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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.

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.

One of the main functions of the autophagy pathway is to control infections. Intracellular micro-organisms or their products once internalized in the host cell can be directly degraded by autophagy, a process called xenophagy. Autophagy is also involved in other innate immune responses and participates to the adaptive immune system. In addition, several autophagy proteins play a role in the development of infectious diseases independently of their role in the autophagy pathway. To replicate efficiently, pathogens have therefore evolved to counteract this process or to exploit it to their own profit. The review focuses on the relationship between autophagy and micro-organisms, which is highly diverse and complex. Many research groups are now working on this topic to find new therapeutics and/or vaccines. Given the large number of data, we have addressed this subject through some representative examples.

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.

The processes of cell attachment and membrane fusion of Herpes Simplex Virus 1 involve many different envelope glycoproteins. Viral proteins gC and gD bind to cellular receptors. Upon binding, gD activates the gH/gL complex which in turn activates gB to trigger membrane fusion. Thus, these proteins must be located at the point of contact between cellular and viral envelopes to interact and allow fusion. Using super-resolution microscopy, we show that gB, gH/gL and most of gC are distributed evenly round purified virions. In contrast, gD localizes essentially as clusters which are distinct from gB and gH/gL. Upon cell binding, we observe that all glycoproteins, including gD, have a similar ring-like pattern, but the diameter of these rings was significantly smaller than those observed on cell-free viruses. We also observe that contrary to cell-free particles, gD mostly colocalizes with other glycoproteins on cell-bound particles. The differing patterns of localization of gD between cell-free and cell-bound viruses indicates that gD can be reorganized on the viral envelope following either a possible maturation of the viral particle or its adsorption to the cell. This redistribution of glycoproteins upon cell attachment could contribute to initiate the cascade of activations leading to membrane fusion., Author summary The envelopes of Herpesvirus particles contain a variety of different proteins that allow them to infect specific cell types. An essential core set of these proteins is designed to allow viral entry into the cell after adsorption by binding to specific receptors and ultimately inducing fusion between the viral and cellular membranes in a regulated way through a succession of interactions between receptor-binding and fusion-triggering viral proteins. We have identified here for the first time the localization patterns of these essential proteins at the surface of purified virions and we describe how their localization changes after cell attachment. These results illustrate how the dynamics of viral proteins at the surface of the viral particle could participate in optimizing the all-important process of cell binding and membrane fusion.

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 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.

One of the main functions of the autophagy pathway is to control infections. Intracellular micro-organisms or their products once internalized in the host cell can be directly degraded by autophagy, a process called xenophagy. Autophagy is also involved in other innate immune responses and participates to the adaptive immune system. In addition, several autophagy proteins play a role in the development of infectious diseases independently of their role in the autophagy pathway. To replicate efficiently, pathogens have therefore evolved to counteract this process or to exploit it to their own profit. The review focuses on the relationship between autophagy and micro-organisms, which is highly diverse and complex. Many research groups are now working on this topic to find new therapeutics and/or vaccines. Given the large number of data, we have addressed this subject through some representative examples.

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.

Macroautophagy is a vacuolar degradation pathway that terminates in the lysosomal compartment after formation of a cytoplasmic vacuole or autophagosome that engulfs macromolecules and organelles. The identification of ATG (autophagy-related) genes that are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of macroautophagy, and its roles in cell function, which extend far beyond degradation and quality control of the cytoplasm. Macroautophagy, which plays a major role in tissue homeostasis, is now recognized as contributing to innate and adaptive immune responses. Recently, several mediators of apoptosis have been shown to control macroautophagy. Deciphering the cross talk between macroautophagy and apoptosis probably should help increase understanding of the role of macroautophagy in human disease and is likely to be of therapeutic importance.

Autophagy and the ubiquitin proteasome system (UPS) mediate the degradation of cellular proteins. However, we are now realizing that autophagy can also be a selective process that degrades various organelles. Peter and co-workers discovered a selective autophagic pathway that targets ribosomes in Saccharomyces cerevisiae. This pathway, which they termed ribophagy, depends on Ubp3 ubiquitin protease and its partner Bre5. This is an important finding, because it suggests that the number of ribosomes can be adjusted to match the needs of the cell.

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.

Autophagy is activated early after human cytomegalovirus (HCMV) infection, but lateron the virus blocks autophagy. Here we characterized 2 HCMV proteins, TRS1 andIRS1, which inhibit autophagy during infection. Expression of either TRS1 or IRS1 wasable to block autophagy in different cell lines, independently of the EIF2S1 kinase,EIF2AK2/PKR. Instead, TRS1 and IRS1 interacted with the autophagy proteinBECN1/Beclin 1. We mapped the BECN1-binding domain (BBD) of IRS1 and TRS1 andfound it to be essential for autophagy inhibition. Mutant viruses that express only IRS1or TRS1 partially controlled autophagy, whereas a double mutant virus expressingneither protein stimulated autophagy. A mutant virus that did not express IRS1 andexpressed a truncated form of TRS1 in which the BBD was deleted, failed to controlautophagy. However, this mutant virus had similar replication kinetics as wild-type virus,suggesting that autophagy inhibition is not critical for viral replication. In fact, usingpharmacological modulators of autophagy and inhibition of autophagy by shRNAknockdown, we discovered that stimulating autophagy enhanced viral replication.Conversely, inhibiting autophagy decreased HCMV infection. Thus, our resultsdemonstrate a new proviral role of autophagy for a DNA virus Fil: Mouna, Lina. Université Paris Sud; Francia Fil: Hernandez, Eva. Université Paris Sud; Francia Fil: Bonte, Dorine. Centre National de la Recherche Scientifique; Francia Fil: Brost, Rebekka. Heinrich Pette Institute; Alemania Fil: Amazit, Larbi. Université Paris Sud; 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 Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Brune, Wolfram. Heinrich Pette Institute; Alemania Fil: Geballe, Adam P.. University of Washington; Estados Unidos Fil: Beau, Isabelle. Université Paris Sud; Francia Fil: Esclatine, Audrey. Université Paris Sud; Francia

Rotaviruses, which are the main cause of viral gastroenteritis in young children, induce structural and functional damages in infected mature enterocytes of the small intestine. To investigate a relationship between rotavirus infection and cell death by apoptosis, we used the human intestinal Caco-2 cell line. We demonstrated by several methods including TUNEL and ELISA detection of cytoplasmic histone-associated DNA fragments that the infection of fully differentiated Caco-2 cells by the RRV rotavirus strain induces apoptosis. Rotavirus infection leads to the loss of mitochondrial membrane potential and the release of cytochrome C from mitochondria. We showed that rotavirus-induced apoptosis was dependent of the multiplicity of infection and increased with time from 4 h to 24 h of infection. Flow cytometric analysis showed that DNA fragmentation occurs in productively infected cells, suggesting that rotavirus induces apoptosis by a direct mechanism. We also demonstrated that non-replicative RRV particles are not sufficient to induce apoptosis and viral gene expression seems required. Intracellular calcium plays a role in RRV-induced apoptosis because treatment with an intracellular calcium ion chelator (BAPTA-AM) partially inhibited apoptosis.

In HSV-1 (herpes simplex virus 1)-infected cells, the U(L)41 gene product carried with the virion has been shown to mediate the degradation of mRNA, leading to the shut-off of cellular protein synthesis. Analysis of the RNAs accumulating in cells infected with HSV-1 revealed the accumulation of RNAs encoding numerous cellular proteins both associated with and independent of activation of the NF-kappaB (nuclear factor kappaB) pathway. Studies on the activation of NF-kappaB and the expression and fate of selected cellular transcripts revealed the following. (i) In HSV-1-infected cells, NF-kappaB is activated by activated protein kinase R. Furthermore, the blockade of NF-kappaB translocation by suppression of protein kinase R activation does not render the cell more susceptible to apoptosis induced by viral gene expression. (ii) A number of mRNA up-regulated in infected cells [e.g. IkappaBalpha (inhibitory kappaBalpha), the immediate-early response protein IEX-1 and c-fos] are partially degraded and not translated. The degradation is U(L)41-dependent and results in deadenylation, endonucleolytic cleavage and 3'-5' degradation. The 5'-portion resulting from the endonucleolytic cleavage tends to linger in the infected cells. To date, the RNAs processed in this manner contained ARE (AU-rich elements) in their 3'-untranslated domains. RNAs lacking ARE were expressed and not degraded in this manner. (iii) Tristetraprolin and T-cell internal antigen-1, cellular proteins involved in the degradation of ARE-containing RNAs, are induced and activated in infected cells and tristetraprolin interacts physically with the U(L)41 protein.

In cells infected with herpes simplex virus 1, the RNA encoded by the stress-inducible immediate early response gene IEX-1 was up-regulated immediately after infection. However, the accumulated RNA was degraded 3′-5′, and the protein was detectable only at very early times after infection. The degradation was dependent on the U L 41 gene encoding the virion host shutoff (vhs) protein and resulted in the accumulation of truncated RNA containing the 5′-end portion of the transcript. IEX-1 contains an AU-rich element (ARE) in its 3′-untranslated domains known to regulate negatively the RNA lifespan. To examine the role of ARE in signaling the degradation, we compared the stability of several RNAs up-regulated during infection to WT virus. These were ARE-containing RNAs encoding IEX-1, c-fos, and IκBα and the non-ARE-containing RNAs GADD45β and tristetraprolin. We report that the ARE-containing RNAs exemplified by IEX-1 RNA are deadenylated and cleaved in the ARE within the 3′ UTR in a U L 41 -dependent manner. In contrast, Northern blot hybridizations and analyses of poly(A) tails revealed no evidence of degradation of GADD45β RNA. GADD45β protein was detected in WT virus-infected cells. These results indicate that the degradation of RNAs and the mechanism by which cellular RNAs are degraded are selective and may be sequence specific. The persistence of partially degraded ARE-containing RNAs may reflect specific targeting of the vhs proteins to the ARE and the modification of the RNA degradation machinery of the cell induced by the presence of the vhs protein.

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.

Human cytomegalovirus (CMV) causes severe disease in immunosuppressed patients and notably infects the gastrointestinal tract. To understand the interaction of CMV with intestinal epithelial cells, which are highly susceptible to CMV infection in vivo, we used the intestinal epithelial cell line Caco-2 and demonstrated that CMV enters predominantly through the basolateral surface of polarized Caco-2 cells. As shown by expression of all three classes of CMV proteins and by visualization of nucleocapsids by transmission electron microscopy, both poorly and fully differentiated Caco-2 cells were permissive to CMV replication. However, infection failed to produce infectious particles in Caco-2 cells, irrespective of the state of differentiation.

Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light of recent advances in our understanding of the formation of autophagosomes, which are double-membrane-bound vacuoles that sequester cytoplasmic cargos and deliver them to lysosomes. In most cases, this final step is preceded by a maturation step during which autophagosomes interact with the endocytic pathway. The discovery of AuTophaGy-related genes has greatly increased our knowledge about the mechanism responsible for autophagosome formation, and there has also been progress in the understanding of molecular aspects of autophagosome maturation. Finally, the regulation of autophagy is now better understood because of the discovery that the activity of Atg complexes is targeted by protein kinases, and owing to the importance of nuclear regulation via transcription factors in regulating the expression of autophagy genes.

Macroautophagy is a vacuolar degradation pathway that terminates in the lysosomal compartment. Macroautophagy is a multistep process involving: (1) signaling events that occur upstream of the molecular machinery of autophagy; (2) molecular machinery involved in the formation of the autophagosome, the initial multimembrane-bound compartment formed in the autophagic pathway; and (3) maturation of autophagosomes, which acquire acidic and degradative capacities. In this chapter we summarize what is known about the regulation of the different steps involved in autophagy, and we also discuss how macroautophagy can be manipulated using drugs or genetic approaches that affect macroautophagy signaling, and the subsequent formation and maturation of the autophagosomes. Modulating autophagy offers a promising new therapeutic approach to human diseases that involve macroautophagy.

Macroautophagy is a vacuolar degradation pathway that terminates in the lysosomal compartment. Macroautophagy is a multistep process involving: (1) signaling events that occur upstream of the molecular machinery of autophagy; (2) molecular machinery involved in the formation of the autophagosome, the initial multimembrane-bound compartment formed in the autophagic pathway; and (3) maturation of autophagosomes, which acquire acidic and degradative capacities. In this chapter we summarize what is known about the regulation of the different steps involved in autophagy, and we also discuss how macroautophagy can be manipulated using drugs or genetic approaches that affect macroautophagy signaling, and the subsequent formation and maturation of the autophagosomes. Modulating autophagy offers a promising new therapeutic approach to human diseases that involve macroautophagy.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that remains the major infectious cause of birth defects, as well as being an important opportunistic pathogen. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved process responsible for the degradation of cytoplasmic macromolecules, and the elimination of damaged organelles via a lysosomal pathway. This process is also triggered in organisms by stressful conditions and by certain diseases. Previous observations have suggested that autophagy (also known as xenophagy in this case) may contribute to innate immunity against viral infections. Recent studies on HSV-1, another herpesvirus, have shown that HSV-1 is able to avoid this cellular defense by means of a viral protein, ICP34.5, which antagonizes the host autophagy response. However, it was not known whether HCMV was also able to counteract autophagy. Here, we show that HCMV infection drastically inhibits autophagosome formation in primary human fibroblasts. Autophagy was assessed by GFP-LC3 redistribution, LC3-II and p62 accumulation and electron microscopy. Inhibition of autophagy occurred early in the infection by a mechanism involving viral protein(s). Indeed, only infected cells expressing viral proteins displayed a striking decrease of autophagy; whereas bystander, non-infected cells displayed a level of autophagy similar to that of control cells. HCMV activated the mTOR signaling pathway, and rendered infected cells resistant to rapamycin-induced autophagy. Moreover, infected cells also became resistant to the stimulation of autophagy by lithium chloride, an mTOR-independent inducer of autophagy. These findings suggest that HCMV has developed efficient strategies for blocking the induction of autophagy during infection.

Herpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the UL41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3′-to-5′ degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking UL41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking UL41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs-dependent degradation of ARE-containing RNAs correlates with the transactivation, cytoplasmic accumulation, and persistence of tristetraprolin in infected cells.

Cellular RNA extracted from quiescent human foreskin fibroblasts harvested at 1, 3, 7, or 12 h after infection was profiled on Affymetrix HG-U95Av2 arrays designed to detect 12,626 unique human transcripts. We also profiled RNA extracted from cells harvested at 1 and 7 h after infection with a mutant lacking the gene (ΔU L 41) encoding a protein ( vhs ) brought into cells by the virus and responsible for nonselective degradation of RNA early in infection. We report the following: ( i ) of the 12 tested genes, up-regulated at least 3-fold relative to the values of mock infected cells, 9 were confirmed by real-time PCR. The microchip assays analyses indicate that there were 475 genes up-regulated ≥3-fold. The up-regulated genes were clustered into 15 groups with respect to temporal pattern of transcript accumulation, and classified into 20 groups on the basis of their function. The preponderance of cellular genes up-regulated early in infection play a predominant role in transcription, whereas those up-regulated at later times respond to intracellular stress or concern themselves with the cell cycle and apoptosis. ( ii ) The number of genes up-regulated early in infection was higher in cells infected with the ΔU L 41 mutant. Conversely, more genes were down-regulated late in infection with wild-type virus than with mutant viruses. Both observations are compatible with the known function of the U L 41 gene product early in infection and with degradation of cellular RNAs in the absence of replenishment by de novo transcription of cellular genes.

Research in autophagy continues to accelerate,1and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response. ©2008 Landes Bioscience.

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 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.