HIV-1 Env induces pexophagy and an oxidative stress leading to uninfected CD4+ T cell death.

The immunodeficiency observed in HIV-1-infected patients is mainly due to uninfected bystander CD4+ T lymphocyte cell death. The viral envelope glycoproteins (Env), expressed at the surface of infected cells, play a key role in this process. Env triggers macroautophagy/autophagy, a process necessary for subsequent apoptosis, and the production of reactive oxygen species (ROS) in bystander CD4+ T cells. Here, we demonstrate that Env-induced oxidative stress is responsible for their death by apoptosis. Moreover, we report that peroxisomes, organelles involved in the control of oxidative stress, are targeted by Env-mediated autophagy. Indeed, we observe a selective autophagy-dependent decrease in the expression of peroxisomal proteins, CAT and PEX14, upon Env exposure; the downregulation of either BECN1 or SQSTM1/p62 restores their expression levels. Fluorescence studies allowed us to conclude that Env-mediated autophagy degrades these entire organelles and specifically the mature ones. Together, our results on Env-induced pexophagy provide new clues on HIV-1-induced immunodeficiency.Abbreviations: Ab: antibodies; AF: auranofin; AP: anti-proteases; ART: antiretroviral therapy; BafA1: bafilomycin A1; BECN1: beclin 1; CAT: catalase; CD4: CD4 molecule; CXCR4: C-X-C motif chemokine receptor 4; DHR123: dihydrorhodamine 123; Env: HIV-1 envelope glycoproteins; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GFP-SKL: GFP-serine-lysine-leucine; HEK: human embryonic kidney; HIV-1: type 1 human immunodeficiency virus; HTRF: homogeneous time resolved fluorescence; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NAC: N-acetyl-cysteine; PARP: poly(ADP-ribose) polymerase; PEX: peroxin; ROS: reactive oxygen species; siRNA: small interfering ribonucleic acid; SQSTM1/p62: sequestosome 1.

HIV-1 Vpr inhibits autophagy during the early steps of infection of CD4 T cells.

BACKGROUND INFORMATION: Autophagy is induced during HIV-1 entry into CD4 T cells by the fusion of the membranes triggered by the gp41 envelope glycoprotein. This anti-HIV-1 mechanism is inhibited by the viral infectivity factor (Vif) neosynthesized after HIV-1 integration to allow viral replication. However, autophagy is very rapidly controlled after HIV-1 entry by a still unknown mechanism. As HIV-1 viral protein R (Vpr) is the only auxiliary protein found within the virion in substantial amount, we studied its capability to control the early steps of HIV-1 envelope-mediated autophagy. RESULTS: We demonstrated that ectopic Vpr inhibits autophagy in both the Jurkat CD4 T cell line and HEK.293T cells. Interestingly, Vpr coming from the virus also blocks autophagy in CD4 T cells, the main cell target of HIV-1. Furthermore, Vpr decreases the expression level of two essential autophagy proteins (ATG), LC3B and Beclin-1, and an important autophagy-related protein, BNIP3 as well as the level of their mRNA. We also demonstrated in HEK.293T cells that Vpr degrades the FOXO3a transcription factor through the ubiquitin proteasome system. CONCLUSION: Vpr, the only well-expressed HIV-1 auxiliary protein incorporated into viruses, is able to negatively control autophagy induced during HIV-1 entry into CD4 T cells. SIGNIFICANCE: We provide insights of how HIV-1 controls autophagy very early after its entry into CD4 T cells and discovered a new function of Vpr. These results open the route to a better understanding of the roles of Vpr during HIV-1 infection through FOXO3a degradation and could be important to consider additional therapies that counteract the role of Vpr on autophagy.

[LAP (LC3-associated phagocytosis): phagocytosis or autophagy?] / La phagocytose associée à LC3 (LAP) - Phagocytose ou autophagie ?

Phagocytosis and macroautophagy, named here autophagy, are two essential mechanisms of lysosomal degradation of diverse cargos into membrane structures. Both mechanisms are involved in immune regulation and cell survival. However, phagocytosis triggers degradation of extracellular material whereas autophagy engulfs only cytoplasmic elements. Furthermore, activation and maturation of these two processes are different. LAP (LC3-associated phagocytosis) is a form of phagocytosis that uses components of the autophagy pathway. It can eliminate (i) pathogens, (ii) immune complexes, (iii) threatening neighbouring cells, dead or alive, and (iv) cell debris, such as POS (photoreceptor outer segment) and the midbody released at the end of mitosis. Cells have thus optimized their means of elimination of dangerous components by sharing some fundamental elements coming from the two main lysosomal degradation pathways.

TITLE: La phagocytose associée à LC3 (LAP) - Phagocytose ou autophagie ? ABSTRACT: Phagocytose et macroautophagie, appelée ici autophagie, sont deux mécanismes essentiels de dégradation lysosomale de divers cargos englobés dans des structures membranaires. Ils sont tous deux impliqués dans la régulation du système immunitaire et la survie cellulaire. Cependant, la phagocytose permet l'ingestion de matériel extracellulaire alors que l'autophagie dégrade des composants intra-cytoplasmiques, avec des mécanismes d'activation et de maturation différents. La LAP (LC3-associated phagocytosis) est une forme particulière de phagocytose qui utilise certains éléments de l'autophagie. Elle permet l'élimination de pathogènes, de complexes immuns, de cellules avoisinantes, mortes ou vivantes, constituant un danger pour l'organisme, et de débris cellulaires, tels que les segments externes des photorécepteurs (POS, photoreceptor outer segment), ou la pièce centrale du pont intercellulaire produit en fin de mitose. Les cellules ont ainsi « optimisé ¼ leurs moyens d'éliminer les composés potentiellement dangereux en partageant certains éléments essentiels des deux voies de dégradation lysosomale.

HIV-1 envelope glycoproteins isolated from Viremic Non-Progressor individuals are fully functional and cytopathic.

In untreated HIV-1-infected individuals, viremia is positively associated with disease progression. However, some viremic non progressors (VNPs) individuals show paradoxical high CD4+ T cell counts. HIV-1 envelope glycoprotein complex (Env) is a major cytopathic determinant in viral replication; therefore, we have deeply characterized Env function in this rare clinical phenotype. Full-length Env clones isolated from individuals with Viral Load (VL) > 10,000 copies/mL classified as VNPs (n = 15) or rapid progressors (RPs, n = 17) were geno- and phenotypically analyzed by determining diversity, expression, CD4 binding/signaling, fusogenicity, infectivity and autophagy induction. Selected Env clones from VNPs and RPs (n = 32) showed similar expression, fusion and infection abilities. Env clones from both groups showed similar affinity for CD4 during cell-to-cell transmission and consistently induced similar levels of CD4 signaling, measured by α-tubulin acetylation. Moreover, we demonstrate for the first time that primary Env clones from VNP and RP induce autophagy in uninfected cells and that this feature correlated with fusogenic capacity but was unrelated to disease progression. In conclusion, our data suggest that Env clones from VNP individuals are fully functional. Therefore, the paradoxical CD4+ T cell count stability coexisting with high levels of viral replication is unrelated to Env function.

[Autophagy, ATG proteins and infectious diseases]. / Autophagie, protéines ATG et maladies infectieuses.

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.

Brief Report: Impaired CD4 T-Cell Response to Autophagy in Treated HIV-1-Infected Individuals.

Autophagy restricts infection of CD4 T lymphocytes by HIV-1, but little is known about autophagy in treated HIV-1-infected individuals. We have analyzed the capability of CD4 T cells from aviremic-treated individuals to trigger autophagy and correlated this response with parameters known to be important for immunological recovery. Autophagy was significantly decreased in CD4 T cells from HIV-1-treated individuals compared with uninfected controls, and this defective autophagic response was more pronounced in individuals with poor CD4 T-cell recovery, suggesting a link between impaired autophagy in CD4 T cells and chronic immunological defects that remain in treated HIV infection.

The HDAC6/APOBEC3G complex regulates HIV-1 infectiveness by inducing Vif autophagic degradation.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) has evolved a complex strategy to overcome the immune barriers it encounters throughout an organism thanks to its viral infectivity factor (Vif), a key protein for HIV-1 infectivity and in vivo pathogenesis. Vif interacts with and promotes "apolipoprotein B mRNA-editing enzyme-catalytic, polypeptide-like 3G" (A3G) ubiquitination and subsequent degradation by the proteasome, thus eluding A3G restriction activity against HIV-1. RESULTS: We found that cellular histone deacetylase 6 (HDAC6) directly interacts with A3G through its C-terminal BUZ domain (residues 841-1,215) to undergo a cellular co-distribution along microtubules and cytoplasm. The HDAC6/A3G complex occurs in the absence or presence of Vif, competes for Vif-mediated A3G degradation, and accounts for A3G steady-state expression level. In fact, HDAC6 directly interacts with and promotes Vif autophagic clearance, thanks to its C-terminal BUZ domain, a process requiring the deacetylase activity of HDAC6. HDAC6 degrades Vif without affecting the core binding factor ß (CBF-ß), a Vif-associated partner reported to be key for Vif- mediated A3G degradation. Thus HDAC6 antagonizes the proviral activity of Vif/CBF-ß-associated complex by targeting Vif and stabilizing A3G. Finally, in cells producing virions, we observed a clear-cut correlation between the ability of HDAC6 to degrade Vif and to restore A3G expression, suggesting that HDAC6 controls the amount of Vif incorporated into nascent virions and the ability of HIV-1 particles of being infectious. This effect seems independent on the presence of A3G inside virions and on viral tropism. CONCLUSIONS: Our study identifies for the first time a new cellular complex, HDAC6/A3G, involved in the autophagic degradation of Vif, and suggests that HDAC6 represents a new antiviral factor capable of controlling HIV-1 infectiveness by counteracting Vif and its functions.

Autophagy restricts HIV-1 infection by selectively degrading Tat in CD4+ T lymphocytes.

UNLABELLED: Autophagy is a ubiquitous mechanism involved in the lysosomal-mediated degradation of cellular components when they are engulfed in vacuoles called autophagosomes. Autophagy is also recognized as an important regulator of the innate and adaptive immune responses against numerous pathogens, which have, therefore, developed strategies to block or use the autophagy machinery to their own benefit. Upon human immunodeficiency virus type 1 (HIV-1) infection, viral envelope (Env) glycoproteins induce autophagy-dependent apoptosis of uninfected bystander CD4(+) T lymphocytes, a mechanism likely contributing to the loss of CD4(+) T cells. In contrast, in productively infected CD4(+) T cells, HIV-1 is able to block Env-induced autophagy in order to avoid its antiviral effect. To date, nothing is known about how autophagy restricts HIV-1 infection in CD4(+) T lymphocytes. Here, we report that autophagy selectively degrades the HIV-1 transactivator Tat, a protein essential for viral transcription and virion production. We demonstrated that this selective autophagy-mediated degradation of Tat relies on its ubiquitin-independent interaction with the p62/SQSTM1 adaptor. Taken together, our results provide evidence that the anti-HIV effect of autophagy is specifically due to the degradation of the viral transactivator Tat but that this process is rapidly counteracted by the virus to favor its replication and spread. IMPORTANCE: Autophagy is recognized as one of the most ancient and conserved mechanisms of cellular defense against invading pathogens. Cross talk between HIV-1 and autophagy has been demonstrated depending on the virally challenged cell type, and HIV-1 has evolved strategies to block this process to replicate efficiently. However, the mechanisms by which autophagy restricts HIV-1 infection remain to be elucidated. Here, we report that the HIV-1 transactivator Tat, a protein essential for viral replication, is specifically degraded by autophagy in CD4(+) T lymphocytes. Both Tat present in infected cells and incoming Tat secreted from infected cells are targeted for autophagy degradation through a ubiquitin-independent interaction with the autophagy receptor p62/SQSTM1. This study is the first to demonstrate that selective autophagy can be an antiviral process by degrading a viral transactivator. In addition, the results could help in the design of new therapies against HIV-1 by specifically targeting this mechanism.

HIV-1 viral infectivity factor interacts with microtubule-associated protein light chain 3 and inhibits autophagy.

OBJECTIVE: Autophagy, an important antiviral process triggered during HIV-1 entry by gp41-dependent membrane fusion, is repressed in infected CD4+ T cells by an unknown mechanism. The aim of this study was to identify the role of viral infectivity factor (Vif) in the autophagy blockade. DESIGN/METHODS: To determine the role of Vif in autophagy inhibition, we used cell lines that express CD4 and CXCR4 and primary CD4+ T cells. Pull-down experiments, immunoprecipitation assays and computational analyses were performed to analyze the interaction between Vif and microtubule-associated protein light chain 3B (LC3B), a major autophagy component, in presence or absence of the antiviral host factor apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G), after HIV-1 infection or ectopic expression of Vif. Autophagy was analyzed after infection by viruses expressing Vif (NL4.3) or not (NL4.3[DELTA]Vif), or after exogenous Vif expression. RESULTS: We demonstrate that the C-terminal part of Vif interacts directly with LC3B, independently of the presence of APOBEC3G.Vif binds to pro-LC3 and autophagy-related protein 4-cleaved LC3 forms, and glycine 120, the amino acid conjugated to phosphatidylethanolamine on autophagosomes, is required. Importantly, we evidence that Vif inhibits autophagy during HIV-1 infection. Indeed, autophagy is detected in target cells infected by NL4.3[DELTA]Vif, but prevented in cells infected by NL4.3. Furthermore, autophagy triggered in NL4.3[DELTA]Vif-infected cells is inhibited when Vif is expressed in trans but is still active when target cells express a mutant of Vif that binds weakly to LC3B. CONCLUSION: Our study unveils that Vif inhibits autophagy independently of its action on APOBEC3G and, therefore, suggest a new function of this viral protein in restricting innate antiviral mechanisms.

High-resolution live-cell imaging reveals novel cyclin A2 degradation foci involving autophagy.

Cyclin A2 is a key player in the regulation of the cell cycle. Its degradation in mid-mitosis relies on the ubiquitin-proteasome system (UPS). Using high-resolution microscopic imaging, we find that cyclin A2 persists beyond metaphase. Indeed, we identify a novel cyclin-A2-containing compartment that forms dynamic foci. Förster (or fluorescence) resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) analyses show that cyclin A2 ubiquitylation takes place predominantly in these foci before spreading throughout the cell. Moreover, inhibition of autophagy in proliferating cells induces the stabilisation of a subset of cyclin A2, whereas induction of autophagy accelerates the degradation of cyclin A2, thus showing that autophagy is a novel regulator of cyclin A2 degradation.

Macroautophagy Regulation during HIV-1 Infection of CD4+ T Cells and Macrophages.

Autophagy is an intracellular mechanism whereby pathogens, particularly viruses, are destroyed in autolysosomes after their entry into targets cells. Therefore, to survive and replicate in host cells, viruses have developed multiple strategies to either counteract or exploit this process. The aim of this review is to outline the known relationships between HIV-1 and autophagy in CD4+ T lymphocytes and macrophages, two main HIV-1 cell targets. The differential regulation of autophagy in these two cell-types is highlighted and its potential consequences in terms of viral replication and physiopathology discussed.

Inhibition of HIV-1 replication with stable RNAi-mediated knockdown of autophagy factors.

Autophagy is a cellular process leading to the degradation of cytoplasmic components such as organelles and intracellular pathogens. It has been shown that HIV-1 relies on several components of the autophagy pathway for its replication, but the virus also blocks late steps of autophagy to prevent its degradation. We generated stable knockdown T cell lines for 12 autophagy factors and analyzed the impact on HIV-1 replication. RNAi-mediated knockdown of 5 autophagy factors resulted in inhibition of HIV-1 replication. Autophagy analysis confirmed a specific defect in the autophagy pathway for 4 of these 5 factors. We also scored the impact on cell viability, but no gross effects were observed. Upon simultaneous knockdown of 2 autophagy factors (Atg16 and Atg5), an additive inhibitory effect was scored on HIV-1 replication. Stable knockdown of several autophagy factors inhibit HIV-1 replication without any apparent cytotoxicity. We therefore propose that targeting of the autophagy pathway can be a novel therapeutic approach against HIV-1.

HIV-1, ubiquitin and ubiquitin-like proteins: the dialectic interactions of a virus with a sophisticated network of post-translational modifications.

The modification of intracellular proteins by ubiquitin (Ub) and ubiquitin-like (UbL) proteins is a central mechanism for regulating and fine-tuning all cellular processes. Indeed, these modifications are widely used to control the stability, activity and localisation of many key proteins and, therefore, they are instrumental in regulating cellular functions as diverse as protein degradation, cell signalling, vesicle trafficking and immune response. It is thus no surprise that pathogens in general, and viruses in particular, have developed multiple strategies to either counteract or exploit the complex mechanisms mediated by the Ub and UbL protein conjugation pathways. The aim of this review is to provide an overview on the intricate and conflicting relationships that intimately link HIV-1 and these sophisticated systems of post-translational modifications.

IRGM is a common target of RNA viruses that subvert the autophagy network.

Autophagy is a conserved degradative pathway used as a host defense mechanism against intracellular pathogens. However, several viruses can evade or subvert autophagy to insure their own replication. Nevertheless, the molecular details of viral interaction with autophagy remain largely unknown. We have determined the ability of 83 proteins of several families of RNA viruses (Paramyxoviridae, Flaviviridae, Orthomyxoviridae, Retroviridae and Togaviridae), to interact with 44 human autophagy-associated proteins using yeast two-hybrid and bioinformatic analysis. We found that the autophagy network is highly targeted by RNA viruses. Although central to autophagy, targeted proteins have also a high number of connections with proteins of other cellular functions. Interestingly, immunity-associated GTPase family M (IRGM), the most targeted protein, was found to interact with the autophagy-associated proteins ATG5, ATG10, MAP1CL3C and SH3GLB1. Strikingly, reduction of IRGM expression using small interfering RNA impairs both Measles virus (MeV), Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1)-induced autophagy and viral particle production. Moreover we found that the expression of IRGM-interacting MeV-C, HCV-NS3 or HIV-NEF proteins per se is sufficient to induce autophagy, through an IRGM dependent pathway. Our work reveals an unexpected role of IRGM in virus-induced autophagy and suggests that several different families of RNA viruses may use common strategies to manipulate autophagy to improve viral infectivity.

Autophagy in HIV-induced T cell death.

HIV infection leads to progressive CD4 T cell depletion, resulting in the development of AIDS. The mechanisms that trigger T cell death after HIV infection are still not fully understood, but a lot of data indicate that apoptosis of uninfected CD4 lymphocytes plays a major role. HIV directly modulates cell death using various strategies in which several viral proteins, in particular the envelope glycoproteins (Env), play an essential role. Importantly, Env, expressed on infected cells, triggers autophagy in uninfected CD4 T cells, leading to their apoptosis. Furthermore, HIV, like other viruses, has evolved strategies to inhibit this autophagic process in HIV-infected cells. This discovery further increases the level of complexity of the cellular processes involved in HIV-induced pathology. Interestingly, HIV protease inhibitors, currently used in highly active antiretroviral therapy (HAART), are able to induce autophagy in cancer cells, leading to a recent repositioning of these drugs as anticancer agents. This review presents an overview of the relationship between HIV, HAART, and autophagy.

Differential role of autophagy in CD4 T cells and macrophages during X4 and R5 HIV-1 infection.

BACKGROUND: HIV-1 can infect and replicate in both CD4 T cells and macrophages. In these cell types, HIV-1 entry is mediated by the binding of envelope glycoproteins (gp120 and gp41, Env) to the receptor CD4 and a coreceptor, principally CCR5 or CXCR4, depending on the viral strain (R5 or X4, respectively). Uninfected CD4 T cells undergo X4 Env-mediated autophagy, leading to their apoptosis, a mechanism now recognized as central to immunodeficiency. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that autophagy and cell death are also induced in the uninfected CD4 T cells by HIV-1 R5 Env, while autophagy is inhibited in productively X4 or R5-infected CD4 T cells. In contrast, uninfected macrophages, a preserved cell population during HIV-1 infection, do not undergo X4 or R5 Env-mediated autophagy. Autophagosomes, however, are present in macrophages exposed to infectious HIV-1 particles, independently of coreceptor use. Interestingly, we observed two populations of autophagic cells: one highly autophagic and the other weakly autophagic. Surprisingly, viruses could be detected in the weakly autophagic cells but not in the highly autophagic cells. In addition, we show that the triggering of autophagy in macrophages is necessary for viral replication but addition of Bafilomycin A1, which blocks the final stages of autophagy, strongly increases productive infection. CONCLUSIONS/SIGNIFICANCE: Taken together, our data suggest that autophagy plays a complex, but essential, role in HIV pathology by regulating both viral replication and the fate of the target cells.

HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells.

Cell-expressed HIV-1 envelope glycoproteins (gp120 and gp41, called Env) induce autophagy in uninfected CD4 T cells, leading to their apoptosis, a mechanism most likely contributing to immunodeficiency. The presence of CD4 and CXCR4 on target cells is required for this process, but Env-induced autophagy is independent of CD4 signaling. Here we demonstrate that CXCR4-mediated signaling pathways are not directly involved in autophagy and cell death triggering. Indeed, cells stably expressing mutated forms of CXCR4, unable to transduce different Gi-dependent and -independent signals, still undergo autophagy and cell death after coculture with effector cells expressing Env. After gp120 binding to CD4 and CXCR4, the N terminus fusion peptide (FP) of gp41 is inserted into the target membrane, and gp41 adopts a trimeric extended pre-hairpin intermediate conformation, target of HIV fusion inhibitors such as T20 and C34, before formation of a stable six-helix bundle structure and cell-to-cell fusion. Interestingly, Env-mediated autophagy is triggered in both single cells (hemifusion) and syncytia (complete fusion), and prevented by T20 and C34. The gp41 fusion activity is responsible for Env-mediated autophagy since the Val2Glu mutation in the gp41 FP totally blocks this process. On the contrary, deletion of the C-terminal part of gp41 enhances Env-induced autophagy. These results underline the major role of gp41 in inducing autophagy in the uninfected cells and indicate that the entire process leading to HIV entry into target cells through binding of Env to its receptors, CD4 and CXCR4, is responsible for autophagy and death in the uninfected, bystander cells.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.

Research in autophagy continues to accelerate,(1) and 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.(2,3) There 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.

Regulation of macroautophagy by mTOR and Beclin 1 complexes.

Macroautophagy or autophagy is a vacuolar degradative pathway terminating in the lysosomal compartment after forming a cytoplasmic vacuole or autophagosome that engulfs macromolecules and organelles. The original discovery that ATG (AuTophaGy related) genes in yeast are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of autophagy, and its role in cell function that extends far beyond non-selective degradation. The regulation of autophagy by signaling pathways overlaps the control of cell growth, proliferation, cell survival and death. The evolutionarily conserved TOR (Target of Rapamycin) kinase complex 1 plays an important role upstream of the Atg1 complex in the control of autophagy by growth factors, nutrients, calcium signaling and in response to stress situations, including hypoxia, oxidative stress and low energy. The Beclin 1 (Atg6) complex, which is involved in the initial step of autophagosome formation, is directly targeted by signaling pathways. Taken together, these data suggest that multiple signaling checkpoints are involved in regulating autophagosome formation.