key: cord-017326-1caeui30
authors: Seay, Montrell; Dinesh-Kumar, Savithramma; Levine, Beth
title: Digesting Oneself and Digesting Microbes: Autophagy as a Host Response to Viral Infection
date: 2005
journal: Modulation of Host Gene Expression and Innate Immunity by Viruses
DOI: 10.1007/1-4020-3242-0_11
sha: 
doc_id: 17326
cord_uid: 1caeui30

Although research in this area is still in a stage of infancy, it seems likely that the lysosomal degradation pathway of autophagy plays an evolutionarily conserved role in antiviral immunity. The interferon-inducible, antiviral PKR signaling pathway positively regulates autophagy, and both mammalian and plant autophagy genes restrict viral replication and protect against virus-induced cell death. Given this role of autophagy in innate immunity, it is not surprising that viruses have evolved numerous strategies to inhibit host autophagy. Different viral gene products can either modulate autophagy regulatory signals or directly interact with components of the autophagy execution machinery. Moreover, certain RNA viruses have managed to “co-apt” the autophagy pathway, selectively utilizing certain components of the dynamic membrane rearrangement system to promote their own replication inside the host cytoplasm. In addition to this newly emerging role of autophagy in innate immunity, autophagy plays an important role in many other fundamental biological processes, including tissue homeostasis, differentiation and development, cell growth control, and the prevention of aging. Accordingly, the inhibition of host autophagy by viral gene products has important implications not only for understanding mechanisms of immune evasion, but also for understanding novel mechanisms of viral pathogenesis. It will be interesting to dissect the role of viral inhibition of autophagy in acute, persistent, and latent viral replication, as well as in the pathogenesis of cancer and other medical diseases.

The cellular pathway of autophagy is as ancient as the origins of eukaryotic life. Derived from the Greek and meaning to eat ("phagy") oneself ("auto"), the term autophagy refers to a lysosomal pathway of selfdigestion, involving dynamic membrane rearrangement to sequester cargo for delivery to the lysosome, where the sequestered material is degraded and recycled. For decades, it has been known that autophagy is the primary intracellular catabolic mechanism for the degradation and recycling of longlived cellular proteins and organelles. For decades, it has also been known that the recycling function of autophagy is an important adaptive response to nutrient deprivation and other forms of environmental stress. However, only recently have we discovered that autophagy may also be an important mechanism for the degradation of intracellular pathogens and that autophagy may also be important in cellular protection against the stress of microbial infection. Not surprisingly, we have also recently learned that some successful intracellular pathogens have devised strategies either to block host autophagy or to subvert the host autophagic process to foster their own replication. In this chapter, we will review recent progress in understanding the interrelationships between viruses, autophagy, and innate immunity (Figures 1 & 2) . 

Before discussing the interrelationships between viruses, autophagy, and innate immunity, we will provide a brief overview of the molecular and cell biology of autophagy. While this subject has been covered extensively in a recent book and numerous recent review articles 2-5 we will highlight the aspects of this subject that may have particular relevance for viral infections.

r r The process of autophagy was first described more than forty years ago, however, for many decades our understanding of autophagy was based largely on morphological observations from electron microscopy (reviewed in 6 ). The field has expanded considerably within the last 15 years after the cloning and molecular characterization of the yeast AuTophagGy (ATG)related genes (reviewed in 7 ) . The analysis of sequenced genomes of higher eukaryotes has identified ATG homologues in mammals, C. elegans, Drosophila, Dictyostelium, and plants, and many of these genes have been shown to be essential for autophagy function in higher eukaryotes (reviewed in 2 ) ( Table 1 ). In addition to the identification of the autophagy genes, significant progress has been made in the past decade in understanding some of the signaling events that regulate autophagy (reviewed in 8, 9 ) . Interestingly, some of the signaling molecules that regulate autophagy, as well as some of the autophagy genes, play a role in the host antiviral innate immune response (Table 1, Figure 2 ).

The initial step of autophagy is the formation and elongation of the isolation membrane. The isolation membrane invaginates and sequesters cytoplasmic constituents including mitochondria, endoplasmic reticulum (ER) and ribosomes, and the edges of the membrane fuse with each other to form a double-membrane structure called an autophagosome. The outer membrane of the autophagosome fuses to the lysosome/vacuole with subsequent delivery of the inner vesicle or autophagic body into the lumen of the degradative compartment. The source of the autophagosomal membrane is still unclear, but presently, it is thought that the preautophagosomal structure (PAS) acts as the site of vesicle formation during autophagy [10] [11] [12] . The PAS is thought to form de novo, but the source of the vesicle membrane is not known. It seems likely that the "typical" autophagosomes observed during viral infection that contain a mix of virions and self-cytoplasmic constituents, originate from the PAS. However, it is not yet known whether the PAS also serves as the site of vesicle formation for the formation of "atypical" autophagic-like double-membrane vesicles that function as replication sites for certain RNA viruses (e.g. poliovirus, mouse hepatitis virus, equine arterivirus) (reviewed in 13 ). More likely, these double-membrane vesicles arise directly from the endoplasmic reticulum (ER) 13 .

Autophagosomes are lipid-rich, protein-poor vesicles that vary in size and membrane thickness depending on the organism and cell type. The composition and abundance of proteins sequestered within autophagosomes reflects the relative composition and abundance of proteins in the surrounding cytoplasm 14 . This observation has led to the concept that autophagosomes indiscriminately sequester cytoplasmic content. However, in yeast, there are well-established pathways of specific autophagy, including the biosynthetic cytoplasm-to-vacuole targeting pathway and pexophagy (reviewed in 15 ), and in mammalian cells, mitochondria-specific autophagy has been reported 16 . Although molecular determinants of cargo recognition have been identified in yeast pathways of specific autophagy, virtually nothing is known about the specificity of cargo recognition in higher eukaryotes. In circumstances where there is degradation of viruses observed inside "typical" autophagosomes that also contain cellular constituents, the sequestration step may lack specificity. However, in circumstances where viruses utilize components of the autophagic machinery for the formation of "autophagic-like" double-membrane structures that exclusively contain viral constituents, the sequestration step is likely to have exquisite specificity. The identification of the viral and cellular determinants of this specificity will be an important advance in understanding the cell biology of these types of RNA virus infections and may eventually lead to the identification of novel antiviral therapeutic targets.

Autophagy is tightly regulated by nutritional, hormonal, and other environmental cues. It occurs as a cellular response to extracellular stimuli (e.g. nutrient starvation, hypoxia, overcrowding, high temperature, hormonal or chemotherapeutic treatment), and intracellular stimuli (e.g. accumulation of damaged, superfluous or unwanted organelles, accumulation of misfolded proteins, invasion of microorganisms). Although it is not yet known whether different stimuli act through parallel, convergent, or divergent pathways to trigger autophagy, significant progress has been made within the past decade in identifying different signaling molecules that function in the positive (e.g. eIF2 kinases, Class III PI-3 kinases, PTEN, death-associated protein kinases) or negative (e.g. Tor, insulin-like growth factor signals, Class I PI-3 kinase, Rho/Ras family of GTPases) regulation of autophagy (reviewed in 8, 9 ) .

The identification of a role for these signaling molecules in autophagy regulation has implications for understanding antiviral immunity, and more speculatively, generates hypotheses about novel principles of virus-host interactions. The recently defined evolutionarily conserved role of the eIF2 kinase signaling pathway in autophagy induction suggests that autophagy regulation may contribute to the antiviral function of the interferon-inducible eIF2 kinase, PKR. PKR and other eIF2 kinases induce a general translational arrest by phosphorylating the serine 51 residue of eIF2 (reviewed in 17 ) . Genetic studies in yeast and mammalian cells have also shown that the eIF2 kinase signaling pathway is required for starvation and herpes simplex virus-induced autophagy 18 . While further analyses are required to dissect the relative contributions of autophagy induction vs. translational arrest in mediating the antiviral effects of PKR, these findings link a new cellular function (i.e. autophagy) with interferon signaling.

Although the eIF2 kinase signaling pathway is the only as-of-yet defined autophagy regulatory signaling pathway that has known antiviral functions, it is interesting to note that most autophagy regulatory signals play a role in other important cellular processes, including cell growth control, cell death, and aging. Some of these effects may be the consequence of divergent downstream targets of these regulatory signals and some of these effects may be directly mediated through autophagy. As will be discussed below, given the evidence for a role of autophagy in innate immunity, it is likely that viruses have evolved different strategies to antagonize host f f autophagy, which, at least in the case of herpes simplex virus (see Sections 4.1 and 5) include the targeting of upstream autophagy regulatory signals f 18 . Therefore, it is tempting to speculate that some of the effects of viruses on cell growth control and cell death may be either direct or indirect consequences of the evolutionary pressure that viruses face to modulate host autophagy.

As one example, the insulin-like/Class I PI-3K/Akt signaling pathway inhibits autophagy 19, 20 , promotes oncogenesis (reviewed in 21 ) , and decreases lifespan (most likely through autophagy-inhibitory effects) 20 . Certain retroviruses have recruited the catalytic subunit of PI3-K and its downstream target Akt and these viral gene products function as oncoproteins (reviewed in 22 ) . The emerging link between these signaling molecules and autophagy inhibition raises the interesting hypothesis that the initial acquisition of these molecules by viruses was perhaps related to the selective advantage of autophagy inhibition in viral growth. In view of recent evidence supporting a role of autophagy in tumor suppression [23] [24] [25] [26] , the presence of these genes in retroviral genomes could contribute to oncogenesis at least, in part, through inhibition of autophagy signaling, as well as through modulation of other downstream pathways.

The ATG genes encode proteins important for responding to upstream signaling pathways as well as proteins needed for the generation, maturation, and recycling of autophagosomes (reviewed in 4, 5, 15, 27 ). The Atg proteins can be grouped into four functional groups, including a protein kinase cascade important for responding to upstream signals, a lipid kinase signaling complex important for vesicle nucleation, ubiquitin-like conjugation pathways important for vesicle expansion, and a recycling pathway important for the disassembly of Atg protein complexes from matured autophagosomes. The role of some of the Atg proteins, including ones that act in the lipid kinase signaling complex and in the ubiquitin-like conjugation pathways, has been studied in plant and mammalian viral infections (see Table 1 ).

Autophagy is a dynamic process that is tightly regulated by protein kinases and phosphatases. One of the first ATG genes identified in yeast, ATG1, encodes a serine/threonine kinase 28 . The Atg1 kinase maintains a weak interaction with a hyperphosphorylated Atg protein, Atg13, in nutrientrich conditions. Upon starvation conditions or stress, Atg13 is dephosphorylated resulting in a tighter association with Atg1 29 . Atg13 binding is essential for autophagy since atg13 mutants unable to bind to Atg1 are completely defective in autophagy 29 . Atg17, which is also thought to play a role in Atg1 activation, also interacts with Atg1 29 . Downstream targets of Atg1 have not been identified, although Atg1 interacts with other proteins independently of its kinase activity 30 . The upstream kinase, Tor (target of rapamycin), indirectly or directly results in Atg13 hyperphosphorylation, which is one presumptive mechanism by which Tor kinase inhibits autophagy. Of note, the Atg1 component of the yeast autophagy induction complex plays a conserved role in autophagy in higher eukaryotes. However, as-of-yet, the role of Atg1 in antiviral immunity has not been evaluated.

Lipid kinase signaling VPS34, which encodes a phosphatidylinositol-3 kinase (PI3-K), d d phosphorylates the 3' hydroxyl group inosotiol ring of phosphoinositides u u 31 . Although there is only one PI3-K in yeast, there are three classes of PI3-K in higher eukaryotes; class III PI3-K has been shown to be analogous to yeast VPS34. The importance of Class III PI3-K signaling in autophagy has been demonstrated pharmacologically and genetically. The nucleotide derivative, 3-methyladenine, inhibits Class III PI3-K activity and is widely used to inhibit autophagosome formation in mammalian cells 32, 33 . A null mutation in VPS34 causes defects in autophagosome formation in yeast 34, 35 and microinjection of an inhibitory antiVps34 antibody blocks autophagy in cultured mammalian cells 36 .

Vps34 functions through the association with other Atg proteins in a large complex that includes Vps15, Atg6/Vps30 and Atg14 35 . This complex is thought to be important in vesicle nucleation by mediating the localization of other Atg proteins at the PAS 10, 35 . Vps34 and Atg6/Vps30 are conserved in higher eukaryotes. Importantly, the mammalian (beclin 1) and plant (BECLIN 1 ( ( ) homologues of yeast ATG6/VPS30 have been the most extensively studied ATG genes in viral infections. As will be discussed in more detail below, mammalian beclin 1 restricts viral replication, protects against virus-induced cell death, and is a target of inhibition by different virally-encoded gene products [37] [38] [39] . Furthermore, both plant BECLIN 1 and its binding partner, Class III PI3-K/VPS34 prevent the spread of programmed cell death during the plant antiviral hypersensitive response 40 . Thus, the lipid kinase complex plays an evolutionarily conserved role in antiviral innate immunity.

Autophagic vesicle expansion and completion involves conjugation machinery analogous to the ubiquitin conjugation needed for proteasomemediated protein degradation. Autophagy utilizes an E1-like enzyme (Atg7), two E2-like enzymes (Atg10 and Atg3) that facilitate the conjugation, and activation and localization of different ubiquitin-like modifiers (Atg5 and t Atg8). The conjugation modification of Atg proteins is necessary for the formation of an autophagosome of appropriate size and shape 41 . However, the precise molecular functions of the conjugation reactions are not known and remain a critical unanswered question in autophagy research. The first conjugation system involves the lipidation of Atg8, a ubiquitinlike protein whose close mammalian homologues have three-dimensional structures very similar to ubiqutin [42] [43] [44] [45] . Both Atg8 and the mammalian homomlogue LC3 are cleaved post-translationally by the cysteine endopeptidase Atg4 46, 47 . The cleavage of Atg8/Lc3 is essential for conjugation and further maturation of the autophagosomes 48 . In yeast and mammalian systems, the cleaved Atg8/LC3 is immediately activated by Atg7, an E1-like enzyme; transferred to Atg3, an E2-like enzyme; and finally conjugated to the lipid molecule phosphatidylethanolamine (PE) 42, 47, 49, 50 .

The second ubiquitin-like reaction is the conjugation of Atg12 to Atg5. Atg12 is an ubiquitin-like protein that is activated by Atg7 (E1-like enzyme), transferred to Atg10 (E2-like enzyme), and subsequently conjugated to Atg5 through an isopeptide bond 41, 49 . The conjugation of Atg5 to Atg12 is necessary for autophagosome formation but not necessary for localization to the PAS 10 .

Almost all of the components of the autophagy machinery that participate in the protein conjugation systems have orthologs in at least some higher eukaryotes. However, only mammalian Atg5 has been studied in the context of its role in viral infections. The contrasting phenotypes of atg5 null cells infected with two different RNA viruses illustrate two distinct mechanisms by which viruses interact with the autophagic machinery. The murine coronavirus, mouse hepatitis virus, which replicates in association with double-membrane vesicles, has severely impaired growth in atg5 null embryonic stem (ES) cells 51 , suggesting that atg5 is required for the formation of coronavirus replication complexes. In contrast, the prototype alphavirus, Sindbis virus, replicates to higher titers in atg5 null murine embryonic fibroblasts (MEFs) than in wildtype controls 38 , suggesting that the autophagic machinery functions to restrict Sindbis virus replication.

In yeast, Atg proteins that act at the stage of vesicle formation are not associated with the completed autophagosome, with the exception of Atg8. This suggests that Atg proteins are retrieved at some point prior to, or upon, vesicle completion, and then reutilized in the generation of new autophagosomes. The process of recycling requires the action of Atg2 and Atg18, which allow the recycling of Atg9, the only transmembrane protein that is part of the autophagic machinery 52, 53 . Atg9 and Atg18 have orthologues in higher eukaryotes, but their function in antiviral responses has not been studied.

The unique association of Atg8/LC3 with the mature autophagosome has led to an important technical advance in autophagy research. Atg8/LC3 is presently the most widely used and reliable marker for labeling autophagosomes 10, 11, 20, 50, 54 and with the recent availability of transgenic mice that express GFP-tagged LC3 54 , it is now possible to study autophagy induction in vivo during viral infections.

In addition to its emerging role in innate immunity, autophagy plays a role in diverse other biological processes, including survival during starvation, differentiation and development, tissue homoeostasis, aging, cell growth control, and certain forms of programmed cell death. These biological functions of autophagy have been reviewed in detail elsewhere 2, 55, 56 . In this section, we will however, briefly discuss selected biological functions of autophagy that have relevance either to understanding the mechanisms by which autophagy protects cells against virus infections ( Figure 1 ) or to understanding the potential consequences for the host of viral evasion of autophagy ( Figure 3 ). 

Perhaps the primordial function of autophagy is its ability to recycle nutrients and help sustain life during periods of starvation. Several decades ago, starvation was noted to be a potent inducer of autophagy in rodent liver (reviewed in 57 ), leading to the hypothesis that autophagy is an adaptive response to starvation. Following the identification of the conserved autophagy genes, genetic studies in different species have confirmed that autophagy genes are required for the maintenance of eukaryotic life in the face of limited environmental nutrient supply. This principle was first demonstrated in yeast, i.e. all ATG gene mutant yeasts grow normally in nutrient rich conditions, but unlike wild-type yeasts, die rapidly during carbon or nitrogen starvation 28 . Similarly, Dictyostelium discoideum that lack ATG genes also grow normally in the presence of their food, nonpathogenic bacteria, but die rapidly when subjected to starvation 58, 59 . During nitrogen or starvation, atg7 and atg9 mutant plants display two phenotypes that are thought to result from a defective ability to mobilize nutrients through autophagic delivery, including enhanced chlorosis (yellowing of leaves due to a loss of chlorophyll) and accelerated leaf senescence 60, 61 . In addition, mammalian cells deleted of the autophagy genes, atg5 or beclin 1, also undergo accelerated cell death in response to starvation as compared to their wild-type counterparts 62 .

The pro-survival function of autophagy during starvation is thought to be related directly related to its ability to recycle nutrients to generate a sufficient pool of amino acids required for the synthesis of essential proteins. While the eIF2 kinase signaling pathway shuts off general translation during starvation, at least in yeast, Gcn2 signaling simultaneously stimulates the transcription of essential starvation response genes, including autophagy genes 63, 64 . Thus, this signaling pathway provides a coordinated method to effectively generate new amino acids by autophagy and redirect the host cell synthetic machinery to use its limited amino acid supply specifically for the synthesis of essential starvation response proteins.

Although a downstream transcription factor like yeast Gcn4 (which is downstream of yeast eIF2 and transcriptionally transactivates autophagy genes), has not yet been identified for mammalian PKR signaling initiated during virus infection, it seems likely that there are functionally homologous molecules that direct virus-infected cells to mount an adaptive and selective transcriptional and translational response during virus infection. Even in the absence of this postulated arm of PKR signaling, the mere recycling of nutrients in virus-infected cells would be predicted to have a beneficial function for the host. Although few studies have compared cellular amino acid pools during nutrient starvation and virus infection, acute viral replication involves the parasitism of not only the host cell's translational machinery, but also the host cell's translational building blocks. Therefore, it seems likely that acute viral replication induces what can be thought of as a state of "pseuodostarvation". According to this model, the prediction is that the nutrient recycling function of autophagy plays a similar protective function during viral infection as it plays during nutrient deprivation.

Differentiation and development both require cells to undergo significant phenotypic changes and must entail a mechanism for the breakdown and recycling of obsolete cellular components. Genetic studies have revealed an essential role for components of the autophagic machinery in differentiation and developmental processes in several different organisms, including sporulation in yeast, multicellular development in Dictyostelium, dauer development in C. elegans, and embryonic development in mice (reviewed in 2 ). In addition, the mammalian autophagy gene, beclin 1, appears to play a role in epithelial cell differentiation, since the mammary glands in beclin 1 heterozygous-deficient mice display striking morphological abnormalities 23 . Since viral gene products can inhibit the autophagy function of Beclin 1 (see below) and potentially other autophagy proteins 65 , it is possible that autophagy blockade represents a mechanism by which viruses can affect cellular differentiation. For example, the Bcl-2-like BHRF1 protein encoded by EBV binds to Beclin 1 39 blocks its autophagy function 39 , and also perturbs epithelial cell differentiation 66 . Further studies are needed to determine the role of Beclin 1 binding in the perturbation of epithelial cell differentiation by BHRF1, as well as to investigate the effects of other viral inhibitors of autophagy on cellular differentiation and multicellular development.

Many different viruses, including retroviruses, gammaherpesviruses, papillomaviruses, and hepatitis viruses are oncogenic. Studies of the mechanisms of viral oncogenesis have largely focused on the ability of viruses to alter mitogenic signaling, cell cycle regulation, and/or apoptosis. However, new evidence is emerging that autophagy plays a role in tumor suppression and that autophagy is antagonized by gene products encoded by certain oncogenic viruses. Accordingly, it will be important to evaluate the role of viral inhibition of autophagy in viral oncogenesis.

Normal cell growth requires a well-coordinated balance between the cell's biosynthetic machinery (e.g. protein synthesis and organelle biogenesis) and its degradative processes (e.g. protein degradation and organelle turnover). In the 1970's, it was first proposed that protein catabolism through autophagy is a major determinant of cell growth 67, 68 . According to this model, both cell mass and the rate of cell growth is a balance between the amount of protein synthesized and the amount of autophagic protein degradation. Although this model has received little attention in recent years, interest in the role of autophagy in cell growth control has reemerged in light of new biochemical and genetic links between autophagy and the negative regulation of tumorigenesis.

As stated above in Section 2.2, several different oncogenic signaling molecules, including members of the insulin signaling pathway (e.g. Class I PI-3K, Akt) and members of the Rho and Ras family of GTPases negatively regulate autophagy in mammalian cells and the PTEN tumor suppressor positively regulates autophagy (reviewed in 69 ). Furthermore, the autophagy inhibitor, Tor, is an important positive regulator of cell growth in diverse organisms, and the Tor inhibitor, rapamycin, has promising anti-tumor effects in human clinical trials (reviewed in 70 ). Oncogenic viruses have developed multiple different strategies to activate autophagy-inhibitory signaling pathways. These strategies include encoding viral oncoproteins that represent activated forms of the corresponding cellular proto-oncogene (reviewed in 22 ) or upregulating Rho/Ras or Class I PI-3K/Akt/TOR signaling through alternative mechanisms [71] [72] [73] [74] [75] [76] [77] .

Components of the autophagic machinery may also play a direct role in tumor suppression. The beclin 1 gene is monallelically deleted in a high percentage of cases of human breast, ovarian, and prostate cancer (reviewed in 78 ) and has tumor suppressor function in cultured mammary carcinoma cells 79, 80 . Heterozygous disruption of beclin 1 in mice increases the frequency of spontaneous tumorigenesis (including papillary lung carcinomas, B cell lymphomas, and hepatocellular carcinomas) and accelerates the development of hepatitis B virus-induced pre-malignant lesions 23, 24 . In addition, atg5 null ES cells are more tumorigenic in mice than their wild-type counterparts and result in teratomas that are less welldifferentiated 81 . Together, these findings lead to the concept that autophagy genes may represent a novel class of tumor suppressor genes and that genetic disruption of autophagy may represent a novel mechanism of tumorigenesis. As will be discussed in more detail below, two different classes of viral gene products have been identified thus far that bind to Beclin 1 and inhibit its autophagy function, including the alphaherpesvirus-encoded neurovirulence protein, HSV-1 ICP34.5, and the gammaherpesvirus-encoded Bcl-2-like proteins, KSHV vBcl-2 and EBV BHRF1 38, 39 . The gammaherpesviruses are oncogenic viruses that are etiologically linked to a variety of different malignancies, including lymphoma, nasopharyngeal carcinoma, and Kaposi's sarcoma. At present, the precise role of gammaherpesvirus Bcl-2like proteins in viral oncogenesis is uncertain. Nonetheless, given the welldefined role of cellular Bcl-2 in oncogenesis and the emerging evidence that Beclin 1 is a tumor suppressor protein, it will be important to evaluate whether viral Bcl-2 antagonism of Beclin 1 function plays a role in gammaherpesvirus oncogenesis. Of note, preliminary data indicates that KSHV may also encode other gene products that interact with other components of the autophagic machinery 65 . Thus, oncogenic gammaherpesviruses may have multiple mechanisms to disarm host autophagy. It will be of interest to determine whether other oncogenic DNA viruses, especially human papillomavirus, also directly inhibit the host autophagic machinery.

In many tissues in the adult organism (especially post-mitotic cells), protein and organelle turnover by autophagy plays an essential cellular homeostatic or housekeeping function, removing damaged or unwanted organelles and proteins. For many decades, it has been presumed that this homeostatic function of autophagy represents an anti-aging mechanism, perhaps by reducing reactive oxidative species and other toxic intracellular substances that contribute to genotoxic stress (reviewed in 82 ). The conserved effects of protein caloric restriction (a dietary inducer of autophagy) on lifespan extension has provided further fuel for this concept (reviewed in 83 ). Recent genetic studies, especially those performed in C. elegans, provide more direct evidence for a role of both autophagy regulatory signals and components of the autophagic machinery in anti-aging pathways. Loss-offunction mutations in autophagy-inhibitory insulin-like signaling pathway extend lifespan (reviewed in 84 ) , and inactivation of the C. elegans ortholog of yeast autophagy gene, ATG6/VPS30, blocks this lifespan extension 20 .

While the precise mechanisms by which autophagy extends lifespan are unknown, one theory is that autophagy selectively removes damaged mitochondria, resulting in decreased levels of intracellular reactive oxygen species and cellular protection against oxidative damage. Viral infections, as well as the inflammatory response to viral infections, can damage mitochondria and/or increase the intracellular generation of reactive oxygen species, and these effects may contribute to viral pathogenesis. For example, the mitochondrial damage that occurs in HIV infection (even in the absence of antiretroviral treatment) is thought to be a major contributory factor to the metabolic abnormalities and cardiomyopathy that occur in patients with AIDS (reviewed in 85 ). As another example, in a transgenic mouse model of hepatitic C virus (HCV) infection, oxidative stress in the absence of inflammation has been implicated in HCV-associated hepatocarcinogenesis 86 . Similarly, studies in transgenic mice and cultured cells indicate that pre-S1/S2 mutant hepatitis B virus surface antigens, which accumulate in late stages of HBV infection, cause oxidative stress and DNA damage 87 . Therefore, it is possible that the mechanisms by which autophagy functions as an anti-aging pathway may be relevant to potential roles that autophagy may play in protecting cells against adverse sequelae of oxidative stress during virus infection.

Diseases associated with an accumulation of misfolded and aggregated proteins, including neurodegenerative disorders and 1 -anti-trypsin liver disease, are associated with an increase in the accumulation of autophagic vacuoles (reviewed in 56 ). In these diseases, it has both been argued that autophagy plays a protective role (i.e. by removing protein aggregates and damaged mitochondria) and a pathologic role (i.e. by promoting liver dysfunction in 1 -anti-trypsin deficiency through excessive mitochondrial autophagy 88 or by promoting autophagic cell death). Although both of these roles may be operative in different diseases or even in different facets of a single disease, recent studies provide compelling evidence that autophagy plays a protective role against the toxic effects associated with protein aggregation. For example, mutant -synuclein (associated with early onset Parkinson's disease), and aggregate-prone proteins with polyglutamine expansions (associated with Huntington's disease) are targeted for autophagic degradation 88, 89 . Rapamycin, which stimulates autophagy, not only enhances the clearance of aggregate-prone proteins but also reduces the appearance of the aggregates and the cell death associated with expression of mutant Huntington's proteins 89 . Furthermore, induction of autophagy with rapamycin protects against neurodegeneration in both a fly and mouse model of Huntington's disease 90 .

Recent advances have also been made in understanding the mechanisms by which autophagy is induced in response to misfolded protein aggregates. In cell models, transgenic mice, and samples from human brains of patients with Huntington's disease, mTOR is sequestered into polyglutamine aggregates. This sequestration impairs its kinase activity, leading to induction of autophagy 90 . Although it has not yet been evaluated, it is likely that the accumulation of misfolded protein aggregates also induces autophagy through activation of the ER stress response, which is mediated by the eIF2 kinase, PKR-like ER resident kinase (PERK) 91, 92 , since other stress stimuli (e.g. starvation and virus infection) that activate other eIF2 kinases (e.g. Gcn2 and PKR) induce autophagy through this same signaling pathway 18 .

These observations are potentially relevant to understanding the role of autophagy in protection against virus-induced diseases in which protein misfolding and ER stress are thought to play pathogenetic roles. Similar to genetic neurodegenerative disorders, there is increasing evidence that murine retrovirus-associated spongiform-like neuronal degeneration is also associated with protein misfolding and ER stress. For example, viral envelope proteins from avirulent strains are processed normally and fail to induce ER stress, whereas envelope proteins from neurovirulent strains are misfolded and activate ER stress response pathways [93] [94] [95] . In addition, it has been proposed that the mechanism by which pre-S mutant HBV surface antigens promote oxidative stress and DNA damage is through the accumulation of misfolded mutant proteins and activation of ER stress 87, 96 . Thus, based on recent studies with non-viral associated neurodegenerative disorders, the prediction is that autophagy induction might be beneficial in attenuating diseases associated with misfolded viral proteins, such as retrovirus-associated spongiform encephalopathy and hepatitis B virusinduced liver damage. As a corollary, the possibility that these viruses might possess mechanisms to evade host autophagy could be an exacerbating factor in the pathogenesis of these infections.

An interesting question is whether viral protein aggregates trigger autophagy by mechanisms that are similar to those involved in autophagy induction initiated by cellular protein aggregates. Different viral glycoproteins are known to activate the ER stress-related eIF2 kinase, PERK 97,98 , although a role for PERK in autophagy induction has not yet been formally demonstrated. It is completely unknown, however, whether viral protein aggregates, like polyglutamine aggregates in Huntington's disease, sequester and thereby inactivate the autophagy-inhibitory kinase, mTOR. If so, this would represent a highly novel mechanism by which viruses trigger intracellular innate immune responses.

Autophagy is emerging as a newly described mechanism of antiviral innate immunity that is targeted by viral virulence gene products. Although there are not yet many published articles in this area, there are several observations that support this concept. First, during herpes simplex virus infection, the interferon-inducible antiviral PKR signaling pathway regulates the autophagic degradation of cellular and viral components 18, 99 . Second, mammalian autophagy execution genes, including beclin 1 and atg5, regulate Sindbis virus replication and Sindbis virus-induced cell death 37, 38 . Third, plant autophagy execution genes, including BECLIN 1, Class III PI3-K/VPS34, ATG3, and ATG7, restrict tobacco mosaic virus replication and limit the spread of cell death during the innate immune response 40 . In this section, each of these observations will be described in more detail.

The interferon-inducible dsRNA-dependent protein kinase R (PKR) plays an important role in innate immunity against viral infections. PKR activation leads to phosphorylation of the subunit of eukaryotic initiation factors 2 (eIF2 ) and a subsequent shutdown of host and viral protein synthesis and viral replication (reviewed in 100 ). To avoid this translational shutdown, many viruses have evolved different strategies to antagonize PKR function. These include interference with the dsRNA-mediated activation of PKR or PKR dimerization; blockade of the kinase catalytic site or PKR-f substrate interactions; alterations in the levels of PKR; direct regulation of eIF2 phosphorylation; and effects on components downstream of eIF2 (reviewed in 100,101 ). The importance of viral antagonism of PKR function in viral pathogenesis has been most clearly demonstrated using a herpes simplex virus type 1 (HSV-1) model system 102, 103 . The HSV-1 neurovirulence protein, ICP34.5, binds to protein phosphatase 1 and causes it to dephosphorylate eIF2 , thereby negating the activity of PKR 104, 105 . A neuroattenuated HSV-1 mutant lacking ICP34.5 exhibits wild-type replication and virulence in mice genetically lacking pkr 103 , proving that the ICP34.5 gene product mediates neurovirulence by antagonizing PKRdependent functions.

In addition to regulating host translation during viral infection, the PKR signaling pathway also regulates the autophagic degradation of host proteins 18 . As mentioned above, molecules in the yeast eIF2 kinase signaling pathway (e.g. the eIF2 kinase, Gcn2, the eIF2 Ser51 residue, and the transcriptional transactivator, Gcn4) are required for nitrogen starvationinduced autophagy. Interestingly, the autophagy defect of gcn2 null yeast can be rescued by mammalian pkr transformation 18 . Direct evidence that viruses can induce PKR-dependent autophagy has been provided by studies done with herpes simplex virus type 1 (HSV-1) infection in genetically engineered MEFs 18 . A herpes simplex virus (HSV-1) mutant virus lacking the ICP34.5 inhibitor of PKR signaling (termed HSV-1 34.5), but not wild-type HSV-1, is able to induce the autophagic breakdown of long-lived cellular proteins in wild-type MEFs. However, HSV-1 34.5 infection is not able to induce the autophagic breakdown of long-lived cellular proteins in MEFs lacking pkr or with a nonphosphorylatable mutation in Ser-51 of eIF2 . These findings indicate the PKR-dependent signaling events regulate the autophagic breakdown of host proteins during viral infection and that this function of PKR is antagonized by the HSV-1 ICP34.5 neurovirulence gene product. As discussed in section 3.1, the breakdown of cellular proteins may help protect host cells against the effects of "pseudostarvation" induced by viral infection.

More recent studies indicate that PKR-dependent signaling also regulates the breakdown of viral components during HSV-1 infection 99 . Ultrastructural analyses of wild-type and pkr-deficient MEFs and sympathetic neurons infected with wild-type and HSV-1 34.5 demonstrate that HSV-1 is degraded in autophagosomes by a pkr-dependent process. In wild-type cells infected with wild-type HSV-1, the majority of intracytoplasmic virions are a either randomly dispersed in the cytoplasm or are contained within "viral vacuoles," a structure thought to represent an important intermediate in the egress of HSV-1 from the nucleus out of the cell 106 . In contrast, in wild-type cells infected with HSV-1 34.5, most cytoplasmic virions are localized within autophagosomes that contain a mix of different cytoplasmic constituents ( Figure 4A ). Pkr-deficient MEFs or pkr-deficient neurons infected with HSV-1 34.5 have very few autophagosomes, and appear similar to wild-type HSV-1-infected wild-type cells, with randomly dispersed intracytoplasmic virions and numerous viral vacuoles. Together, these observations demonstrate that HSV-1 is degraded by autophagy, that HSV-1 ICP34.5 antagonizes the cellular autophagic degradation of HSV-1, and that this process requires PKR.

Recent biochemical analyses confirm that PKR signaling and HSV-1 ICP34.5 regulate viral protein degradation 99 . HSV-1 protein degradation is significantly accelerated in wild-type MEFs infected with HSV-1 t t 34.5 as compared to wild-type MEFs infected with wild-type HSV-1, indicating that ICP34.5 delays viral protein degradation. However, in autophagy-deficient pkr -/-MEFs or eIF2 S51A mutant MEFs, the rate of HSV-1 protein degradation is similar in HSV-1-and HSV-1 34.5-infected cells, indicating that HSV-1 protein degradation is positively regulated by the PKR signaling pathway. Thus, the eIF2 kinase-dependent autophagy signaling pathway not only regulates the degradation of long-lived cellular proteins but also regulates the degradation of viral proteins. Accordingly, it seems logical to speculate that PKR-dependent autophagic degradation of viruses inhibits viral replication and is an antiviral defense mechanism. However, the relative contributions of the effects of PKR on viral protein synthesis and the effects of PKR on viral protein degradation in the regulation of the HSV-1 replication have not yet been assessed. For this purpose, it will be necessary to selectively inhibit the autophagic protein degradation machinery and/or have HSV-1 mutant viruses that selectively block specific downstream functions regulated by PKR. It will also be important to determine whether PKR-dependent autophagy degrades and inhibits the replication of viruses other than HSV-1. Preliminary observations indicate that PKR-dependent autophagy does lead to the degradation of another neurotropic virus, the enveloped, positive-strand RNA virus in the alphavirus genus, Sindbis virus 107 ( Figure 4B ).

The role of PKR in antiviral innate immunity is well established, but PKR regulates many different cellular processes, and it is not yet known exactly what role autophagy induction plays in the antiviral effects of PKR. However, the concept that autophagy is important in innate immunity is more directly supported by studies involving components of the mammalian and plant autophagic machinery.

The first identified mammalian autophagy gene product, Beclin 1, was isolated in a yeast two-hybrid screen, in the context of studies of the mechanism by which the antiapoptotic protein, Bcl-2, protects mice against lethal Sindbis virus encephalitis 37 . Similar to the neuroprotective effects of Bcl-2 108 , enforced neuronal expression of wild-type Beclin 1 in a recombinant chimeric Sindbis virus vector reduces Sindbis virus replication, reduces Sindbis virus-induced apoptosis, and protects mice against lethal Sindbis virus encephalitis 37 . Mutations in the Bcl-2-binding domain of Beclin 1 and mutations in other regions of Beclin 1 that block its autophagy function also block its protective effects during Sindbis virus infection 37, 62 .

Thus, it appears that both the interaction with Bcl-2 and the autophagy function may be required for the antiviral effects of Beclin 1. However, further studies are required to define the precise mechanism of how Beclin 1 inhibits viral replication and virus-induced apoptosis and to identify the precise role of Bcl-2-Beclin 1 interactions in these processes.

Preliminary studies with beclin 1 null ES cells and atg5 null MEFs indicate a role for these two endogenous autophagy genes in innate immunity against Sindbis virus infection. Sindbis virus replicates to higher titers and results in accelerated death in beclin 1 null ES cells as compared to wild-type control ES cells and in atg5 null MEFs as compared to wild-type control MEFs 38 . In the case of Sindbis virus infection, it is not known whether the acceleration of virus-induced death in beclin 1 null or atg5 null cells is a result of increased viral replication or of independent effects of atg gene deficiency on cell death. However, studies comparing HSV-1 infection in wild-type and beclin 1 -/-ES cells suggest that beclin 1 can protect against virus-induced cell death in the absence of inhibitory effects on viral replication 38 .

Together, the studies of Sindbis virus infection in neurons overexpressing beclin 1 or in cultured cells lacking beclin 1 or atg5 demonstrate a role for mammalian autophagy genes in both restricting viral replication and in protection against virus-induced cell death. It will be important to examine whether other autophagy genes have a similar antiviral function and to examine whether autophagy genes also protect against other types of virus infections. The mechanisms by which autophagy genes exert protective effects in Sindbis virus infection are not yet known. Presumably, the autophagic breakdown of viral components leads to decrease viral yields. However, it is also possible that autophagy leads to the breakdown of cellular components required for viral replication. As noted above, the protective effects against cell death may be secondary to inhibitory effects on viral replication. Alternatively, the protective effects may relate to the nutrient recycling functions or "damage control" functions of autophagy, or in the case of beclin 1, to interactions between autophagy proteins and antiapoptotic pathways. It is also possible that autophagy may protect against cell death by degrading specific viral proteins (e.g. the Sindbis virus E1 and E2 envelope glycoprotein's 109 ) that are involved in triggering the apoptotic pathway.

The protective effects of beclin 1 and atg5 on virus-induced cell death are consistent with the "pro-survival" effects of autophagy during nutrient starvation and other forms of environmental stress. It is not yet clear how to reconcile these pro-survival effects with the view that autophagy represents an alternative form of non-apoptotic programmed cell death (reviewed in 2, 25, 110 ). While the primary basis for this view has been morphologic correlations between the presence of autophagic vacuoles and dying cells (reviewed in 2,111 ), recent genetic experiments establish a more direct role for autophagy genes in certain types of programmed cell death. Mammalian Atg7 and beclin 1 RNAi blocks cell death in fibroblast and macrophage cell lines treated with the caspase inhibitor, zVAD 112 , and atg 6 , 7 , and 12 RNAi blocks salivary gland destruction during Drosophila development 113 . Thus, the relationship between autophagy, cell survival, and cell death is quite complex and likely varies according to the cell type and the specific physiological or pathophysiological setting. It remains to be determined whether autophagy genes primarily play a protective role in preventing cell death during virus infection, or whether they also participate directly in cell death that is induced by certain viruses.

The plant homologue of beclin 1 also functions in antiviral host defense in plants. Similar to mammalian beclin 1, plant BECLIN 1 restricts viral replication; tobacco mosaic virus replication is increased in BECLIN 1silenced tobacco plants as compared to vector-treated control plants 40 . However, in contrast to mammalian beclin 1, which prevents the death of Sindbis virus-and HSV-1-infected cells, plant BECLIN 1 plays an interesting role in preventing the death of uninfected cells 40 .

In plants, the innate immune response during virus infection is characterized by a hypersensitive response which is a programmed cell death response that occurs around the infected areas (reviewed in [114] [115] [116] ). This hypersensitive response limits virus spread and confers pathogen resistance. It is triggered by a pathogen-encoded avirulence protein, which is recognized by a specific plant cognate resistance protein, termed an R protein. In plants lacking R proteins, there is uncontrolled virus spread and pathogen sensitivity.

A tobacco mosaic virus infection model has recently been used to study the role of autophagy genes in plant innate immunity. During tobacco mosaic virus infection, the hypersensitive response is triggered by tobacco mosaic virus protein, TMV p50, which is the helicase domain of the viral replicase 117 . TMV p50 is recognized by an R protein (called the N protein) of N. benthamiana, which is composed of a Toll/interleukin 1 receptor domain, a nucleotide binding domain, and a leucine-rich repeat domain 118 . Therefore, in tobacco plants containing the N protein (N ( ( +/+ N N ), there is local cell death in cells that are either infected with TMV or that express the TMV p50 protein but no systemic illness is observed. BECLIN 1 silencing in N +/+ N N tobacco plants reveals a striking role for this autophagy gene in limiting the spread of cell death during the hypersensitive response 40 . During TMV infection of N N plants, cell death begins as discreet and defined foci but continues to spread beyond the site of TMV infection until there is death of the entire inoculated leaf and other uninoculated leaves. A similar spreading cell death phenotype is seen with local expression of the TMV p50 protein, suggesting that the cell death occurs in response to a specific signal triggered by the pathogen encoded avirulence protein, and is not due to increased TMV replication or altered virus movement. In addition, in plants that lack the N gene, BECLIN 1 silencing does not lead to cell death after TMV infection. Moreover, BECLIN 1 silencing also results in spreading cell death during the hypersensitive response triggered by bacterially-encoded pathogen avirulence proteins. Together, these observations demonstrate that the spreading cell death phenotype in BECLIN 1-silenced plants is mediated by R gene-mediated innate immune responses and that BECLIN 1 is an important negative regulator of cell death during the plant innate immune response.

A similar role for other autophagy genes in limiting the spread of programmed cell death during TMV infection has also been observed. As discussed in Section 2.3.3, PI3-K/Vps34 is a protein that physically interacts with Atg6/Beclin 1 in yeast and mammals and is essential for proper autophagosome formation. Interestingly, silencing of the plant class III PI-3K/VPS34 in N +/+ N N plants results in a spreading cell death phenotype during TMV infection that is similar to that observed with BECLIN 1 silencing. As discussed in Section 2.3.3, yeast and mammalian ATG3 and ATG7 are essential for conjugation reactions needed for autophagosome formation, and d silencing of the plant homologues of these genes also results in a spreading cell death phenotype after TMV infection. Thus, multiple different f f autophagy genes, including those that act in the vesicle nucleation stage (e.g. t BECLIN 1, Class III PI3-K/VPS34) and those that act in the vesicle expansion stage (e.g. ATG3 and ATG7) are necessary to prevent the spread of cell death during the plant innate immune response.

While plant autophagy genes protect uninfected cells against death whereas mammalian autophagy genes protect infected cells against death during virus infection, the plant data nonetheless further support a "prosurvival", rather than "pro-death" function of autophagy genes during viral f f infections. At present, it is not yet clear how autophagy genes protect uninfected cells against death during the plant hypersensitive response. One possibility is that the absence of autophagy genes in uninfected cells somehow modifies the R gene-mediated signal transduction pathway in a way that instructs uninfected cells to die. An alternative, perhaps more likely, possibility is that the absence of autophagy genes in uninfected cells f renders them more susceptible to pro-death signals emitted from infected cells. Regardless of the mechanism, this newly defined role for autophagy genes in preventing the spread of cell death during plant innate immunity has significant implications for understanding the role of autophagy in systemic protection against viral infections. An important question is whether autophagy genes play a similar role during animal virus infections.

The evolutionarily conserved function of both mammalian and plant autophagy genes in restricting viral replication and/or protection against cell death suggests an essential role for autophagy in innate immunity. This concept is further supported by recent observations indicating that the herpes simplex virus neurovirulence protein, ICP34.5, possesses multiple mechanisms to disarm host autophagy. It can both antagonize the PKR signaling pathway required for autophagy induction and inhibit the function of one component of the autophagic machinery, Beclin 1.

As discussed in Section 4.1, ICP34.5 blocks PKR-dependent, eIF2 Ser-51-dependent autophagic degradation of cellular and viral components in f HSV-1-infected MEFs and neurons 99 . One predicted mechanism by which ICP34.5 blocks PKR-dependent autophagy is through its known ability to promote the dephosphorylation of eIF2 via interactions with PP1 105 . However, new evidence also suggests a second potential mechanism. Roizman et al. isolated the mammalian autophagy protein, Beclin 1, in a yeast two-hybrid screen using ICP34.5 as a bait 119 . Subsequent studies have shown that ICP34.5 directly interacts with Beclin 1 in mammalian cells and inhibits the ability of Beclin 1 to rescue autophagy in autophagy-defective atg6 null yeast and in autophagy-defective human MCF7 breast carcinoma cells 38 . Since ICP34.5 binds to Beclin 1 via a domain that is distinct from its PP1 -binding domain, it should be possible to construct HSV-1 viruses containing mutations in ICP34.5 that help differentiate between the role of PP1 -binding (and eIF2 phosphorylation) and the role of Beclin 1-binding in HSV-1 ICP34.5-mediated neurovirulence.

Besides HSV-1 ICP34.5, there are numerous other viral proteins or RNAs that suppress PKR signaling through a variety of different mechanism (reviewed in 100,101 ). For example, vaccinia virus E3, influenza virus NS1, HSV-1 Us11, reovirus 3, and rotavirus NSP3 encode double-stranded (ds) RNA-binding proteins that prevent PKR activation. Adenovirus VAI RNAs and HIV Tar RNAs bind to dsRNA substrates and inhibit PKR. Hepatitis C virus NS5A protein inhibits the dimerization of PKR, and influenza virus recruits a cellular protein, P58 IPK , that directly interacts with PKR and inhibits its dimerization. The vaccinia virus K3L, hepatitis C virus E2, and HIV Tat proteins act as pseudosubstrates of PKR. As-of-yet, the role of these other viral RNAs and proteins in autophagy inhibition has not been investigated. However, given the evolutionarily conserved requirement for an intact eIF2 kinase signaling pathway in autophagy induction, the prediction is that these other viral inhibitors of PKR, like HSV1 ICP34.5, also function as antagonists of host autophagy. Further studies are needed to test this prediction and to study the role of this predicted antagonism of host autophagy in the pathogenesis of diseases caused by these other important viral pathogens that encode putative autophagy inhibitors.

Not only may other viruses antagonize the autophagy function of PKR, but other viral gene products may also antagonize the autophagy function of specific mammalian atg genes. Beclin 1 was originally isolated in a yeast two-hybrid screen with the cellular anti-anti-apoptotic protein, Bcl-2 37 . Subsequently, Beclin 1 has also been shown to interact with viral Bcl-2-like proteins encoded by different gammaherpesviruses, including EBV-encoded BHRF1, KSHV-encoded v-Bcl-2, and murine HV68-encoded M11 39 . Like ICP34.5, these viral proteins can also inhibit the autophagy function of Beclin 1 in yeast and mammalian assays. In addition, preliminary evidence indicates that other KSHV-encoded proteins may interact with other specific Atg proteins 65 .

An as-of-yet explored area is whether viruses also inhibit autophagy by activating autophagy inhibitory signaling pathways. As noted in Section 2.2 and 3.2, the Class I PI-3K/Akt signaling pathway negatively regulates autophagy in both mammalian cells and C. elegans 19, 20, 120 and several different viruses activate this pathway. Certain oncogenic retroviruses encode the catalytic subunit of PI3-K and Akt (reviewed in 22 ). In addition, the EBV latent membrane proteins, LMP1 and 2A, the hepatitis B virus protein, HBx, the Kaposi's sarcoma virus protein, K1, and the hepatitis C virus protein, NS5A all activate the PI3-K/Akt signaling pathway [72] [73] [74] [75] [76] [77] . Presumably, such activation plays a role in autophagy inhibition, although this has not yet been formally tested.

While further studies are required to more precisely define the interactions between viral gene products and autophagy regulatory signals and autophagy proteins, there is, however, accumulating evidence that viruses do target multiple different steps of the host autophagy pathway. This observation strongly suggests an evolutionary advantage for viruses to inhibit host autophagy, and by extrapolation, a beneficial role for host autophagy in defense against viral infections.

Some viruses appear to have even further outsmarted host autophagy. Rather than merely devising strategies to block host autophagy, certain positive-strand RNA viruses have figured out ways to "co-opt" elements of the autophagy pathway to promote their own replication. This subject has been recently reviewed in detail elsewhere 13 and will therefore only be briefly summarized in this section.

As early as 1965, electron microscopic studies of poliovirus-infected cells demonstrated the presence of large numbers of membranous vesicles that were postulated to develop by an autophagic-like mechanism 121 . More recently, work by Kirkegaard et al, has extended these findings to further show that poliovirus replication complexes are associated with doublemembrane vesicles that resemble autophagosomes, in that they (1) have similar double membrane-bound morphology; (2) have low buoyant density; and (3) label with the autophagosome marker, GFP-LC3, and the lysosome marker, LAMP1 13, 122 . Unlike classical autophagosomes, these autophagiclike vesicles do not appear to have a destructive role or mature into degradative compartments. In support of this, treatment with autophagy r r inducers, rapamycin or tamoxifen, both increase, rather than decrease poliovirus growth 13 . Furthermore, these double-membrane vesicles are also different from classical autophagosomes in that they contain Sec13 and Sec31, components of the anterograde transport system that bud from the ER 123 .

Therefore, it is possible that poliovirus-induced vesicles arise from an alternate source rather than the PAS, but still share some of the same characteristics of classical autophagosomes (e.g. GFP-LC3 and labeling, augmentation with rapamycin treatment). Similar to the replication vacuoles that are associated with certain intracellular bacterial pathogens (e.g. Legionella pneumophila) 124 , the poliovirus-induced double-membrane vesicles likely originate from the ER 123 . Furthermore, these poliovirusinduced vesicles seem to have an alternate function than autophagosomes (i.e. they are pro-replicative, rather than degradative compartments). These r observations suggest that poliovirus may promote its own replication by inducing dynamic membrane rearrangements that share in common certain features of the autophagy pathway (e.g. formation of sequestering doublemembrane vesicles, presence of overlapping markers) but avoid, other unwanted features of the autophagy pathway (e.g. maturation into degradation compartments). Of note, specific poliovirus proteins, including 2BC and 3A, have been identified that are sufficient for the induction of t these "autophagic-like" double-membrane bound vesicles 122 . However, the mechanisms by which these proteins induce the formation of such vesicles are not yet known.

A recent study with the coronavirus, murine hepatitis virus, has provided more direct evidence that components of the autophagic machinery can be utilized for RNA virus replication 51 . MHV replication complexes localize to double-membrane vesicles (that are also thought to arise from the ER) ( Figure 5A ) and they co-localize with certain autophagy proteins, including LC3 and Atg12. In MHV-infected atg5 -/-ES cells, double-membrane vesicles are not detected ( Figure 5B) , and viral replication is dramatically reduced. These observations provide the first genetic demonstration that proteins necessary for autophagic vacuole formation are also required for maximal levels of viral replication. Thus, MHV, and potentially other viruses that replicate in association with double-membrane vesicles (e.g. poliovirus, equine arterivirus), utilize components of cellular autophagy to foster their f own growth. Presumably, the Atg protein conjugation system (involving Atg5) that plays a role in autophagic vesicle expansion and completion also plays a role in the formation of double-membrane vesicles involved in viral replication. It is not yet known whether the entire autophagic machinery or only selective components of the autophagic machinery are used for the formation of double-membrane vesicles that are associated with viral replication complexes.

These observations with poliovirus and MHV represent two examples of how viruses can "subvert" elements of the host autophagy pathway to promote their own intracellular growth. In these infections, RNA replication complexes are observed in association with "autophagic-like" doublemembrane vesicles but not in association with degradative autophagosomes. It is not clear whether this represents fundamental differences in the host pathways leading to the formation of "autophagic-like" double-membrane vesicles and classical degradative autophagosomes, the diversion of the autophagic machinery towards the formation of "autophagic-like" doublemembrane vesicles from the formation of classical degradative autophagosomes, or specific viral mechanisms to antagonize the maturation of "autophagic-like" double-membrane vesicles into mature degradative autophagosomes. However, interestingly, MHV infection does lead to the induction of atg-5-dependent long-lived cellular protein degradation, ruling out the hypothesis that the autophagic machinery is entirely diverted to form membranes required for viral replication complexes. Perhaps MHV possesses as-of-yet defined mechanisms to shield its replication complexes from autophagic degradation.

Although research in this area is still in a stage of infancy, it seems likely that the lysosomal degradation pathway of autophagy plays an evolutionarily conserved role in antiviral immunity. The interferon-inducible, antiviral PKR signaling pathway positively regulates autophagy, and both mammalian and plant autophagy genes restrict viral replication and protect against virus-induced cell death. Given this role of autophagy in innate immunity, it is not surprising that viruses have evolved numerous strategies to inhibit host autophagy. Different viral gene products can either modulate autophagy regulatory signals or directly interact with components of the autophagy execution machinery. Moreover, certain RNA viruses have managed to "co-apt" the autophagy pathway, selectively utilizing certain components of the dynamic membrane rearrangement system to promote their own replication inside the host cytoplasm.

In addition to this newly emerging role of autophagy in innate immunity, autophagy plays an important role in many other fundamental biological processes, including tissue homeostasis, differentiation and development, cell growth control, and the prevention of aging. Accordingly, the inhibition of host autophagy by viral gene products has important implications not only for understanding mechanisms of immune evasion, but also for understanding novel mechanisms of viral pathogenesis. It will be interesting to dissect the role of viral inhibition of autophagy in acute, persistent, and latent viral replication, as well as in the pathogenesis of cancer and other medical diseases.

Development by self-digestion: molecular mechanisms and biological functions of autophagy

Development by self-digestion: molecular mechanisms and biological functions of autophagy

Autophagy: a regulated bulk degradation process inside cells. d

The molecular mechanism of autophagy

Functions of lysosomes

A unified nomenclature for yeast autophagy-related genes

Diversity of Signaling Controls of Macroautophagy in Mammalian Cells

The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation

Convergence of multiple autophagy and cytoplasm to vacuole targeting components to a perivacuolar membrane compartment prior to de novo vesicle formation

Yeast autophagosomes: de novo formation of a membrane structure

Cellular autophagy: surrender, avoidance and subversion by microrganisms

Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes

Autophagy as a regulated pathway of cellular degradation

The mitochondrial permeability transition initiates autophagy in rat hepatocytes

PKR: a sentinel kinase for cellular stress

Regulation of starvation-and virus-induced autophagy by the eIF2a kinase signaling pathway

Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells

Autophagy genes are essential for dauer development and lifespan extension in C. elegans

Oncogenic kinase signalling

Promotion of tumorigenesis by heterozygous disruption of the beclin 1 gene

Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor

Autophagy as a cell death and tumor suppressor mechanism

Defective autophagy leads to cancer

Molecular dissection of autophagy: two ubiquitin-like systems

Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae

Tor-mediated induction of autophagy via an Apg1 protein kinase complex

Chemical genetic analysis of Apg1 reveals a non-kinase role in the induction of autophagy

Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for prtoein sorting

3-methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes

The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes

The Hansenula polymorpha PDD1 gene product, essential for the selective degradation of peroxisomes, is a homologue of Saccharomyces cerevisiae Vps34p

Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae

Inhibition of autophagy in mitotic animal cells

Protection against fatal Sindbis virus encephalitis by Beclin, a novel Bcl-2-interacting protein

Unpublished data

Unpublished data

Autophagy genes are essential for limiting the spread of programmed cell death associated with plant innate immunity

A protein conjugation system essential for autophagy

A ubiquitin-like system mediates protein lipidation

The X-ray crystal structure and putative ligand-derived peptide binding t t properties of gamma-aminobutyric acid receptor type A receptor-associated protein

Solution structure of human GAGA(A) receptor-associated protein GABARAP: implications for biological function and its regulation

The cyrstal structure of microtubule-associated protein light chain 3, a mammalian homolgue of Saccharomyces cerevisiae Atg8

Formation process of autophagosome is traced with Apg8/Aut7p in yeast

HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three humans Atg8 homologues and delipidates LC3-and GABARAP-phospholipid conjugates

The reversible modifcation regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway

Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy

LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing

Coronavirus replication complex formation utilizes components of cellular autophagy

Apg9/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways

The Atg1-Atg13 complex regulates Atg9 and Atg23 retrieval transport from the preautophagosomal structure

In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a r r fluorescent autophagy marker

Autophagy in health and disease: a double-edged sword

Functions of lysosomes

Macroautophagy is required for multicellular development of the social amoeba Dictyostelium discoideum

Dictyostelium macroautophagy mutants vary in the severity of their developmental defects

Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene

The APG8/12-activating enzyme APG7 is required for proper nutrientrecycling and sensescence in Arabidopsis thaliana

Unpublished data

Translation initiation: adept at adapting

Trascriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast

BHRF1, a viral homologue of the Bcl-2 oncogene, disturbs epithelial cell r differentiation

Reduced rates of proteolysis in transformed cells

Role of the vacuolar apparatus in augmented protein degradation in cultured fibroblasts

Inhibitors of mammalian target of rapamycin as novel antitumor agents: from bench to clinic

Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation

Epstein-Barr virus LMP2A transforms epithelial cells, inhibits cell differentiation, and activates Akt

Latent membrane protein 2A inhibits transforming growth factor-beta 1-induced apoptosis through the phosphatidylinositol 3-kinase/Akt pathway

Differential signaling pathways are activated in the Epstein-Barr virus-associated malignancies nasopharyngela carcinoma and Hodgin lymphoma

Hepatitis B viral HBx induces matrix metalloproteinase-9 gene expression trhough activation of ERK and PI-3K/Akt pathways: involvement of invasive potential

The K1 protein of Kaposi's sarcoma-associated herpesvirus activates the Akt signaling pathway

The hepatitis C virus NS5A protein activates a phosphoinosite 3-kinase-dependent survival signaling cascade

Cloning and genomic organization of beclin 1, a candidate tumor suppressor gene on chromosome 17q21

Induction of autophagy and inhibition of tumorigenesis by beclin 1

Beclin 1 contains a leucine-rich nuclear export signal that is reguired for its autophagy and tumor suppressor function

Unpublished data

Autophagy and aging--when "all you can eat" is yourself

The anti-ageing effects of caloric restriction may involve stimulation of macroautophagy and lysosomal degradation, and can be intensifid pharmacologically

Genetic pathways that regulate ageing in model organisms

Mitochondria and HIV infection: the first decade

Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated carcinogenesis

Pre-S mutant surface antigens in chronic hepatitis B virus infection induce oxidative stress and DNA damage

Mitochondrial autophagy and injury in the liver in 1 -antitrypsin deficiency

Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy

Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models off Huntington disease

Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control

Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase

Endoplasmic reticulum stress is a determinant of retrovirus-induced spongiform neurodegeneration

Activation of endoplasmic reticulum stress signaling pathway is associated with neuronal degeneratio in MoMuLV-ts1-induced spongiform encephalopathy

Endoplasmic reticulum (ER) stress induced by a neurovirulent mouse retrovirus is associated with prolonged BiP binding and retention of a viral protein in the ER

Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress

Replication of a cytopathic strain of bovine viral diarrhea virus activates PERK and induces endoplasmic reticulum stress-mediated apoptosis of MDBK cells

Possible involvment of both endoplasmic reticulum-and mitochondriadependent pathways in MoMuLV-ts1-induced apoptosis in astrocytes

PKR-dependent autophagic degradation of herpes simplex virus type 1

Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase

com: maneuvering the internetworks of viral neuropathogenesis and evasion of the host defense

Mapping of herpes simplex virus-1 neurovirulence to g134.5, a gene nonessential for growth in culture

Specific phenotypic restoration of an attenuated virus by knockout of a hostresistance gene

Association of a M(r) 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2 alpha and premature shutoff of protein synthesis after infection with gamma 134.5-mutants of herpes simplex virus 1

The (1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1 to dephosphorylate the subunit of the eukaryotic translation initiation factor 2 and preculde the shutoff of protein synthesis by double-stranded RNA-activated protein kinase

Electron microscopic observations on the development of herpes simplex virus

Unpublished data

Bcl-2 protects mice against fatal alphavirus encephalitis

The transmembrane domains of Sindbis virus envelope glycoproteins induce cell death

The autophagosomal-lysosomal compartment in programmed cell death

Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase 8

American Society for Cell Biology 44th Annual Meeting

Plant pathogens and integrated defence responses to infection

Plant innate immunity -direct and indirect recognition of general and specific pathogen-associated molecules

Controlled cell death, plant survival and development

The helicase domain of the TMV replicase proteins induces the Nmediated defense response in tobacco

The product of the tobacco mosaic virus resistance gene N: similarity to toll and the interleukin-1 receptor

The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway

Electron microscopic study of the formation of poliovirus

Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles

Cellular COOPII proteins are involved in production of the vesicles that form the poliovirus replication complex

Apg1p, a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae

Apg14p and Apg6/Vps30p form a protein complex essential for autophagy in the yeast, Saccharomyces cerevisiae

The Drosophila homolog of Aut1 is essential for autophagy and development

Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hAPG5p

The loss of Drosophila APG4/AUT2 function modifies the phenotypes of cut and Notch signaling pathway pathway mutants

A single protease, Apg4B, is specific for the autophagy-related ubiquitin-like proteins GATE-16, MAP1-LC3, GABARAP, and Apg8L

Dissection of autophagosome formation using Apg5deficient mouse embryonic stem cells

A new protein conjugation system in human. The counterpart of the yeast Apg12p conjugation system essential for autophagy

Structural and functional analyses of APG5, a gene involved in autophagy in yeast

The human homolog of Saccharomyces cerevisiae Apg7p is a protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3

Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein

Mouse Apg10 as an Apg12conjugating enzyme: analysis by the conugation-mediated yeast two-hybrid method

Apg10p, a novel protein-conjugating enzyme essential for autophagy in yeast

Murine Apg12p has a substrate preference for murine Apg7p over three Apg8p homologs

Apg16p is required for the function of the Apg12p-Apg5p conjugate in the yeast autophagy pathway

Cvt18/Gas12 is required for cytoplasm-to-vacuole transport, pexophagy, and autophagy in Saccharomyces cerevisiae and Pichia Pastoris

The work done in the authors' laboratories was supported by NIH RO1 grants AI51367 and AI44157 to B.L, and an NSF plant genome grant DBI-0116076 and NIH RO1 grant GM62625 to S.P.D-K.