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International audience; CD4 + T lymphocytes play a major role in the establishment and maintenance of immunity. They are activated by antigenic peptides derived from extracellular or newly synthesized (endogenous) proteins presented by the MHC-II molecules. The pathways leading to endogenous MHC-II presentation remain poorly characterized. We demonstrate here that the autophagy receptor, T6BP, influences both autophagy-dependent and-independent endogenous presentation of HIV-and HCMV-derived peptides. By studying the immunopeptidome of MHC-II molecules, we show that T6BP affects both the quantity and quality of peptides presented. T6BP silencing induces the mislocalization of the MHC-II-loading compartments and rapid degradation of the invariant chain (CD74) without altering the expression and internalization kinetics of MHC-II molecules. Defining the interactome of T6BP, we identify calnexin as a T6BP partner. We show that the calnexin cytosolic tail is required for this interaction. Remarkably, calnexin silencing replicates the functional consequences of T6BP silencing: decreased CD4 + T cell activation and exacerbated CD74 degradation. Altogether, we unravel T6BP as a key player of the MHC-II-restricted endogenous presentation pathway, and we propose one potential mechanism of action.

CD4+ T lymphocytes play a major role in the establishment and maintenance of immunity. They are activated by antigenic peptides derived from extracellular or newly synthesized (endogenous) proteins presented on the surface of antigen presenting cells (APCs) by the MHC-II molecules. The pathways leading to endogenous MHC-II presentation remain poorly characterized. We demonstrate here that the autophagy receptor, T6BP, influences both autophagy-dependent and -independent endogenous presentation of HIV- and HCMV-derived peptides. By studying the immunopeptidome of MHC-II molecules, we show that T6BP affects both the quantity and quality of peptides presented. T6BP silencing induces the mislocalization of the MHC-II-loading compartments and a rapid degradation of the invariant chain (CD74) without altering the expression and internalization kinetics of MHC-II molecules. We determined the interactome of T6BP, identified calnexin as a T6BP partner and show that CANX cytosolic tail is required for this interaction. Remarkably, calnexin silencing replicates the functional consequences of T6BP silencing: decreased CD4+ T cell activation and exacerbated CD74 degradation. Altogether, we unravel T6BP as a key player of the MHC-II-restricted endogenous presentation pathway and we propose one potential mechanism of action.

Mitochondria respond to many cellular functions and act as central hubs in innate immunity against viruses. This response is notably due to their role in the activation of interferon (IFN) signaling pathways through the activity of MAVS (mitochondrial antiviral signaling protein) present at the mitochondrial surface. Here, we report that the BHRF1 protein, a BCL2 homolog encoded by Epstein-Barr virus (EBV), inhibits IFNB/IFN-β induction by targeting the mitochondria. Indeed, we have demonstrated that BHRF1 expression modifies mitochondrial dynamics and stimulates DNM1L/Drp1-mediated mitochondrial fission. Concomitantly, we have shown that BHRF1 is pro-autophagic because it stimulates the autophagic flux by interacting with BECN1/Beclin 1. In response to the BHRF1-induced mitochondrial fission and macroautophagy/autophagy stimulation, BHRF1 drives mitochondrial network reorganization to form juxtanuclear mitochondrial aggregates known as mito-aggresomes. Mitophagy is a cellular process, which can specifically sequester and degrade mitochondria. Our confocal studies uncovered that numerous mitochondria are present in autophagosomes and acidic compartments using BHRF1-expressing cells. Moreover, mito-aggresome formation allows the induction of mitophagy and the accumulation of PINK1 at the mitochondria. As BHRF1 modulates the mitochondrial fate, we explored the effect of BHRF1 on innate immunity and showed that BHRF1 expression could prevent IFNB induction. Indeed, BHRF1 inhibits the IFNB promoter activation and blocks the nuclear translocation of IRF3 (interferon regulatory factor 3). Thus, we concluded that BHRF1 can counteract innate immunity activation by inducing fission of the mitochondria to facilitate their sequestration in mitophagosomes for degradation. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; BCL2: BCL2 apoptosis regulator; CARD: caspase recruitment domain; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CI: compaction index; CQ: chloroquine; DAPI: 4ʹ,6-diamidino-2-phenylindole, dihydrochloride; DDX58/RIG-I: DExD/H-box helicase 58; DNM1L/Drp1: dynamin 1 like; EBSS: Earle’s balanced salt solution; EBV: Epstein-Barr virus; ER: endoplasmic reticulum; EV: empty vector; GFP: green fluorescent protein; HEK: human embryonic kidney; IFN: interferon; IgG: immunoglobulin G; IRF3: interferon regulatory factor 3; LDHA: lactate dehydrogenase A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOM: mitochondrial outer membrane; PINK1: PTEN induced kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Human Cytomegalovirus (HCMV) is a frequent opportunistic pathogen in immunosuppressed patients, which can be involved in kidney allograft dysfunction and rejection. In order to study the pathophysiology of HCMV renal diseases, we concentrated on the impact of HCMV infection on human renal tubular epithelial HK-2 cells. Our aim was to develop a model of infection of HK-2 cells by using the viral strain TB40/E, that contains the extended cell tropism of clinical isolates and the efficient viral multiplication in cell culture of laboratory-adapted strains. We observed that HK-2 cells can be infected by HCMV and expressed viral antigens, but they do not produce extracellular viral particles. We then studied the interplay of HCMV with ciliogenesis and autophagy. Primary cilium (PC) is a stress sensor important to maintain renal tissue homeostasis that projects from the apical side into the lumen of tubule cells. PC formation and length were not modified by HCMV infection. Autophagy, another stress response process critically required for normal kidney functions, was inhibited by HCMV in HK-2 cells with a reduction in the autophagic flux. HCMV classically induces an enlargement of infected cells in vivo and in vitro, and we observed that HCMV infection led to an enlargement of the HK-2 cell volume. Our results constitute therefore an excellent starting point to further explore the role of these mechanisms in renal cells dysfunction. Fil: López Giuliani, Ana C.. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Hernández, Eva. Centre D'etudes de Saclay; Francia Fil: Tohmé Chapini, María Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Taisne, Clémence. Centre D'etudes de Saclay; Francia Fil: Roldan, Julieta Suyay. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: García Samartino, Clara. Universidad Nacional de Cuyo; Argentina Fil: Lussignol, Marion. Centre D'etudes de Saclay; Francia Fil: Codogno, Patrice. Sorbonne University; Francia Fil: Colombo, María Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Esclatine, Audrey. Centre D'etudes de Saclay; Francia Fil: Delgui, Laura Ruth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina

During its life cycle, Human cytomegalovirus (HCMV) tightly modulates autophagy, a vesicular pathway allowing degradation and recycling of cellular components. To study the interplay between autophagy and the viral life cycle, we established various autophagy-deficient human fibroblastic cell lines. By knocking down the expression or activity of five autophagy-related proteins, we confirmed the proviral function that the autophagic machinery exerts on HCMV production. Using 3D reconstruction from confocal microscopy and electron microscopy, we demonstrated that lipidated LC3-positive vesicles accumulated at the viral assembly compartment (vAC). The vAC is a juxtanuclear ring-shaped structure containing several organelles and membranes, where assembly and final envelopment of HCMV particles occur. Two LC3 homologs, GABARAPL1 and GATE16, also accumulated during HCMV infection and were associated with the vAC, in proximity with fragmented Golgi stacks. Additionally, we observed the formation of a pre-assembly compartment (PrAC) in infected cells, which consists of a juxtanuclear structure containing both fragmented Golgi and LC3-positive vesicles. Finally, we showed that highly purified extracellular viral particles were associated with various autophagy proteins. Our results thus suggest that autophagy machinery participates to the final cytoplasmic envelopment of HCMV viral particles into the vAC and that autophagy-related proteins can be spotted in the virions.

Autophagy is an essential vacuolar process of the cell, leading to lysosomal degradation and recycling of proteins and organelles, which is extremely important in maintaining homeostasis. Multiple roles have been now associated with autophagy, in particular a pro-survival role in nutrient starvation or in stressful environments, a role in life span extension, in development, or in innate and adaptive immunity. This cellular process can also take over microorganisms or viral proteins inside autophagosomes and degrade them directly in autolysosomes and is then called xenophagy and virophagy, respectively. Several Herpesviruses have developed strategies to escape this degradation, by expression of specific anti-autophagic proteins. However, we are increasingly discovering that Herpesviruses hijack autophagy, rather than just fight it. This beneficial effect is obvious since inhibition of autophagy will lead to decreased viral titers for human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) or Varicella-Zoster virus (VZV), for example. Conversely, autophagy stimulation will improve viral multiplication. The autophagic machinery can be used in whole or in part, and can optimize viral propagation or persistence. Some viruses block maturation of autophagosomes to avoid the degradation step, then autophagosomal membranes are used to contribute to the envelopment and/or the egress of viral particles. On the other hand, VZV stimulates the whole process of autophagy to subvert it in order to use vesicles containing ATG (autophagy-related) proteins and resembling amphisomes for their transport in the cytoplasm. During latency, autophagy can also be activated by latent proteins encoded by different oncogenic Herpesviruses to promote cell survival and achieve long term viral persistence in vivo. Finally, reactivation of gammaherpesvirus Murid Herpesvirus 68 (MHV68) in mice appears to be positively modulated by autophagy, in order to control the level of inflammation. Therefore, Herpesviruses appear to behave more like thieves than fugitives.

Autophagy is a cellular degradation pathway that exerts numerous functions in vital biological processes. Among these, it contributes to both innate and adaptive immunity. On the other hand, pathogens have evolved strategies to manipulate autophagy for their own advantage. By monitoring autophagic markers, we showed that HSV-1 transiently induced autophagosome formation during early times of the infection of monocytic THP-1 cells and human monocytes. Autophagy is induced in THP-1 cells by a mechanism independent of viral gene expression or viral DNA accumulation. We found that the MyD88 signaling pathway is required for HSV-1-mediated autophagy and it is linked to the toll-like receptor 2 (TLR2). Interestingly, autophagy inhibition by pharmacological modulators or siRNA knockdown impaired viral replication in both THP-1 cells and human monocytes, suggest that the virus exploits the autophagic machinery to its own benefit in these cells. Taken together, these findings indicate that the early autophagic response induced by HSV-1 exerts a proviral role, improving viral production in a semi-permissive model such as THP-1 cells and human monocytes.

The Epstein-Barr virus (EBV) is a ubiquitous human virus whose latent persistence is associated with several cancers, including Burkitt lymphoma (BL). BHRF1 is a BCL-2 ortholog, expressed during latency and the productive cycle. This viral protein is expressed in 15% of EBV-related BL, making them particularly resistant to treatment. Initially, BHRF1 was known for its anti-apoptotic activity. In this work, we focused on better characterizing the role of BHRF1, notably regarding autophagy, a recycling process for intracellular components. We have identified that BHRF1 stimulates autophagy and alters mitochondrial morphodynamics. More precisely, BHRF1 increases mitochondrial fission and uses the microtubule network to induce the formation of a mito-aggresome in the perinuclear region. All of these cellular alterations lead to the selective degradation of mitochondria by autophagy, a process called mitophagy. We also demonstrated that BHRF1 constitutes a restriction factor of antiviral immunity, since it blocks the production of antiviral cytokines called interferon. In the last part of this work, we investigated whether these functions were found in EBV⁺ LB cells expressing BHRF1. In this model, we observed that BHRF1 induces the formation of mito-aggresomes, and stimulates cytoprotective autophagy against apoptosis. In conclusion, we propose that these new functions of BHRF1 could participate in the chemoresistance and the lymphomagenesis of EBV-related BL., Le virus Epstein-Barr (EBV) est un virus humain ubiquitaire dont la persistance à l’état latent est associée à de nombreux cancers, tels que le lymphome de Burkitt (LB). BHRF1 est une orthologue de BCL-2, qui s’exprime au cours de la latence et du cycle productif. Cette protéine virale est retrouvée dans 15 % des LB liés à l’EBV, les rendant particulièrement résistants au traitement. Initialement, BHRF1 était connue pour son activité anti-apoptotique. Dans ce travail, nous avons souhaité mieux caractériser le rôle de BHRF1, notamment au regard de l’autophagie, un processus de recyclage de constituants cellulaires. Nous avons identifié que BHRF1 stimule l’autophagie et altère la morphodynamique des mitochondries. Plus précisément, BHRF1 augmente la fission mitochondriale et exploite le réseau de microtubules pour induire la formation de mito-agrésomes dans la région périnucléaire. L’ensemble de ces altérations cellulaires conduisent à la dégradation sélective des mitochondries par autophagie, un processus appelé mitophagie. Nous démontrons aussi que BHRF1 constitue un facteur de restriction de l’immunité antivirale puisqu’elle bloque la production de cytokines antivirales, les interférons. Dans la dernière partie de ce travail, nous avons recherché si ces fonctions étaient retrouvées dans des lymphomes EBV⁺ en utilisant des cellules de LB exprimant BHRF1. Dans ce modèle, BHRF1 induit des mito-agrésomes et est aussi à l’origine d’une autophagie cytoprotectrice vis-à-vis de l’apoptose. En conclusion, nous proposons que ces nouvelles fonctions de BHRF1 pourraient participer à la chimiorésistance et à la lymphomagenèse des LB liés à l’EBV.

The Human cytomegalovirus (HCMV) belongs to the Herpesviridae family. This virus is responsible for congenital infections and for severe illnesses in immunocompromised patients. We have previously shown interactions between HCMV and autophagy, a cellular process involved in the degradation and recycling of components. This is a constitutive process responsible for maintaining cellular homeostasis. Autophagy is induced by different stresses and can be used by the cell as a defense mechanism against viral or bacterial infection. We have shown that HCMV infection induces the accumulation of autophagic structures but the virus blocks the final step of autophagy to avoid its own degradation. To characterize the role of autophagy in viral multiplication, we used genetic approaches tomodulate autophagy and we demonstrated its beneficial role on HCMV multiplication. We therefore established knock-down or knock-out stable cell lines for different autophagic proteins (ULK1, BECN1, ATG5, LC3B and ATG4B). Using these tools, we investigated the role of autophagy on different steps of the HCMV life cycle: entry, protein expression, viral genome replication, assembly and egress. Our results showed that autophagic proteins are present and involved in the assembly compartment organized by HCMV in the cytoplasm. Several of these proteins are consequently found in the extracellular viral particles, as LC3 and ATG5. Taken together, these results demonstrate that HCMV hijacks autophagic membranes to acquire its envelope and this seems to improve its infectivity and its transmission., Le cytomégalovirus humain (HCMV) appartient à la famille des Herpesviridae. Ce virus est responsable d’infections congénitales et d’infections graves chez les personnes immunodéprimées. Nous avons précédemment montré l’interaction entre le HCMV et l’autophagie, un processus cellulaire de dégradation et de recyclage. Ce processus est constitutif et permet le maintien de l’homéostasie cellulaire. L’autophagie est induite en cas de stress et peut être utilisée par la cellule pour se défendre des infections virales ou bactériennes. Nous avons montré que le HCMV induit la formation de structures autophagiques mais que le virus bloque l’étape finale du processus pour empêcher sa propre dégradation. Afin de caractériser le rôle de l’autophagie sur la multiplication virale, nous avons utilisé une approche génétique qui a montré que l’autophagie était bénéfique pour la multiplication du HCMV. Nous avons donc établi des lignées cellulaires stables, déficientes ou invalidées pour des protéines autophagiques (ULK1, BECN1, ATG5, LC3B et ATG4B). Grâce à ces outils, nous avons ensuite étudié comment l’autophagie module les différentes étapes du cycle du HCMV, à savoir : l’entrée, la synthèse protéique, la réplication du génome, l’assemblage et la sortie des virus. Nos résultats montrent que les protéines de l’autophagie sont présentes et agissent au niveau du compartiment d’assemblage viral dans le cytoplasme. Plusieurs de ces protéines sont conservées au sein des particules virales extracellulaires, notamment LC3 et ATG5. L’ensemble de nos résultats démontrent que le HCMV détourne les membranes autophagiques pour acquérir son enveloppe ce qui semble favoriser son infectivité et sa propagation.