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Purpose: Evaluation of marketed eye drops with or without trehalose, a nonreducing natural osmoprotector disaccharide, in autophagy modulation and its role in cell survival during desiccation. Methods: Eye drops containing either sodium hyaluronate (SH) (Hyabak®, Thea, France) or a combination of SH with trehalose (Thealose Duo®, Thea, France) were compared with control conditions to evaluate the ability to modulate autophagy in human epithelial cells in vitro. Autophagy was monitored using LC3, a marker of the autophagic machinery, by fluorescence microscopy and immunoblot analysis. Control and autophagy-deficient cells treated with eye drops were exposed to desiccation to mimic dry eyes and cell survival was evaluated by thiazolyl blue tetrazolium bromide (MTT) assay. Trehalose, a known autophagy inducer was used as a positive control. Results: Artificial tears containing SH with and without trehalose induce a complete autophagic flux, as indicated by an increase in the number of autophagosomes and autolysosomes, and the accumulation of the lipidated form of LC3 associated with complete autophagy. In addition, there was a synergistic effect of SH for autophagy induction when combined with trehalose, compared with each of the components alone. Survival of cells treated with both eye drops and exposed to desiccation was decreased in autophagy-deficient cells, demonstrating the essential role of autophagy on eye drop protection. Conclusions: Autophagic flux is induced by SH-containing eye drops, and this phenomenon is enhanced in combination with trehalose. We also demonstrated that autophagy induction is involved in the osmoprotective effects of both trehalose and SH-containing eye drops, to maintain epithelial cell homeostasis in dry conditions.

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.

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 interferon-stimulated genes (ISGs) whose products interact with viral components or alter cellular physiology to suppress viral replication (1–3). Among the ISGs that can inhibit influenza A virus (IAV) (4) are the myxovirus resistance 1 (MX1) GTPase (5) and IFN-induced transmembrane protein 3 (IFITM3) (6, 7). Here we use ectopic expression and gene knock-out 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 (HCV). 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 (vRNPs). NCOA7, therefore, provides a mechanism for immune regulation of endo-lysosomal 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.

Hepatitis C virus (HCV) is a unique enveloped virus that assembles as a hybrid lipoviral particle by tightly interacting with host lipoproteins. As a result, HCV virions display a characteristic low buoyant density and a deceiving coat, with host-derived apolipoproteins masking viral epitopes. We previously described methods to produce high-titer preparations of HCV particles with tagged envelope glycoproteins that enabled ultrastructural analysis of affinity-purified virions. Here, we performed proteomics studies of HCV isolated from culture media of infected hepatoma cells to define viral and host-encoded proteins associated with mature virions. Using two different affinity purification protocols, we detected four viral and 46 human cellular proteins specifically copurifying with extracellular HCV virions. We determined the C terminus of the mature capsid protein and reproducibly detected low levels of the viral nonstructural protein, NS3. Functional characterization of virion-associated host factors by RNAi identified cellular proteins with either proviral or antiviral roles. In particular, we discovered a novel interaction between HCV capsid protein and the nucleoporin Nup98 at cytosolic lipid droplets that is important for HCV propagation. These results provide the first comprehensive view to our knowledge of the protein composition of HCV and new insights into the complex virus-host interactions underlying HCV infection.

Autophagy is a well-described vacuolar degradative pathway which contributes to the homeostasis of the cell and to the response to environmental stresses. An increasing number of studies describe the modulation of autophagy by a large subset of viruses, either to avoid a host defense mechanism (potentially degrading viral particles before they can escape the host cell) or to hijack the autophagic machinery for their own benefit. Human cytomegalovirus (HCMV) has the capacity to fine tune the autophagic process during its own life cycle, and finally block the autophagic flux to promote its own replication and presumably avoid the degradation of cytoplasmic viral components. At least two anti-autophagic viral proteins encoded by HCMV have been described, but future studies may reveal new viral modulators. The purpose of this chapter is to discuss the significance of these findings in the light of the general knowledge on the autophagic pathway and the interplay between the viruses and this conserved homeostatic pathway. The importance of such information is supported by the recent publication of the 12-volume series (Elsevier) discussing autophagy.

Human cytomegalovirus modulates macroautophagy in two opposite directions. First, HCMV stimulates autophagy during the early stages of infection, as evident by an increase in the number of autophagosomes and a rise in the autophagic flux. This stimulation occurs independently of de novo viral protein synthesis since UV-inactivated HCMV recapitulates the stimulatory effect on macroautophagy. At later time points of infection, HCMV blocks autophagy (M. Chaumorcel, S. Souquere, G. Pierron, P. Codogno, and A. Esclatine, Autophagy 4:1–8, 2008) by a mechanism that requires de novo viral protein expression. Exploration of the mechanisms used by HCMV to block autophagy unveiled a robust increase of the cellular form of Bcl-2 expression. Although this protein has an anti-autophagy effect via its interaction with Beclin 1, it is not responsible for the inhibition induced by HCMV, probably because of its phosphorylation by c-Jun N-terminal kinase. Here we showed that the HCMV TRS1 protein blocks autophagosome biogenesis and that a TRS1 deletion mutant is defective in autophagy inhibition. TRS1 has previously been shown to neutralize the PKR antiviral effector molecule. Although phosphorylation of eIF2α by PKR has been described as a stimulatory signal to induce autophagy, the PKR-binding domain of TRS1 is dispensable to its inhibitory effect. Our results show that TRS1 interacts with Beclin 1 to inhibit autophagy. We mapped the interaction with Beclin 1 to the N-terminal region of TRS1, and we demonstrated that the Beclin 1-binding domain of TRS1 is essential to inhibit autophagy.

In the version of Supplementary Fig. 5a originally published with this Letter, the authors mistakenly duplicated images of LAMP1 staining in place of CD63 staining; this has now been amended to the correct version shown below.

Autophagy is an evolutionarily conserved vacuolar mechanism for the autodigestion of recycling cellular components. It is involved in cellular homeostasis, and in the response to stressful environmental conditions. Autophagy also plays an important role during viral infections, when it may act either as part of the immune defense system, or as a proviral mechanism. Numerous viruses have developed mechanisms that modulate autophagy. In this chapter, we focus on the most recent data from the Herpesviridae family, and in particular the autophagy-regulating proteins that are encoded by these viruses. For several of these viruses, autophagy contributes to the host antiviral defenses, either by enhancing innate immunity or by helping antigen presentation. In order to counteract autophagy, herpesviruses have evolved various proteins that are able to inhibit this cellular mechanism. We highlight the four main steps in the autophagic process that are targeted by herpesvirus proteins. However, herpesviruses also encode proteins that activate autophagy at specific points in the virus life cycle, i.e., during lytic replication, latency, or reactivation. Interestingly, autophagy can be triggered not only by proteins, but also by the viral genome. Studies of autophagy regulation by herpesviruses help to give us a clearer understanding of the positive or negative impact of autophagy on viral infection, and on the long-term persistence of the virus in the host organism.

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.

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.