Virus-responsive signaling pathways that induce alpha/beta interferon production and engage intracellular immune defenses influence the outcome of many viral infections. The processes that trigger these defenses and their effect upon host permissiveness for specific viral pathogens are not well understood. We show that structured hepatitis C virus (HCV) genomic RNA activates interferon regulatory factor 3 (IRF3), thereby inducing interferon in cultured cells. This response is absent in cells selected for permissiveness for HCV RNA replication. Studies including genetic complementation revealed that permissiveness is due to mutational inactivation of RIG-I, an interferon-inducible cellular DExD/H box RNA helicase. Its helicase domain binds HCV RNA and transduces the activation signal for IRF3 by its caspase recruiting domain homolog. RIG-I is thus a pathogen receptor that regulates cellular permissiveness to HCV replication and, as an interferonresponsive gene, may play a key role in interferon-based therapies for the treatment of HCV infection.Hepatitis C virus (HCV) is a major public health problem, infecting nearly 200 million people worldwide and causing hepatic fibrosis, end-stage cirrhosis, and hepatocellular carcinoma (16). A member of the Flaviviridae, HCV's positivesense RNA genome contains highly structured 5Ј and 3Ј nontranslated regions (NTRs) flanking a large open reading frame (ORF) encoding a polyprotein that is processed into both structural (core-E2) and nonstructural (NS) proteins (Fig. 1A). The NS3-NS5B proteins support viral genome replication, which is also dependent upon conserved RNA sequences within the 5ЈNTR and 3ЈNTR that are highly structured and required for both protein translation and RNA replication (15,20). HCV infection is treated with alpha interferon (IFN-␣)-based therapy, but treatment is effective at best in only 50% of patients (10). The nearly unique ability of HCV to establish persistent infections in humans has been attributed, in part, to a variety of strategies to evade host immune and IFN-induced defenses (12). Epidemiological studies suggest that 25 to 50% of all persons resolve acute HCV infection without treatment (16), however, indicating that innate and/or adaptive immune responses are indeed capable of controlling the outcome of HCV infection. Processes that regulate innate intracellular antiviral responses may therefore serve as pivotal points of control, potentially limiting host permissiveness for HCV replication and favorably modulating subsequent adaptive immune responses.Virus-induced production of IFN-␣ and IFN-␤ and the subsequent expression of IFN-stimulated genes (ISGs) are central to these antiviral defenses (22). This host response is initiated by cellular recognition of a pathogen-associated molecular pattern (PAMP) presented by the infection, in which a host protein receptor is engaged by the PAMP ligand and signals downstream components to activate intracellular immune defenses. In mammalian cells, replicating viral RNAs present features of nucleic...

The lysosomal degradation pathway of autophagy has a crucial role in defence against infection, neurodegenerative disorders, cancer and ageing. Accordingly, agents that induce autophagy may have broad therapeutic applications. One approach to developing such agents is to exploit autophagy manipulation strategies used by microbial virulence factors. Here we show that a peptide, Tat–beclin 1—derived from a region of the autophagy protein, beclin 1, which binds human immunodeficiency virus (HIV)-1 Nef—is a potent inducer of autophagy, and interacts with a newly identified negative regulator of autophagy, GAPR-1 (also called GLIPR2). Tat–beclin 1 decreases the accumulation of polyglutamine expansion protein aggregates and the replication of several pathogens (including HIV-1) in vitro, and reduces mortality in mice infected with chikungunya or West Nile virus. Thus, through the characterization of a domain of beclin 1 that interacts with HIV-1 Nef, we have developed an autophagy-inducing peptide that has potential efficacy in the treatment of human diseases.

Persistent infections with hepatitis C virus (HCV) are likely to depend on viral inhibition of host defenses. We show that the HCV NS3/4A serine protease blocks the phosphorylation and effector action of interferon regulatory factor-3 (IRF-3), a key cellular antiviral signaling molecule. Disruption of NS3/4A protease function by mutation or a ketoamide peptidomimetic inhibitor relieved this blockade and restored IRF-3 phosphorylation after cellular challenge with an unrelated virus. Furthermore, dominant-negative or constitutively active IRF-3 mutants, respectively, enhanced or suppressed HCV RNA replication in hepatoma cells. Thus, the NS3/4A protease represents a dual therapeutic target, the inhibition of which may both block viral replication and restore IRF-3 control of HCV infection.

SUMMARY The removal of unwanted or damaged mitochondria by autophagy, a process called mitophagy, is essential for key events in development, cellular homeostasis, tumor suppression, and prevention of neurodegeneration and aging. However, the precise mechanisms of mitophagy remain uncertain. Here, we identify the inner mitochondrial membrane protein, prohibitin 2 (PHB2), as a crucial mitophagy receptor involved in targeting mitochondria for autophagic degradation. PHB2 binds the autophagosomal membrane-associated protein LC3 through an LC3-interaction region (LIR) domain upon mitochondrial depolarization and proteasome-dependent outer membrane rupture. PHB2 is required for Parkin-induced mitophagy in mammalian cells and for the clearance of paternal mitochondria after embryonic fertilization in C. elegans. Our findings pinpoint a conserved mechanism of eukaryotic mitophagy and demonstrate a function of prohibitin 2 that may underlie its roles in physiology, aging, and disease.

A long-standing controversy is whether autophagy is a bona fide cause of mammalian cell death. We used a cell-penetrating autophagy-inducing peptide, Tat-Beclin 1, derived from the autophagy protein Beclin 1, to investigate whether high levels of autophagy result in cell death by autophagy. Here we show that Tat-Beclin 1 induces dose-dependent death that is blocked by pharmacological or genetic inhibition of autophagy, but not of apoptosis or necroptosis. This death, termed "autosis," has unique morphological features, including increased autophagosomes/autolysosomes and nuclear convolution at early stages, and focal swelling of the perinuclear space at late stages. We also observed autotic death in cells during stress conditions, including in a subpopulation of nutrient-starved cells in vitro and in hippocampal neurons of neonatal rats subjected to cerebral hypoxia-ischemia in vivo. A chemical screen of ∼5,000 known bioactive compounds revealed that cardiac glycosides, antagonists of Na

Summary Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic process including autophagy, and aberrant activation of such receptors is a common feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds to the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated Class III phosphatidylinositol-3 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors, and restores autophagy in non-small cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance.

Autophagy functions in antiviral immunity. However, it is not yet known whether endogenous autophagy genes protect against viral disease in vertebrates. Using three different approaches to inactivate the autophagy gene Atg5 in virally-infected neurons, we found that loss of Atg5 function increases mouse susceptibility to lethal Sindbis virus CNS infection. This phenotype is associated with delayed clearance of viral proteins, increased accumulation of the cellular p62 adaptor protein, and increased cell death in neurons, but not with altered levels of CNS viral replication. In vitro, p62 interacts with Sindbis virus capsid protein and genetic knockdown of p62 blocks the targeting of viral capsid to autophagosomes. Moreover, p62 or autophagy gene knockdown increases viral capsid accumulation and accelerates virus-induced cell death without affecting virus replication. These results suggest a novel function for autophagy in mammalian antiviral defense: a cell-autonomous mechanism in which p62 adaptor-mediated autophagic viral protein clearance promotes cell survival.

Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction1–4. Yeast genetic screens have identified proteins required for different forms of selective autophagy, including cytoplasm-to-vacuole targeting, pexophagy, and mitophagy, and mammalian genetic screens have identified proteins required for autophagy regulation5. However, there have been no systematic approaches to identify molecular determinants of selective autophagy in mammalian cells. To identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide siRNA screen to detect genes required for the colocalization of Sindbis virus capsid protein with autophagolysosomes. We identified 141 candidate genes required for viral autophagy, which were enriched for cellular pathways related to mRNA processing, interferon signaling, vesicle trafficking, cytoskeletal motor function, and metabolism. Ninety-six of these genes were also required for Parkin-mediated mitophagy, indicating that common molecular determinants may be involved in autophagic targeting of viral nucleocapsids and autophagic targeting of damaged mitochondria. Murine embryonic fibroblasts lacking one of these gene products, the C2-domain containing protein, Smurf1, are deficient in the autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria. Moreover, Smurf1-deficient mice display an accumulation of damaged mitochondria in heart, brain, and liver. Thus, our study identifies candidate determinants of selective autophagy, and defines Smurf1 as a newly recognized mediator of both viral autophagy and mitophagy.