Marion Lussignol

L’autophagie est un mécanisme vacuolaire de dégradation de matériel cytoplasmique permettant le maintien de l’homéostasie cellulaire, mais elle peut être également activée par de nombreux stress, comme l’infection virale. Le virus de l’Herpès Simplex de type 1 (HSV 1) est capable de contrecarrer ce mécanisme de défense antivirale. HSV-1 possède une protéine ICP34.5 capable d’inhiber l’autophagie en se liant à Beclin 1, une protéine de la machinerie autophagique. Nous avons mis en évidence une deuxième protéine d’HSV-1 capable d’inhiber l’autophagie, la protéine tardive Us11, qui pourrait avoir un rôle complémentaire à celui d’ICP34.5 dans le contrôle de l’autophagie par le virus.Nous montrons que l’expression ectopique d’Us11 permet de bloquer l’autophagie induite par différents stimuli, et ce de manière similaire à ICP34.5. De plus, dans un contexte viral, l’expression précoce d’Us11 dans des cellules infectées par un virusICP34.5 permet un contrôle de l’autophagie comparable à celui d’un virus sauvage. Nous avons ensuite recherché le mécanisme d’action d’Us11. La protéine Us11 a été décrite comme pouvant interagir avec la kinase dépendante de l’ARN double brin PKR, empêchant ainsi la phosphorylation de son substrat eIF2, un facteur d’initiation de la traduction. Nous avons observé qu’en l’absence de PKR, Us11 n’est plus capable d’inhiber l’autophagie. Nous avons pu confirmer qu’Us11 a besoin de se lier à PKR pour exercer son activité inhibitrice par la construction de formes tronquées d’Us11, permettant de montrer l’importance de son domaine d’interaction avec PKR dans l’inhibition de l’autophagie. L’étude des formes tronquées d’Us11 a soulevé le fait que le domaine N-terminal était également nécessaire. Aucune interaction de ce domaine avec une protéine cellulaire n’a été identifiée à ce jour, mais il pourrait permettre l’interaction d’Us11 avec une autre protéine de la machinerie autophagique. Cependant, nous avons montré qu’Us11 n’interagissait pas avec Beclin 1 et n’avait pas d’effet sur la kinase mTOR, une autre voie importante de l’autophagie. Enfin, nous avons étudié la modulation de la voie PKR/eIF2 lors de la stimulation de l’autophagie par la carence, et nos résultats suggèrent que cette voie joue un rôle sous-estimé dans la réponse à la carence.Le mécanisme d’action de la protéine Us11, qui consiste en un blocage de l’autophagie en inhibant PKR, n’avait jamais été décrit auparavant. Ce travail ouvre de nombreuses perspectives dans l’étude de la voie PKR/eIF2 vis à vis de la régulation de l’autophagie, ainsi que dans la compréhension de l’implication de l’autophagie dans la neurovirulence d’HSV-1.

Autophagy is a complex and highly regulated degradative process, which acts as a survival pathway in response to cellular stress, starvation and pathogen infection. Ricin toxin is a plant toxin produced by the castor bean and classified as a category B biothreat agent. Ricin toxin inhibits cellular protein synthesis by catalytically inactivating ribosomes, leading to cell death. Currently, there is no licensed treatment for patients exposed to ricin. Ricin-induced apoptosis has been extensively studied; however, whether its intoxication via protein synthesis inhibition affects autophagy is not yet resolved. In this work, we demonstrated that ricin intoxication is accompanied by its own autophagic degradation in mammalian cells. Autophagy deficiency, by knocking down ATG5, attenuates ricin degradation, thus aggravating ricin-induced cytotoxicity. Additionally, the autophagy inducer SMER28 (Small Molecule Enhancer 28) partially protects cells against ricin cytotoxicity, an effect not observed in autophagy-deficient cells. These results demonstrate that autophagic degradation acts as a survival response of cells against ricin intoxication. This suggests that stimulation of autophagic degradation may be a strategy to counteract ricin intoxication.

Innate immunity constitutes the first line of defense against viruses, in which mitochondria play an important role in the induction of the interferon (IFN) response. BHRF1, a multifunctional viral protein expressed during Epstein-Barr virus reactivation, modulates mitochondrial dynamics and disrupts the IFN signaling pathway. Mitochondria are mobile organelles that move through the cytoplasm thanks to the cytoskeleton and in particular the microtubule (MT) network. MTs undergo various post-translational modifications, among them tubulin acetylation. In this study, we demonstrated that BHRF1 induces MT hyperacetylation to escape innate immunity. Indeed, the expression of BHRF1 induces the clustering of shortened mitochondria next to the nucleus. This “mito-aggresome” is organized around the centrosome and its formation is MT-dependent. We also observed that the α-tubulin acetyltransferase ATAT1 interacts with BHRF1. Using ATAT1 knockdown or a non-acetylatable α-tubulin mutant, we demonstrated that this hyperacetylation is necessary for the mito-aggresome formation. Similar results were observed during EBV reactivation. We investigated the mechanism leading to the clustering of mitochondria, and we identified dyneins as motors that are required for mitochondrial clustering. Finally, we demonstrated that BHRF1 needs MT hyperacetylation to block the induction of the IFN response. Moreover, the loss of MT hyperacetylation blocks the localization of autophagosomes close to the mito-aggresome, impeding BHRF1 to initiate mitophagy, which is essential to inhibiting the signaling pathway. Therefore, our results reveal the role of the MT network, and its acetylation level, in the induction of a pro-viral mitophagy.

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.

Interferons (IFNs) mediate cellular defence against viral pathogens by upregulation of IFN-stimulated genes whose products interact with viral components or alter cellular physiology to suppress viral replication^1,2,3. Among the IFN-stimulated genes that can inhibit influenza A virus (IAV)⁴ are the myxovirus resistance 1 GTPase⁵ and IFN-induced transmembrane protein 3 (refs ^6,7). Here, we use ectopic expression and gene knockout to demonstrate that the IFN-inducible 219-amino acid short isoform of human nuclear receptor coactivator 7 (NCOA7) is an inhibitor of IAV as well as other viruses that enter the cell by endocytosis, including hepatitis C virus. NCOA7 interacts with the vacuolar H⁺-ATPase (V-ATPase) and its expression promotes cytoplasmic vesicle acidification, lysosomal protease activity and the degradation of endocytosed antigen. Step-wise dissection of the IAV entry pathway demonstrates that NCOA7 inhibits fusion of the viral and endosomal membranes and subsequent nuclear translocation of viral ribonucleoproteins. Therefore, NCOA7 provides a mechanism for immune regulation of endolysosomal physiology that not only suppresses viral entry into the cytosol from this compartment but may also regulate other V-ATPase-associated cellular processes, such as physiological adjustments to nutritional status, or the maturation and function of antigen-presenting cells.

With more than 240 million people infected, hepatitis B virus (HBV) is a major health concern. The inability to mimic the complexity of the liver using cell lines and regular primary human hepatocyte (PHH) cultures pose significant limitations for studying host/pathogen interactions. Here, we describe a 3D microfluidic PHH system permissive to HBV infection, which can be maintained for at least 40 days. This system enables the recapitulation of all steps of the HBV life cycle, including the replication of patient-derived HBV and the maintenance of HBV cccDNA. We show that innate immune and cytokine responses following infection with HBV mimic those observed in HBV-infected patients, thus allowing the dissection of pathways important for immune evasion and validation of biomarkers. Additionally, we demonstrate that the co-culture of PHH with other non-parenchymal cells enables the identification of the cellular origin of immune effectors, thus providing a valuable preclinical platform for HBV research.

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.

Significance: Virion proteomics represents a powerful and unbiased approach to gain insights into the process of virus assembly and the host factors important for infection. For hepatitis C virus (HCV), it is known that infectious virions are assembled alongside endogenous lipoproteins to give rise to a chimeric lipoviral particle. Host-derived apolipoproteins coat the exterior of circulating particles, making for a veiled pathogen. The protein composition of HCV remains, however, undefined. Here, using MS, we identified novel viral and cellular proteins specifically associated with HCV virions and discovered an essential interaction between the capsid protein and the nucleoporin Nup98 that is required for virion biogenesis.

Autophagy is now known to be an essential component of host innate and adaptive immunity. Several herpesviruses have developed various strategies to evade this antiviral host defense. Herpes simplex virus 1 (HSV-1) blocks autophagy in fibroblasts and in neurons, and the ICP34.5 protein is important for the resistance of HSV-1 to autophagy because of its interaction with the autophagy machinery protein Beclin 1. ICP34.5 also counteracts the shutoff of protein synthesis mediated by the double-stranded RNA (dsRNA)-dependent protein kinase PKR by inhibiting phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) in the PKR/eIF2α signaling pathway. Us11 is a late gene product of HSV-1, which is also able to preclude the host shutoff by direct inhibition of PKR. In the present study, we unveil a previously uncharacterized function of Us11 by demonstrating its antiautophagic activity. We show that the expression of Us11 is able to block autophagy and autophagosome formation in both HeLa cells and fibroblasts. Furthermore, immediate-early expression of Us11 by an ICP34.5 deletion mutant virus is sufficient to render the cells resistant to PKR-induced and virus-induced autophagy. PKR expression and the PKR binding domain of Us11 are required for the antiautophagic activity of Us11. However, unlike ICP34.5, Us11 did not interact with Beclin 1. We suggest that the inhibition of autophagy observed in cells infected with HSV-1 results from the activity of not only ICP34.5 on Beclin 1 but also Us11 by direct interaction with PKR.