by activating the ERK signaling pathway [350]. It was shown that LMP2A also induces the formation of autophagosomes and increases the expression of proteins of the autophagosome pathway, which eventually resulted in abnormal acinus formation by promoting resistance to anoikis in non-malignant epithelial cells [351]. Autophagy inhibitors, including 3-MA and CQ, induce caspase 3 activation and impair the abnormal growth to restore the normal acinus formation [351].

 58 Latent protein EBNA3C, which acts as a prerequisite for inhibition of apoptosis and maintenance of cell growth, has also been reported to induce autophagy [352].

EBNA3C recruits several histone activation epigenetic marks (H3K4me1, H3K4me3,

H3K9ac, and H3K27ac) for transcriptional activation of a number of autophagy modulators which are essential for autophagosome biogenesis (ATG3, ATG5, ATG7)

[352] .

Although B lymphocytes and epithelial cells are the two major targets of EBV, the virus also infects dendritic cells (DCs) and their precursors under different circumstances.

EBV-infected plasmacytoid DCs (pDCs) express viral latency genes and release high amounts of type I IFNs by a mechanism requiring TLRs and a functional autophagic machinery [353]. EBV has been reported to inhibit the development of DCs by reducing cell survival of their monocyte precursors [354]. It has recently been shown that this effect correlated with autophagy, reactive oxygen species (ROS) and mitochondrial biogenesis reduction [355].

1.9.2 Autophagy modulation during EBV lytic cycle

During the lytic cycle, extensive viral gene expression occurs and progeny viruses are produced. The function of autophagy during this phase of the cycle is complex and depends on the stage of lytic cycle.

IE proteins ZEBRA (Zta) and Rta are transcription factors that control the switch from latency to the lytic stage. The induction of EBV lytic cycle promotes the accumulation of lipidated LC3 in EBV-positive BL cell lines and LCLs [356,357]. When ectopically expressed in 293T cells, Hung and collaborators observed that Rta induces complete autophagic flux including the formation of autophagosomes and their maturation into autolysosomes. This effect was not observed when ZEBRA was expressed in the same system [356]. Controversial result has been reported regarding the function of ZEBRA by Granato and collaborators in the same year [357]. Autophagy is supposed to be regulated both at the posttranscriptional and transcriptional levels [288]. The induction

 59 of autophagy by Rta correlates with its transacting ability, which correlates with an increase in the transcription of genes that are involved in the formation of autophagosomes (LC3A, LC3B, and ATG9B) as well as the regulation of autophagosome formation (TNF, immunity related GTPase M [IRGM], and TRAIL)

[356]. Rta-induced autophagy requires Atg5, which participates to the formation of autophagosomes and is independent of mTOR signaling but via ERK signaling pathway

[356].

The increased amount of LC3-II that has been measured by immunoblot in reactivated

EBV-positive BL cells in several studies could result either from the stimulation of autophagosomes formation or from an inhibition of autophagosome maturation [356].

Using 3-MA, an inhibitor of autophagosome maturation, it was further demonstrated that the autophagy was blocked at late stages in cells undergoing lytic cycle [357].

ZEBRA was not involved in the autophagic block. On the opposite, it was shown that this block requires the expression of the complete set of lytic genes [357], indicating that still unidentified lytic viral protein(s) might be involved in this inhibition. Rab7 plays an essential role in the autophagosome maturation, complete autophagic flux and lysosome biogenesis [358–360], and its downregulation in cells undergoing EBV lytic cycle might contribute to the autophagic block [357]. Furthermore, EBV subverts the fusion of autophagosomes with lysosomes and redirect the autophagic vesicles toward the plasma membrane (Figure 32), suggesting that EBV might hijack the autophagic vesicles for viral transportation and avoid lysosomal degradation of viral components.

Inhibition of autophagy leads to the accumulation of EBV particles in the cytosol suggesting autophagic vesicles might contribute to the cytosolic maturation of EBV

[361]. It has also observed that lipidated LC3-II gets incorporated into EBV viral particles. This led to the assumption that EBV uses LC3-coupled membranes for secondary envelopment in the cytosol [361]. This question is still largely open. Indeed, unpublished results from our group demonstrated that an almost complete silencing of

 60 LC3B do not significantly abrogates the production of virions. Current experiments are conducted to evaluate the infectivity of these virions.

Figure 32. EBV subverts autophagy and hijacks the autophagic vesicles for transportation towards the plasma membrane. (A) B95-8 cells induced into lytic cycle: electron microscopy analysis shows nucleocapsid (arrowhead), autophagic vesicles containing viral particles at different stages (arrows,

1-5). Au, empty autophagosomes. (B) Schematic model of how EBV may subvert autophagy during replication. The induction of the complete EBV lytic program inhibits autophagy, impairing the delivery of the autophagic vesicles, which might contain viral particles, to the lysosomes,

Concomitantly, the virus might deploy this strategy to reroute the autophagic vesicles towards the plasma membrane for release progeny viruses. Other data suggested that early steps of autophagy might be stimulated, therefore potentially providing autophagic membranes and other components that may be required for the maturation of the viral particles. (Adapted from Ref. 359)

 61 In addition, inhibiting autophagy by various drugs or using genetic tools (by genetic

autophagy modulators including Atg5 small interfering RNA (siRNA), BECN1 siRNA,

Atg12 small hairpin RNA (shRNA) and Atg16 shRNA as well as pharmacological

inhibitor 3-MA ) reduces the ability of EBV to express lytic genes and the production

of viral particles. Accordingly, opposite effect on the viral cycle were observed upon

pharmacological stimulation of autophagy by rapamycin [356,357,361]. This model

may not be definitive since De Leo and collaborators observed that inhibition of

autophagy with Bafilomycin A1 or BECN1 shRNA resulted in an increased expression

of EBV lytic genes, accumulation of intracellular viral DNA and highest release of

progeny virus [362].

1.10 Herpesvirus and autophagy: a lesson to explore the contribution of autophagy to EBV infection

A multitude of viruses appropriates the autophagic machinery for their own benefits

including replication and survival. Enhanced autophagy, for example, results in the

increased yield of poliovirus, hepatitis C virus and HIV [363–366]. As known,

autophagy is negatively regulated by several herpesviruses (Figure 33).

1.10.1 Subversion of autophagy by Herpesviruses

HSV-1 encodes for two proteins exhibiting anti-autophagic activities. Us11 is a late gene product of HSV-1 that inhibits autophagy by direct interaction with PKR [367].

HSV-1-encoded neurovirulence protein ICP34.5 binds to the Beclin 1 via the Beclin- binding domain (BBD) resulting in inhibition of autophagy [368]. HSV-2 maintains basal autophagy levels mostly unchanged during productive infection and inhibiting basal autophagy results in a reduced viral replication and diminished infection [369].

HCMV encodes two proteins, TRS1 and IRS1, which inhibit autophagy during infection [370,371]. In different cell lines, expression of either TRS1 or IRS1 is able to

 62 block autophagy by interaction with Beclin 1, which requires the essential BBD. Their

co-expression blocks the maturation process through the PI3K-Beclin 1-UVRAG

complex [370,371]. In order to counteract autophagy deleterious effects, KSHV has

evolved several viral genes whose products modulate autophagic pathways at different

steps. KSHV encodes vFLIP, a viral homolog of cellular FLICE-like inhibitor protein,

which represses autophagy by preventing Atg3 from binding and processing LC3 [372].

During latency, vFLIP inhibits autophagy and senescence induced by v-cyclin of

KSHV, a homolog of cellular D-type cyclins, in a manner that requires intact vFLIP

ATG3-binding domains [373]. In addition, the K7 protein of KSHV interacts with

Rubicon autophagy protein and inhibits the autophagosome maturation step by

blocking Vps34 enzymatic activity [374]. On the other hand, the inhibition of

autophagy affects lytic gene expression and viral DNA replication mediated by RTA,

a replication and transcription activator of KSHV, indicating that RTA induces

autophagy to facilitate KSHV lytic replication [375]. In VZV-infected cells, autophagy

plays a pro-viral role which maintains cellular homeostasis and facilitates VZV

glycoprotein biosynthesis and processing [376,377].

1.10.2 Herpesvirus-encoded vBcl-2s inhibit autophagy

There is accumulating lines of evidence that some cells can switch between apoptosis and autophagy in a mutually exclusive manner, indicating these two responses might be controlled by common albeit adverse upstream signals [378]. It is therefore no surprising that the Bcl-2 family proteins play an essential role in these two processes.

It has been reported that cellular Bcl-2 inhibits autophagy through an interaction with

Beclin 1 under normal conditions, while Beclin 1 dissociates from Bcl-2 upon stress allowing the activation of Vps34 and resulting in the subsequent induction of autophagy

[379]. The BH3 domain of Beclin 1 binds to the hydrophobic groove in Bcl-2 proteins formed by their BH1, BH2 and BH3 domain [380,381].

 63

Figure 33. Herpesviruses subvert autophagy. The autophagic machinery is inhibited by various

viral factors. HSV-1 inhibits autophagy by direct interaction with PKR through Us11 and binding

to the Beclin 1 by ICP34.5, respectively. HCMV encodes two proteins, TRS1 and IRS1, which inhibit autophagy by interaction with Beclin 1 and UVRAG. KSHV encodes vFLIP, which represses autophagy by preventing Atg3 from binding and processing LC3, and K7 protein of KSHV interacts with Rubicon autophagy protein and inhibits the autophagosome maturation step. Two vBcl-2s,

MHV68 M11 and KSHV Ks-Bcl-2 bind to Beclin 1 to inhibit autophagy. (Figure courtesy of Prof

A. Esclatine, Université Paris-Sud)

Autophagy contributes to the microbial clearance mechanism to protect eukaryotic cells against intracellular pathogens. Reciprocally some pathogens have evolved strategies to avoid immune control, or to promote their own replication by manipulating the autophagic pathway for their own benefits. For instance, A179L, the vBcl-2 of African swine fever virus, manipulates autophagy through interaction with Beclin 1 of its BH3 domain resulting in inhibition of autophagosome formation [382]. E1B19K, the adenovirus BH3 domain protein, interacts with Beclin 1 to initiate autophagy [383].

Herpesviruses also encode vBcl-2 homologs which mimic the anti-apoptotic Bcl-2 proteins and could modulate autophagy. Of these, KSHV open reading frame 16 (orf16) encodes Ks-Bcl-2, which shares sequence and functional homology with members of

 64 the Bcl-2 family [207,220]. As cellular Bcl-2 and some other vBcl-2s, Ks-Bcl-2 binds to Beclin-1 and inhibits autophagy [379,384]. The negative regulation of autophagy is mediated through the association between Ks-Bcl-2 and the Beclin-1–PI3KC3–

UVRAG autophagic complex, which, unlike the case of cellular Bcl-2, is not disrupted by Jun N-terminal protein kinase 1 (JNK1) phosphorylation [379,385,386]. The vBcl-

2 of MHV68, namely M11, has been implicated in preventing Fas-, TNFα- and Sindbis virus infection-induced apoptosis [211]. M11 has also been demonstrated to inhibit autophagy [387]. As shown for other vBcl2, the BH3 domain of Beclin 1 is necessary and sufficient for binding to a hydrophobic groove on M11 which results in the downregulation of Beclin 1-dependent autophagy [384]. Whereas EBV encodes two vBcl-2s, their functions on autophagy are still not known.

1.11 Aims and objectives

BALF0/1 is a putative EBV protein that has been described as a modulator of apoptosis

[104,105]. Two in-frame methionine codons were found near the beginning of

BALF0/1 ORF suggesting that several proteins may be encoded with different N- termini, namely BALF0 and BALF1 [105]. So far, the existence of BALF0 and/or

BALF1 in naturally infected cells could not be assessed due to the lack of specific immunological reagents. An essential prerequisite for generating such reagents is the production of purified soluble forms of BALF0/1 that could eventually be used for immunization. In order to overcome this arduous obstacle, a recombinant form of

BALF0/1 has been produced therefore to generate specific polyclonal antiserum for characterization of the BALF0/1 expression under physiologic conditions.

Autophagy is an essential catabolic process that degrades cytoplasmic components within the autolysosome for cell survival and homeostasis. Autophagy also acts as a microbial clearance mechanism to protect eukaryotic cells against intracellular pathogens. In addition, autophagy provides an alternative pathway to present microbial

 65 antigens to the immune cells. A multitude of herpesviruses examined so far has evolved various strategies to hijack the autophagic machinery for their own benefits i.e. to promote viral persistence, replication and survival [388]. Recent studies indicate that

EBV can modulate autophagy both during latency and reactivation. In an attempt to identify lytic viral proteins that could modulate autophagy during EBV reactivation, we looked for EBV proteins that are orthologs to proteins interfering with the autophagic machinery as observed in other herpesviruses. Beclin 1, a cellular protein that is required for phagophore nucleation [388], has been demonstrated to be targeted by cellular Bcl-2 [379] as well as by vBcl-2s encoded by KSHV and MHV68. We herein inferred that Bcl-2 orthologues from EBV, namely BALF0, BALF1and BHRF1, could modulate autophagy. In the present manuscript, I will mainly present my contribution to the characterization of BALF0 and BALF1. I also participated to the elucidation of

BHRF1 impact on autophagy, that will be presented as well.

 66 2. Results

2.1 Detection of IgG directed against a recombinant form of Epstein-Barr virus BALF0/1 protein in patients with nasopharyngeal carcinoma

EBV is a ubiquitous human γ-herpesvirus which infects more than 95% of adults worldwide. EBV primary infection is asymptomatic when occurring in children whereas it may be responsible for infectious mononucleosis in young adults. EBV is transmitted by saliva mainly and establishes lifelong infection after primary infection.

This persistent infection alternates latent infection, mainly in memory B cells, and sporadic reactivations that eventually lead to virus production and subsequent transmission. EBV infection is associated with the pathogenesis of a number of human malignancies of lymphoid and epithelial origin including BL, HL and NPC. The close association between EBV and the undifferentiated form NPC is illustrated by the consistent expression of EBV gene products in epithelial NPC cells and a specific serological response to defined EBV antigens in NPC patients.

BALF0/1 is a putative EBV protein that has been described as a modulator of apoptosis.

Two in-frame methionine codons were found near the beginning of BALF0/1 ORF suggesting that several proteins may be encoded with different N-termini. BALF1 protein would be encoded by the shorter ORF, while the protein encoded from the first non-conserved methionine is referred to as BALF0. Previous work has shown that NPC patients may produce antibodies recognizing a 31 kDa protein in BALF0/1-transfected

NIH3T3 cells [389], which was compatible with BALF0 expected size. Nonetheless the existence of BALF1 could not be confirmed in the same context. So far, the existence of BALF0/1 in naturally infected cells cannot be assessed due to the lack of specific immunological reagents. An essential prerequisite for generating such reagents is the production of purified soluble forms of BALF0/1 that could eventually be used for immunization.

 67 We initially tried to express full length BALF0/1 from two prokaryotic expression

vectors (pET-22b and pGEX-2T) with a C-terminal poly-histidine tag and a N-terminal

glutathione S- tag, respectively. However, despite trying a wide variety of

expression conditions, the growth of the host bacteria was impaired, and the protein

was not expressed. We assumed that hydrophobic domains may impair BALF0/1

expression in E. coli. Structural analysis predicted the presence of 2 α-helices with high hydrophobicity at BALF0/1 C-terminus. This study describes the expression and purification of a truncated form of BALF0/1 (tBALF0, amino acid 1 to 140), a mutant with deletion of the C-terminal transmembrane domain, using a heterologous bacterial

expression plasmid (pET-22b-tBALF0). pET-22b-tBALF0 was transformed into

Rosetta (DE3) pLysS for recombinant protein production. After induction with

isopropyl β-D-1-thiogalactopyranoside (IPTG), tBALF0 was purified under denaturing

conditions in the presence of urea by nickel affinity chromatography and eluted by

discontinuously decreasing the pH of the elution buffer. Following SDS-PAGE analysis,

only one band was observed at the expected molecular weight (MW) of tBALF0 under

reducing condition. Conversely at least seven bands were observed under non-reducing

condition, suggesting that oxidization might promote tBALF0 multimerization either

during protein expression, purification or gel analysis. The identity of recombinant

tBALF0 in oligomeric forms was confirmed by peptide mass fingerprinting. tBALF0

was further used as an antigen in an indirect Enzyme-linked Immunosorbent Assay

(ELISA) that unraveled the presence of low titer IgGs to BALF0/1 during primary

(10.0%) and past (13.3%) EBV infection. Conversely high-titer IgGs to BALF0/1 were

detected in 33.3% of NPC patients suggesting that BALF0/1 and/or humoral response

against it may contribute to NPC pathogenesis. The presence of antibodies to BALF0/1

in patients infected by EBV could therefore be considered as an important indirect

evidence for the existence of BALF0 and/or BALF1 in vivo.

 68 3URWHLQ([SUHVVLRQDQG3XULILFDWLRQ  ²

Contents lists available at ScienceDirect

Protein Expression and Purification

journal homepage: www.elsevier.com/locate/yprep

Detection of IgG directed against a recombinant form of Epstein-Barr virus BALF0/1 protein in patients with nasopharyngeal carcinoma 7

∗ Zhouwulin Shaoa, , Chloé Bordea, Christophe H. Marchandb,c, Stéphane D. Lemaireb, Pierre Bussond, Joël-Meyer Gozlana, Alexandre Escargueila, Vincent Maréchala a Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine, F-75012, Paris, France b CNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, UMR8226, 75005, Paris, France c CNRS, Institut de Biologie Physico-Chimique, FR550, Plateforme de Protéomique, 75005, Paris, France d Université Paris-Saclay, CNRS, UMR 8126, Gustave Roussy, 94805, Villejuif, France

ARTICLE INFO ABSTRACT

Keywords: BALF0/1 is a putative Epstein-Barr virus (EBV) protein that has been described as a modulator of apoptosis. So Epstein-Barr virus far, the lack of specific immunological reagents impaired the detection of native BALF0/1 in EBV-infected cells. Recombinant protein This study describes the expression and purification of a truncated form of BALF0/1 (tBALF0) using a hetero- BALF0/1 logous bacterial expression system. tBALF0 was further used as an antigen in an indirect Enzyme-linked ELISA Immunosorbent Assay (ELISA) that unraveled the presence of low titer IgGs to BALF0/1 during primary (10.0%) Nasopharyngeal carcinoma and past (13.3%) EBV infection. Conversely high-titer IgGs to BALF0/1 were detected in 33.3% of nasophar- yngeal carcinoma (NPC) patients suggesting that BALF0/1 and/or humoral response against it may contribute to NPC pathogenesis.

1. Introduction reading frame (ORF) suggesting that several proteins may be encoded with different N-termini. BALF1 protein would be encoded by the Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus which shorter ORF, while the protein encoded from the first non-conserved infects about 95% of adults worldwide [1]. EBV primary infection is methionine is referred to as BALF0. Importantly only BALF1 would be asymptomatic when occurring in children whereas it may be re- conserved among primate lymphocryptoviruses [14]. The genetic in- sponsible for infectious mononucleosis in young adults [2]. EBV is activation of both vBcl-2 genes severely impairs the ability of EBV to transmitted by saliva mainly and establishes lifelong infection after transform primary resting B lymphocytes and evade apoptosis in in- primary infection. This persistent infection alternates latent infection, fected cells [15]. BHRF1 has been characterized as an anti-apoptotic mainly in memory B cells, and sporadic reactivations that eventually protein [16] whereas the function of BALF0/1 is still equivocal. BALF0/ lead to virus production and subsequent transmission [3]. EBV infection 1 is transcribed both in lytic stage and latency in EBV-positive Burkitt is associated with the pathogenesis of a number of human malignancies lymphoma's cell lines and NPC biopsies [17] and promotes tumor for- of lymphoid and epithelial origin including Burkitt's lymphoma (BL), mation and metastasis in nude mice [18]. BALF0/1 has been proposed Hodgkin's lymphoma (HD) and nasopharyngeal carcinoma (NPC) [4,5]. to play a role in inhibiting apoptosis through association with Bax and The close association between EBV and NPC is illustrated by the con- Bak [13]. Conversely BALF1 fails to protect against Sindbis virus- or sistent expression of EBV gene products in epithelial NPC cells [6] and BAX-induced apoptosis and antagonizes the anti-apoptotic activity of the distinct serological responses to defined EBV antigens in NPC pa- BHRF1 [14]. BALF0 may also antagonize the anti-apoptotic activity of tients [7–10]. BHRF1 but does not co-immunoprecipitate with BHRF1 as BALF1 [14]. Cellular Bcl-2 is an anti-apoptotic protein of the B-cell lymphoma-2 Previous work has shown that NPC patients may produce antibodies (Bcl-2) family [11]. Whereas members of γ-herpesviruses usually en- recognizing a 31 kDa protein in BALF0/1-transfected NIH3T3 cells code for only one viral Bcl-2 homolog (vBcl-2), EBV potentially encodes [17], which was compatible with BALF0 expected size. Nonetheless the for two vBcl-2 proteins, namely BHRF1 and BALF0/1 [12,13]. Two in- existence of BALF1 could not be confirmed in the same context. So far, frame methionine codons were found near the beginning of BALF1 open the existence of BALF0/1 in naturally infected cells cannot be assessed

∗ Corresponding author. Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75012, Paris, France. E-mail address: [email protected] (Z. Shao). https://doi.org/10.1016/j.pep.2019.05.010 Received 4 April 2019; Received in revised form 23 May 2019; Accepted 27 May 2019

$YDLODEOHRQOLQH0D\ ‹(OVHYLHU,QF$OOULJKWVUHVHUYHG Z. Shao, et al. 3URWHLQ([SUHVVLRQDQG3XULILFDWLRQ  ² due to the lack of specific immunological reagents. An essential pre- antibiotics and was grown at 37 °C, 300 rpm, until an OD600 of 0.5 was requisite for generating such reagents is the production of purified so- reached. The expression of tBALF0 was induced by addition of 1 mM luble forms of BALF0/1 that could eventually be used for immunization. isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma-Aldrich, USA). The present work describes the production of a purified form of After 3 h induction at 37 °C, bacterial pellets were harvested by cen- BALF0/1 and its subsequent evaluation as an antigen for the ser- trifugation at 12,000 g for 20 min and resuspended in 500 μL of lysis oepidemiological survey in various EBV-infected populations. buffer (100 mM NaH2PO4, 10 mM Tris·HCl, 6 M GuHCl, pH 8). To re- move cellular debris, lysates were centrifuged for 20 min at 12,000 g, 2. Material and methods and the supernatants were transferred to fresh tubes. Twenty micro- liters of Ni-NTA resin (Qiagen, Germany) were added to each tube and 2.1. Cell line, bacterial strains and media incubated with cleared lysate under constant rotation at 4 °C for 2 h. The resins were harvested by centrifugation at 12,000 g for 5 min and

The EBV-positive B-cell line B95.8 (purchased from ATCC) was washed three times by wash buffer A (100 mM NaH2PO4, 10 mM grown in RPMI-1640 media (Gibco, USA) supplemented with 10% fetal Tris·HCl, 8 M urea, pH 7). After an incubation on ice for 5 min with the bovine serum (Thermo Scientific, USA). E. coli strains DH5α and addition of wash buffer A containing 100 mM ethylenediaminete- Rosetta (DE3) pLysS (EMD Millipore, USA) were used for transforma- traacetic acid (EDTA; Sigma-Aldrich, USA), the eluate was collected tion steps by heat shock method. Bacteria were cultured in Luria- following centrifugation at 12,000 g for 5 min. Small-scale preparations Bertani (LB) broth [1% (w/v) peptone 140, 0.5% (w/v) yeast extract, of tBALF0 protein were obtained and analyzed by SDS-PAGE. and 0.5% NaCl], which was supplemented with ampicillin (100 μg/mL) Large scale production of recombinant tBALF0 protein was per- (Sigma-Aldrich, USA) for the production of plasmid DNA in DH5α and formed by inoculating a single colony into 10 mL LB medium con- both ampicillin and chloramphenicol (34 μg/mL) (Sigma-Aldrich, USA) taining antibiotics, and cultured overnight at 37 °C under agitation at for the expression of the recombinant protein in Rosetta (DE3) pLysS. 300 rpm. The overnight pre-culture was inoculated to 1 L LB medium containing antibiotics in a 2.5 L glass Erlenmeyer flask. The culture was

2.2. Plasmid construction grown at 37 °C, 300 rpm, until an OD600 of 0.5 was reached. Protein expression was induced with the addition of 1 mM IPTG. After 3 h of The DNA sequence corresponding to truncated BALF0/1 (tBALF0, induction at 37 °C, cultures were harvested by centrifugation at amino acid 1 to 140), a mutant with deletion of the C-terminal trans- 12,000 g for 30 min, 4 °C, and cell pellets were stored at −80 °C until membrane domain, was amplified by PCR using viral genomic DNA purification. The pellets (around 6 g wet weight from 1 L of liquid (EBV B95.8 strain) as template. Primers were designed according to the culture) were then resuspended in 30 mL of pre-chilled lysis buffer and manufacturer's instructions of Cold Fusion Cloning Kit (SBI, USA). The incubated overnight at 4 °C under constant rotation. The lysate was sequences of primers were as follows: 5′-GAAGGAGATATACATATGA centrifuged at 4 °C, 12,000 g for 30 min, and the pellet discarded. The ACCTGGCCATTGCTCT-3’ (forward) and 5′-GTCGACGGAGCTCGAGTT supernatant was added to 1 mL 50% Ni-NTA resin slurry pre-equili- GTACACTGCGCGCAGGA-3’ (reverse), with the underlined regions re- brated with lysis buffer, and incubated under constant rotation at 4 °C presenting the sequences from the expression vector pET-22b for 2 h. The resins were harvested by centrifugation at 12,000 g for

(Novagen, USA). Both primers were synthesized and purchased from 5 min and washed extensively by wash buffer B (100 mM NaH2PO4, Eurogentec (Belgium) and used without further purification. The total 10 mM Tris·HCl, 8 M urea, pH 6.3). In the presence of a low pH, tBALF0 PCR reaction was prepared according to the manufacturer's instructions protein was eluted in batch under denaturing condition. The elution of PfuUltra DNA polymerase (Agilent Technologies, USA). The PCR was buffer (100 mM NaH2PO4, 10 mM Tris·HCl, 8 M urea, pH 4.5) was performed under the following conditions: 2 min at 95 °C, 35 cycles added to the resin, and the eluates were collected in 1 mL fractions. (20 s at 95 °C, 20 s at 61 °C, 15 s at 72 °C), and 3 min at 72 °C. Following Fractions were boiled in sample buffer with and without 10% β-mer- PCR amplification, the PCR product was analyzed by electrophoresis on captoethanol (Sigma-Aldrich, USA), respectively, prior to be analyzed a 1% agarose gel, then stained with ethidium bromide and visualized by by SDS-PAGE (15%). The tBALF0-containing fractions were pooled and UV illumination. dialyzed against phosphate-buffered saline (PBS; EUROMEDEX, The PCR product and the linearized expression vector pET-22b, France). Following dialysis, the protein fractions were centrifuged at which was digested with NdeI and EcoRI (NEB, USA), were purified by 4 °C, 12,000 g for 15 min to remove insoluble aggregates and stored in QIAquick Gel Extraction Kit (Qiagen, Germany). The cloning reaction of aliquots at −80 °C. tBALF0 DNA insert and linearized pET-22b was incubated 5 min at room temperature, followed by 10 min on ice according to the manu- 2.4. Peptide mass fingerprinting facturer's instructions. The recombinant plasmid pET-22b-tBALF0 was transformed into chemically competent E. coli DH5α. Several colonies All reagents were purchased from Sigma-Aldrich (Saint Quentin- were screened for the presence of tBALF0 DNA by colony PCR, and then Fallavier, France) unless otherwise specified. Trypsin Gold (proteomics positive colonies were grown in liquid culture and plasmids were pur- grade) was obtained from Promega (Charbonnières, France). Purified ified using QIAprep Spin Miniprep Kit (Qiagen, Germany). The presence tBALF0 was subjected to both in-gel and in-solution trypsin digestion. of tBALF0 DNA in the purified plasmids was confirmed by restriction For in-gel digestion, Coomassie Blue stained bands corresponding to digestion by PstI (NEB, USA), and plasmids were verified by DNA se- tBALF0 monomer and higher molecular weight species were excised quencing (Eurofins Genomics, Germany). manually from 15% acrylamide gels, destained, dried by vacuum and digested as previously described [19]. Briefly, in-gel trypsin digestion 2.3. Protein expression and purification was performed overnight at 37 °C in 50 mM ammonium bicarbonate using 125 ng of trypsin per band. Preliminary steps for reduction and The recombinant expression plasmid pET-22b-tBALF0 was trans- alkylation of disulfides bonds were omitted. The supernatants were kept formed into chemically competent Rosetta (DE3) pLysS cells for protein and bands were subjected to a peptides extraction step using 60 μL of a expression. For the small-scale analysis, freshly transformed cells were 60% acetonitrile solution containing 1% trifluoroacetic acid. Super- plated onto solid LB medium (supplemented with ampicillin, 100 μg/ natants were combined and reduced to 10 μL using a centrifugal va- mL and chloramphenicol, 34 μg/mL). One positive colony was chosen cuum concentrator before analysis by Matrix-Assisted Laser Deso- randomly and inoculated into 2 mL LB medium containing antibiotics, rption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). grown at 37 °C overnight with shaking at 300 rpm. Fifty microliters of For in-solution digestion, 3 μL of recombinant tBALF0 (900 ng) was first overnight pre-culture were added to 5 mL of fresh LB media containing incubated at 25 °C for 30 min with 2.5 mM DTT in 50 mM ammonium

 Z. Shao, et al. 3URWHLQ([SUHVVLRQDQG3XULILFDWLRQ  ²

Fig. 1. Construction of an expression plasmid encoding truncated BALF0/1 with a C-terminal histidine tag. (A) Prediction of transmembrane helices in BALF0/1 protein. Hydrophobicity plot of BALF0/1 was obtained by using the TMpred algorithm (https://embnet.vital-it.ch/software/ TMPRED_form.html) showing probability of transmembrane domains. The putative mem- brane-spanning regions are marked by arrows. (B) Schematic representation of the full length BALF0/1 and truncated BALF0 protein. BALF0 consists of 220 amino acids, and contains puta- tive transmembrane (TM) domains at the C-ter- minus. The BALF1 protein is proposed to be translated from the second methionine at amino acid 39. tBALF0 (amino acid 1 to 140) was ob- tained by removing the C-terminal TM domain and was fused with a C-terminal poly-histidine tag. (C) Schematic diagram of pET-22b-tBALF0 expression plasmid. The recombinant gene en- coding tBALF0 was cloned into a pET-22b(+) vector containing a C-terminal poly-histidine tag and expressed in E. coli Rosetta (DE3) pLysS under the control of the T7 promoter. (D) Amplification of the truncated BALF0/1 gene from viral genomic DNA (EBV B95.8 strain) by PCR. The PCR product was analyzed by electro- phoresis on a 1% agarose gel; lane 1: 1 kb DNA ladder, lane 2: tBALF0 fragment (450 bp).

bicarbonate (total volume 10 μL). Then, 2 μL of trypsin prepared at volume of these samples. The cancer patient panel consisted of plasma 12.5 ng/μL in 1 mM HCl were added. Trypsin digestion was performed samples from 27 histologically-confirmed NPC patients and 8 EBV-po- at 37 °C for 4h and was diluted 10 times in LC-MS grade water before sitive patients bearing non-EBV-related head and neck squamous cell MALDI-TOF MS analysis. One microliter of tryptic peptides was mixed carcinomas (HNSCCs), which was obtained at Institut Gustave Roussy with 3 μL of a solution of α-cyano-4-hydroxycinnamic acid prepared at (Villejuif, France) with informed consent of patients entering a protocol half-saturation in a mixture containing 50% acetonitrile, 49.7% grade approved by the ethics committee (exoplasma, CPP Tarnier-Cochin water and 0.3% trifluoroacetic acid. Two microliters of this premix N°2746, 2010). All samples were stored at −80 °C prior to be processed were spotted onto the sample plate and allowed to dry under a gentle anonymously. air stream. Spectra were acquired in positive reflectron mode on the Axima Performance MALDI-TOF/TOF mass spectrometer (Shimadzu- Kratos, Manchester, UK) with a pulse extraction fixed at 2500 as pre- 2.7. Development of an indirect ELISA assay for detecting antibodies against viously described [20]. BALF0/1 in patients with EBV infection and NPC

2.5. Mass spectrometry analysis of intact tBALF0 96-well ELISA plates (BD Biosciences, USA) were coated with 100 ng tBALF0 in denaturing coating buffer (100 mM NaH2PO4, 10 mM Two microliters of tBALF0 protein (34 pmoles) were incubated at Tris·HCl, 8 M urea, pH 8). After overnight incubation at room tem- 20 °C for 1 h with 1 μL of 20 mM DTT and 2 μL of 25 mM ammonium perature, antigen was discarded and 150 μL blocking buffer [10% skim bicarbonate. Then, 1 μL was taken and mixed with 2 μL of a solution of milk powder (Régilait, France) in PBS containing 0.2% Tween 20 sinapinic acid prepared at full saturation in a mixture containing 30% (PBST)] was added to each well. After incubation at 37 °C for 1 h, the acetonitrile, 49.7% grade water and 0.3% trifluoroacetic acid. Two wells were emptied and washed five times with PBST. All plasma microliters of this premix were spotted onto the sample plate and al- samples, 1:10-diluted in blocking buffer, were added in duplicate and lowed to dry under a gentle air stream at room temperature. Spectra incubated for 1 h at 37 °C. After five washes with PBST, horseradish were acquired in positive linear mode on a Shimadzu Axima peroxidase (HRP) conjugated rabbit anti-human IgG (1:1000-diluted in Performance MALDI-TOF/TOF mass spectrometer with a pulse extrac- blocking buffer) (Rockland Immunochemicals, USA) was added and tion fixed at 20000. Mass determination of tBALF0 was performed after incubated for 1 h at 37 °C. After five washes with PBST, 100 μL of ABTS external calibration using mono-charged and dimer ions of equine (Roche Diagnostic, Germany) was added per well and incubated apomyoglobin. avoiding light for 20 min. The optical density was determined at 405 nm (OD405) using an ELISA plate reader (Infinite 200 PRO; Tecan 2.6. Plasmas Life Sciences, Switzerland). All OD405 values from wells coated with tBALF0 were normalized by subtracting the background value of wells A panel of plasma samples from EBV negative controls (n = 10), coated with bovine serum albumin (BSA; EUROMEDEX, France) which EBV primary infection patients (n = 10), and healthy EBV carriers (past loaded with same plasma sample. To discriminate between negative ff infection) (n = 60) was collected at Hôpital Saint-Antoine (Paris, and positive samples, a cut-o value was determined by selecting the France). All these samples were tested for EBV serology as part of their mean plus three standard deviations of the normalized OD405 of the medical follow-up, and BALF0/1 ELISA was performed on excess plasma samples from EBV-negative controls.

 Z. Shao, et al. 3URWHLQ([SUHVVLRQDQG3XULILFDWLRQ  ²

3. Results 3.3. Characterization of recombinant tBALF0 by mass spectrometry

3.1. Construction of an expression plasmid encoding for tBALF0, a The band migrating at the expected molecular weight for tBALF0 truncated form of BALF0/1 and the six other bands corresponding to higher molecular weight species were excised from the non-reducing gel (Fig. 2B, right panel). We initially tried to express full length BALF0/1 from two prokar- They were submitted to an in-gel trypsin digestion to generate peptide yotic expression vectors (pET-22b and pGEX-2T) with a C-terminal mass fingerprints (PMFs) by MALDI-TOF MS. Mass spectra of the seven poly-histidine tag and a N-terminal glutathione S-transferase tag, re- bands were highly similar to the one performed after in-solution trypsin spectively. However, despite trying a wide variety of expression con- digestion of recombinant tBALF0 (Fig. 3A, inset). These PMFs allowed ditions, the growth of the host bacteria was inhibited and the protein us to cover around 80.4% of the tBALF0 primary sequence (Fig. 3B). was not expressed. We assumed that hydrophobic domains may impair They also confirmed that higher molecular weight species were likely BALF0/1 expression in E. coli. Structural analysis predicted the pre- disulfide-linked oligomeric forms of tBALF0 as they completely dis- sence of 2 α-helices with high hydrophobicity at BALF0/1 C-terminus appeared within the gel performed under reducing conditions (Fig. 2B). (Fig. 1A). Therefore, we constructed an expression plasmid encoding a We also analyzed the intact protein by MALDI-TOF MS (Fig. 3A) to truncated form of BALF0/1 (tBALF0) in which amino acids 141 to 220 confirm the integrity of recombinant tBALF0. The experimental mass of were removed (Fig. 1B). The tBALF0 gene was amplified by PCR from tBALF0 was in good agreement with the calculated molecular weight viral genomic DNA (EBV B95.8 strain) and cloned into pET-22b vector confirming that recombinant tBALF0 was correctly produced and pur- (Fig. 1C and D). The recombinant plasmid pET-22b-tBALF0 was further ified. characterized by specific restriction digestion and DNA sequencing which demonstrated that the amplified target sequence was inserted into the correct ORF in the vector. 3.4. Detection of anti-BALF0/1 IgG antibodies by indirect Enzyme linked Immunosorbent Assay (ELISA)

3.2. Expression and purification of recombinant tBALF0 Human antibodies directed against key EBV antigens are commonly used to distinguish between non-infected patients, primary infection or fi pET-22b-tBALF0 was transformed into Rosetta (DE3) pLysS. past infection [21]. IgG and IgA antibodies directed against speci c Expression was induced with the addition of 1 mM IPTG at 37 °C. In this EBV antigens have been associated with EBV positive NPC diagnosis condition, tBALF0 expression was associated with a marked reduction and monitoring [22,23]. Therefore, we wondered whether patients with in bacterial growth. A protein of 20 kDa apparent mobility could be EBV-positive NPC could produce antibodies against BALF0/1. observed following SDS-PAGE and Coomassie Blue staining (theoretical For this purpose, we designed an ELISA using tBALF0 as an antigen. MW of His-tagged tBALF0 = 17.7 kDa) (Fig. 2A). Large scale protein The baseline OD405 for negative samples was calculated as the mean expression was performed in the same condition and bacterial pellets OD405 plus three standard deviations obtained on 10 plasmas from EBV were harvested 3 h post-induction. tBALF0 was purified under dena- negative individuals. Samples with OD405 greater than or equal to the ff turing conditions in the presence of 8 M urea by nickel affinity chro- cuto value were considered positive. In order to search for the pre- ff matography and eluted from the Ni-NTA resin by discontinuously de- sence of anti-BALF0/1 IgG antibodies in di erent forms of EBV infec- creasing the pH of the solution. Following SDS-PAGE analysis, only one tion, ELISA was performed on a panel of plasmas from primary infec- band was observed at the expected MW under reducing condition tion patients and healthy EBV carriers (past infection) as well. 10% of (Fig. 2B, left panel). Conversely at least seven bands were observed the plasmas from primary infection patients showed IgG reactivity to under non-reducing condition (Fig. 2B, right panel) suggesting that BALF0/1 and a minority of healthy EBV carriers (8 of 60, 13.3%) had a oxidization may promote tBALF0 multimerization either during protein positive IgG response (Fig. 4). We then searched for anti-BALF0/1 IgGs expression, purification or gel analysis. in a panel of NPC patients (n = 27). A group of plasma samples col- lected from EBV-positive patients with HNSCCs (Head and Neck

Fig. 2. Expression and purification of recombinant tBALF0 protein. (A) Small-scale expression and pur- ification of His-tagged tBALF0 protein. Protein ex- pression was induced with the addition of 1 mM IPTG at 37 °C for 3 h. The purified fractions of tBALF0 were analyzed by SDS-PAGE (15%) and Coomassie Blue staining. (B) SDS-PAGE analysis of purified fractions of tBALF0. Prior to loading onto gels, fractions were boiled in sample buffer with and without 10% β-mercaptoethanol (β-ME), respec- tively.

 Z. Shao, et al. 3URWHLQ([SUHVVLRQDQG3XULILFDWLRQ  ²

Fig. 3. Mass spectrometry analysis of recombinant tBALF0 protein. (A) Matrix-assisted laser desorption/ionization mass spectrum of intact tBALF0 protein. The experimental mass (17703.3 Da) of tBALF0 is within the expected experimental error (calculated mass: 17722.5 Da). Peaks labelled “Matrix adduct” correspond to tBALF0 with a sinapinic acid adduct. Peptide mass fingerprinting (PMF) of recombinant tBALF0 after in-solution trypsin digestion is shown in the inset. Sequence of peptides belonging to tBALF0 is indicated in brackets. (B) Protein sequence of tBALF0 showing regions covered by MALDI-TOF mass spectrometry analysis. squamous cell carcinomas) was used as non-NPC controls (n = 8). IgG affinity chromatography and eluted from the Ni-NTA resin in batch by antibodies to BALF0/1 were detectable in 9 of 27 (33.3%) NPC patients, discontinuously decreasing the pH of the buffer. Several attempts have compared to 1 of 8 (12.5%) in non-NPC patient controls (Fig. 4). It is to been done to renature tBALF0 on the resin by gradually decreasing urea note that high frequency of IgGs against BALF0/1 in NPC patients was concentration until 0 in the washing buffers. However, we could not also associated with a significant higher average OD405, suggesting that succeed in eluting native tBALF0 in these conditions even in the pre- high level of anti-BALF0/1 IgG antibodies might circulate in this spe- sence of 250 mM imidazole. This suggested that tBALF0 may form ag- cific subpopulation. gregates in these conditions, which was further confirmed by SDS-PAGE analysis (data not shown). The identity of recombinant tBALF0 in oli- gomeric form was confirmed by peptide mass fingerprinting. Two cy- 4. Discussion steines are present in tBALF0 suggesting that disulfide bridges may likely contribute to the multimerization. In the definitive procedure, The growth of bacterial hosts can be inhibited by the expression of tBALF0 was purified in batch under denaturing conditions and dialyzed heterologous proteins. Gene products that affect the growth rate of against PBS in the absence of reducing agents. A fraction of the protein bacterial hosts are considered to be toxic. They include membrane precipitated during dialysis and was discarded by centrifugation. We proteins, proteins interacting with DNA or interfering with electron estimated that 2.8 mg tBALF0 could be obtained in a purified and so- transport [24]. Lower transformation efficiency and marked reduction luble form from a 74 mg (1 L culture) total E.coli protein extract. in bacterial growth of constructs encoding full length BALF0/1 were The existence of EBV BALF0/1 in cells that are infected by EBV has observed (data not shown) suggesting that the BALF0/1 gene product never been confirmed so far due to the absence of dedicated im- was toxic to the host. Structural analysis predicted the presence of 2 α- munological reagents. The presence of antibodies to BALF0/1 in pa- helices with high hydrophobicity at BALF0/1 C-terminus showing pu- tients infected by EBV could therefore be considered as an important tative transmembrane domains. A recombinant protein with mem- indirect evidence for the existence of BALF0/1 in vivo. brane-spanning domains may have a toxic effect on a bacterial host, Seroepidemiological studies were performed by ELISA in EBV primary probably due to the association between the protein and bacterial infection patients, healthy EBV carriers and NPC patients. IgG directed membranes [25]. Removal of putative transmembrane domains in against tBALF0 were detected in different forms of EBV infection (pri- BALF0/1 led to a 17.7 kDa polypeptide that could be expressed in E. mary and past infections) and in NPC patients suggesting that either coli. High expression levels of recombinant proteins in bacterial ex- BALF0 or BALF1 are expressed under physiological conditions. Among pression systems can lead to the formation of insoluble inclusion bodies 60 healthy EBV carriers, 13.3% of individuals had a weak positive IgG [26], which can be completely solubilized by strong denaturing agents, response to tBALF0. The weak immunogenicity of EBV lytic protein has such as 6 M guanidine hydrochloride or 8 M urea. tBALF0 was purified also been reported in a recent evaluation of IgG antibody responses to a under denaturing conditions in the presence of 8 M urea by nickel

 Z. Shao, et al. 3URWHLQ([SUHVVLRQDQG3XULILFDWLRQ  ²

investigations on the role of BALF0/1 in NPC pathogenesis.

Funding

Z.S was the recipient of a Ph.D. fellowship from the China Scholarship Council. This work was supported by LABEX DYNAMO (ANR-LABX-011) and EQUIPEX (CACSICE ANR-11-EQPX-0008), no- tably through funding of the Proteomic Platform of IBPC (PPI).

Conflicts of interest

The authors declare that they have no conflict of interest.

Acknowledgements

We are grateful to Dr. Frédérique Quignon for the assistance of plasmid construction and the provision of viral genomic DNA of EBV B95.8 strain. We also thank Marion Hamon for the technical assistance of mass spectrometry.

References

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 2.2 Epstein-Barr virus BALF0 and BALF1 modulate autophagy

Autophagy is an essential catabolic process that degrades cytoplasmic components within the autolysosome for cell survival and homeostasis. As a metabolic and intracellular biomass and organelle quality and quantity control pathway, autophagy also acts as a microbial clearance mechanism to protect eukaryotic cells against intracellular pathogens. However, microbes are vulnerable to autophagic destruction and some pathogens have evolved successful strategies to escape immune control or to promote their own replication by manipulating autophagy for their own benefit. Recent studies indicate that EBV can modulate autophagy both during latency and reactivation.

During latency, LMP1 induces autophagy to control its own degradation, LMP2A can induce autophagy to promote abnormal acinus formation and EBNA3C, which acts as a prerequisite for inhibition of apoptosis and maintenance of cell growth, has also been reported to induce autophagy. During the lytic cycle, Rta stimulates the expression of autophagy related genes following an ERK-dependent pathway, a process that is proposed to be beneficial to the production of virus particles. Others have reported that autophagy is blocked at the final degradative steps to possibly favor the acquisition of the viral envelop by EBV. However, the viral proteins that are responsible for EBV- mediated subversion of the autophagy pathways are still unknown.

Cellular Bcl-2 and two Herpesviridae-encoded Bcl-2 homologs, namely M11 (MHV68) and Ks-Bcl-2 (KSHV) have been shown to inhibit autophagy via an interaction with

Beclin 1. During lytic cycle, EBV encodes for two to three putative vBcl-2 proteins, namely BHRF1, BALF0 and/or BALF1, whose contribution to autophagy has not been investigated so far. Whereas BHRF1 has been extensively characterized as an anti- apoptotic protein, the function of BALF0 and/or BALF1 is still equivocal. BALF0/1 has been proposed to play a role in inhibiting apoptosis through association with Bax and Bak. Conversely BALF1 fails to protect against Sindbis virus- or Bax-induced apoptosis and antagonizes the anti-apoptotic activity of BHRF1. BALF0 may also antagonize the anti-apoptotic activity of BHRF1 but does not co-immunoprecipitate

 76 with BHRF1 as BALF1 [105]. BALF0/1 is transcribed both in lytic stage and latency in EBV-positive Burkitt lymphoma's cell lines and NPC biopsies. So far, the existence of BALF0 and/or BALF1 in cells that are naturally infected by EBV has never been confirmed due to the absence of dedicated immunological reagents.

We provide here initial evidences that BALF0/1 ORF indeed encodes for two proteins in latently and reactivated EBV-positive BL cell line Akata. BALF0 and BALF1 were barely detectable in non-reactivated cells but accumulated during the early phase of the lytic cycle as shown by immunoblot using the specific polyclonal antiserum directed against tBALF0. Surprisingly, BALF1 accumulation preceded BALF0’s whereas

BALF0 accumulation was conversely associated with a marked decrease in the level of

BALF1. Based on the use of plasmids expressing BALF0 and BALF1 separately, we could confirm that BALF1 overexpression promoted BALF0 accumulation which in turn inhibited BALF1’s in a dose-dependent manner, therefore providing an explanation for the unbalanced kinetics of both proteins during EBV reactivation.

Since BALF1 and BALF0 are sequentially expressed during EBV reactivation, autophagy was analyzed in HeLa cells expressing either BALF1 alone or co-expressing

BALF0 and BALF1. Due to its very weak expression level, the impact of BALF0 on autophagy could not be evaluated. In HeLa cells that stably expressed GFP-LC3,

BALF1 induced a significant increase in the number of GFP-LC3 containing puncta, which marks autophagosomes, that accumulated in the presence of lysosomal inhibitor

(CQ). This observation suggested that BALF1 stimulated the autophagic flux. In addition, BALF1 expression induced a significant increase in LC3-II, a lipidated form of LC3 that is tightly associated with the autophagosome membranes, which was markedly amplified in the presence of CQ. This experiment therefore confirmed that

BALF1 stimulates the formation of autophagosomes in HeLa cells. As to investigate a later phase of autophagy, i.e. the fusion between autophagosomes and lysosomes, we used HeLa cells stably expressing a tandem mRFP-GFP-LC3 probe. In such cells, autophagosomes are dually labelled with GFP and mRFP whereas acidic

 77 autolysosomes, resulting from the fusion between autophagosomes (neutral) and lysosomes (acidic), are only labelled with mRFP because of the quenching of GFP that occurs at low pH. In these cells, BALF1 induced the concomitant accumulation of both dually labelled (autophagosomes) and red-only (autolysosomes) vesicles, confirming that it significantly increased the autophagic flux until autolysosome formation. As previously demonstrated, BALF0 tempered the expression of BALF1. In contrast to the pro-autophagic effect of BALF1, co-expression of BALF0 and BALF1 simultaneously from same expression plasmid resulted in the blockage in autophagic flux, since the number of GFP-LC3 vesicles and the level of LC3-II did not increase in the presence of CQ. Altogether, these results led to the conclusion that BALF1 stimulates the autophagic flux which, in turn, was limited in the presence of BALF0.

Confocal microscopy analysis unrevealed that BALF1 formed discrete cytoplasmic puncta that colocalized with GFP-LC3 positive vesicles. This led us to postulate that

BALF1 might be associated with GFP-LC3 containing vesicles, possibly through a LIR motif. This hypothesis was further supported by the presence of a putative LIR motif between amino-acids 146 to 149 (146-WSRL-149). Two punctual BALF1 mutants were generated to evaluate the contribution of the putative LIR-domain (1) to BALF1 localization in LC3-positive vesicles and (2) to BALF1 ability to promote autophagy.

These mutations had a dramatic effect both on the subcellular localization of BALF1 and did partly or totally abrogate BALF1 ability to promote autophagosomes formation.

Whereas we could not provide additional evidences for the direct interaction between

ATG8 family proteins and BALF1 through the putative LIR motif, we could demonstrate herein that this domain was required both for efficiently targeting of

BALF1 to GFP-LC3 vesicles as well as for the pro-autophagic effect of BALF1.

Previous work demonstrated that BALF1 and BHRF1 could interact with each other therefore modulating BHRF1 ability to inhibits apoptosis [105]. This led us to wonder whether these two vBcl-2s of EBV could work in concert to modulate autophagy. As shown in supplementary results (Figure 34), co-expression of BALF1 and BHRF1

 78 resulted in an additional accumulation of LC3-II compared to the BALF1 and BHRF1 alone. However, the addition of CQ did not modify LC3-II level, demonstrating that autophagy-mediated degradation of LC3-II was inhibited when both proteins were co- expressed. In addition, immunoblot analysis revealed that BHRF1 promotes the accumulation of BALF0 and BALF1 whereas BALF0 and BALF1 were both able to dramatically reduce BHRF1 expression.

 79

1 Article 2 Epstein-Barr virus BALF0 and BALF1 modulate 3 autophagy

4 Zhouwulin Shao *, Chloé Borde, Frédérique Quignon, Alexandre Escargueil and Vincent 5 Maréchal

6 Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine, F-75012 Paris, France; 7 [email protected] (C.B.); [email protected] (F.Q.); 8 [email protected] (A.E.); [email protected] (V.M.) 9 * Correspondence: [email protected] (Z.S.) 10 Received: date; Accepted: date; Published: date

11 Abstract: Autophagy is an essential catabolic process that degrades cytoplasmic components within 12 the lysosome therefore ensuring cell survival and homeostasis. A growing number of viruses 13 including members of the Herpesviridae family have been shown to manipulate autophagy to 14 facilitate their persistency or to optimize their replication. Previous works have shown that Epstein- 15 Barr virus (EBV), a human transforming gammaherpesvirus, hijacked autophagy during the lytic 16 phase of its cycle possibly to favor the formation of viral particles. However, the viral proteins that 17 are responsible for EBV-mediated subversion of the autophagy pathways are still to be 18 characterized. Here we provide first evidences that BALF0/1 open reading frame encodes for two 19 conserved proteins of the Bcl-2 family, namely BALF0 and BALF1, that are expressed during the 20 early phase of the lytic cycle and can modulate autophagy. A putative LC3-interacting region (LIR) 21 has been identified and is required for both BALF1 colocalizing with autophagosomes and 22 stimulating autophagy.

23 Keywords: Epstein-Barr virus; BALF0/1; BALF1; BALF0; autophagy; vBcl-2 24

25 1. Introduction 26 Autophagy is a homeostatic “self-eating” process involved in the degradation and recycling of 27 cytoplasmic components through a lysosomal pathway. Autophagy was first identified as a 28 physiological pathway that promotes cell survival [1]. In addition to its role as a metabolic and 29 intracellular biomass and organelle quality and quantity control pathway, autophagy also acts as a 30 microbial clearance mechanism that protects eukaryotic cells against intracellular pathogens. 31 Autophagy also emerged as an alternative pathway to present microbial antigens to the immune 32 system [2]. Accordingly, some pathogens have evolved successful strategies to escape immune 33 control or to promote their replication by manipulating autophagy for their own benefit [3,4]. 34 Epstein-Barr virus (EBV) is a human enveloped DNA virus from the Herpesviridae family [5]. 35 EBV primary infection occurs usually during childhood with no symptoms whereas it can be 36 associated with infectious mononucleosis in young adults. EBV establishes a latent, lifelong persistent 37 infection in more than 95% of the adult population. Although it is usually tightly controlled by the 38 immune system, EBV persistency has been related to a number of malignancies including some forms 39 of Burkitt’s lymphomas, Hodgkin’s disease, post-transplant lymphoproliferative diseases as well as 40 epithelial tumors such as undifferentiated nasopharyngeal carcinoma (NPC) and gastric carcinomas 41 [6–9]. EBV expression patterns alternate latency programs that ensure EBV persistency in B 42 lymphocytes mainly, and lytic phases that allow the production of virions from B lymphocytes and 43 epithelial cells [10]. The induction from latency to the lytic cycle is called reactivation. Whereas the 44 stimuli that induce the transition from latency to lytic phase are poorly characterized in vivo,

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45 reactivation can be investigated ex vivo notably by treating type I latently infected B-cells with various 46 chemical or biological inducers [11]. Recent studies have shown that EBV could modulate autophagy 47 both during latency and reactivation. During latency the latent membrane protein 1 (LMP1) induces 48 autophagy to control its own degradation [12], the latent membrane protein 2 (LMP2A) induces 49 autophagy to promote abnormal acinus formation [13] and the EBV nuclear antigen 3C (EBNA3C) 50 activates autophagosome formation through transcriptional induction of several autophagy 51 regulators including ATG3, ATG5, and ATG7 [14]. During EBV lytic cycle, autophagy would be 52 modulated in a complex bimodal way that combines the stimulation of the early phase (i.e. 53 autophagosome formation) with the inhibition of the latest phase of autophagy (i.e. the degradation 54 of the autophagosomes content following the fusion between autophagosomes and lysosomes). 55 Accordingly, De Leo and colleagues have shown that autophagy was transiently induced by EBV 56 reactivation and then inhibited during the latest step of the lytic cycle [15]. Blocking autophagy at the 57 final step may possibly favor the acquisition of viral envelops and components of the autophagic 58 machinery by the neosynthesized virions [16,17]. Except for Rta, an immediate early protein that 59 stimulates the expression of autophagy-related genes through an ERK-dependent pathway [18], the 60 viral proteins that modulate autophagy during the lytic cycle are still poorly characterized. 61 In the present work, we assumed that EBV protein(s) whose viral or cellular orthologs have been 62 shown to act on autophagy could also modulate this process. Cellular Bcl-2 is a well-known anti- 63 apoptotic protein of the B-cell lymphoma-2 (Bcl-2) family [19,20]. Bcl-2 and two Herpesviridae-encoded 64 Bcl-2 orthologs, namely M11 (murine γ-herpesvirus 68, MHV68) and Ks-Bcl-2 (Kaposi’s sarcoma 65 herpesvirus, KSHV) inhibit autophagy through their interaction with Beclin 1, a cellular protein that 66 is required for initiating autophagosome formation [21,22]. Interestingly EBV encodes for two viral 67 Bcl-2 homolog (vBcl-2) proteins, namely BHRF1 and BALF0/1 [23,24]. Both BHRF1 and BALF0/1 68 prevent apoptosis during early infection of primary B cells but may be dispensable once a latent 69 infection is established [25]. Whereas BHRF1 anti-apoptotic activity has been extensively studied [26], 70 the expression and function of BALF0/1 are still equivocal. Indeed, two in-frame methionine codons 71 are present near the beginning of BALF0/1 open reading frame (ORF) suggesting that two proteins 72 with different N-termini may be encoded [27]. BALF1 protein would be encoded by the shorter ORF, 73 while the protein encoded from the first non-conserved methionine was referred to as BALF0. 74 BALF0/1 is transcribed both in lytic stage and latency in EBV-positive Burkitt lymphoma's cell lines 75 and NPC biopsies [28] and promotes tumor formation and metastasis in nude mice [29]. Whereas 76 initial work suggested that BALF0/1 could inhibit apoptosis through association with Bax and Bak 77 [24], Bellows and co-workers have shown that BALF1 failed to protect against Sindbis virus- or Bax- 78 induced apoptosis and antagonized the anti-apoptotic activity of BHRF1 [27]. BALF0 also 79 antagonized the anti-apoptotic activity of BHRF1 but did not associate with either BHRF1 or BALF1 80 [27]. Most importantly, the existence of BALF0 and/or BALF1 in cells naturally infected by EBV has 81 never been confirmed due to the absence of specific antibodies. Previous work has shown that NPC 82 patients may produce antibodies recognizing a 31 kDa protein in BALF0/1-transfected NIH3T3 cells 83 [28], which was compatible with BALF0 expected size. Nonetheless the existence of BALF1 could not 84 be confirmed in the same study. More recently, we designed an ELISA to detect anti-BALF0/1 85 antibodies in human plasma. This assay led to the detection of low titer IgGs to BALF0/1 during 86 primary (10.0%) and past (13.3%) EBV infection whereas high-titer IgGs were detected in 33.3% of 87 NPC patients. Thus, this work provided an important indirect evidence for the expression of BALF0 88 and/or BALF1 in vivo [30]. 89 In the present study we were able to confirm that both BALF0 and BALF1 are expressed in 90 lytically infected B-cells and provided evidences that BALF0 and BALF1 modulate autophagy.

91 2. Materials and Methods

92 2.1. Sequence alignment 93 All the sequences used in this study are listed in Table S1. A search for proteins homologous to 94 BALF1 and BALF0 was performed on the NCBI database (www.ncbi.nlm.nih.gov). Alignments of

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95 protein sequences were performed using the ClustalW method using MacVector (version 17.0.2) with 96 default settings. Phylogenetic trees were generated using UPGMA by MacVector (version 17.0.2).

97 2.2. Cell culture, treatments and transient transfection 98 HeLa cells and EBV-positive Burkitt’s lymphoma cell line Akata (purchased from ATCC) were 99 cultured at 37°C under 5% CO2 in RPMI-1640 (Gibco) supplemented with 10% fetal calf serum (FCS, 100 Thermo Scientific). HeLa cells with stable expression of GFP-LC3 and mRFP-GFP-LC3 were kindly 101 provided by Aviva Tolkovsky [31] and David Rubinsztein [32], respectively. These cells were grown 102 in RPMI-1640 supplemented with 10% FCS and G418 (500 µg/mL; Invivogen). In order to induce the 103 EBV lytic cycle, Akata cells were treated with 7.5 µg/mL of polyclonal rabbit anti-human IgG (Dako). 104 Autophagic flux was monitored by addition of chloroquine (50 µM) 4h prior to cell lysis. Starvation- 105 induced autophagy was carried out by replacing the complete medium with Earle’s Balanced Salt 106 Solution (EBSS, Gibco) for 4h before performing immunofluorescence staining. DNA plasmid 107 transfections were performed by using Fugene HD transfection reagent (Promega) according to the 108 manufacturer’s instructions.

109 2.3. RNA extraction and qRT-PCR 110 To determine the relative mRNA expression of BALF0/1, Akata cells were harvested at the 111 indicated time post-induction and total RNA was extracted by using TRIzol reagent (Invitrogen). Five 112 micrograms of purified total RNA were treated with DNase from TURBO DNA-free kit (Life 113 Technologies). Then 2 µg of DNase-treated RNA was reverse-transcribed into cDNA by High- 114 Capacity cDNA Reverse Transcription Kit (Thermo Scientific). Transcriptional expression was 115 measured by quantitative real-time PCR (qRT-PCR) with KAPA SYBR Fast Universal Readymix Kit 116 (Kapa Biosystems) and specific primers listed in Table S2. Cellular cyclophilin was used as a control 117 to normalize viral mRNA expression. Experiments were performed on the CFX96 Touch Real-Time 118 PCR Detection System (Bio-Rad) and analyzed with the 2−ΔΔCT method [33].

119 2.4. Plasmid construction and mutagenesis 120 The DNA sequence corresponding to the open reading frame of BALF0/1 was amplified by PCR 121 using viral genomic DNA (EBV B95.8 strain) as template. Primers were designed according to the 122 manufacturer's instructions of Cold Fusion Cloning Kit (SBI). The sequence encoding the 123 hemagglutinin (HA) tag has been added to the reverse primer to generate a C-terminal HA-tag fused 124 with BALF0/1 from the expression vector pcDNA3.1 (Invitrogen). The amplified PCR product was 125 inserted into the linearized pcDNA3.1 (EcoRI, NEB) by ligation using the Cold Fusion Cloning kit. 126 Mutant constructions were generated by site-directed mutagenesis from the template pcDNA3.1- 127 BALF0/1-HA. All PCR for plasmid construction were performed under standard conditions by using 128 PfuUltra DNA polymerase (Agilent Technologies) and plasmids were verified by sequencing. The 129 sequences of primers for plasmid construction and mutagenesis are listed in Table S2.

130 2.5. Immunoblotting 131 Transfected cells were collected at 48h post-transfection and reactivated Akata cells were 132 harvested at the indicated time. Cell pellets were lysed in lysis buffer (50 mM Tris·HCl pH 6.8, 2% 133 SDS, 2% β-mercaptoethanol), subjected on SDS-PAGE and transferred onto a PVDF membrane 134 (Amersham). The membranes were blocked with 5% BSA or skim milk powder and incubated at 4°C 135 overnight with the indicated antibodies. The anti-ZEBRA (sc-53904), anti-β-actin (sc-47778) and anti- 136 HA (sc-805) antibodies were purchased from Santa Cruz; anti-LC3B (L7543) and anti- SQSTM1/p62 137 (5114T) were obtained from Sigma and Cell Signaling Technology, respectively. Anti-sera against 138 BALF0/1 were prepared by immunizing a rabbit with the recombinant protein of BALF0/1 which 139 produced as previously described [30]. Horseradish peroxidase conjugated goat antibodies directed 140 against mouse (Cell Signaling Technology) or rabbit (Amersham) immunoglobulins were used as

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141 secondary antibodies. Immunodetection was performed using the ECL detection system according 142 to manufacturer’s instructions (Amersham).

143 2.6. Immunofluorescence analysis 144 Cells were grown on 8-well Lab-Tek chamber slide (Thermo Scientific) and fixed 24h after 145 transfection with paraformaldehyde (4%) in PBS for 10 min at room temperature (RT). Fixed cells 146 were washed with PBS twice and permeabilized with 0.2% Triton X-100 for 5 min at RT, blocked with 147 5% FCS and incubated with anti-HA rabbit antibody, or anti-BALF0/1 rabbit sera for 1h at 37°C. Then, 148 the cells were washed with PBS and incubated with the secondary antibody (Alexa Flour 555 goat 149 anti-rabbit IgG or Alexa Flour 647 goat anti-rabbit IgG, Thermo Scientific). Next, the cells were 150 washed with PBS and the nuclei were counterstained with Hoechst 33342 (Thermo Scientific). 151 Coverslips were mounted in Glycergel mounting medium (Dako) and observed by using Zeiss 152 AxioObserver Z1 or Leica SP8 confocal laser microscope. Images were resized, organized and labeled 153 using ImageJ software. Three-dimensional reconstruction was established by IMARIS (Bitplane) 154 software.

155 2.7. Statistics 156 Data from three independent experiments are presented as mean ± standard error of the mean 157 (SEM) which were analyzed with Prism software (GraphPad) by using Student’s t test or one-way 158 analysis of variance (ANOVA) test comparisons. P values less than 0.05 were considered statistically 159 significant.

160 3. Results

161 3.1. BALF0 and BALF1 are both expressed during EBV reactivation and cross-regulate each other 162 A search for proteins homologous to BALF1 and BALF0 was performed. This led to the 163 identification of 6 homologous proteins in gammaherpesviruses from , that were closely 164 related to EBV BALF1 and BALF0, as well as 13 proteins of gammaherpesviruses from non-primate 165 hosts (Figure 1A). Interestingly, BALF0 and BALF1 from EBV were more closely related to BALF1 166 from other gammaherpesviruses than to EBV BHRF1, which belongs to a homology group containing 167 vBcl-2s from herpesvirus saimiri and Kaposi’s Sarcoma herpesvirus (KSHV). BALF0/1 ORF encodes 168 for a putative 220 amino acids protein, named BALF0. Due to the presence of a conserved internal 169 putative start codon at position 39, this ORF is also predicted to encode for a 182-amino-acid 170 polypeptide thereafter referred to as BALF1 (Figure 1B). Sequences alignment revealed that amino 171 acids 1 to 38 from BALF0 were unique to Epstein-Barr virus among BALF1 homologues from primate 172 gammaherpesviruses (Figure 1B). Due to the lack of specific antibodies, the existence of BALF0 and 173 BALF1 proteins could not be assessed in naturally infected cells so far. To overcome this limit, we 174 generated a specific polyclonal antiserum directed against a recombinant form of BALF0/1. For this 175 purpose, a truncated form of BALF0 encoding for amino acid 1 to 140 was expressed and purified 176 from E. coli [30] and used as an antigen to obtain rabbit anti-BALF0/1 antibodies. The resulting 177 antiserum could specifically detect polypeptides whose size was compatible with BALF0 and BALF1 178 following immunoblotting analysis of HeLa cells transfected with pcDNA3.1-BALF0/1-HA, an 179 expression vector expressing BALF0/1 mRNA (Figure 2A left panel). As expected BALF0 and/or 180 BALF1 were also detected by immunofluorescence in the cytoplasm of transfected cells as previously 181 reported [27] (Figure 2A right panel). 182 Akata is a Burkitt’s lymphoma-derived cell line in which EBV establishes a type I latent infection. 183 EBV can be reactivated from Akata cells following B-cell receptor cross-linking with anti-surface 184 immunoglobulins [11]. In a first attempt to investigate BALF0/1 expression pattern, Akata cells were 185 reactivated and BALF0/1 mRNA was analyzed by qRT-PCR. In agreement with previous work [28] 186 BALF0/1 mRNA was barely detectable in non-reactivated cells but progressively accumulated from 187 8 to 48 hours following reactivation (Figure 2B). Immunoblot analysis demonstrated that both BALF0 188 and BALF1 were expressed during early lytic infection and accumulated shortly after the expression

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189 of ZEBRA, an immediate early protein that is essential for the virus to enter the lytic cycle (Figure 2C) 190 [34]. Surprisingly, BALF1 accumulation preceded BALF0’s whereas BALF0 accumulation was 191 conversely associated with a marked decrease in the level of BALF1 (Figure 2C). This suggested that 192 BALF0 and BALF1 synthesis and/or degradation were cross-regulated. To test this hypothesis, 193 expression vectors encoding for HA-tagged BALF0 and BALF1 (pcDNA3.1-BALF0/1-HA), BALF0 194 alone (pcDNA3.1-BALF0-HA) or BALF1 alone (pcDNA3.1-BALF1-HA) were constructed by 195 mutating the first (BALF1) or the second (BALF0) start codon (Figure 2D). Comparable amounts of 196 BALF0 and BALF1 were expressed in HeLa cells transfected with pcDNA3.1-BALF0/1-HA. Whereas 197 BALF1 was expressed at high level from pcDNA3.1-BALF1-HA, BALF0 was barely detectable when 198 expressed alone from pcDNA3.1-BALF0-HA either by immunofluorescence (Figure 2E) or by 199 immunoblot (Figure 2F). This led us to hypothesize that BALF0 accumulation may require BALF1 200 expression. This was further confirmed since BALF0 accumulation was restored at least in part when 201 pcDNA3.1-BALF0-HA was co-transfected with pcDNA3.1-BALF1-HA (Figure 2F). To confirm this 202 result, we analyzed BALF0 expression from pcDNA3.1-BALF0-HA in the presence of increasing 203 amounts of pcDNA3.1-BALF1-HA. As expected, BALF0 accumulation positively correlated with the 204 amounts of pcDNA3.1-BALF1-HA (Figure 2G). Conversely transfecting increasing amounts of 205 pcDNA3.1-BALF0-HA in the presence of a constant amount of pcDNA3.1-BALF1-HA led to a 206 significant decrease in BALF1 accumulation (Figure 2H). Since the total amount of transfected 207 plasmids was constant in each experiment, it was concluded that BALF1 promoted BALF0 208 accumulation which in turn inhibited BALF1 expression, therefore providing an explanation for the 209 unbalanced kinetics of both proteins during EBV reactivation.

210

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211 212 Figure 1. BALF1 of primate and non-primate herpesviruses. (A) Phylogenetic tree generated using 213 UPGMA method from amino acid sequences of the indicated human and viral Bcl-2 family members 214 as well as BALF1 from primate and non-primate herpesviruses. (B) ClustalW alignment of amino acid 215 sequences analyzed in (A). Identical amino acids are marked in dark shading. The putative LIR (LC3- 216 interacting region) motif of BALF1 is marked by box. The GenBank accession numbers of sequences 217 used in this study are listed in Table S1. The analysis was performed by MacVector software.

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218 219 Figure 2. Characterization of BALF0 and BALF1 expression. (A) Characterization of the rabbit anti- 220 sera against BALF0/1. Hela cells were transfected with the BALF0/1 expression vector (pcDNA3.1- 221 BALF0/1-HA) or the corresponding negative control (EV, empty vector). The expression of BALF0/1 222 was observed at 48h post transfection (p.t.) by immunoblot (left panel) and immunofluorescence 223 (right panel) using the rabbit anti-sera against BALF0/1. In immunoblot analysis, two polypeptides 224 whose relative mobility following SDS-PAGE corresponding to the predicted size of BALF0 (26 kDa) 225 and BALF1 (22 kDa) can be recognized respectively. Scale bar = 20µm. (B) Time-course of BALF0/1 226 mRNA expression in EBV-positive BL cell line. Akata cells were induced into the lytic cycle by cross- 227 linking of surface immunoglobulin for 2 to 48 hours. At the indicated time post-induction, mRNA 228 encoding for BALF0/1 was quantified by qRT-PCR. (C) Protein expression of BALF0 and BALF1 in 229 EBV-positive BL cell line. Total protein was extracted from Akata cells as described in (B) and 230 analyzed by immunoblot (left panel) using the rabbit anti-sera against BALF0/1. ZEBRA is an 231 immediate early protein of EBV that is used herein as a marker for viral reactivation. Relative 232 expression of BALF0 and BALF1, shown at right panel, was analyzed by using ImageJ and compared 233 to that of the β-actin loading control at each time point. (D) Schematic diagram of different expression 234 vectors encoding for BALF0/1 (pcDNA3.1-BALF0/1-HA), BALF0 (pcDNA3.1-BALF0-HA) and BALF1 235 (pcDNA3.1-BALF1-HA). BALF0 and BALF1 alone were obtained by replacing the methionine at 236 amino acid 39 and 1 by glycine and isoleucine, respectively. (E) Immunofluorescence staining of HA- 237 tagged BALF0/1, BALF0 and BALF1. HeLa cells were fixed 24h p.t. and the expression of BALF0/1, 238 BALF0 and BALF1 were detected by anti-HA antibody. Scale bar = 10µm. (F) Immunoblot analysis of 239 ectopic expression of BALF0/1, BALF0 and BALF1. HeLa cells were co-transfected with 0.25 µg of EV 240 and the same amount of BALF0/1, BALF0 and BALF1 as well as 0.25 µg of BALF0 and BALF1 so that 241 the effector plasmids totaled 0.5 µg. The ectopic expression was analyzed 48h p.t. by Western blot

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242 using the rabbit anti-sera against BALF0/1. (G) and (H) Does-dependent cross-regulation between 243 BALF0 and BALF1. Hela cells were transfected with constant amount (0.25 µg) of BALF0 or BALF1 244 plasmid with increasing amount of BALF1 or BALF0 (0 to 0.5 µg) respectively. Western blot analysis 245 was carried out as described in (F). Relative expression of BALF0 and BALF1 was evaluated by 246 densitometric analysis through ImageJ and compared to that of the β-actin loading control. Shown 247 values represent the mean ± SEM of three independent experiments. One representative set of 248 immunoblotting results is shown. *P<0.05.

249 3.2. BALF1 and BALF0/1 modulate autophagy 250 Previous work has shown that cellular Bcl-2 could inhibit autophagy. Indeed Bcl-2 interacts with 251 Beclin 1 which prevents Beclin 1 from assembling the pre-autophagosomal structure [35]. Since 252 BALF0 and BALF1 share noticeable homology with cellular Bcl-2, we wondered whether they may 253 modulate autophagy as well [21]. Considering that BALF1 and BALF0 were sequentially expressed 254 during EBV reactivation, autophagy was analyzed in HeLa cells expressing either BALF1 alone or co- 255 expressing BALF0 and BALF1. Due to its very weak expression level, the impact of BALF0 alone 256 could not be evaluated in the same assays. 257 Microtubule-associated protein light chain 3 (LC3) is a widely used marker for autophagosomes. 258 During autophagy, cytosolic LC3-I is conjugated to phosphatidylethanolamine to form LC3-II, which 259 is subsequently incorporated onto the autophagosomal membrane. Therefore, LC3-II level positively 260 correlates with the number of autophagosomes. To investigate the impact of BALF1 on 261 autophagosomes formation, BALF1-HA expression vector was transfected into HeLa cells that stably 262 expressed a GFP-labeled form of LC3B (GFP-LC3). In these cells, autophagosomes and 263 autolysosomes appear as small cytoplasmic vesicles. Chloroquine (CQ) neutralizes the lysosomal pH 264 and causes the accumulation of GFP-LC3 positive vesicles by inhibiting endogenous protein 265 degradation [36]. As illustrated (Figure 3A and 3B), BALF1 induced a significant increase in the 266 number of GFP-LC3 containing vesicles, suggesting that BALF1 either stimulates autophagosomes 267 formation or alternatively inhibits the fusion of autophagosomes with lysosomes. Importantly we 268 noticed that BALF1 concentrated in GFP-LC3 positive puncta. To explore the autophagic flux, which 269 reflects the autophagic degradation activity, we analyzed the accumulation of LC3-II by immunoblot 270 in cells that were treated with or without CQ. As shown in Figure 3C, BALF1 expression induced a 271 significant increase in LC3-II that was markedly amplified in the presence of CQ, indicating that 272 BALF1 indeed stimulates the autophagic flux. Next, we used an expression vector encoding for a 273 tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3B) probe. This probe is a convenient tool for 274 monitoring autophagic flux based on the fact that EGFP and mRFP fluorescent proteins have a 275 different stability in response to a low pH. Indeed, the fusion between autophagosomes and 276 lysosomes is associated with a decrease in pH that affects EGFP but not mRFP fluorescence [37]. As 277 a result, autophagosomes are dually labelled with EGFP and mRFP whereas acidic autolysosomes 278 are only labelled with mRFP. As illustrated in Figure 3D and 3E, CQ inhibits pH decrease which 279 induced the accumulation of vesicles that are labelled both with mRFP and EGFP (autophagosomes). 280 On the contrary, BALF1-HA induced the concomitant accumulation of both dually labelled 281 (autophagosomes) and red-only (autolysosomes) vesicles, confirming that it significantly increased 282 the autophagy flux up to the formation of autolysosomes. 283 Since it was previously demonstrated that BALF0 limited BALF1 accumulation, we wondered 284 whether the stimulation of autophagy by BALF1 was similar in the presence of BALF0. For this 285 purpose, autophagy was investigated in HeLa cells expressing both proteins from the same vector 286 (pcDNA3.1-BALF0/1-HA). As before, BALF0/1 expression was associated with an increase in the 287 number of GFP-LC3 positive puncta (Figure 4A and 4B) as well as LC3-II accumulation (Figure 4C), 288 albeit to a lower extent compared to BALF1 alone. This effect however could not be observed 289 anymore in the presence of CQ (in comparison to empty vector and pcDNA3.1-BALF0/1-HA in the 290 presence of CQ), indicating that the autophagic flux induced by BALF1 was reduced in the presence 291 of BALF0.

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292 Altogether, these results led to the conclusion that BALF1 stimulates the autophagic flux which, 293 in turn, is limited in the presence of BALF0. In both instances, the expression of BALF1 alone or in 294 association with BALF0 was associated with accumulation of proteins that are targeted by 295 autophagy-mediated proteolysis, such as Sequestosome1 (SQSTM1/p62). p62, a multidomain protein 296 that is targeted to LC3-containing membranes, is both an effector and a substrate for autophagy. As 297 shown on Figure 3C and 4C, p62 accumulated in BALF1 and BALF0/1 expressing cells which 298 suggested that BALF1-mediated increase in autophagosomes formation was not associated with an 299 increase in autophagy-mediated proteolysis.

300

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301 Figure 3. BALF1 stimulates the formation of autophagosome and the autophagic flux in HeLa cells. 302 (A) Representative images of GFP-LC3 HeLa cells transfected with EV or BALF1-HA encoding 303 plasmids. 24h p.t., cells were cultured in complete medium with or without chloroquine (CQ). Ectopic 304 expression of BALF1 was detected by anti-HA antibody and observed by fluorescence microscopy. 305 Scale bar = 20µm. (B) Autophagosomes formation was evaluated by counting the number of GFP-LC3 306 puncta per cell. The results are the means ± SEM of three independent experiments, and 50 cells were 307 analyzed per assay. **P<0.01. (C) Immunoblot analysis of cellular LC3 and p62 proteins in HeLa cells 308 transfected with EV or BALF1-HA plasmids. 48h p.t., cells were cultured in complete medium with 309 or without CQ. The level of β-actin was used as a loading control. The LC3-II and p62 to β-actin ratio 310 were evaluated by densitometric analysis through ImageJ. (D) Representative images of HeLa cells 311 with stable expression of mRFP-GFP-LC3 transfected with EV and BALF1-HA plasmids, starved or 312 treated with CQ. 24h p.t., cells were fixed and imaged by confocal microscopy. Scale bar = 20µm and 313 5µm for insets. (E) Quantification of yellow puncta (mRFP-GFP-LC3 positive) and red puncta (mRFP- 314 LC3 positive) per cell. Shown values represent the mean ± SEM of three independent experiments, 315 and 50 cells were analyzed per assay. **P<0.01.

316 317 Figure 4. BALF0 limits the function of BALF1 to stimulate autophagic flux. (A) Representative images 318 of GFP-LC3 HeLa cells transfected with EV or BALF0/1 plasmid for 24h and then fixed after 4 h of CQ 319 treatment. BALF0/1-transfected cells were visualized by an anti-HA antibody (red) and nuclei were 320 subsequently stained with Hoechst 33342 (blue). (B) The number of GFP-LC3 puncta in EV and 321 BALF0/1-expressing cells was quantified. The results are the mean ± SEM of three independent 322 experiments, and 50 cells were analyzed per assay. ns: not significant; *P<0.05. (C) Immunoblot 323 analysis of cellular LC3 and p62 proteins in HeLa cells transfected with EV or BALF0/1-HA plasmids. 324 48h p.t., cells were cultured in complete medium with or without CQ. The β-actin was used as a 325 loading control. The LC3-II and p62 to β-actin ratio were evaluated by densitometric analysis through 326 ImageJ.

327 3.3.A LIR-like motif is required for BALF1 stimulation of the autophagic flux 328 BALF0/1 protein(s) were previously described as diffuse cytoplasmic proteins [27]. Since B cells 329 have a reduced cytoplasmic volume, BALF0/1 subcellular localization was investigated herein by

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330 expressing HA-tagged proteins in HeLa cells. BALF0/1-HA (Figure 2E) as well as BALF0-HA and 331 BALF1-HA mainly accumulated in the cytoplasm although low levels BALF0-HA and BALF1-HA 332 could also be detected in the nucleus. Confocal microscopy analysis revealed that BALF1 (Figure 5A) 333 and BALF0 (data not shown) could also form discrete cytoplasmic puncta that colocalized with GFP- 334 LC3 positive vesicles (Figure 5A) as well as with endogenous LC3 containing vesicles (data not 335 shown). Z-stacked confocal images were collected, and a three-dimensional reconstruction was 336 performed to more precisely investigate BALF1 and LC3 subcellular colocalization. As illustrated on 337 Figure 5B, BALF1 and GFP-LC3-positive vesicles were in close contact or overlapped each other. This 338 led us to hypothesize that BALF1 might be addressed to GFP-LC3 containing vesicles, possibly 339 through a LC3-interacting region (LIR) motif. LIR motifs are short amino acid sequences that allows 340 the targeting of autophagy receptors to LC3 anchored in the phagophore membrane [38]. A subset of 341 verified LIR motifs led to the identification of a core consensus sequence [W/F/Y]xx[L/I/V], which 342 consists of four key amino acids including an aromatic residue (W/F/Y) at the first position and a 343 hydrophobic residue (L/I/V) at the fourth position, while x may be any other residue [38,39]. Sequence 344 analysis of BALF1 revealed the presence of a putative LIR motif between amino-acids 146 to 149 (146- 345 WSRL-149) that closely resembles known LIR motifs (Figure 5C). This putative LIR motif was highly 346 conserved among BALF1 orthologs from primate gammaherpesviruses (Figure 1B, box), with the 347 noticeable exception of BALF1 from Callitrichine gammaherpesvirus 3 in which the second and the 348 fourth amino acids were different. Nonetheless the resulting sequence (WFRV) still matched with the 349 consensus LIR motif. To evaluate its effective contribution to BALF1 targeting to LC3 containing 350 vesicles, discrete mutations were generated in which one (W146A) or two (W146A and L149A) 351 essential amino acids of the putative LIR motif were modified. As shown on Figure 5A, these 352 mutations had a dramatic effect on the subcellular localization of BALF1 inducing either a partial 353 (W146A) or a total (W146A and L149A) relocalization of the modified proteins into the nucleus. To 354 evaluate the impact of these mutations on BALF1 ability to stimulate autophagy, we measured the 355 average number of autophagosomes (GFP-LC3 puncta) in Hela cells expressing BALF1 single 356 (W146A) or double mutant (W146A/L149A). As shown in Figure 5A and 5D, these mutations 357 dramatically reduced BALF1 ability to promote autophagosomes formation. These experiments led 358 to the conclusion that this region was required both for efficiently targeting BALF1 to GFP-LC3 359 vesicles and for BALF1 ability to promote autophagy.

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360

361 Figure 5. BALF1 modulates autophagy through a LIR-like motif. (A) Representative images of GFP- 362 LC3 HeLa cells transfected with EV and plasmids encoding BALF1-HA or the indicated mutants. 24h 363 p.t., cells were fixed and imaged by confocal microscopy. (B) The GFP-LC3 HeLa cells transiently 364 transfected with BALF1-HA. 3D volume rendering (top) and the relative IMARIS isosurface 3D 365 rendering (middle) are shown. Scale bar = 5µm. Magnification of the boxed area is shown in the inset 366 on the bottom (scale bar = 2 µm). (C) Typical LIR sequences were aligned alongside BALF1. The 367 underlined regions indicate highly conserved residues in the putative LIR of BALF1. (D) The number 368 of GFP-LC3 puncta in cells transfected with plasmids encoding wild-type (wt) or LIR-like mutants of 369 BALF1 were quantified with ImageJ. mean± SEM; n=50 cells from three independent experiments; ns: 370 not significant; **P<0.01.

371 4. Discussion 372 The majority of herpesviruses examined so far have evolved various strategies to manipulate 373 autophagy, either for improving their persistence or to optimize their replication [40]. Previous 374 studies indicated that EBV reactivation and autophagy were intimately intricated, in a possibly 375 bimodal way. On the one hand, early steps of autophagy are induced during EBV reactivation and 376 molecules that can inhibit autophagy such as chloroquine, ammonium chloride (Quignon et al. in 377 prep) or 3-methyladenine [18] repress the accumulation of lytic proteins and reduce the production 378 of viral particles. On the other hand, the final steps of autophagy, i.e. the fusion between

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379 autophagosomes and lysosomes and the subsequent degradation of the cargos within autolysosomes, 380 are inhibited [16]. Taken together, these processes may eventually lead to the accumulation of 381 autophagic material, such as autophagic machinery components and vesicle membranes, that could 382 be used to produce viral particles. Concomitantly, this would limit the degradation of viral 383 components as well as viral particles by autophagy. In agreement with this model, Nowag and co- 384 authors provided first evidences that LC3-associated membranes might be readdressed to virion 385 envelopes [17]. In a first attempt to identify lytic viral proteins that could modulate autophagy during 386 EBV reactivation, we looked for EBV proteins that were orthologs to proteins interfering with the 387 autophagic machinery in other herpesviruses. During lytic replication, Herpes simplex type 1 (HSV- 388 1) [41], MHV68 [42], KSHV [21] and Human cytomegalovirus (HCMV) [43,44] encode for proteins 389 that can inhibit early steps of autophagy by binding to and inactivating Beclin-1, a cellular protein 390 that is required for phagophore formation [40]. Since Beclin-1 was demonstrated to be targeted and 391 inhibited by cellular Bcl-2 [21], we inferred that Bcl-2 orthologues from EBV, namely BHRF1, BALF0 392 and BALF1, could modulate autophagy as well. Database analysis showed that proteins orthologous 393 to BALF0 and BALF1 can be identified in most gammaherpesviruses. In addition, BALF0 and BALF1 394 proved to be more related to each other than to EBV BHRF1, KSHV vBcl-2 (Ks-Bcl-2) and Saimiriine 395 gammaherpesvirus 2 (Figure 1). Although BALF1 from primate hosts were closely related, we could 396 not identify BALF0 orthologs even in the closest relatives of EBV BALF1 proteins as previously [27]. 397 The production of specific antibodies to BALF0/1 led to the detection of both BALF0 and BALF1 398 during EBV reactivation. Using plasmids encoding both proteins together or separately, we could 399 demonstrate that BALF1 (Figure 3) and BALF0 (not shown) increased the number of autophagosomes 400 when expressed individually. This correlated with the accumulation of the membrane bound form 401 of LC3 that is the core autophagic machinery required for the elongation of the phagophore. 402 Importantly, the number of LC3-positive vesicles as well as the accumulation of LC3-II significantly 403 increased in the presence of CQ in BALF1 expressing cells, indicating that BALF1 has a global positive 404 impact on the autophagic flux. This result was reminiscent of previous work indicating that the 405 adenovirus protein E1B19K could activate autophagy. Indeed E1B19K, a Bcl-2 orthologue, could 406 replace Bcl-2 in the Beclin-1 complexes, thereby activating PI3KC3 and promoting early phase of 407 autophagy [45]. Overexpression of both BALF0 and BALF1 resulted in the accumulation of LC3- 408 positive vesicles, whereas LC3-II did not accumulate in the presence of CQ, suggesting that BALF0 409 tempered BALF1 pro-autophagic activity. This agreed with the fact that BALF1 accumulation 410 decreased in the presence of BALF0. Additional experiments showed that both proteins were 411 stabilized in the presence of the proteasome inhibitor MG132 (data not shown). Thus, it is possible 412 that BALF0 and BALF1 proteins act directly or indirectly on the proteasomal degradation pathway, 413 a hypothesis that is currently under investigation. These results are in agreement with the unbalanced 414 accumulation of BALF0 and BALF1 that was observed during EBV reactivation as well as with 415 previous observations by De Leo and colleagues who observed the transient accumulation of LC3-II 416 in Akata cells 8 hours after reactivation [15], a time point where BALF1 accumulated in these cells. 417 The differential impact of BALF0 and BALF1 on autophagy confirms that BALF1 and BALF0 may 418 exhibit slightly different biological activities. Accordingly, Bellows and collaborators have already 419 noticed that whereas both BALF0 and BALF1 could antagonize BHRF1 anti-apoptotic activities, only 420 BALF1 could interact with BHRF1 in vitro [27]. 421 The stimulation of autophagy by BALF1 was shown to require a conserved motif (amino acid 422 146-WSRL-149) that is reminiscent of a LIR motif that has been identified in known partners of LC3, 423 such as sequestosome1 (SQSTM1/p62) (WTHL). SQSTM1/p62, a multidomain protein, has been 424 identified as the first selective receptor for autophagic degradation of ubiquitylated protein 425 aggregates. SQSTM1/p62 is also a selective autophagy substrate, whose interaction with phagophore 426 membranes is mediated through a LIR domain [46–48]. Herein, it is demonstrated that BALF1 427 expression (as well as co-expression of BALF1 with BALF0) induced the accumulation of p62 (Figure 428 3C and 4C). This suggested that p62 escaped autophagic degradation in the presence of BALF1. Since 429 we demonstrated that BALF1 accumulated in LC3 positive vesicles, a process that also depends on 430 the putative LIR motif, we propose that BALF1 may compete with other LIR-containing proteins for

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431 targeting to the autophagosome membranes, therefore preventing them from being degraded by 432 autophagy. This might be especially important in the case of p62 since it has multiple domains that 433 mediate its interactions with various partners. As such, p62 serves as an essential signaling hub and 434 is involved in autophagy, oxidative stress signaling and cancer [49]. LC3 and BALF1 were copurified 435 in cell fractionation experiments. However, both proteins were mainly insoluble under mild 436 extraction conditions and could not be subjected to immunoprecipitation. The use of stronger 437 detergents or chaotropic agents solubilized both LC3 and BALF1 but were not compatible with 438 immunoprecipitation procedures. Other biochemical approaches are therefore needed to explore 439 putative LC3-BALF1 interaction. To date, only two viral proteins have been reported to interact 440 directly with LC3 family proteins. These include human immunodeficiency virus type 1 (HIV-1) viral 441 infectivity factor (Vif) and influenza A virus (IAV) Matrix 2 (M2) [50,51]. M2 is the only one that 442 contains an experimentally validated LIR motif, which was required for the re-distribution of LC3 to 443 plasma membrane in IAV-infected cell [51]. Last but not least, the modulation of autophagy by 444 BALF0 and BALF1 may directly or indirectly contribute to virion morphogenesis. Although initial 445 work by Johannsen and colleagues did not identify BALF0 and BALF1 in purified EBV virions [52], 446 another study led to the co-purification of BALF0 and BALF1 with BSRF1 [53], an EBV tegument 447 protein that is homologous to HSV-1 unique long 51 (UL51) and HCMV UL71 that is involved in 448 virion egress. This result, which shed a new light on putative function of BALF0 and BALF1 during 449 EBV lytic cycle, is strongly reminiscent of a recent report indicating that vBcl-2 from KSHV could 450 similarly interact with tegument protein ORF55 [54]. However, KSHV vBcl-2 interaction with ORF55 451 was shown to be critical for KSHV lytic cycle, whereas BALF0 and BALF1 proteins may be 452 dispensable for virus production as shown by Altman and co-workers [25]. Although BALF0 and 453 BALF1 proteins may not contribute to virion release, they may affect virion infectivity, which would 454 deserve further investigations. Similarly, genetic studies will be required to precisely delineate 455 BALF0 and BALF1 regions that are required for apoptosis inhibition, autophagy stimulation and 456 virion morphogenesis, three functions that have recently been dissected in KSHV vBcl-2 [54]. 457 Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1: GenBank 458 accession numbers of sequences used for alignment in this study, Table S2: Primer sequences used in this study. 459 Author Contributions: Z.S. performed the major experiments of this study. C.B. and F.Q. provided technical 460 assistance for the experiments. V.M., Z.S. and C.B. analyzed the data and wrote the paper. V.M. conceived and 461 designed this research. A.E. provided substantial assistance during the experiment and critical comments on the 462 manuscript. 463 Funding: Z.S. was the recipient of a Ph.D. fellowship from the China Scholarship Council. This research was 464 funded by recurrent funding from SU and INSERM. C.B.’s support as an ATER from SU. 465 Acknowledgments: We are grateful to Prof. Audrey Esclatine for the provision of experimental materials. We 466 also thank Grégoire Stym-Popper for the assistance of IMARIS operation. 467 Conflicts of Interest: The authors declare no conflict of interest.

468 References

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Table S1. GenBank accession numbers of sequences used for alignment in this study Group Name Organism Accession number Human Bcl-2 homolog Bak Homo sapiens NP_001179 Bax Homo sapiens NP_620116 Bcl-2 Homo sapiens NP_000624

Bcl-xL Homo sapiens CAA80661 Viral Bcl-2 homolog BHRF1 Human gammaherpesvirus 4 YP_401646 Ks-Bcl-2 Human gammaherpesvirus 8 YP_001129368 vBcl-2 Saimiriine gammaherpesvirus 2 CAC84311 BALF1 homolog BALF0 Human gammaherpesvirus 4 CAA24810 BALF1 Human gammaherpesvirus 4 YP_401718 BALF1 Gorilline gammaherpesvirus 1 AAK60342 BALF1 Macaca arctoides gammaherpesvirus 1 AYA49869 BALF1 Macacine herpesvirus 4 YP_068013 BALF1 Panine gammaherpesvirus 1 AAK01917 BALF1 Papiine gammaherpesvirus 1 AAK01916 ORF1 Callitrichine gammaherpesvirus 3 NP_733853 BALF1 Alcelaphine gammaherpesvirus 1 NP_597933 BALF1 Dolphin gammaherpesvirus 1 YP_009388510 BALF1 NP_042601 BALF1 Equid gammaherpesvirus 5 YP_009118395 BALF1 Myotis gammaherpesvirus 8 YP_009229842 Bcl-2 Rhinolophus gammaherpesvirus 1 YP_009551815 orf4 Alcelaphine gammaherpesvirus 2 YP_009044392 Ov4.5 YP_438132 v-bcl2 Porcine lymphotropic herpesvirus 1 YP_009505335 v-bcl2 Porcine lymphotropic herpesvirus 2 YP_009505382 v-bcl2 Porcine lymphotropic herpesvirus 3 AAO12311 vBcl-2 Bovine gammaherpesvirus 6 YP_009041986 vbcl-2b Phascolarctid gammaherpesvirus 1 AZB49183

 97 Table S2. Primer sequences used in this study Program Target Forward primer 5’-3’ Reverse primer 5’-3’ qRT-PCR BALF0/1 GAAACTACCTGGATGACCAC CAAACCAGAGTCTGCGATA AA GAG  Cyclophilin GCCTTAGCTACAGGAGAGA TTTCCTCCTGTGCCATCTC A Plasmid BALF0/1 AGTCCAGTGTGGTGGAGCG GATATCTGCAGAATTTTAAG ATGAACCTGGCCATTGCT CGTAATCTGGAACATCGTAT GGGTACAAAGATTTCAGGA AGTC Mutagenesis BALF0 GCCTGACGAGACCGGTAGG GACTTGGCTGGCCTACCGG CCAGCCAAGTC TCTCGTCAGGC  BALF1 GTGGTGGAGCGATATCCCT GAGCAATGGCCAGGGATAT GGCCATTGCTC CGCTCCACCAC  W146A CACTACGACTACGCTAGCCG CACCCTGAGCCGGCTAGCG GCTCAGGGTG TAGTCGTAGTG  W146A-L149A GACTACGCTAGCCGGGCGC GTAGCACAGCACCACGCGC GCGTGGTGCTGTGCTAC GCCCGGCTAGCGTAGTC 

 98 Supplementary Results

BHRF1 stabilizes BALF0 and BALF1 proteins, and cooperates with BALF1 to

modulate autophagy

BHRF1, BALF0 and BALF1 are concomitantly expressed during the early phase of the

lytic cycle. Recent work from our group (Vilmen et al., submitted; presented in this thesis) provided first evidences that BHRF1, in addition to its well-known role as an anti-apoptotic protein, could also stimulate mitophagy, a process that prevents the initiation of the innate immune response mediated by mitochondrial pathways. Bellows and collaborators provided preliminary evidences that BHRF1 and BALF1 interacted with together therefore modulating BHRF1 ability to inhibits apoptosis [105].

Considering that BALF1 stimulated autophagy, we wondered whether BHRF1 could modulate the impact of BALF1 on autophagy as well. In a first set of experiments, LC3-

II accumulation was evaluated in BALF1 expressing cells in the presence or absence of

BHRF1. As shown in figure 34A, both BALF1 and BHRF1 independently promoted

LC3-II accumulation. Importantly, LC3-II accumulation was even higher in the presence of CQ, demonstrating that each protein stimulates autophagy. Accordingly, co-expressing BALF1 and BHRF1 resulted in an additional accumulation of LC3-II.

However, the addition of CQ did not modify LC3-II level, demonstrating that autophagy-mediated degradation of LC3-II was inhibited when both proteins were co- expressed. This observation was reminiscent of the effect that was observed when

BALF0 was co-expressed with BALF1.

More surprisingly, immunoblot analysis revealed that BHRF1 promotes the accumulation of BALF0 and BALF1 whereas BALF0 and BALF1 were both able to dramatically reduce BHRF1 expression (Figure 34B). The stabilizing effect of BHRF1 on BALF0 and BALF1 was specific since BHRF1 could not exhibit same effect in other proteins such as GFP, viral proteins ZEBRA and EB2 (Figure 34C, 34D and 34E).

BHRF1 activity is likely to act on the proteasome-dependent degradation pathway for

 99 two reasons. First, we demonstrated that BALF0 and BALF1 can both be degraded by the proteasome, since these proteins accumulate in the presence of the proteasome inhibitor MG132 (Figure 34F). Second, the accumulation of BALF0 and BALF1 in

BHRF1 expressing cells was not affected in the presence of MG132, suggesting that they both inhibit the proteasome-mediated degradation of BALF0 and BALF1 (Figure

34G). This hypothesis is currently under investigation.

Figure 34. BHRF1 stabilization effect and cooperation with BALF1 to modulate autophagy. (A)

Immunoblot analysis of the cooperation between BALF1 and BHRF1 to stimulate autophagy. HeLa cells were transfected with EV (empty vector), BALF1-HA, and BHRF1-HA, respectively; or co- transfected with BALF1-HA and BHRF1-HA for 48 hours and cultured in complete medium with or without CQ for 4 h before cell lysis. The level of β-actin was used as a loading control. The LC3-

II to β-actin ratio was evaluated by densitometric analysis through ImageJ. (B) Immunoblot analysis of BALF0 and BALF1 stabilization by BHRF1. HeLa cells transfected with BALF0/1-HA, BALF0-

HA and BALF1-HA in the absence or the presence of BHRF1-HA plasmid. Transfected cells were subjected to immunoblot after 48h transfection. The level of β-actin was used as a loading control.

(C-E) The effect of stabilization by BHRF1 is specific for BALF0 and BALF1. HeLa cells transfected with GFP; ZEBRA or EB2 plasmids in the absence or the presence of BHRF1-HA.

Immunoblot analysis was performed after 48h transfection. (F) Treatment of HeLa cells with proteasome inhibitor MG132 alleviates degradation of BALF0 and BALF1. HeLa cells were transfected with BALF0/1-HA, BALF0-HA and BALF1-HA. Forty-two hours after transfection, transfected cells were treated with 10 μM MG132 for 6h and Western blot analysis was carried out by using anti-BALF0/1 sera, anti-β-actin. Relative expression of BALF0 and BALF1 was evaluated by densitometric analysis through ImageJ and compared to that of the β-actin loading control (right panel). Shown values represent the mean ± SEM of three independent experiments. One representative set of immunoblotting results is shown. (G) HeLa cells with co-transfection of

BHRF1-HA and BALF0/1-HA, BALF0-HA and BALF1-HA, respectively, were treated with 10

μM MG132 for 6h and Western blot analysis was carried out by using anti-BALF0/1 polyclonal sera, anti-HA and anti-β-actin.

 100 A B BALF1 BALF0/1 BALF0 BALF1 + EV BALF1 BHRF1 BHRF1 BHRF1 - + - + - + CTRL CQ - + - + - + - + anti-BALF0/1

anti-BALF0/1 BALF0 BALF1 HA HA BHRF1 β-actin Long exposure

LC3I LC3 II β-actin LC3 II/ β- actin 1.0 7.9 3.8 7.5 3.8 9.5 5.2 8.6

C D E GFP + + ZEBRA + + EB2 + + BHRF1 - + BHRF1 - + BHRF1 - +

GFP ZEBRA EB2

BHRF1 BHRF1 BHRF1

β-actin β-actin β-actin

F BALF0/1 BALF0 BALF1

MG132 - + - + - + expression No treatment BALF0 MG132 BALF1 protein β-actin Relative BALF0 BALF1 BALF0 BALF1 BALF0/1

G BHRF1

BALF0/1 BALF0 BALF1

MG132 - + - + - + - + anti-BALF0/1

BALF0 HA BALF1 BHRF1

β-actin

Figure 34

 101 2.3 BHRF1, a Bcl-2 viral homolog, disturbs mitochondrial dynamics and stimulates mitophagy to dampen type I IFN induction

BHRF1, one of the well-characterized Bcl-2 homologs of EBV, has been described as

an anti-apoptotic modulator in different experimental cell systems. It has been

previously regarded as an early protein although it also expressed during some latency

programs. The mechanism by which BHRF1 exerts its anti-apoptotic function, as its

mammalian counterparts, is thought to be through the binding and sequestration of a

sub-set of cellular pro-apoptotic Bcl-2 family proteins. This is strongly reminiscent of

what has been described for mammalian pro-survival protein Bcl-xL where interactions involved the BH3 peptide of pro-apoptotic proteins into a surface groove formed by α- helices of Bcl-xL. In addition, BHRF1, like Bcl-2, resides mainly in the mitochondrial membrane.

Mitochondria are involved in many cellular functions such as energy production, maintenance of calcium homeostasis, generation of reactive oxygen species (ROS) and apoptosis initiation. In addition, mitochondria also act as central hubs in innate immunity against viruses. This is notably due to their role in the activation of interferon

(IFN) signaling pathways through the activity of the mitochondrial antiviral signaling

(MAVS) proteins present at the mitochondrial surface. Functions of mitochondria are intertwined with the morphology and the number of mitochondria. Indeed, mitochondria are dynamic and mobile organelles that constantly undergo membrane remodeling through repeated cycles of fusion and fission and their length is determined by the balance between fission/fusion rates. Maintenance of mitochondrial homeostasis also includes the control of the number of mitochondria through stimulation of their biogenesis, and removal of damaged mitochondria by mitophagy.

The ability of BHRF1 to block apoptosis is well documented, whereas no information is available so far regarding its putative role on mitochondrial dynamics and autophagy.

 102 In collaboration with Prof. A. Esclatine (Université Paris-Sud), we demonstrated that

ectopic expression of BHRF1 leads to a -related protein 1 (Drp1) dependent

mitochondrial fission. Subsequently, BHRF1 induces the accumulation of

autophagosomes most likely by interacting with Beclin 1, an autophagic machinery

protein that is known to interact with and to be inhibited by Bcl-2. These cellular

modifications result in the formation of a mito-aggresome, a perinuclear mitochondrial

clustering that precedes mitophagy. Given the central role of mitochondria in innate

immunity, the contribution of BHRF1 in the control of innate immunity was explored,

which led to the demonstration that BHRF1-mediated mitochondrial degradation

resulted in the inhibition of type I IFN induction in response to various stimuli.

My main contribution to this study was to assess the impact of EBV reactivation on mitochondrial morphological phenotype. Mitochondria were homogenously distributed in the cytoplasm of non-reactivated Akata cells. However, once lytic cycle induced, mitochondria formed mito-aggresomes in reactivated cells. Although BHRF1 was not expressed in latent Akata cells, it accumulated in reactivated cells where it colocalized with mito-aggresomes. Drp1 is a GTPase that plays a critical role in mitochondrial fission. During the viral lytic cycle, Drp1 was similarly required for the formation of mito-aggresomes since treatment of Akata cells by Mdivi-1, a chemical inhibitor of Drp1, impeded the formation of mito-aggresomes.

 103 0DQXVFULSWZLWKIXOODXWKRUGHWDLOV

1 BHRF1, a Bcl-2 viral homolog, disturbs mitochondrial dynamics and stimulates

2 mitophagy to dampen type I IFN induction

3

4 Géraldine Vilmen1, 2¶, Damien Glon1¶, Gabriel Siracusano2, Marion Lussignol1, Zhouwulin Shao2, Eva

5 Hernandez1, Daniel Perdiz3, Frédérique Quignon2, Lina Mouna1, Christian Poüs3, 4, Vincent Maréchal2#

6 and Audrey Esclatine1#*

7 1. I2BC, Institute for Integrative Biology of the Cell, UMR9198, Faculté de Pharmacie, Université Paris- 8 Sud, 92296 Châtenay-Malabry, France 9 2. CRSA, Centre de Recherche Saint-Antoine, UMRS 938, INSERM, Sorbonne Université, 75012 Paris, 10 France 11 3. INSERM UMR-S 1193, Université Paris-Sud, Université Paris-Saclay, 92296 Châtenay-Malabry, 12 France 13 4. Biochimie-Hormonologie, APHP, Hôpitaux Universitaires Paris-Sud, Site Antoine Béclère, 92141 14 Clamart, France

15 * Corresponding author 16 Email: [email protected] 17 18 ¶ These authors contributed equally to this work. 19 # These authors share senior authorship. 20

21 Abstract

22 Mitochondria respond to many cellular functions and act as central hubs in innate immunity against 23 viruses. This is notably due to their role in the activation of interferon (IFN) signaling pathways 24 through the activity of the mitochondrial antiviral signaling (MAVS) protein present at the 25 mitochondrial surface. Here we report that the BHRF1 protein, a Bcl-2 homolog encoded by Epstein- 26 Barr virus (EBV), inhibits IFN-ɴ induction by targeting mitochondria. Indeed, we have demonstrated 27 that BHRF1 expression modifies mitochondrial dynamics and stimulates Drp1-mediated 28 mitochondrial fission. Concomitantly, we have showed that BHRF1 is pro-autophagic since it 29 stimulates the autophagic flux and interacts with BECN1/Beclin 1. In response to BHRF1-induced 30 mitochondrial fission and autophagy stimulation, BHRF1 drives mitochondrial network reorganisation 31 to form juxtanuclear mitochondrial aggregates known as mito-aggresomes. Mitophagy is a cellular

1

32 process that can specifically sequester and degrade mitochondria. Our confocal studies uncovered 33 that numerous mitochondria are present in autophagosomes and in acidic compartments in BHRF1- 34 expressing cells. Moreover, mito-aggresome formation allows the induction of mitophagy by 35 recruitment of PINK1/Parkin at the mitochondria. As BHRF1 modulates the mitochondrial fate, we 36 explored the effect of BHRF1 on innate immunity and showed that BHRF1 expression is able to 37 prevent IFN-ɴ induction. Indeed, BHRF1 inhibits IFN-ɴ promoter activation and blocks the nuclear 38 translocation of interferon regulatory factor 3 (IRF3). We concluded that BHRF1 is able to counteract 39 innate immunity activation by inducing fission of mitochondria to facilitate their sequestration in 40 mitophagosomes for degradation.

41 Abbreviations

42 ACTB, actin beta ; ATG, autophagy-related ; BECN1, Beclin 1 ; CARD, caspase recruitment domain ; 43 CCCP, carbonyl cyanide 3-chlorophenylhydrazone ; CQ, chloroquine ; DAPI, 4',6-diamidino-2- 44 phenylindole, dihydrochloride ; Drp1, dynamin-related protein 1 ; EBSS, EĂƌůĞ͛ƐďĂůĂŶĐĞĚƐĂůƚƐŽůƵƚŝŽŶ 45 ; EBV, Epstein-Barr virus ; ER, endoplasmic reticulum ; EV, empty vector ; GFP, green fluorescent 46 protein ; IFN, interferon ; IRF3, interferon regulatory factor 3 ; LC3, microtubule-associated protein 47 light chain 3 ; LDH, lactate dehydrogenase ; MAVS, mitochondrial antiviral-signaling ; MDA5, 48 melanoma differentiation-associated protein 5 ; MOM, mitochondrial outer membrane ; PINK1, 49 PTEN-induced putative kinase 1 ; RIG-I, retinoid acid-inducible gene ; RFP, red fluorescent protein ; 50 ROS, reactive oxygen species ; SQSTM1/p62, sequestosome ; STING, stimulator of interferon genes ; 51 TBK1, TANK-binding kinase 1 ; VDAC, voltage-dependent anion channel ; 3-MA, 3-methyladenine

52 Keywords

53 EBV, BHRF1, autophagy, mitophagy, Bcl-2, BECN1, mitochondrial dynamics, Drp1, IFN, MAVS

2

54 Introduction

55 The Epstein-Barr virus (EBV) is an ubiquitous virus belonging to the Herpesviruses family. Although its 56 prevalence is high in populations worldwide, EBV primary infection is asymptomatic when occurring 57 in children whereas it may be responsible for infectious mononucleosis in young adults. EBV 58 essentially infects epithelial cells and B cells, in which it establishes a latent lifelong persistence. 59 Several latency programs exist, corresponding to the expression of different viral proteins without 60 production of infectious viral particles [1, 2]. Some forms of latency promote cell transformation and 61 EBV infection has therefore been associated with cancers, such as lymphomas and carcinomas [3]. It 62 has been reported that EBV has the ability to block programmed cell death, thus prolonging the 63 lifespan of infected cells, maximizing the production of progeny viruses, and facilitating the 64 establishment of virus persistence [4, 5].

65 EBV has a large 172 kpb DNA genome that can encode for more than 100 different proteins together 66 with noncoding RNAs [4]. Among these proteins, BHRF1 is expressed during the viral production cycle 67 but also during some latency programs, and its only known function is to protect cells against 68 apoptosis [6] notably during primary infection [7]. BHRF1 is a 17kDa putative transmembrane protein 69 which is highly conserved among different EBV isolates and shows strong functional homology with 70 the human Bcl-2 [8]. This 191-amino-acid viral protein owns two motifs referred to as Bcl-2 homology 71 domains 1 and 2 (BH1 and BH2). Cellular Bcl-2 belongs to a family of more than 20 members with 72 anti-apoptotic properties, such as Bcl-2 or Bcl-xL, and pro-apoptotic proteins, such as Bax, Bak, Bad 73 or Bim [9]. BHRF1 anti-apoptotic activity may at least in part rely on its ability to interact with 74 proapoptotic BH3-only proteins (especially Bim) and with the executioner Bak [10, 11]. In addition, 75 BHRF1, like Bcl-2, seems to reside mainly in the mitochondrial membrane [12, 13].

76 Mitochondria fulfill multiple cellular functions, such as energy production, maintenance of calcium 77 homeostasis, generation of reactive oxygen species (ROS) and apoptosis initiation. In addition, 78 mitochondria also actively participate to innate immunity in order to limit viral infections. Indeed, the 79 innate immune signaling adaptor MAVS (mitochondrial antiviral-signaling) is located at the 80 mitochondrial outer membrane (MOM) and plays a pivotal role in signaling cascades that lead to 81 type I interferons (IFNs) and pro-inflammatory cytokine production. MAVS is activated following 82 interaction with RIG-I (retinoid acid-inducible gene) or MDA5 (melanoma differentiation-associated 83 protein 5), two cytoplasmic pattern recognition receptors that detect viral genome during infection. 84 Functions of mitochondria are intertwined with the morphology and the number of mitochondria. 85 Indeed, mitochondria are dynamic and mobile organelles that constantly undergo membrane 86 remodeling through repeated cycles of fusion and fission and their length is determined by the

3

87 balance between fission/fusion rates. Maintenance of mitochondrial homeostasis also includes the 88 control of the number of mitochondria through stimulation of their biogenesis, and removal of 89 damaged mitochondria by selective autophagy, a process called mitophagy. Selective autophagy 90 consists in the recognition of a specific cargo by a molecular adaptor that links the cargo to the 91 autophagosome membrane, leading to its sequestration into this double-membrane vesicle [14]. 92 Autophagy can also be non-selective and randomly sequester bulk cytosol and other cytoplasmic 93 components into autophagosomes [15]. In both cases, maturation of autophagosomes ends with 94 their fusion with lysosomes, creating autolysosomes, and the digestion of the contents by proteases.

95 Whereas BHRF1 ability to block apoptosis is well documented, no information is available so far 96 regarding its putative role on mitochondrial dynamics and autophagy. Interestingly, several studies 97 reported that EBV can subvert autophagy to improve viral replication and final envelopment of viral 98 particles [16-18]. Here, we demonstrate that ectopic expression of BHRF1 leads to a Dynamin-related 99 protein 1 (Drp1) dependent mitochondrial fission. Subsequently, BHRF1 induces the accumulation of 100 autophagosomes by interacting with BECN1/Beclin 1, an autophagy machinery protein, which is 101 known to interact with Bcl-2. These cellular modifications result in the formation of a mito- 102 aggresome, a perinuclear mitochondrial clustering that precedes mitophagy. Given the central role of 103 mitochondria in innate immunity, we explored the contribution of BHRF1 in the control of innate 104 immunity and demonstrated that BHRF1-mediated mitochondrial degradation resulted in the 105 inhibition of type I IFN induction.

106 Materials and methods

107 Antibodies, plasmids and reagents 108 References of materials used in this study are summarized in Table 1.

109 Cell culture and treatments

110 HeLa and HEK293T cells were cultured at 37°C under 5% CO2 in DMEM supplemented with 10% fetal 111 calf serum (FCS). Drp1 knock-down (sh-Drp1) and corresponding control (sh-NT) cell lines were

112 generated from HeLa or HEK293T cells and were cultured at 37°C under 5% CO2 in DMEM 113 supplemented with 10% FCS and Puromycin (2.5µg/mL). GFP-LC3 stably transfected HeLa cells were 114 kindly provided by Aviva Tolkovsky [19] and mRFP-GFP-LC3 stably transfected HeLa cells were kindly 115 provided by David Rubinsztein [20]. They were grown in DMEM supplemented with 10% FCS and 116 G418 (500 µg/mL). The EBV-positive Burkitt lymphoma cell line Akata was purchased from ATCC and

117 cultured at 37°C under 5% CO2 in RPMI-1640 supplemented with 10% FCS.

4

118 For stress induction, culture media was replaced by freshly prepared media containing indicated 119 drugs. Mitochondrial fission was induced by culturing cells in media supplemented with NaCl 120 (125mM) for 30min and mitophagy was promoted by CCCP treatment (10µM) for 4h. Starvation- 121 ŝŶĚƵĐĞĚĂƵƚŽƉŚĂŐLJǁĂƐĐĂƌƌŝĞĚŽƵƚďLJƌĞƉůĂĐŝŶŐƚŚĞĐĞůůƐŝŶĂƌůĞ͛ƐĂůĂŶĐĞĚ^Ăůƚ^ŽůƵƚŝŽŶ;^^ͿĨŽƌ 122 4h before cell lysis. Autophagic flux was monitored by addition of chloroquine (50µM) 4h before cell 123 lysis. Autophagy inhibition was performed by two different ways: addition of spautin-1 (20µM) or 3- 124 methyladenine (5mM) in culture media for 6h.

125 Cell transfection 126 DNA plasmids transfections were performed using Fugene HD transfection reagent (Promega) 127 ĂĐĐŽƌĚŝŶŐ ƚŽ ƚŚĞ ŵĂŶƵĨĂĐƚƵƌĞƌ͛Ɛ ƉƌŽƚŽĐŽů ;https://www.promega.com/techserv/tools/Fugene 128 HdTool/). One-day prior transfection, the cells were seeded in 24-well plates and incubated at 37°C. 129 On the day of transfection, DNA plasmids were diluted in opti-MEM and Fugene HD was added to the 130 mix (ratio DNA/Fugene equals 3:1). After incubation for 10min at room temperature (RT), the 131 transfection mix was added to the cells. The cells were incubated at 37°C and 6h post-transfection 132 DMEM supplemented with 10% FCS was added. Depending on the expression plasmids, cells were 133 fixed 24h or 48h post-transfection. 134 Cells transfection with linear dsDNA was performed using Oligofectamine transfection reagent 135 (Thermo Fisher Scientific) according to ƚŚĞŵĂŶƵĨĂĐƚƵƌĞƌ͛ƐƉƌŽƚŽĐŽů͘KŶĞ-day prior transfection, the 136 cells were seeded in 24-well plates and incubated at 37°C. On the day of transfection, linear dsDNA 137 (final concentration of 5µg/mL) and oligofectamine were diluted in opti-MEM and separately 138 incubated for 5min at RT. The two mixes were then pooled and incubated for 20min at RT. After one 139 PBS wash, the culture medium was changed to opti-MEM and transfection mix was added. The cells 140 were incubated at 37°C and 4h post-transfection, DMEM supplemented with 30% FCS was added to 141 the cells. Finally, cells were fixed 8h post-transfection.

142 Co-immunoprecipitation assays 143 To study the interaction between BHRF1 and BECN1, HeLa cells cultured in 6-well plates were 144 transfected to co-express BECN1, and different molecular partners tested (EV, BHRF1-HA or Flag- 145 ICP34.5). Two day (48h) after transfection, cells were washed with cold, sterile PBS and then lysed at 146 4°C for 2h in lysis buffer (50mM Tris HCl, 50mM NaCl,0.5% Triton X-100, 0.5% deoxycholic acid, 0.2%

147 bovine serum albumin (BSA), 25mM NaPPi, 50mM NAF, 1mM Na3VO4) followed by centrifugation at 148 20,000g at 4°C for 30min to remove cell debris. BECN1 was immunoprecipitated with a goat anti- 149 BECN1 antibody (20µg/mL). The interaction between BHRF1-HA and endogenous BECN1 was also 150 explored by immunoprecipitation with a rabbit anti-HA antibody (20µg/mL). Immunoprecipitations 151 were performed overnight at 4°C. Protein G-Sepharose beads were added for 2h at 4°C and were

5

152 washed 3 times with lysis buffer and twice with wash buffer (20mM Tris HCl, 50mM NaCl, 0.2% BSA). 153 The immune complexes were finally boiled for 5min in loading buffer (62.5mM Tris, pH6.8, 10% 154 ŐůLJĐĞƌŽů͕ ϭ͘ϱй ^^͕ Ϭ͘ϬϮϱй ďƌŽŵŽƉŚĞŶŽů ďůƵĞ͕ ϴй ɴ-mercaptoethanol) before being analyzed by 155 SDS-PAGE.

156 Cytosol extraction and purification of mitochondrial fractions 157 Mitochondrial fractions were prepared using a digitonin-based subcellular fractionation technique, as 158 described previously [21]. Briefly, HeLa cells transfected with an EV or BHRF1-HA plasmids were 159 plated 48h before the experiment in 6-well plates. After being washed twice with PBS, cells were

160 permeabilized with lysis buffer (250mM sucrose, 70mM KCl, 137mM NaCl, 4.3mM Na2HPO4, 1.4mM

161 KH2PO4 pH7.2, supplemented with 200µg/mL digitonin, and a cocktail of protease/phosphatase 162 inhibitors) for 5min on ice and then collected before being centrifugated at 1,000g for 10min. The 163 cytosolic fraction consisted of the supernatant whereas the pellet, containing mitochondria, was 164 resuspended in mitochondrial lysis buffer (50mM Tris-HCl pH7.4, 150mM NaCl, 2mM EDTA, 2mM 165 EGTA, 0,2% TritonX-100, 0.3% NP-40, and a cocktail of protease/phosphatase inhibitors) and 166 incubated for 5min on ice. After centrifugation at 10,000g for 10min, the pellet was discarded and 167 the supernatant corresponded to the mitochondrial fraction. Purity of the mitochondrial fraction was 168 confirmed by presence of the mitochondrial marker voltage-dependent anion channel (VDAC) and 169 quasi-absence of a cytosolic marker, the lactate dehydrogenase (LDH).

170 EBV reactivation and Drp1-inhibition in Akata cells 171 In order to assess the impact of EBV reactivation on mitochondrial phenotype, Akata cells were used. 172 This cell line corresponds to EBV-positive Burkitt lymphoma B cells, where EBV gene expression is 173 limited to type I latency. Akata cells were pretreated with Mdivi-ϭ;ϱϬʅDͿĨŽƌϭŚ͕ĂƐƉĞcific inhibitor 174 of Drp1. To induce the EBV lytic cycle, cells were treated with polyclonal rabbit anti-human IgG at 7.5 175 µg/mL for 8h. Then, cells were washed with PBS and incubated at 37°C with MitoTracker CMX ROS 176 (250nM) for 30min. Following incubation, cells were washed with PBS, fixed with 4% 177 paraformaldehyde in PBS for 10min, and permeabilized in PBS, Triton X-100 (0.2%) for 5min. The cells 178 were pelleted and resuspended with PBS. The cell suspension was then fixed to microscope slides via 179 cytocentrifugation using a Cytospin (A78300003; Thermo Scientific, USA) at 800rpm under medium 180 acceleration mode for 1 min. Cells were blocked and immunostained as described below.

181 Establishment of stable Drp1 knock-down cell lines 182 Stable HeLa and HEK293T cells knockdown for Drp1 expression were established using lentiviral 183 transduction. Drp1 and non-target shRNA (sh-NT) were purchased from Sigma (MISSION lentiviral 184 transduction particles). One-day before transduction, the cells were seeded in 96-well plates. On the

6

185 day of transduction, the cells were placed in transduction media (DMEM supplemented with 10% 186 FCS, Hepes 10mM and polybrene 8µg/mL). Then, the cells were infected with lentiviruses at a 187 multiplicity of infection of 0.5, 1 or 5. One-day post-transduction, the culture medium was replaced 188 by DMEM supplemented with 10% FCS. Two days post-transduction, sh-NT and sh-Drp1-expressing 189 cells were selected by addition of Puromycin (5µg/mL) in culture medium. To assess the knockdown, 190 proteins level in cells stably expressing the sh-Drp1 were compared to cells expressing a sh-NT by 191 immunoblot.

192 Immunoblot analysis 193 ĞůůƐǁĞƌĞůLJƐĞĚŝŶůLJƐŝƐďƵĨĨĞƌ;ϲϱŵDdƌŝƐ͕Ɖ,ϲ͘ϴ͕ϰй^^͕ϭ͘ϱйɴ-mercaptoethanol and a cocktail of 194 protease/phosphatase inhibitors) and held at 100°C for 10min. Protein extracts were resolved on 195 SDS-PAGE (12.5%) and electro-transferred onto a polyvinylidene difluoride membrane (Amersham). 196 After 1h of incubation in blocking buffer (PBS, 0.1% Tween20 and 5% BSA or nonfat dry milk), the 197 membranes were probed overnight at 4°C with primary antibodies. Horseradish peroxidase-labeled 198 antibodies were used as secondary antibody, and revelation was performed using the ECL detection 199 system according to manufĂĐƚƵƌĞƌ͛ƐŝŶƐƚƌƵĐƚŝŽŶƐ;/ŵŵŽďŝůŽŶtĞƐƚĞƌŶ͕DĞƌĐŬDŝůůŝƉŽƌĞͿ͘ Scanning for 200 quantification of protein levels was monitored using ImageJ software. An anti-ACTB antibody was 201 used to ensure equal loadings and normalize quantification.

202 Immunofluorescence microscopy 203 For indirect immunofluorescence, cells were cultured on glass coverslips in 24-well plates. Cell 204 monolayers were washed with PBS and cells were fixed with paraformaldehyde (4%) in PBS or

205 methanol or acetone/water, depending on the antibodies. The cells were treated with PBS, NH4Cl 206 (50mM) for 10min, permeabilized using PBS, Triton X-100 (0.2%) for 4min, washed twice with PBS 207 and then incubated for 1h in PBS, gelatin (0.2%) supplemented with FCS (5%) for blocking. Then the 208 cells were incubated for 1h with appropriate primary antibody diluted in PBS, gelatin (0.2%) at RT. 209 Then cells were washed 3 times with PBS and then incubated with appropriate secondary antibody 210 diluted in PBS, gelatin (0.2%) for 1h at RT. After washing, the nuclei were counterstained with DAPI or 211 Hoechst 33342. Coverslips were mounted in Glycergel and observed by microscopy as described 212 below. 213 For labelling of autophagic vesicles, cells were fixed with paraformaldehyde (4%) in PBS.

214 Permeabilisation and blocking were performed by treating the cells with PBS, NH4Cl (50mM) for 215 10min followed by an incubation of 30min with PBS, saponin (0.075%), BSA (1%) and FCS (5%). Then 216 the appropriate primary antibody diluted in PBS, saponin (0.075%) and BSA (1%) was incubated for 217 1h at RT. The cells were then washed twice in PBS, saponin (0.075%), BSA (1%), one time with PBS,

7

218 saponin (0.037%), BSA (0.5%) and twice in PBS. Then, cells were incubated with appropriate 219 secondary antibody diluted in PBS, saponin (0.075%), BSA (1%). After washing, the nuclei were 220 counterstained with DAPI. Coverslips were mounted in Glycergel and observed using a Nikon Eclipse 221 80i epifluorescence microscope (Nikon Instruments) or a Leica TCS SP8 X inverted confocal 222 microscope (Leica). Photographic images were resized, organized and labeled using ImageJ software 223 or LAS AF Lite. Photographic images were resized, organized and labeled using ImageJ software or 224 LAS AF Lite. For colocalization analyzes, line profiles were performed using LAS AF Lite software and 225 Manders split coefficients were determined using ImageJ software.

226 Luciferase reporter assays 227 HEK293T cells were cultured in 24-well plates and transfected for 24h using Fugene HD, with 228 plasmids encoding IFN-ɴ ůƵĐŝĨĞƌĂƐĞ ƌĞƉŽƌƚĞƌ ;ĨŝƌĞĨůLJ ůƵĐŝĨĞƌĂƐĞ͖ ϭϬϬŶŐͿ͕ ƉZ>-TK (renilla luciferase 229 plasmid; 25ng) and increasing concentrations (0, 250, 500 or 750ng) of plasmids expressing BHRF1- 230 HA. EV was used to maintain equal amounts of DNA among wells. To study the IFN-ɴ ƉƌŽŵŽƚĞƌ 231 activation dependent on MAVS pathway, a transfection with 150ng of a plasmid encoding a positive 232 dominant form of RIG-I (Flag-ȴZ/'-I (2xCARD)) was performed. In order to study the IFN promoter 233 activation dependent on STING pathway, a stimulation by salmon sperm dsDNA (5µg/mL) was 234 performed for 8h using Oligofectamine as previously described [22]. 24h post-transfection, cells were 235 lysed and measurement of firefly and renilla luciferase activities was performed using the Dual- 236 luciferase reporter assay system (Promega) according to the manufacturer's protocol. Relative 237 expression levels were calculated by dividing the firefly luciferase values by those of renilla luciferase 238 and normalized to the control condition (without BHRF1-HA expression).

239 Statistical analysis 240 Data are expressed as mean ± standard error of the mean (SEM) and were analyzed with Prism 241 ƐŽĨƚǁĂƌĞ ;'ƌĂƉŚWĂĚͿ ďLJ ƵƐŝŶŐ ^ƚƵĚĞŶƚ͛Ɛ ƚ ƚĞƐƚ Žƌ ŽŶĞ-way analysis of variance (ANOVA) test 242 comparisons. P values less than 0.05 were considered statistically significant. Experiments were 243 performed three times.

244 Results

245 BHRF1 expression induces mitochondrial fission and the formation of mito-aggresomes 246 BHRF1 subcellular localization was investigated by confocal microscopy in HeLa cells following 247 transfection with a BHRF1-HA expression vector for 24 hours. Mitochondria were counterstained by 248 MitoTracker red CMX Ros dye. In accordance with previous reports, we observed an important 249 colocalization of BHRF1 with mitochondria (Figure 1A), corresponding to a perinuclear staining of

8

250 BHRF1 and a mitochondrial distribution pattern similar to Bcl-2 [12, 23]. Moreover, BHRF1 seemed to 251 induce a modification of the shape of the nucleus. More interestingly, mitochondrial labeling with an 252 antibody directed against the mitochondrial import receptor subunit TOM20 confirmed that the 253 mitochondrial network morphology was dramatically altered in BHRF1 expressing cells (Figure 1B). 254 The mitochondrial average length was measured (Figure 1C) and confirmed that BHRF1 expression 255 induced a marked reduction in mitochondrial length which was indicative of a mitochondrial fission. 256 While the mitochondrial population exhibited a tubular network or an intermediate phenotype in the 257 majority of control cells, BHRF1 caused fragmentation of mitochondria, with almost 75% of them 258 displaying a size under 1µm (Figure 1C). BHRF1-expressing cells also showed abnormal and 259 juxtanuclear mitochondrial aggregates, while these organelles were homogenously distributed as a 260 network in the cytoplasm of control cells (Figure 1B). These rough aggregates were strongly 261 reminiscent of structures previously described and classically called mito-aggresomes [24]. Based on 262 the crescent pattern of mitochondrion distribution, used herein as the criterion for judging 263 mitochondrion clustering, virtually 80% of BHRF1-expressing cells showed a mito-aggresome (Figure 264 1D). To confirm this observation in the context of native EBV-infection, we analyzed mitochondrial 265 morphology in EBV-positive Akata B cells during latency or following viral reactivation. Mitochondria 266 were homogenously distributed in the cytoplasm of latent (non-reactivated) Akata cells (Figures 267 1E,F). However, after reactivation of the viral lytic cycle by IgG treatment, they formed mito- 268 aggresomes in reactivated cells that were characterized by the expression of immediate early protein 269 ZEBRA (Figure 1E). Although BHRF1 was not expressed in latent Akata cells, it accumulated in 270 reactivated cells where it colocalized with mito-aggresomes (Figure 1F). More than 90 % of BHRF1- 271 positive cells displayed mito-aggresomes (Figure 1F).

272 Drp1 is required for BHRF1-induced mitochondrial fission and mito-aggresome formation 273 Mitochondria change their overall morphology by fusion and fission in response to cellular stress. 274 Damaged mitochondria can trigger their fission to get rid of the unhealthy parts. Drp1 is a GTPase 275 that plays a critical role in mitochondrial fission [25]. Drp1 mostly localizes in the cytoplasm and is 276 translocated to the MOM to regulate mitochondrial fission [26]. Since BHRF1 expression was 277 associated with an increased mitochondrial fission, we monitored Drp1 localization in BHRF1- 278 expressing cells by confocal microscopy (Figure 2A). Whereas Drp1 was diffusely distributed in the 279 cytoplasm in control cells, it relocated upon BHRF1 expression into BHRF1-positive mito-aggresomes. 280 Cell fractionation experiments were performed to separate mitochondria associated proteins [21]. 281 This experiment confirmed that BHRF1 is specifically present at the mitochondria and demonstrated 282 that Drp1 was slightly more abundant in the mitochondrial fraction in BHRF1-expressing cells than in 283 control cells (Figure 2B). Overall Drp1 accumulation increased by 40% in the mitochondrial fraction in

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284 BHRF1-expressing cells (Figure 2B). Post-translational modifications of Drp1, such as 285 phosphorylation, sumoylation, ubiquitination, and S-nitrosylation regulate mitochondrial fission in 286 response to diverse cellular stimuli [26]. We analyzed the level of phosphorylation of Drp1 in position 287 Ser-637 in BHRF1 expressing cells (Figure 2C). Indeed, this phosphorylation site depends on cAMP- 288 dependent protein kinase A (PKA) activation and impedes mitochondrial fission. Conversely, 289 dephosphorylation of Drp1, by the phosphatase calcineurin for example, induces translocation of 290 Drp1 and fragmentation of the organelle [27]. Whereas no modification of the total level of Drp1 291 occurred in BHRF1-expressing cells compared to control cells, a strong downregulation of Drp1 Ser- 292 637 phosphorylation was observed following BHRF1 expression (Figure 2C), which corroborates the 293 translocation of Drp1 to mitochondria and the mitochondrial fragmentation that was previously 294 observed (Figures 2B,1C). As to confirm that Drp1 was indeed required for BHRF1-mediated 295 mitochondrial fission, we generated knockdown HeLa cells for Drp1 expression, using lentiviruses 296 which delivered sh-RNA specifically targeting human DRP1 mRNA, and characterized them (Figure 297 S1). In HeLa cells expressing sh-Drp1, mitochondria formed a highly connected network (Figure S1A) 298 along with an 83% decrease in Drp1 protein expression (Figure 2D). Moreover, BHRF1 did not induce 299 mitochondrial reorganization in sh-Drp1 expression cells. Accordingly, mitochondrial length was 300 unaffected by BHRF1 expression and Drp1 deficiency mostly abrogated BHRF1 ability to induce the 301 formation of mito-aggresomes (Figures 2E,G). During the viral lytic cycle, Drp1 was similarly required 302 for the formation of mito-aggresomes since treatment of Akata cells by Mdivi-1, a chemical inhibitor 303 of Drp1, impeded the formation of mito-aggresomes (Figure 2H). Altogether, our results 304 demonstrated that BHRF1 expression induces Drp1-dependent mitochondrial fission, which is 305 required for mitochondrial clustering.

306 BHRF1 triggers autophagy 307 Cellular Bcl-2 is known to inhibit autophagy. Since BHRF1 shares notable homology with cellular Bcl- 308 2, we wondered whether it might modulate autophagy as well [13]. To study the impact of BHRF1 on 309 autophagy, we first used HeLa cells that stably express GFP-LC3, a classical marker of 310 autophagosomes, and added chloroquine (CQ) four hours before harvesting the cells [19]. CQ 311 neutralizes the lysosomal pH and causes the accumulation of GFP-LC3 positive vesicles by inhibiting 312 endogenous protein degradation [28]. As shown in Figures 3A,B BHRF1-expressing cells displayed 313 increased numbers of GFP-LC3 dots compared to control cells, with and without CQ. Because an 314 accumulation of autophagosomes in cells can result either from an increase in the rate of their 315 formation or a decrease in their fusion with lysosomes, we next explored the autophagic flux, which 316 reflects the autophagic degradation activity. We therefore analyzed the accumulation of the LC3 317 lipidated form (LC3 form II) by immunoblotting in cells that were treated or not with CQ. An

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318 increased level of LC3-II was observed in BHRF1-expressing cells (Figure 3C) which was even higher in 319 presence of CQ. This led us to conclude that BHRF1 stimulates the autophagic flux. We also 320 monitored the abundance of the autophagy substrate SQSTM1 by immunoblot and concordantly 321 observed a decrease in SQSTM1 when BHRF1 was expressed (Figure 3C). To confirm our data, the 322 autophagic flux was investigated using the mRFP-GFP-LC3 HeLa cells (Figures 3D,S2) [20]. Based on 323 the fact that the GFP fluorescence is quenched in acidic compartments, such as autolysosomes, this 324 probe makes it possible to differentiate between autophagosomes (GFP+ RFP+ or yellow dots) and 325 autolysosomes (GFP- RFP+ or red dots) [29]. Cells were treated with CQ to block the autophagic flux, 326 or maintained in EBSS (starvation) to induce autophagy. We observed an increase in the total amount 327 of autophagic vacuoles (autophagosomes and autolysosomes) in BHRF1-expressing cells (Figure S2B). 328 Furthermore, BHRF1, as well as starvation, led to a significant percentage of autolysosomes over 329 autophagosomes, whereas CQ-induced vacuoles were mainly autophagosomes (>95%) (Figure 330 3D,S2B). Altogether, these findings clearly indicated that BHRF1 stimulates the biosynthesis of 331 autophagosomes and the autophagic flux in HeLa cells. BECN1 is a critical component of highly- 332 regulated complexes that control the formation and maturation of autophagosomes [30, 31]. Since 333 Bcl-2 and several viral proteins have been described to interact with BECN1, we investigated the 334 putative interaction between BECN1 and BHRF1 [32-34]. First, we analyzed, by confocal microscopy, 335 the distribution of BHRF1 and BECN1 in HeLa cells and observed a partial colocalization of the two 336 proteins in the cytoplasm (Figure 3E), which suggests an interaction. To confirm that co- 337 immunoprecipitation experiments were performed using HeLa cells transiently transfected by 338 plasmids encoding for HA-tagged BHRF1 and BECN1 (Figure 3F). Since Herpes simplex virus type 1 339 (HSV-1) ICP34.5 has been previously reported to interact with BECN1, we used a Flag-tagged ICP34.5 340 plasmid as a positive control [34, 35]. Immunoprecipitation of BECN1 pulled down both BHRF1 and 341 ICP34.5. Reversely, endogenous BECN1 was co-immunoprecipitated with HA-tagged BHRF1 using an 342 anti-HA antibody (Figure 3G). Therefore, we can conclude that BHRF1 bound to BECN1. Altogether, 343 these findings suggested that BHRF1 stimulated the autophagy machinery through its interaction 344 with BECN1.

345 Interplay between BHRF1-induced autophagy and mitochondrial morphology alterations 346 Since BHRF1 induced both mitochondrial fission and autophagy in an apparently concomitant 347 manner, we wondered whether fission was required for the induction of autophagy. To answer this 348 question, we monitored the level of BHRF1-induced autophagy in Drp1-deficient cells by staining 349 endogenous LC3 (Figure 4A). We first demonstrated that Drp1 deficiency did not impair starvation- 350 induced autophagy, since EBSS treatment of sh-Drp1 cells induced LC3 dot accumulation as well as 351 SQSTM1 degradation (Figure S3). Then we confirmed that BHRF1 induced the accumulation of

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352 endogenous LC3 dots in sh-NT control cells, and even more after CQ treatment (Figures 4A,B). 353 However, BHRF1 did not promote accumulation of LC3 dots in sh-Drp1 deficient cells, even in the 354 presence of CQ (Figure 4B). These results demonstrated that Drp1, and by extension Drp1-mediated 355 mitochondrial fission, is required for BHRF1-induced autophagy. In order to seek whether autophagy 356 is required to form mito-aggresomes, HeLa cells expressing BHRF1 were treated with two different 357 autophagy inhibitors i.e. Spautin-1 and 3-methyladenine (3-MA) and immunostained mitochondrial 358 network and BHRF1 (Figure 4C). When autophagy was blocked, mitochondria were homogenously 359 distributed in the cytoplasm and were not organized into mito-aggresomes even in the presence of 360 BHRF1 (Figure 4D). However, autophagy was not required for BHRF1 to induce mitochondrial fission 361 since a significant reduction in the length of the mitochondria was observed in BHRF1 expressing 362 cells both in the presence and in the absence of autophagy inhibitors. (Figure 4E). Altogether, these 363 findings demonstrated that mitochondrial fission induced by BHRF1 leads to autophagy activation, 364 which in turn promotes mito-aggresomes formation.

365 BHRF1 expression induces mitophagy 366 Considering that BHRF1 induces mitochondrial fission and stimulates autophagy, we wondered 367 whether BHRF1 was able to induce mitophagy. Indeed, the fragmentation of mitochondria facilitates 368 their envelopment into autophagosomes, which eventually leads to their degradation into lysosomes 369 via mitophagy [36]. Moreover, mito-aggresomes were previously described following depolarization 370 of mitochondria and their appearance preceded mitophagy in a Parkinson͛Ɛ disease model [24]. 371 Finally, during mitophagy it has been observed that dysfunctional mitochondria undergo retrograde 372 transport to the perinuclear region where they cluster to be degraded [37]. A sensitive dual 373 fluorescence reporter expressing mito-mRFP-EGFP fused in-frame with a mitochondrial targeting 374 sequence was employed to monitor the completion of the mitophagic process [38]. In the presence 375 of BHRF1, numerous mitochondria were detected in acidic compartments (mitochondria labeled in 376 red) suggesting that BHRF1 triggered mitophagy (Figure 5A). Confocal microscopy analysis confirmed 377 that mitochondria (TOM20 labelling) and autophagosomes (GFP-LC3 labelling) significantly 378 colocalized in the presence of BHRF1 (Figure 5B). CQ treatment increased the colocalization intensity, 379 measured by DĂŶĚĞƌƐ͛ ĐŽĞĨĨŝĐŝĞŶƚ͕ possibly because it inhibited autophagic degradation of 380 sequestered mitochondria (Figure 5C). Since autophagosomes are expected to fuse with lysosomes, 381 we also monitored the impact of BHRF1 on the colocalization between mitochondria and lysosomes, 382 using LAMP1 labelling as a lysosomal marker (Figure S4). In BHRF1-expressing cells, the presence of 383 LAMP1-positive vesicles containing mitochondria could be detected both in presence and in absence 384 of CQ (Figures S4A,B), together with an increased LAMP1 expression (Figure S4C). Mitochondria need 385 to recruit autophagy receptor proteins for mitophagy to occur. One of the best-studied mitophagy

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386 mechanisms in mammalian cells is the PINK1-Parkin-mediated mitophagy pathway [39]. PTEN- 387 induced putative kinase 1 (PINK1) is constitutively imported into healthy mitochondria through the 388 TOM and TIM ( of the Inner Membrane) membrane translocation complexes, where it is 389 degraded by the mitochondrial inner membrane protease PARL. When mitochondria are 390 dysfunctional, PINK1 is not imported anymore and starts to accumulate on the MOM, triggering the 391 recruitment of the cytosolic E3 ubiquitin ligase Parkin [39]. Once activated, Parkin ubiquitinates 392 proteins on the MOM surface, this initiates the engulfment of mitochondria into autophagosomes 393 and finally complete mitophagy. We wondered whether BHRF1 induced mitophagy through a PINK1- 394 Parkin pathway. First, we monitored the expression of PINK1 protein in the cytosolic and 395 mitochondrial fractions of BHRF1-expressing cells. As shown on Figure 5D, PINK1 was significantly 396 translocated to the mitochondrial fraction following BHRF1 expression. Then, HeLa cells, which lack 397 detectable endogenous Parkin, were co-transfected with CFP-Parkin and BHRF1 plasmids for 48 398 hours. Since a treatment with the protonophore CCCP (carbonyl cyanide 3-chlorophenylhydrazone) 399 for 24 hours leads to mitochondrial depolarization, it was herein used as a positive control for 400 mitophagy induction. Parkin distribution was diffuse in the cytoplasm of control cells, whereas Parkin 401 was clearly recruited to mitochondrial clusters in cells expressing CFP-Parkin and treated with CCCP 402 (Figure 5F). Similarly, in cells expressing both CFP-Parkin and BHRF1, CFP-Parkin was recruited to 403 BHRF1-positive structures and mito-aggresomes (Figure 5F). Moreover, in cells expressing CFP-Parkin 404 and BHRF1, mitochondrial population seemed decreased compared to the population present in 405 absence of CFP-Parkin (Figure 5F). These results demonstrated that BHRF1 expression induced 406 mitochondrial translocation of CFP-Parkin in HeLa cells. Finally, we observed a clear decrease in 407 TOM20 accumulation in the mitochondrial fraction upon BHRF1 expression, which confirmed a 408 partial degradation of mitochondria (Figure 5E). Altogether, these results confirmed that BHRF1 409 induces mitophagy.

410 BHRF1 expression blocks type I IFN induction via MAVS/STING signaling pathway 411 BHRF1-induced mitochondrial alterations and mitophagy suggested that BHRF1 might impact 412 mitochondria-dependent signaling pathways, such as antiviral signaling and IFN production. Indeed, 413 MAVS, an innate immunity adaptor, is localized at the surface of mitochondria and its interaction 414 with STING (stimulator of interferon genes), an endoplasmic reticulum (ER)-resident adaptor, plays a 415 crucial role in the induction of type I IFN production [40]. MAVS and STING recruit and activate TANK- 416 binding kinase 1 (TBK1), which phosphorylates the transcription factor interferon regulatory factor 3 417 (IRF3), leading to its nuclear translocation and subsequent activation of type I IFN promoter [41]. It 418 has been also previously reported that fragmentation of mitochondrial network disrupted interaction 419 between MAVS and STING, thus leading to a reduced signaling by IRF3 [40]. Moreover, Ding et al

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420 demonstrated that sequestration of mitochondria in autophagosomes during infection with human 421 parainfluenza virus type 3 (HPIV3) blocked IFN response [42].

422 To investigate the potential significance of BHRF1-mediated mitochondrial network alterations on 423 innate immunity, we performed an IFN-ɴ promoter-driven luciferase reporter assay (Figure 6) [43]. 424 We explored the effect of BHRF1 expression on IFN-ɴ promoter activation in HEK293T cells. To 425 activate IFN-signaling pathway in a MAVS-dependent manner we used a dominant-positive mutant 426 form of RIG-I ('RIG-I) (Figure 6A) that contains two caspase recruitment domains (CARD), which 427 mimic its oligomerization, but lacks the helicase domain [44]. As MAVS contains one CARD domain 428 [45], it becomes constitutively activated by CARD-CARD interaction in the presence of ȴZ/'-I. 429 Transfection of double stranded DNA (dsDNA) was used to activate cGAS, a DNA sensor, to stimulate 430 the STING signaling cascade [22] (Figure 6B). BHRF1 expression dramatically blocked IFN-ɴ promoter 431 activation by either pathway in a dose-dependent manner (Figures 6A,B). We next examined 432 whether BHRF1 modulated the activation of transcription factor IRF3. IRF3 staining was diffuse in the 433 cytoplasm of non-stimulated cells, but it became mainly nuclear in response to 'RIG-I or to dsDNA in 434 cells expressing an empty control vector (Figure 6D). Conversely IRF3 nuclear translocation was 435 clearly abrogated in BHRF1-expressing cells following MAVS or STING activation. We measured the 436 percentage of cells that exhibited nuclear IRF3 in EV or BHRF1-expressing cells and observed a 437 tenfold decrease when BHRF1 was expressed (Figure 6E). Taken together, these observations 438 showed that BHRF1 prevented MAVS and STING-mediated IFN-ɴ activation pathway.

439 Mitochondrial fission and autophagy induction are both necessary to block IFN-ɴ induction 440 We speculated that mitochondrial fission might be required for BHRF1 to downregulate IFN-ɴ 441 response. To test this hypothesis, we generated sh-Drp1 HEK293T cells and measured IFN-ɴ 442 promoter activity in this cell line, as before (Figures 6A,B). We observed that Drp1 knockdown 443 abolished the inhibitory effect of BHRF1 on IFN-ɴ promoter activation triggered by 'RIG-I or dsDNA 444 (Figure 7A). Similarly, nuclear translocation of IRF3 was abrogated by BHRF1 in sh-NT cells, whereas 445 BHRF1 lost this ability in sh-Drp1 cells (Figures 7B,C). Altogether these results showed that BHRF1 446 required Drp1-dependent mitochondrial fission to block IFN-ɴ induction. Since we previously 447 demonstrated that BHRF1 activated the autophagic flux, we speculated that autophagy might be 448 required for BHRF1 to block IFN-ɴ induction as well. To test this hypothesis, cells were treated with 449 two different autophagy inhibitors, Spautin-1 and 3-MA. As shown in Figure 7D both treatments 450 totally abrogated the ability of BHRF1 to inhibit IFN-ɴ induction, suggesting that autophagy induction 451 is necessary for BHRF1 to inhibit the IFN-ɴ system. This was further confirmed by the inability of

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452 BHRF1 to block nuclear IRF3 translocation in response to 'RIG-I or dsDNA activation when autophagy 453 was inhibited by Spautin-1 or 3-MA (Figures 7E,F).

454 Discussion 455 Whereas it has been reported that BHRF1, an EBV-encoded protein, acts on mitochondria to control 456 apoptosis, only one study previously noticed that BHRF1 might modify the distribution of 457 mitochondria [23]. However, the mechanisms leading to BHRF1-induced mitochondrial 458 reorganization as well as their functional consequences have not been explored so far. In the present 459 study, we shed a new light on the role of BHRF1 and provide evidences that contribute to innate 460 immunity escape. It is shown here that BHRF1 dampens type I IFN activation pathway by promoting 461 the fragmentation and subsequent sequestration of mitochondria in autophagosomes, which 462 eventually leads to their degradation by mitophagy. This could be explained by the fact that MAVS, 463 which is localized on the mitochondrial surface, might become unavailable for a functional 464 interaction with STING.

465 Throughout evolution, Herpesviruses such as EBV, HSV-1 or Human cytomegalovirus (HCMV) have 466 developed numerous strategies to escape innate immune response [46] and several EBV-encoded 467 proteins have been described to block type I IFN production by different mechanisms. Hahn et al. 468 initially reported that the trans-activator protein BZLF1 (or ZEBRA, Zta) interfered with type I IFN 469 production. During EBV reactivation, ZEBRA counteracts IFN-Į4 and IFN-ɴ production by interacting 470 with IRF7, although ZEBRA had no effect on the nuclear localization of IRF7 [47]. The other 471 immediate early activator BRLF1 (or Rta) is also able to suppress IFN-ɴ production by decreasing both 472 IRF3 and IRF7 expression in reactivated Akata cells [48]. EBV-encoded BGLF4 kinase can interact with 473 IRF3 and inhibits its phosphorylation [49]. The tegument protein LF2 blocks IRF7 dimerization directly 474 inside the nucleus [50]. Finally, it has been recently demonstrated that EBV reactivation is associated 475 with the induction of cellular TRIM29, which leads to the proteosomal degradation of STING and 476 therefore inhibits the downstream signaling cascade [51]. In this report, we discovered that BHRF1 477 inhibits type I IFN activation pathway through an original mechanism, which involves mitochondrial 478 dynamics and autophagy.

479 Besides its role in the maintenance of cellular homeostasis, autophagy is a process with multiple 480 effects on immunity. Indeed, during viral infections autophagy plays a crucial role by promoting the 481 clearance of viral components but also by activating innate immunity in order to produce antiviral 482 cytokines. However, some viruses have developed different strategies to impair immune response by 483 subverting autophagy [52]. One mechanism relies on the suppression of signal transduction related 484 to the autophagic degradation of signaling proteins such as MAVS or RIG-I. Following viral infection,

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485 tetherin recruits MARCH8, an E3 ubiquitin ligase, in order to mediate K27-ubiquitination of MAVS for 486 NDP52-dependent degradation [53]. LRRC25, another key negative regulator of type I IFN, stimulates 487 the interaction between RIG-I and the autophagic cargo receptor SQSTM1 therefore promoting RIG-I 488 degradation by selective autophagy [54]. Autophagic proteins can also interfere with innate 489 immunity by directly inhibiting signaling molecules. To that respect ATG5-ATG12 conjugate 490 downregulates RIG-I signaling by direct binding to RIG-I and MAVS CARD domains [55]. J. Cui and 491 collaborators have demonstrated that BECN1 is also able to block the interaction between RIG-I and 492 MAVS [56]. They have shown that the ubiquitin-specific protease 19 (USP19), which stabilizes BECN1 493 by deubiquitination, allows its interaction with the MAVS CARD domain and therefore inhibits RIG-I- 494 mediated type I IFN signaling. Members of the IRF family are also targeted by autophagic proteins to 495 downregulate type I IFN production. Kim et al. have reported that Rubicon, an autophagic inhibitor, 496 can suppress IRF3 dimerization by interacting with the IRF association domain [57]. Mitochondrial 497 sequestration into autophagosomes may be an additional mechanism to downregulate type I IFN 498 signaling [42]. Indeed, it has been recently reported that the HPIV3-encoded matrix protein M 499 triggers mitochondrial sequestration and inhibits the type I IFN response. However, the final 500 degradation of mitochondria does not occur in infected cells, thanks to the expression of the 501 phosphoprotein P that inhibits the maturation of autophagosomes [42]. Here we show that BHRF1 502 protein induces the sequestration of fragmented mitochondria in autophagosomes which leads to 503 their degradation by mitophagy, thereby suppressing type I IFN production. Considering that both 504 EBV-encoded Rta and ZEBRA have been described to induce autophagy when ectopically expressed. 505 However, their involvement regarding the IFN signaling pathway was not investigated [17, 58].

506 Modifications of mitochondrial distribution, mitochondrial fission and/or mitophagy have been 507 reported to occur during several viral infections. Indeed other Herpesviruses than EBV, such as HSV- 508 1, Pseudo rabies virus (PRV) and HCMV have been reported to disrupt the mitochondrial network and 509 promote aberrant mitochondrial dynamics [59, 60]. Interestingly, Hepatitis (HBV) and 510 Hepatitis C virus (HCV) also induce formation of mito-aggresomes in a juxtanuclear region, 511 mitochondrial fission and subsequent mitophagy [38, 61]. The activation of mitophagy during HBV or 512 HCV infection seems essential for the clearance of damaged mitochondria, contributes to cell survival 513 and attenuates the virus-induced mitochondrial apoptotic cell death. Expression of proteins encoded 514 by different viral families is also able to reproduce the effects on mitochondria observed during viral 515 infection. For example, expression of HBx encoded by HBV promotes mitochondrial fragmentation 516 and Parkin-dependent mitophagy [38]. The anti-apoptotic protein vMIA (viral mitochondrion- 517 localized inhibitor of apoptosis), encoded by HCMV, induces mitochondrial fragmentation either 518 alone or in the context of viral infection [59]. Nevertheless, vMIA induces only minor modifications of

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519 the membrane potential and seems unable to initiate mitophagy [59, 62]. Until now, the latent 520 membrane protein 2A (LMP2A), is the only EBV protein that has been reported to modify the 521 mitochondrial network and to increase Drp1-dependent mitochondrial fission [63]. BHRF1 is 522 therefore the first EBV protein described to trigger mitophagy. It is tempting to speculate that BHRF1 523 participates to cell survival in EBV-infected cells by attenuation of mitochondrial apoptosis, similarly 524 to HBV and HCV, by inducing mitophagy and autophagy. Moreover, due to its expression during 525 some latency programs and by inhibiting type I IFN activation pathways, BHRF1 may favor the 526 establishment of latent EBV infection. Indeed, BHRF1, currently described as the tenth latent protein, 527 is constitutively expressed during Wp-restricted latency and from Wp-initiated transcripts in Latency 528 III [1]. Since type I and II IFN were described for their antiproliferative properties, BHRF1 could also 529 participate to EBV-related cancer development by impairing type I IFN production [64]. However, 530 further studies are required to confirm the significance of our findings in the context of latency.

531 Acknowledgments 532 We would like to thank D. Rubinsztein for providing us the mRFP-GFP-LC3 HeLa cells. We are also 533 very grateful to Andreas Till for providing us the p-mito-mRFP-EGFP plasmid. We wish to thank 534 Valérie Nicolas for her technical assistance from the cellular imaging MIPSIT facility. Finally, special 535 thanks to Barbara Trimbach for her critical reading of the manuscript.

536 Disclosure statement

537 No potential conflict of interest was reported by the authors.

538 Funding

539 This work was supported by institutional funding from CNRS, from Univ. Paris-Sud, DIM MALINF 540 Région Ile de France to GV and GS and grants from the Agence Nationale de la Recherche (ANR-14- 541 CE14-0022) to AE.

542 References

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704 Supplementary material

705 - Table 1. Materials used in this study 706 - Figure S1. Mitochondrial fission is reduced in Drp1-deficient cell line 707 - Figure S2. BHRF1 stimulates the autophagic flux 708 - Figure S3. Knockdown of Drp1 does not impact autophagy 709 - Figure S4. Localization of mitochondria in LAMP1-positive vesicles under BHRF1 expression

710 Figure Legends

711 Figure 1. BHRF1 induces the formation of mito-aggresomes in HeLa cells and in EBV-reactivated B 712 cells. (A) BHRF1 localizes to mitochondria. Confocal images of BHRF1-HA transfected HeLa cells 713 immunostained for BHRF1 and mitochondria (MitoTracker). Scale bars = 10µm and 4µm for inset. (B- 714 D) BHRF1 induces mitochondrial fission in HeLa cells. Cells were transfected with an empty vector 715 (EV) or a plasmid encoding BHRF1-HA for 24h. Cells were fixed and immunostained for TOM20, to 716 visualize mitochondrial network, and HA to visualize BHRF1-expressing cells. Nuclei were 717 subsequently stained with DAPI. (B) Representative images. Scale bars = 20µm and 10µm for insets. 718 (C) Left: mitochondrial average length. Right: mitochondrial phenotype. (D) Percentage of cells 719 presenting a mito-aggresome. (E and F) EBV reactivation induces mito-aggresome formation. EBV- 720 reactivated Akata cells were labeled with MitoTracker to visualize mitochondrial phenotype and 721 reactivated cells were detected by labeling EBV antigens. Nuclei were stained with Hoechst. Scale 722 bar = 10µm. (E) Confocal images of MitoTracker and ZEBRA co-staining. (F) Left: confocal images of 723 MitoTracker and BHRF1 co-staining. Right: percentage of EBV-reactivated Akata cells presenting a 724 mito-aggresome. Data represent the mean ± SEM of three independent experiments. *** P < 0.001 725 ;^ƚƵĚĞŶƚ͛ƐƚƚĞƐƚͿ͘ Mitochondria average length was determined using ImageJ software. Images were 726 converted into 8-bit, and brightness/contrast was adjusted in order to better visualize isolated 727 mitochondria. For each condition the length of 20 isolated mitochondria per cell was measured on 10 728 random cells from three independent experiments. The proportion of cells presenting a mito- 729 aggresome was determined by counting at least 50 random cells in each condition from three 730 independent experiments.

731 Figure 2. BHRF1 induces Drp1-dependent mitochondrial fission. (A) Confocal images of HeLa cells 732 transfected with EV or BHRF1-HA plasmids for 24h and immunostained for Drp1 and HA. Nuclei were 733 stained with DAPI. Cells outline was drawn to better visualize the recruitment of Drp1 into mito- 734 aggresomes. Scale bar = 10µm. (B) Subcellular fractionation of HeLa cells transfected for 48h with EV 735 or BHRF1-HA plasmids. VDAC and LDH were used as markers for mitochondrial and cytosolic fractions

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736 respectively. Left: immunoblot analysis of Drp1, LDH and VDAC. Right: quantification of Drp1 737 expression in the mitochondrial fraction. (C) Immunoblot analysis of phosphorylated (Serine 637) and 738 total Drp1 upon BHRF1-HA expression. ACTB was used as a loading control. (D) Silencing level of Drp1 739 in HeLa knockdown cell line (sh-Drp1), compared to control cell line (sh-NT). ACTB was used as a 740 loading control. See Figure S1 for deficient cell line characterization. (E-G) sh-NT and sh-Drp1 HeLa 741 cells were transfected with EV or BHRF1-HA plasmids for 24h and immunostained for TOM20 and HA. 742 Nuclei were stained with DAPI. (E) Representative images of BHRF1-HA expressing cells. Scale 743 bar = 10µm. (F) Mitochondrial average length. (G) Percentage of cells presenting a mito-aggresome. 744 (H) Confocal images of EBV-reactivated Akata cells treated or not with Drp1-inhibitor Mdivi-1 and 745 labeled with MitoTracker and an antibody against EBV (Ea-D). Nuclei were stained with Hoechst. 746 Scale bar = 5µm. Data represent the mean ± SEM of three independent experiments. 747 ns = non-significant; * P < 0.05; *** P < 0.001 (Student͛ƐƚƚĞƐƚͿ͘

748 Figure 3. BHRF1 stimulates autophagy and interacts with BECN1. (A and B) GFP-LC3 HeLa cells were 749 transfected with EV or BHRF1-HA plasmids for 48h and fixed after chloroquine (CQ) treatment when 750 indicated. Nuclei were stained with DAPI. (A) Representative images. Scale bar = 20µm. (B) Number 751 of GFP-LC3 dots. (C) Immunoblot analysis of LC3 and SQSTM1 expression in HeLa cells treated with 752 CQ when indicated. ACTB was used as a loading control. (D) To study the autophagic flux, mRFP-GFP- 753 LC3 HeLa cells were transfected with BHRF1-HA plasmids, or treated with CQ to block the autophagic 754 flux, or maintained in EBSS (starvation) to induce the autophagic flux. Representative images are 755 presented in Figure S2. Results are shown as the percentage of total autophagic vesicles. (E) Confocal 756 images of BHRF1-HA transfected HeLa cells immunostained for BECN1 and BHRF1. Scale bar = 20µm. 757 (F) HeLa cells were transiently transfected to express BECN1 and either BHRF1-HA or ICP34.5-Flag 758 vector. After immunoprecipitation with a goat polyclonal anti-BECN1 antibody, proteins were 759 detected by immunoblotting with anti-BECN1, anti-Flag or anti-HA antibodies. (G) BHRF1-HA from 760 transfected cells was immunoprecipitated with an anti-HA antibody or control beads. BECN1 and 761 BHRF1-HA proteins were detected by immunoblotting. Data represent the mean ± SEM of three 762 independent experiments. * P < 0.05; *** P < Ϭ͘ϬϬϭ;^ƚƵĚĞŶƚ͛ƐƚƚĞƐƚͿ͘

763 Figure 4. Interplay between BHRF1-induced autophagy and mito-aggresome formation. (A and B) 764 Mitochondrial fission is required for BHRF1-induced autophagy. sh-NT and sh-Drp1 HeLa cells were 765 transfected with EV or BHRF1-HA plasmids for 24h and treated with CQ when indicated. LC3-positive 766 vesicles and BHRF1-HA transfected cells were visualized respectively using anti-LC3 and anti-BHRF1 767 antibodies. Nuclei were stained with DAPI. See Figure S3 for deficient cell line characterization. (A) 768 Representative images. Scale bar = 20µm. (B) Quantification of LC3 dots. (C-E) Autophagy is 769 necessary to BHRF1-induced mito-aggresome formation. HeLa cells were transfected with EV or

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770 BHRF1-HA plasmids for 24h. When indicated, cells were treated with Spautin-1 or 3-MA to inhibit 771 autophagy. After fixation, cells were immunostained for TOM20 and HA. (C) Representative images 772 of BHRF1-HA expressing cells. Scale bar = 20µm. (D) Percentage of BHRF1 positive cells presenting a 773 mito-aggresome. (E) Mitochondrial average length. Data represent the mean ± SEM of three 774 independent experiments. (B and E) ns = non-significant; ** P < 0.01; *** P < Ϭ͘ϬϬϭ;^ƚƵĚĞŶƚ͛ƐƚƚĞƐƚͿ͘ 775 (D) *** P < 0.001 (One-way ANOVA test). For each condition, the number of LC3 dots was count in at 776 least 25 cells from three independent experiments.

777 Figure 5. BHRF1 induces mitophagy and recruits Parkin to mitochondria. (A) Representative images 778 of HeLa cells co-transfected for 24h with mRFP-EGFP probe targeting mitochondria and EV or BHRF1- 779 HA plasmids. Cells were immunostained for BHRF1. Insets show part of the cytoplasm at a higher 780 magnification. Red staining corresponds to the delivery of mitochondria to acidic compartments. 781 Scale bar = 20µm and 5 µm for insets. (B and C) GFP-LC3 HeLa cells were transfected with EV or 782 BHRF1-HA plasmids for 24h and treated with CQ when indicated. Cells were immunostained for 783 TOM20 and HA. Nuclei were stained with DAPI. (B) Representative images of cells. Insets show part 784 of the cytoplasm at a higher magnification to better visualize colocalization (see arrows), between 785 mitochondria (TOM20) and autophagosomes (GFP-LC3), assessed by confocal microscopy. Scale 786 bars = 10µm and 4µm for insets. (C) To assess the colocalization level between mitochondria and 787 autophagosomes, the Manders split coefficient was calculated for each condition with ImageJ 788 software, ƵƐŝŶŐ ƚŚĞ ͞ĐŽůŽĐĂůŝnjĂƚŝŽŶ ƚŚƌĞƐŚŽůĚ͟ ƉůƵŐ-in. All images were taken with the same 789 microscope settings to be compared to each other. For each condition, 10 random cells were 790 analyzed. (D and E) Subcellular fractionation of BHRF1-HA and EV-transfected HeLa cells. VDAC and 791 LDH were used as markers for mitochondrial and cytosolic fractions respectively. (D) Immunoblot 792 analysis of PINK1 showing its accumulation at outer mitochondrial membrane. (E). Left: immunoblot 793 analysis of TOM20. Right: quantification of TOM20 expression in the mitochondrial fraction, showing 794 that BHRF1 decreases TOM20 protein level. (F) BHRF1 recruits CFP-Parkin to mitochondria. 795 Representative images of HeLa cells co-transfected with CFP-Parkin and BHRF1-HA plasmids for 48h 796 and immunostained for TOM20 and HA. Treatment with CCCP was used as a positive control of 797 mitophagy. Cells outline was drawn to better visualize the degradation of mitochondria under BHRF1 798 and CFP-Parkin expression. Scale bar = 20µm. See Figure S4 for visualization of mitochondria inside 799 LAMP1-positive vesicles in BHRF1-HA expressing cells. Data represent the mean ± SEM of two 800 independent experiments. (C) ns = non-significant; *** P < 0.001 (one-way ANOVA test). (E) 801 * P < 0.05 ;^ƚƵĚĞŶƚ͛ƐƚƚĞƐƚͿ͘

802 Figure 6. BHRF1 inhibits type I IFN induction via MAVS and STING signaling pathways. (A and B) 803 Luciferase reporter assay on HEK293T cells co-transfected with plasmids expressing BHRF1-HA, the

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804 reporter plasmid expressing firefly luciferase under the control of IFN-ɴ ƉƌŽŵŽƚĞƌ ;/&E-ɴ-Luc) and 805 renilla luciferase (RL-TK; to standardize the transfection efficiency). The activation of IFN-ɴƉƌŽŵŽƚĞƌ 806 was analyzed 24h after transfection. (A) To study the MAVS pathway, a transfection with Flag-ȴZ/'-I 807 (2xCARD) was performed to express a dominant positive form of RIG-I. (B) To study the STING 808 pathway, cells were stimulated with dsDNA for the last 8 hours to activate cGAS/STING axis. 809 Firefly/renilla luciferase ratios were calculated and normalized to control condition (EV) to assess the 810 activation level of interferon-ɴƉƌŽŵŽƚĞƌ͘(C) Immunoblot analysis of BHRF1-HA from HEK293T cells 811 transfected with increasing amounts of BHRF1-HA plasmid. ACTB was used as a loading control. (D 812 and E) HeLa cells were transfected with EV or BHRF1-HA for 24h. Cells were also transfected with 813 Flag-ȴZ/'-I (2xCARD) plasmid or stimulated with dsDNA. After fixation, cells were immunostained for 814 IRF3 and BHRF1. Nuclei were stained with DAPI. (D) Representative images. Scale bar = 20µm. (E) 815 Percentage of cells with IRF3 nuclear localization. Data represent the mean ± SEM of three 816 independent experiments. (A and B) *** P < 0.001 (one-way ANOVA test). (E) ** P < Ϭ͘Ϭϭ;^ƚƵĚĞŶƚ͛Ɛƚ 817 test). Proportion of cells presenting an IRF3 nuclear localization was determined by counting at least 818 50 random cells in each condition from three independent experiments.

819 Figure 7. Mitochondrial fission and autophagy are required for BHRF1-inhibitor effect on type I IFN 820 induction. (A) Luciferase reporter assay on sh-Drp1 HEK293T cells and its corresponding control cell 821 line (sh-NT). Cells were co-transfected with EV or BHRF1-HA, IFN-ɴ-Luc, and RL-TK plasmids for 24h. 822 Cells were also transfected with Flag-ȴZ/'-I (2xCARD) plasmid or stimulated with dsDNA. 823 Firefly/renilla luciferase ratios were calculated and normalized to control condition (EV). (B and C) sh- 824 NT and sh-Drp1 HeLa cells were transfected with EV or BHRF1-HA plasmids. Cells were also 825 transfected with Flag-ȴZ/'-I (2xCARD) plasmid or stimulated with dsDNA and immunostained for 826 IRF3 and BHRF1. Nuclei were staining with DAPI. (B) Representative images of BHRF1-HA expressing 827 cells. Scale bar = 20µm. (C) Quantification of IRF3 nuclear localization, normalized to EV condition. 828 (D) Luciferase reporter assay as described in Figure 6. To inhibit autophagy, cells were treated by 829 Spautin-1 or 3-MA. Firefly/renilla luciferase ratios were calculated and normalized to control 830 condition (EV). (E and F) HeLa cells were co-transfected with Flag-ȴRIG-I (2xCARD) and EV or BHRF1- 831 HA plasmids. Cells were treated with Spautin-1 or 3-MA, and immunostained for IRF3 and BHRF1. 832 Nuclei were staining with DAPI. (E) Representative images. Scale bar = 20µm. (F) Quantification of 833 IRF3 nuclear localization, normalized to EV condition. Data represent the mean ± SEM of three 834 independent experiments. ns = non-significant; ** P < 0.01; *** P < 0.ϬϬϭ;^ƚƵĚĞŶƚ͛ƐƚƚĞƐƚͿ͘

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DAPI Merge DAPI Merge

EV BHRF1-HA dsDNA

Figure 6 A B ǻRIG-I (2xCARD) + BHRF1-HA sh-NT sh-Drp1 IRF3 BHRF1 IRF3 BHRF1

DAPI Merge DAPI Merge

C IRF3 BHRF1 IRF3 BHRF1

DAPI Merge DAPI Merge

sh-NT sh-Drp1 dsDNA + BHRF1-HA D E Spautin-1 EV BHRF1-HA IRF3 BHRF1 IRF3 BHRF1

DAPI Merge DAPI Merge

F IRF3 BHRF1 IRF3 BHRF1

DAPI Merge DAPI Merge

EV BHRF1-HA Figure 7 3-MA A TOM20 / DAPI B

No treatment CCCP NaCl

NT

-

sh

-Drp1 sh

Figure S1. Mitochondrial fission is reduced in Drp1-deficient cell line. (A and B) sh-NT and sh-Drp1 HeLa cells were treated by CCCP or NaCl to induce mitochondrial fission. Then, cells were immunostained for TOM20 and nuclei were stained with DAPI. (A) Representative images. Insets (5X) show part of the cytoplasm at a higher magnification to observed mitochondrial phenotype. Scale bars = 20µm and 3µm for insets. (B) Mitochondrial average length. Mitochondria display a hyperfused phenotype in sh-Drp1 cells and they stay tubular after fission-inducing treatments.

Figure S1 A B Complete medium Starvation

EV BHRF1-HA CQ EV

BHRF1

GFP

mRFP

Merge Inset (3X)

Figure S2. BHRF1 stimulates the autophagic flux. mRFP-GFP-LC3 HeLa cells were transfected with EV or BHRF1-HA plasmids. As controls, cells were treated with CQ or starved in EBSS for 4 hours. (A) Representative images of autophagosomes (yellow=RFP+/GPF+ dots) and autolysosomes (red=RFP+/GFP- dots) assessed by confocal microscopy. Insets show part of the cytoplasm at a higher magnification. Arrows indicate autolysosomes. CQ was able to suppress the autophagic flux and to induce accumulation of autophagosomes (>95% yellow dots are observed), whereas starvation with EBSS induced the autophagic flux, and only part of the LC3-positive autophagic vacuoles were yellow. Scale bars = 20µm and 5µm for insets. (B) Results are expressed as absolute numbers of individuals vesicles (total autophagic vesicles=all RFP+ dots). We observed that CQ, BHRF1 expression and EBSS all increased the number of autophagic vesicles. Histogram represents the mean “ SEM of three independent experiments. * P < 0.05 (One-way ANOVA test).

Figure S2 ABLC3 / DAPI C CM Starvation sh-NT sh-Drp1

EBSS -+-+ NT

- SQSTM1 sh SQSTM1/ACTB 1.00 0.75 0.81 0.42 LC3-I LC3-II LC3-II/ACTB 1.00 0.59 0.64 0.43

-Drp1 ACTB sh

Figure S3. Knockdown of Drp1 does not impact autophagy. (A and B) sh-NT and sh-Drp1 HeLa cells were cultured in EBSS to carry out starvation-induced autophagy or maintained in complete medium (CM) and immunostained for LC3 to assess autophagy. Nuclei were stained with DAPI. (A) Representative images. Scale bar = 20µm. (B) Number of LC3 dots. (C) Immunoblot analysis of LC3 and SQSTM1 expression in sh- NT and sh-Drp1 HeLa cells. ACTB was used as a loading control.

Figure S3 A No treatment Chloroquine B

EV BHRF1-HA EV BHRF1-HA

MitoTracker

LAMP1 BHRF1

C

BHRF1 -+-+ Merge Chloroquine --++

3 4 LAMP1 2 LAMP1/ACTB 1.00 1.76 1.87 2.94

1 ACTB Inset (3X)

Figure S4. Localization of mitochondria in LAMP1-positive vesicles under BHRF1 expression. (A and B) HeLa cells were transfected with EV or BHRF1-HA plasmids for 48h and treated with CQ when indicated. Cells were labeled with MitoTracker and immunostained for LAMP1 to visualize lysosomes and late endosomes. Nuclei were stained with DAPI. (A) Confocal images of cells. Insets show part of the cytoplasm at a higher magnification to better visualize mitochondria inside LAMP1-positive vesicles. Scale bars = 10µm. Insets of confocal images show lysosomes surrounding mitochondria upon BHRF1-HA expression. Line profiles through these structures were performed (1 to 4). (B) Graphical analyzes show the fluorescence intensity of each channel (red=mitochondria, green=lysosomes/late endosomes) along the line. Under BHRF1 expression, LAMP1-positive vesicles contained mitochondria. Moreover, treatment by CQ inhibited mitochondria degradation inside lysosomes. (C) Immunoblot analysis of HeLa cells transfected with EV or BHRF1-HA plasmids for 48h and treated with CQ when indicated. Accumulation of LAMP1 was observed under BHRF1 expression. ACTB was used as a loading control.

Figure S4 7DEOH

Table 1. Materials used in this study

Antibodies Source Identifier

Mouse Anti-Human SQSTM1 Monoclonal Antibody, Unconjugated, Abnova Corporation Cat#H00008878-M01 Clone 2C11 IgG antibody Agilent Cat#A0423 Beclin antibody BD Biosciences Cat#612112 Drp1 antibody BD Biosciences Cat#611738 Tim23 antibody BD Biosciences Cat#611223

Tom20 antibody BD Biosciences Cat#612278 DRP1 (D6C7) Rabbit mAb antibody Cell Signaling Technology Cat#8570 LAMP1 (D2D11) XP Rabbit Antibody Cell Signaling Technology Cat#9091 Phospho-DRP1 (Ser637) (D3A4) Rabbit mAb antibody Cell Signaling Technology Cat#6319 Alexa Fluor 488-AffiniPure Goat Anti-Mouse IgG (H+L) antibody Jackson ImmunoResearch Cat#115-545-003 Alexa Fluor 488-AffiniPure Goat Anti-Rabbit IgG (H+L) antibody Jackson ImmunoResearch Cat#111-545-003 Peroxidase-AffiniPure Goat Anti-Mouse IgG (H + L) antibody Jackson ImmunoResearch Cat#115-035-003

Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) antibody Jackson ImmunoResearch Cat#111-035-003 Anti-Human LC3 Polyclonal Antibody MBL International Cat#PM036

Anti-Actin, clone C4 antibody Millipore Cat#MAB1501 Anti-EBV EA-R-p17, clone 5B11 antibody Millipore Cat#MAB8188 Beclin 1 human, mouse Novus Cat#NB500-249

PINK1 Antibody Novus Cat#BC100-494 BECN1 (D-18) antibody Santa Cruz Biotechnology Cat#sc-10086 EBV Ea-D (0261) antibody Santa Cruz Biotechnology Cat#sc-58121 EBV ZEBRA (BZ1) antibody Santa Cruz Biotechnology Cat#sc-53904 HA-probe (Y-11) antibody Santa Cruz Biotechnology Cat#sc-805

IRF-3 (SL-12) antibody Santa Cruz Biotechnology Cat#sc-33641 IRF-3 (FL-425) antibody Santa Cruz Biotechnology Cat#sc-9082 Anti-LC3B antibody produced in rabbit Sigma-Aldrich Cat#L7543 Monoclonal ANTI-FLAG® M2 antibody Sigma-Aldrich Cat#F3165

Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Thermo Fisher Scientific Cat#A-31571 Antibody, Alexa Fluor 647 Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Thermo Fisher Scientific Cat#A-11045 Alexa Fluor 350 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Thermo Fisher Scientific Cat#A-21424 Antibody, Alexa Fluor 555 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Thermo Fisher Scientific Cat#A-21429 Antibody, Alexa Fluor 555 LDHA Polyclonal Antibody Thermo Fisher Scientific Cat#PA5-27406

VDAC antibody Provided by Catherine N/A Brenner-Jan

Drugs Source Identifier

Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) Sigma-Aldrich Cat#C2759 Chloroquine diphosphate salt (CQ) Sigma-Aldrich Cat#C6628 Dimethyl sulfoxide sterile (DMSO) Sigma-Aldrich Cat#D2650

ĂƌůĞ͛ƐďĂůĂŶĐĞĚƐĂůƚƐŽůƵƚŝŽŶ;^^Ϳ Gibco Cat#24010-043 G418 InvivoGen Cat#GNL-35-012 Mdivi-1 Sigma-Aldrich Cat#M0199 Puromycin InvivoGen Cat#ant-pr-1 Spautin-1 Sigma-Aldrich Cat#SML0440 3-Methyladenine (3-MA) Sigma-Aldrich Cat#M9281

Dye and markers Source Identifier

DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) Thermo Fisher Scientific Cat#D1306 Hoechst 33342 Thermo Fisher Scientific Cat#H3570 MitoTracker red CMXRos Life technologies Cat#M7512 Protein ladder prestained Euromedex Cat#06P-0111

Oligonucleotides and DNA Source Identifier

BHRF1 ORF forward primer: AGTCCAGTGTGGAAGATGGCCT This paper N/A ATTCAACA BHRF1 ORF reverse primer:GATATCTGCAGAATTTTAAGCGTAATCT This paper N/A GGAACATCGTATGGGTAGTGTCTTCCTCTGGAGAT Deoxyribonucleic acid, low molecular weight from salmon sperm Sigma-Aldrich Cat#31149-10G-F

Plasmids Source Identifier

BECN1 expression vector Provided by Beth Levine [31] N/A BHRF1-HA expression vector This paper N/A CFP-Parkin Addgene Cat#47560 pFlag-ICP34.5 Provided by Bin He [35] N/A pFlag-ȴZ/'-I (2xCARD) [44] N/A pIFN-ɴ-Luc [43] N/A pMito mRFP-EGFP Provided by Andreas Till [38] N/A pcDNA3.1 empty vector (EV) Invitrogen N/A pRL Renilla Luciferase Control Reporter Vectors Promega Corporation Cat#E2241

Reagents and kit Source Identifier

Bovine serum albumin (BSA) Euromedex Cat#04-100-812-C Deoxycholic acid Sigma Cat#D-6750

ƵůďĞĐĐŽ͛ƐŵŽĚŝĨŝĞĚĞĂŐůĞŵĞĚŝƵŵ;DDͿ Gibco Cat#41965-039 Dual-luciferase reporter assay system Promega Corporation Cat#E1910 Fetal calf serum (FCS) Dominique Dutscher Cat#S181B-500 Fugene HD transfection reagent Promega Corporation Cat#E2311 Glycergel Mounting Medium Dako Cat#C0563

Hepes, Sodium Sigma-Aldrich Cat#H-3662

Hexadimethrine bromide (Polybrene) Sigma-Aldrich Cat#28728-55-4 Immobilon western Merck Millipore Cat#MA01821 Oligofectamine Thermo Fisher Scientific Cat# 12252011 Opti-MEM Gibco Cat#31985-047 Phosphate buffered saline (PBS) Gibco Cat#14200-067 Protease inhibitor cocktail Sigma-Aldrich Cat#P8340 RPMI medium 1640 Gibco Cat#A10491-01 Triton X-100 Sigma-Aldrich Cat#T-8787

Software and algorithms Source Identifier

Prism (GraphPad 5.00) GraphPad Software https://www.graphpad .com/ ImageJ (Fiji) ImageJ software https://fiji.sc/

LAS AF Lite Leica Microsystems CMS https://www.leica- GmbH microsystems.com/fr/

Viruses Source Identifier

MISSION pLKO.1-puro non-target shRNA control transduction Sigma-Aldrich Cat#SHC016V particles MISSION lentiviral transduction particles (Drp1 sh-RNA) Sigma-Aldrich Cat#SHCLNV

3. Discussion

3.1 The existence of EBV BALF0 and BALF1 in vivo

The BALF0/1 ORF has been first identified by a blast search in 1999 [104]. It was further suggested that this ORF might potentially encode for two proteins, namely

BALF0 and BALF1. These proteins could be evidenced when expressed from two separate plasmids that comprised mutations abrogating either the first (BALF1) or the second start codon (BALF0). Over subsequent decades, multiple BALF1-like proteins have been identified in herpesviruses from primate and non-primate hosts. The characterization of EBV BALF0 and BALF1 proteins is still matter to controversy, and the name of both proteins has been changing in the NCBI (National Center for

Biotechnology Information) database. The function(s) of BALF0 and/or BALF1 proteins have been analyzed in a very limited number of publications but none of them provided direct evidences for the existence of BALF0 and/or BALF1 in naturally infected cells due to the absence of specific antibodies.

In order to overcome this arduous obstacle, a recombinant soluble truncated form of

BALF0 (tBALF0) has been produced in E. coli that could eventually be used for immunization to generate specific polyclonal antiserum. Expressing the full-length protein was not possible, as already mentioned by others (Minfei Su, “Structural studies of the mechanism by which bcl-2 and Beclin proteins regulate autophagy and apoptosis”, PhD thesis of North Dakota State University). This was not surprising since the growth of bacterial hosts can be inhibited by the expression of heterologous proteins.

Gene products that affect the growth rate of bacterial hosts are considered to be toxic.

They include membrane proteins, proteins interacting with DNA or interfering with electron transport [390]. Lower transformation efficiency and marked reduction in bacterial growth of constructs encoding full length BALF0/1 were observed (data not shown) suggesting that the BALF0/1 gene product was toxic to the bacterial host.

Structural analysis predicted the presence of 2 α-helices with high hydrophobicity at

 142 BALF0/1 C-terminus showing putative transmembrane domains by using the TMpred

algorithm (https://embnet.vital-it.ch/software/TMPRED_form.html). A recombinant

protein with membrane-spanning domains may have a toxic effect on a bacterial host,

probably due to the association between the protein and bacterial membranes [391].

Removal of putative transmembrane domains in BALF0/1 led to a 17.7 kDa

polypeptide (amino acids 1 to 140) that could be easily expressed in E. coli but accumulated in a mainly insoluble form. High expression levels of recombinant proteins in bacterial expression systems can lead to the formation of insoluble inclusion bodies [392], which can be completely solubilized by strong denaturing agents, such as

6 M guanidine hydrochloride or 8 M urea. Therefore, tBALF0 was purified under denaturing conditions in the presence of 8 M urea by nickel affinity chromatography and eluted from the Ni-NTA resin in batch by discontinuously decreasing the pH of the buffer. Several attempts have been done to renature tBALF0 on the resin by gradually decreasing urea concentration until 0 in the washing buffers. However, we could not succeed in eluting native tBALF0 in these conditions even in the presence of 250 mM imidazole. This suggested that tBALF0 may form aggregates in these conditions, which was further confirmed by SDS-PAGE analysis (data not shown). The identity of recombinant tBALF0 in oligomeric form was confirmed by peptide mass fingerprinting.

Two cysteines are present in tBALF0 suggesting that disulfide bridges may likely contribute to the multimerization. We do not have evidences that such oligomerization occurred in eukaryotic cells as well, although BALF1 and BALF0 may form homodimers (BALF1 and BALF0 can form homodimers in Bellows et al., 2002, figure

6A and 6B [105]. No evidences have been found whether they could form heterodimers). In the definitive procedure, tBALF0 was purified in batch under denaturing conditions and dialyzed against PBS in the absence of reducing agents. A fraction of the protein precipitated during dialysis and was discarded by centrifugation.

We estimated that 2.8 mg tBALF0 could be obtained in a purified and soluble form from a 74 mg (1 L culture) total E. coli protein extract.

 143 The presence of antibodies to tBALF0 in patients infected by EBV could be considered

as an important indirect evidence for the existence of BALF0/1 in vivo.

Seroepidemiological studies were performed by ELISA in EBV primary infection patients, healthy EBV carriers and NPC patients. IgG directed against BALF0/1 were detected in different forms of EBV infection (primary and past infections) and in NPC patients. Among 60 healthy EBV carriers, 13.3% of individuals had a weak positive

IgG response to BALF0/1. The weak immunogenicity of EBV lytic protein has also been reported in a recent evaluation of IgG antibody responses to a large spectrum of

EBV proteins. In this study one early lytic protein (BMRF1) and 4 late lytic proteins

(BGRF1/BDRF1, BFLF2, BRFR1A and BcLF1) elicit positive IgG responses in less than 5% of individuals [393]. NPC patients have strong and characteristic immune responses against EBV proteins, including VCA and EBNA1 [394]. For several decades, IgG and IgA antibodies against EBV antigens have been investigated for their potential as biomarkers for NPC early diagnosis [395]. Results have often been disappointing due to a lack of sensitivity. Currently, a number of researchers give preference to the detection of circulating EBV-DNA as a biomarker suitable for early

NPC detection, especially when using next-generation sequencing (NGS) and size assessment of the viral DNA [396]. However, serodiagnosis has probably not said its last word in that a large number of EBV proteins, including early and late lytic proteins, may be the target of circulating antibodies in NPC patients. So far, only 10% of the

EBV proteins have been investigated as potential biomarkers for NPC diagnosis

[397,398]. The potential of these antibodies especially circulating IgA is well highlighted by a recent report based on peptide micro-arrays. In this study, IgG and IgA antibody responses against 199 sequences from 86 EBV proteins have been measured and combined with the VCA/EBNA1 IgA assay which is the current standard for NPC serodiagnosis. This combination represented a significant improvement in the risk prediction analysis of NPC by comparison with the VCA/EBNA1 IgA assay alone

[399]. In the present work, one third of NPC patients exhibited high IgG titers to

 144 BALF0/1, compared to healthy adults with past EBV infection. Due to the mucosal

origin of NPC [394], it would be well advised to evaluate the IgA responses to BALF0/1

in further investigations. Even if the circulating antibodies to BALF0/1 do not

contribute to the early diagnosis and/or monitoring of EBV-positive NPC, their

presence in a fraction of NPC patients will encourage future investigations on the role

of BALF0/1 in NPC pathogenesis.

3.2 EBV vBcl-2s modulate autophagy

A multitude of herpesviruses examined so far has evolved various strategies to appropriate the autophagic machinery for their own benefits including viral persistence, replication and survival [388]. Previous studies indicated that EBV reactivation and autophagy are intimately intricated, possibly in bimodal way. Early steps of autophagy are induced during EBV reactivation and molecules that can inhibit autophagy such as

CQ, ammonium chloride (Quignon et al., in prep) or 3-MA [356] repress the accumulation of lytic proteins and reduce the production of viral particles. On the other hand, the final steps of autophagy, i.e. the fusion between autophagosomes and lysosomes and the subsequent degradation of the cargos within autolysosomes, would be inhibited [357]. Altogether, these coordinated processes would eventually lead to the accumulation of autophagic components, such as autophagic machinery and vesicle membranes that could be used to produce viral particles, and concomitantly limit the lysosomal degradation of viral components and particles by autophagy. Accordingly,

Nowag and collaborators provided initial evidences that LC3-associated membranes are readdressed to envelopes of EBV virion [361].

In a first attempt to identify lytic viral proteins that could modulate autophagy during

EBV reactivation, we looked for EBV proteins that are orthologs to proteins interfering with the autophagic machinery as observed in other herpesviruses. During lytic replication, HSV-1 [368], KSHV [379], HCMV [370,371] and MHV68 [384] encode

 145 for proteins that can inhibit early steps of autophagy by binding to and inactivating

Beclin 1, a cellular protein that is required for phagophore nucleation [388]. Since

Beclin 1 was initially demonstrated to be targeted by cellular Bcl-2 [379], we inferred that Bcl-2 orthologues from EBV, namely BHRF1, BALF0 and BALF1, could modulate autophagy as well. In our study, BHRF1 stimulates the autophagic flux and interacts with Beclin 1. Although the interaction of BALF0 and BALF1 proteins with

Beclin-1 was not investigated in the present study, we could demonstrate that BALF1 and BALF0 (Figure 35) increased the number of autophagosomes when expressed individually, which correlated with the accumulation of the membrane-bound form of

LC3 that is the core autophagic machinery involving in the elongation of phagophore.

Importantly, the number of LC3-positive vesicles as well as the accumulation of endogenous LC3-II significantly increased in the presence of CQ in BALF1-expressing cells, indicating that BALF1 has a global positive impact on the autophagic flux.

Overexpression of both BALF0 and BALF1 resulted in the accumulation of LC3- positive vesicles, whereas LC3-II did not accumulate in the presence of CQ, suggesting that BALF0 tempered the pro-autophagic activity of BALF1. This agreed with other experiments of this study showing that BALF1 expression level decreased in the presence of BALF0. Since both proteins were stabilized in the presence of the proteasome inhibitor MG132, it is possible that BALF0 and BALF1 proteins act directly or indirectly on the proteasomal degradation pathway, a hypothesis that is currently under investigation. One may suggest investigating BALF0 activity following its stabilization by MG132. Unfortunately, this is not possible since MG132 has been proved to induce an endoplasmic reticulum stress that eventually stimulated autophagy

[400]. Our results are also in agreement with the unbalanced accumulation of BALF0 and BALF1 proteins during EBV reactivation described herein as well as with previous observations by De Leo and colleagues who noticed a transient accumulation of LC3-

II in Akata cells 8 hours after reactivation [362], a time point where BALF1 accumulated in these cells. The expression of BHRF1 is detectable both in latency and

 146 lytic cycle whereas BALF0 and BALF1 are barely detectable in non-reactivated cells but accumulated during the early phase of the lytic cycle. To some extent, it would be more interesting to investigate the impact of BHRF1 in BALF1 and BALF0-expressing cells, which might reflect the life cycle of EBV. Bellows and collaborators provided preliminary evidences that BHRF1 and BALF1 interacted with each other therefore modulating BHRF1 ability to inhibits apoptosis [105]. Accordingly, co-expressing

BALF1 and BHRF1 resulted in an additional accumulation of LC3-II. However, the addition of CQ did not modify LC3-II level, demonstrating that autophagy-mediated degradation of LC3-II was inhibited when both proteins were co-expressed. This observation was reminiscent of the effect that was observed when BALF0 was co- expressed with BALF1. Based on our results, we propose that EBV-encoded vBcl-2s modulate autophagy at different steps of autophagy (Figure 36). Indeed, BALF1 and

BHRF1 simulate the autophagic flux especially the autophagosomes formation whereas

BALF0 also increase the autophagosomes formation, which is slightly enhanced in the presence CQ as well. The combination of vBcl-2s (BALF1+BALF0 or

BALF1+BHRF1) impaired the accumulation of LC3-II in the presence of CQ, suggesting that the combinations act on a late step to inhibit the autophagic degradation.

Since Beclin 1 acts at both early and late stages, a possible model would be proposed in which (1) BALF1, BALF0 or BHRF1 stimulates early phase alone and (2) a complex between BALF1-BALF0 or BALF1-BHRF1 may inhibit the last degradative step, while not preventing the early step (Figure 36).

The stimulation of autophagy by BALF1 is shown to require a conserved domain

(amino acid 146-WSRL-149, underlined amino acids exhibiting essential role) that is reminiscent of LIR motif which has been identified in known partners of LC3, such as p62 (WTHL). p62, a multidomain protein, has been identified as the first selective autophagy receptor for autophagic degradation of ubiquitylated protein aggregates as well as a selective autophagy substrate, whose interaction with phagophore membranes is mediated through the LIR domain [330,333,401]. Additionally, experiments

 147 demonstrated that the expression of BALF1, as well as co-expression of BALF1 with

BALF0, resulted in the accumulation of p62, which suggested that p62 escaped autophagic degradation in the presence of BALF1. Since we demonstrated that BALF1 accumulated in LC3-positive vesicles, a process that also depends on the putative LIR- domain, we propose that BALF1 may compete with other LIR-containing proteins for targeting to the autophagosome membranes, therefore preventing them from being degraded by autophagy. This might be especially important in the case of p62 since it has multiple domains that mediate its interactions with various partners. As such, it serves as a signaling hub and is involved in autophagy, oxidative stress signaling and cancer [402].

In addition, the modulation of autophagy by BALF0 and BALF1 proteins may directly or indirectly contribute to the virion morphogenesis. Although initial work by

Johannsen and colleagues did not identify BALF0 and BALF1 in purified EBV virions

[403], more recent work reported that co-purification of BALF0 and BALF1 with

BSRF1, an EBV tegument protein that is homologous to HSV-1 unique long 51 (UL51) and HCMV UL71 [233], which is involved in virion egress. This result, which shed a new light on the function of BALF0 and BALF1 during EBV lytic cycle, is strongly reminiscent of a recent report indicating that vBcl-2 from KSHV could interact similarly with tegument protein ORF55 [404]. Whereas vBcl-2 interaction with ORF55 was shown to be critical for KSHV lytic cycle, BALF0 and BALF1 proteins may be dispensable for virus production [216]. Nonetheless additional investigations should be performed to more carefully evaluate the contribution of BALF0 and BALF1 to the virion infectivity. Similarly, it would be important to conduct genetic studies to characterize domains of BALF0 and BALF1 that are required for apoptosis inhibition, autophagy stimulation and virion morphogenesis, three functions that could be genetically separated in vBcl-2 from KSHV [404].

 148

Figure 35. Autophagic modulation by BALF0. (A) Representative images of GFP-LC3 HeLa cells transfected with EV or BALF0-HA plasmids for 24h and then fixed after 4h of CQ treatment.

BALF0-transfected cells were visualized by an anti-HA antibody (red) and nuclei were subsequently stained with Hoechst 33342 (blue). Scale bars = 20μm. Fluorescent intensities of

BALF0-expressing cells have been modified for visibility. (B) Autophagosomes formation was evaluated by quantifying the number of GFP- LC3 puncta per cell in EV and BALF0-expressing cells. The results are the mean± SEM of three independent experiments, and 50 cells were analyzed per assay. **P<0.01; *P<0.05.

 149

Figure 36. Viral proteins encoded by EBV involved in the regulation of autophagy. Latent proteins

(LMP1, LMP2A, EBNA3C), transcription factor (Rta) and vBlc-2 upregulate autophagy at the early stage (autophagosome formation), whereas the viral protein(s) that inhibit the late stages (fusion of autophagosome with lysosome and degradation of cargos within autolysosome) are still not known.

Our results suggest BALF1, BALF0 or BHRF1 could stimulate autophagosome formation (arrows) and possibly autolysosome (late step). This late step would be inhibited in turn in the presence of

BALF0 or BHRF1 (dot lines). (Adapted from Ref. 390)

3.3 The interplay between vBcl-2s of EBV

Bellows and collaborators reported that BALF0 and BALF1 could antagonize the anti- apoptotic activity of BHRF1 and suggested that BHRF1 function regulated by BALF1 may occur through competition for the same downstream cellular machinery [105]. No colocalization has been found in previous studies. This is in agreement with our data showing that BHRF1 colocalized with mitochondria and drove mitochondrial network reorganization to form mito-aggresomes whereas BALF1 concentrated into peri- nuclear structures that partly colocalized with ER (Figure 37). Interestingly, both

BALF0/1 and BHRF1 might target proteins of the Notch pathway and interact with

EGFR [234], which needs further experimental investigations.

 150

Figure 37. Subcellular localization of BALF1 in HeLa cells. Representative images of subcellular

localization of BALF1 observed by confocal microscopy. HeLa cells were transfected with the

plasmid of BALF1-HA for 24h and immunostained for BALF1 (anti-HA), mitochondria (anti-

TOM20) and Golgi apparatus (anti-GM130). Co-transfection of BALF1-HA with mammalian expression vector (pDsRed2-ER) designed to label the endoplasmic reticulum. Scale bars = 20μm and 10μm for insets.

The expression of BHRF1 is associated with the accumulation of BALF0 and BALF1, respectively, which in turn repressed the level of BHRF1. The stabilization effects to

BALF0 and BALF1 were specific since BHRF1 could not exert same effect in other proteins such as GFP, viral proteins ZEBRA and EB2. Considering that BHRF1,

BALF1 and BALF0 are likely to be concomitantly expressed, we could propose a schema illustrating the interplay between vBcl-2s of EBV (Figure 38). This shows that

BALF1 stabilizes BALF0, which in turn repressed BALF1 accumulation; BHRF1 is inhibited by both BALF0 and BALF1, which in turn accumulated in the presence of

BHRF1. Since both proteins were stabilized in the presence of the proteasome inhibitor

MG132, it is possible that BALF0 and BALF1 proteins act directly or indirectly on the proteasomal degradation pathway, and BHRF1 might stabilize BALF0 and BALF1 by counteracting the proteasomal degradation, a hypothesis that is currently under

 151 investigation. BHRF1 is initially postulated as a global inhibitor of the degradation of

cellular and viral proteins, which non-specifically block the proteasomal pathway. In

order to confirm this hypothesis, experiments are conducting to analyze the effects of

BHRF1 on proteasome-regulated proteins, including c-Jun, p53 and T-cell antigen

receptor chain α [405] as well as the uncoating factor E5 of Vaccinia virus [406]. In

addition, to explore which step is impaired by BHRF1, we will first analyze the labeling

of BALF0 and BALF1 with a tagged ubiquitin. Next, we will evaluate the impact of

BHRF1 either on the ubiquitination of BALF0 and BALF1 or on their degradation. The

whole EBV genome could be propagated in E. coli by using a bacterial artificial chromosome (BAC) system [407]. The recombinant EBV BACmids are maintained in

HEK293T cells under antibiotic selection, which can be induced into lytic cycle by either overexpressing the IE protein, ZEBRA [357], or treating cells with chemical stimuli, such as TPA together with sodium butyrate [408,409]. Any desired mutations can be introduced into a specific viral gene locus, which provides us a strategy to be utilized for investigating the interplay of BALF0, BALF1 and BHRF1. By using these

BACmids, we could compare the protein expression levels in wild type and the knock- outs (KO) of vBcl-2s (BHRF1-KO, BALF1-KO and BALF0-KO), which also could be used for investigating their effects on autophagic modulation during primary infection.

 152

Figure 38. Complex interplay between BALF0, BALF1 and BHRF1. BALF1 stabilizes BALF0, and vice versa; BHRF1 is inhibited by both BALF0 and BALF1, which in turn accumulated. BALF0 and BALF1 were stabilized in the presence of the proteasome inhibitor MG132, it is possible that both proteins act directly or indirectly on the proteasomal degradation pathway, and BHRF1 might stabilize BALF0 and BALF1 by counteracting the proteasomal degradation. solid line: experimentally confirmed; dot line: postulated.

 153 4. Take home message

The EBV BALF0/1 ORF potentially encodes for two proteins, namely BALF0 and

BALF1. The existence and functions of both BALF0 and BALF1 were equivocal. This

thesis presents a series of studies to characterize the BALF0 and BALF1.

Initially, a recombinant soluble truncated form of BALF0/1 (tBALF0) has been

produced in E. coli. tBALF0 was further used as an antigen in an indirect ELISA that unraveled the presence of low titer IgGs to BALF0/1 during primary (10.0%) and past

(13.3%) EBV infection. Conversely high-titer IgGs to BALF0/1 were detected in 33.3% of NPC patients suggesting that BALF0/1 and/or humoral response against it may contribute to NPC pathogenesis. The presence of antibodies to BALF0/1 in patients infected by EBV could therefore be considered as an important indirect evidence for the existence of BALF0 and/or BALF1 in vivo.

Thereafter, the existence of BALF0 and BALF1 was confirmed in EBV-positive BL

cell line. BALF0 and BALF1 were barely detectable in non-reactivated cells but

accumulated during the early phase of the lytic cycle revealed by immunoblot using the specific polyclonal antiserum directed against tBALF0. Interestingly, BALF1 overexpression promoted BALF0 accumulation which in turn inhibited BALF1’s in a dose-dependent manner, therefore providing an explanation for the unbalanced kinetics of both proteins observed during EBV reactivation.

In addition, BALF1 stimulated the autophagic flux which, in turn, was limited in the presence of BALF0. A putative LIR motif was required both for efficiently targeting of

BALF1 to GFP-LC3 vesicles as well as for the pro-autophagic effect of BALF1. In this thesis, it also presented that BHRF1 stimulates mitophagy, a process that prevents the initiation of the innate immune response mediated by mitochondrial pathways.

Surprisingly, co-expression of both BHRF1 and BALF1 resulted in a slight blockage in the degradative step of autophagy. A possible model would be proposed that (1)

BALF1, BALF0 or BHRF1 stimulates early phase alone and (2) a complex between

 154 BALF1-BALF0 or BALF1-BHRF1 may inhibit the last degradative step, while not preventing the early step.

Finally, immunoblot analysis revealed that BHRF1 promotes the accumulation of

BALF0 and BALF1 whereas BALF0 and BALF1 were both able to dramatically reduce

BHRF1 expression. BALF0 and BALF1 were stabilized in the presence of the proteasome inhibitor MG132, it is possible that both proteins act directly or indirectly on the proteasomal degradation pathway, and BHRF1 might stabilize BALF0 and

BALF1 by counteracting the proteasomal degradation, a hypothesis that is currently under investigation.

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 192 Lee, J. Lee, J.H. Lee, M. Lee, M.-S. Lee, P.J. Lee, S.W. Lee, S.-J. Lee, S.-J. Lee, S.Y. Lee, S.H. Lee, S.S. Lee, S.-J. Lee, S. Lee, Y.-R. Lee, Y.J. Lee, Y.H. Lee, C. Leeuwenburgh, S. Lefort, R. Legouis, J. Lei, Q.-Y. Lei, D.A. Leib, G. Leibowitz, I. Lekli, S.D. Lemaire, J.J. Lemasters, M.K. Lemberg, A. Lemoine, S. Leng, G. Lenz, P. Lenzi, L.O. Lerman, D. Lettieri Barbato, J.I.-J. Leu, H.Y. Leung, B. Levine, P.A. Lewis, F. Lezoualc’h, C. Li, F. Li, F.-J. Li, J. Li, K. Li, L. Li, M. Li, M. Li, Q. Li, R. Li, S. Li, W. Li, W. Li, X. Li, Y. Li, J. Lian, C. Liang, Q. Liang, Y. Liao, J. Liberal, P.P. Liberski, P. Lie, A.P. Lieberman, H.J. Lim, K.-L. Lim, K. Lim, R.T. Lima, C.-S. Lin, C.-F. Lin, F. Lin, F. Lin, F.-C. Lin, K. Lin, K.-H. Lin, P.-H. Lin, T. Lin, W.-W. Lin, Y.-S. Lin, Y. Lin, R. Linden, D. Lindholm, L.M. Lindqvist, P. Lingor, A. Linkermann, L.A. Liotta, M.M. Lipinski, V.A. Lira, M.P. Lisanti, P.B. Liton, B. Liu, C. Liu, C.-F. Liu, F. Liu, H.-J. Liu, J. Liu, J.-J. Liu, J.-L. Liu, K. Liu, L. Liu, L. Liu, Q. Liu, R.-Y. Liu, S. Liu, S. Liu, W. Liu, X.-D. Liu, X. Liu, X.-H. Liu, X. Liu, X. Liu, X. Liu, Y. Liu, Y. Liu, Z. Liu, Z. Liu, J.P. Liuzzi, G. Lizard, M. Ljujic, I.J. Lodhi, S.E. Logue, B.L. Lokeshwar, Y.C. Long, S. Lonial, B. Loos, C. López-Otín, C. López-Vicario, M. Lorente, P.L. Lorenzi, P. Lõrincz, M. Los, M.T. Lotze, P.E. Lovat, B. Lu, B. Lu, J. Lu, Q. Lu, S.-M. Lu, S. Lu, Y. Lu, F. Luciano, S. Luckhart, J.M. Lucocq, P. Ludovico, A. Lugea, N.W. Lukacs, J.J. Lum, A.H. Lund, H. Luo, J. Luo, S. Luo, C. Luparello, T. Lyons, J. Ma, Y. Ma, Y. Ma, Z. Ma, J. Machado, G.M. Machado-Santelli, F. Macian, G.C. MacIntosh, J.P. MacKeigan, K.F. Macleod, J.D. MacMicking, L.A. MacMillan-Crow, F. Madeo, M. Madesh, J. Madrigal-Matute, A. Maeda, T. Maeda, G. Maegawa, E. Maellaro, H. Maes, M. Magariños, K. Maiese, T.K. Maiti, L. Maiuri, M.C. Maiuri, C.G. Maki, R. Malli, W. Malorni, A. Maloyan, F. Mami-Chouaib, N. Man, J.D. Mancias, E.-M. Mandelkow, M.A. Mandell, A.A. Manfredi, S.N. Manié, C. Manzoni, K. Mao, Z. Mao, Z.-W. Mao, P. Marambaud, A.M. Marconi, Z. Marelja, G. Marfe, M. Margeta, E. Margittai, M. Mari, F.V. Mariani, C. Marin, S. Marinelli, G. Mariño, I. Markovic, R. Marquez, A.M. Martelli, S. Martens, K.R. Martin, S.J. Martin, S. Martin, M.A. Martin- Acebes, P. Martín-Sanz, C. Martinand-Mari, W. Martinet, J. Martinez, N. Martinez- Lopez, U. Martinez-Outschoorn, M. Martínez-Velázquez, M. Martinez-Vicente, W.K. Martins, H. Mashima, J.A. Mastrianni, G. Matarese, P. Matarrese, R. Mateo, S. Matoba, N. Matsumoto, T. Matsushita, A. Matsuura, T. Matsuzawa, M.P. Mattson, S. Matus, N. Maugeri, C. Mauvezin, A. Mayer, D. Maysinger, G.D. Mazzolini, M.K. McBrayer, K. McCall, C. McCormick, G.M. McInerney, S.C. McIver, S. McKenna, J.J. McMahon,

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