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Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The FA proteins have functions in genome maintenance and in the cytoplasmic process of selective autophagy, beyond their canonical roles of repairing DNA interstrand cross-links. FA core complex proteins FANCC, FANCF, FANCL, FANCA, FANCD2, BRCA1 and BRCA2, which previously had no known direct functions outside the nucleus, have recently been implicated in mitophagy. Although mutations in FANCL account for only a very small number of cases in FA families, it plays a key role in the FA pathophysiology and might drive carcinogenesis. Here, we demonstrate that FANCL protein is present in mitochondria in the control and Oligomycin and Antimycin (OA)-treated cells and its ubiquitin ligase activity is not required for its localization to mitochondria. CRISPR/Cas9-mediated knockout of FANCL in HeLa cells overexpressing parkin results in increased sensitivity to mitochondrial stress and defective clearing of damaged mitochondria upon OA treatment. This defect was reversed by the reintroduction of either wild-type FANCL or FANCL(C307A), a mutant lacking ubiquitin ligase activity. To summarize, FANCL protects from mitochondrial stress and supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner.

Fanconi anemia (FA) patients are at higher risk of developing bone marrow failure (BMF) or cancer upon exposure to potential threats like viral infections, bacterial infections, carcinogens, and mutagens. Deleterious mutations in FA genes cause multiple cellular defects which include impaired ability to repair DNA interstrand crosslink lesions in the nucleus and impaired removal of damaged mitochondria via mitophagy in the cytoplasm. Fancc-deficient mice exhibit enhanced inflammatory response and are hypersensitive to lipopolysaccharide-induced septic shock as a result of hematopoietic suppression. Considering the role of inflammation in pathogenesis of Fanconi anemia and bone marrow failure diseases in general, we asked whether inflammasome inhibition could be a promising therapeutic for the treatment of FA. Our studies demonstrate that MCC950, a small molecule inhibitor of the NLRP3 inflammasome, can protect Fancc null mice from LPS-induced hemopoietic suppression and septic shock as evidenced by increased survival rate of Fancc−/− mice. MCC950 partially rescues the thrombocytopenia in peripheral blood and partially restores the loss of megakaryocyte-erythroid progenitor (MEP) and granulocyte-macrophage progenitor (GMP) pools upon LPS challenge. Further, Treatment with MCC950 significantly improved the bone marrow colony-forming capacity of Fancc−/− bone marrow cells in vitro. Our studies demonstrate that MCC950, a small molecule inhibitor of the NLRP3 inflammasome, can protect Fancc null mice from LPS-induced hemopoietic suppression and septic shock and warrants further evaluation as a potential therapeutic strategy in FA.

Fanconi anemia (FA) is a rare disease, in which homozygous or compound heterozygous inactivating mutations in any of 21 genes lead to genomic instability, early-onset bone marrow failure and increased cancer risk. The FA pathway is essential for DNA damage response (DDR) to DNA interstrand crosslinks. However, proteins of the FA pathway have additional cytoprotective functions that may be independent of DDR. We have shown that many FA proteins participate in the selective autophagy pathway that is required for the destruction of unwanted intracellular constituents. In this Cell Science at a Glance and the accompanying poster, we briefly review the role of the FA pathway in DDR and recent findings that link proteins of the FA pathway to selective autophagy of viruses and mitochondria. Finally, we discuss how perturbations in FA protein-mediated selective autophagy may contribute to inflammatory as well as genotoxic stress.

PEX13 is an integral membrane protein on the peroxisome that regulates peroxisomal matrix protein import during peroxisome biogenesis. Mutations in PEX13 and other peroxin proteins are associated with Zellweger syndrome spectrum (ZSS) disorders, a subtype of peroxisome biogenesis disorder characterized by prominent neurological, hepatic, and renal abnormalities leading to neonatal death. The lack of functional peroxisomes in ZSS patients is widely accepted as the underlying cause of disease; however, our understanding of disease pathogenesis is still incomplete. Here, we demonstrate that PEX13 is required for selective autophagy of Sindbis virus (virophagy) and of damaged mitochondria (mitophagy) and that disease‐associated PEX13 mutants I326T and W313G are defective in mitophagy. The mitophagy function of PEX13 is shared with another peroxin family member PEX3, but not with two other peroxins, PEX14 and PEX19, which are required for general autophagy. Together, our results demonstrate that PEX13 is required for selective autophagy, and suggest that dysregulation of PEX13‐mediated mitophagy may contribute to ZSS pathogenesis.

SummaryCell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic processes, 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 the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated VPS34 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.

SummaryThe mammalian intestine is colonized with a diverse community of bacteria that perform many beneficial functions but can threaten host health upon tissue invasion. Epithelial cell-intrinsic innate immune responses are essential to limit the invasion of both commensal and pathogenic bacteria and maintain beneficial host-bacterial relationships; however, little is known about the role of various cellular processes, notably autophagy, in controlling bacterial interactions with the intestinal epithelium in vivo. We demonstrate that intestinal epithelial cell autophagy protects against tissue invasion by both opportunistically invasive commensals and the invasive intestinal pathogen Salmonella Typhimurium. Autophagy is activated following bacterial invasion of epithelial cells through a process requiring epithelial cell-intrinsic signaling via the innate immune adaptor protein MyD88. Additionally, mice deficient in intestinal epithelial cell autophagy exhibit increased dissemination of invasive bacteria to extraintestinal sites. Thus, autophagy is an important epithelial cell-autonomous mechanism of antibacterial defense that protects against dissemination of intestinal bacteria.

Loss-of-function mutations in 19 Fanconi anemia (FA) pathway genes result in a variety of clinical manifestations such as birth defects, cognitive impairment, bone marrow failure (in up to 90% of FA patients within the first two decades of life), increased cancer risk, and accelerated aging [1]. Following DNA damage, proteins of the FA pathway act in a complex cascade to repair interstrand crosslinks (ICLs), which are caused by reactive oxygen species (ROS) or exposure to reactive aldehydes [2]. Although FA is a rare disease, somatic mutations or epigenetic silencing of several FA genes are found commonly in cancers in the general population (e.g. breast, ovarian, and pancreatic cancers). Furthermore, monoallelic germline mutations in at least five genes in the FA pathway, FANCD1/BRCA2, FANCS/BRCA1, BRIP1/FANCJ, PALB2/FANCN, and RAD51C/FANCO are associated with the development of cancer. Selective autophagy is a homeostatic “cellular housekeeping” pathway in which unwanted cytoplasmic cargos are targeted for lysosomal destruction. We discovered that Fanconi anemia genes represent a new class of selective autophagy factors [3, 4]. Our findings may help to explain some of the pathophysiological features of FA, particularly in regard to “environmental factors” [1] that modulate FA disease progression, and may lead to the identification of novel therapeutic targets for FA. We found that, while Fancc is dispensable for starvation-induced general macroautophagy, it is required for virophagy of two genetically unrelated neuronotropic viruses, Sindbis virus and herpes simplex virus type 1, and for host defense in vivo against lethal CNS infection with these viruses. FANCC is also required for Parkin-mediated mitophagy in vitro and for mitochondrial quality control in vivo. Thus, FANCC is required for two distinct forms of selective autophagy: virophagy and mitophagy (Figure 1). Rather than playing an indirect role in selective autophagy, FANCC is likely to function as an adaptor protein in both virophagy and mitophagy. FANCC is contained within membrane-bound autophagolysosomal structures together with Sindbis viral nucleocapsids and (similar to previously identified virophagy factors p62 [5] and SMURF1 [4]) FANCC interacts biochemically with Sindbis virus capsid protein. FANCC also interacts biochemically with Parkin and colocalizes with damaged mitochondria in a Parkin-dependent manner. Thus, Editorial

The ubiquitin-proteasome system degrades viral oncoproteins and other microbial virulence factors; however, the role of endolysosomal degradation pathways in these processes is unclear. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, and a constitutively active viral G protein-coupled receptor (vGPCR) contributes to the pathogenesis of KSHV-induced tumors. We report that a recently discovered autophagy-related protein, Beclin 2, interacts with KSHV GPCR, facilitates its endolysosomal degradation, and inhibits vGPCR-driven oncogenic signaling. Furthermore, monoallelic loss of Becn2 in mice accelerates the progression of vGPCR-induced lesions that resemble human Kaposi's sarcoma. Taken together, these findings indicate that Beclin 2 is a host antiviral molecule that protects against the pathogenic effects of KSHV GPCR by facilitating its endolysosomal degradation. More broadly, our data suggest a role for host endolysosomal trafficking pathways in regulating viral pathogenesis and oncogenic signaling.

We recently identified physical exercise as a newly defined inducer of autophagy in vivo. Exercise induced autophagy in multiple organs involved in metabolic regulation, such as muscle, liver, pancreas and adipose tissue. To study the physiological role of exercise-induced autophagy, we generated mice with a knock-in nonphosphorylatable mutation in BCL2 (Thr69Ala, Ser70Ala and Ser84Ala) (BCL2 AAA) that are defective in exercise- and starvation-induced autophagy but not in basal autophagy. We found that BCL2 AAA mice could not run on a treadmill as long as wild-type mice, and did not undergo exercise-mediated increases in skeletal glucose muscle uptake. Unlike wild-type mice, the BCL2 AAA mice failed to reverse high-fat diet-induced glucose intolerance after 8 weeks of exercise training, possibly due to defects in signaling pathways that regulate muscle glucose uptake and metabolism during exercise. Together, these findings suggested a hitherto unknown important role of autophagy in mediating exercise-induced metabolic benefits. In the present addendum, we show that treadmill exercise also induces autophagy in the cerebral cortex of adult mice. This observation raises the intriguing question of whether autophagy may in part mediate the beneficial effects of exercise in neurodegeneration, adult neurogenesis and improved cognitive function.

Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction1,2,3,4. 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. Here, to identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide small interfering RNA 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 messenger RNA processing, interferon signalling, 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 accumulate damaged mitochondria in the 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.

Autophagy is a conserved catabolic stress response pathway that is increasingly recognized as an important component of both innate and acquired immunity to pathogens. The activation of autophagy during infection not only provides cell-autonomous protection through lysosomal degradation of invading pathogens (xenophagy), but also regulates signaling by other innate immune pathways. This review will focus on recent advances in our understanding of three major areas of the interrelationship between autophagy and innate immunity, including how autophagy is triggered during infection, how invading pathogens are targeted to autophagosomes, and how the autophagy pathway participates in “tuning” the innate immune response.

SummaryAutophagy functions in antiviral immunity. However, the ability of endogenous autophagy genes to protect against viral disease in vertebrates remains to be causally established. Here, we report that the autophagy gene Atg5 function is critical for protection against lethal Sindbis virus (SIN) infection of the mouse central nervous system. Inactivating Atg5 in SIN-infected neurons results in delayed clearance of viral proteins, increased accumulation of the cellular p62 adaptor protein, and increased cell death in neurons, but the levels of viral replication remain unaltered. In vitro, p62 interacts with SIN 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 function for autophagy in mammalian antiviral defense: a cell-autonomous mechanism in which p62 adaptor-mediated autophagic viral protein clearance promotes cell survival.

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 interferon-responsive gene, may play a key role in interferon-based therapies for the treatment of HCV infection.

Hepatitis C virus (HCV) infection is treated with interferon (IFN)-based therapy. The mechanisms by which IFN suppresses HCV replication are not known, and only limited efficacy is achieved with therapy because the virus directs mechanisms to resist the host IFN response. In the present study we characterized the effects of IFN action upon the replication of two distinct quasispecies of an HCV replicon whose encoded NS5A protein exhibited differential abilities to bind and inhibit protein kinase R (PKR). Metabolic labeling experiments revealed that IFN had little overall effect upon HCV protein stability or polyprotein processing but specifically blocked translation of the HCV RNA, such that the replication of both viral quasispecies was suppressed by IFN treatment of the Huh7 host cells. However, within cells expressing an NS5A variant that inhibited PKR, we observed a reduced level of eukaryotic initiation factor 2 alpha subunit (eIF2α) phosphorylation and a concomitant increase in HCV protein synthetic rates, enhancement of viral RNA replication, and a partial rescue of viral internal ribosome entry site (IRES) function from IFN suppression. Assessment of the ribosome distribution of the HCV replicon RNA demonstrated that the NS5A-mediated block in eIF2α phosphorylation resulted in enhanced recruitment of the HCV RNA into polyribosome complexes in vivo but only partially rescued the RNA from polyribosome dissociation induced by IFN treatment. Examination of cellular proteins associated with HCV-translation complexes in IFN-treated cells identified the P56 protein as an eIF3-associated factor that fractionated with the initiator ribosome-HCV RNA complex. Importantly, we found that P56 could independently suppress HCV IRES function both in vitro and in vivo, but a mutant P56 that was unable to bind eIF3 had no suppressive action. We conclude that IFN blocks HCV replication through translational control programs involving PKR and P56 to, respectively, target eIF2- and eIF3-dependent steps in the viral RNA translation initiation process.

Cells keep themselves healthy by breaking down unneeded or damaged internal structures via a process called autophagy. This process also helps a cell to survive if it is starved of nutrients. For example, if a cell does not receive enough amino acids, it cannot make new proteins. Autophagy can break down existing non-essential proteins so that their amino acids can be re-used to build other proteins that the cell needs to survive. Autophagy is performed by a set of proteins that is found in many different species, ranging from yeast to humans and plants. How these proteins are activated when a cell is starved of amino acids is not fully understood. However, evidence suggests that activating one of these proteins, called Beclin 1, by adding phosphate groups to it controls the extent to which autophagy occurs. It is also known from previous work that less autophagy occurs when Beclin 1 binds to another protein called BCL2. Wei, An et al. identified two enzymes that attach a phosphate group to a specific site on Beclin 1 to activate it, and revealed that autophagy is defective in cells that lack these enzymes. Furthermore, Wei, An et al. found the BCL2 protein prevents autophagy by binding to Beclin 1 in such a way that stops these two enzymes from activating Beclin 1. Beclin 1 is also known to prevent the growth of malignant tumors. Wei, An et al. found that to do so, Beclin 1 must have a phosphate group added to the same site that activates the protein during autophagy. This suggests that drugs that enhance the addition of this phosphate group to Beclin 1 could help activate autophagy and have anti-cancer effects.

The virus–host interactions that influence hepatitis C virus (HCV) replication are largely unknown but are thought to involve those that disrupt components of the innate intracellular antiviral response. Here we examined cellular antiviral pathways that are triggered during HCV RNA replication. We report that ( i ) RNA replication of HCV subgenomic replicons stimulated double-stranded RNA (dsRNA) signaling pathways within cultured human hepatoma cells, and ( ii ) viral RNA replication efficiency corresponded with an ability to block a key cellular antiviral effector pathway that is triggered by dsRNA and includes IFN regulatory factor-1 (IRF-1) and protein kinase R (PKR). The block to dsRNA signaling was mapped to the viral nonstructural 5A (NS5A) protein, which colocalized with PKR and suppressed the dsRNA activation of PKR during HCV RNA replication. NS5A alone was sufficient to block both the activation of IRF-1 and the induction of an IRF-1-dependent cellular promoter by dsRNA. Mutations that clustered in or adjacent to the PKR-binding domain of NS5A relieved the blockade to this IRF-1 regulatory pathway, resulting in induction of IRF-1-dependent antiviral effector genes and the concomitant reduction in HCV RNA replication efficiency. Our results provide further evidence to support a role for PKR in dsRNA signaling processes that activate IRF-1 during virus infection and suggest that NS5A may influence HCV persistence by blocking IRF-1 activation and disrupting a host antiviral pathway that plays a role in suppressing virus replication.

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(+),K(+)-ATPase, inhibit autotic cell death in vitro and in vivo. Furthermore, genetic knockdown of the Na(+),K(+)-ATPase α1 subunit blocks peptide and starvation-induced autosis in vitro. Thus, we have identified a unique form of autophagy-dependent cell death, a Food and Drug Administration-approved class of compounds that inhibit such death, and a crucial role for Na(+),K(+)-ATPase in its regulation. These findings have implications for understanding how cells die during certain stress conditions and how such cell death might be prevented.

Complete 1H and 13C nuclear magnetic resonance (NMR) signal assignments have been established for 5 alpha-cholestan-3 beta-ol, 22 unsaturated C27 sterols, and their acetate derivatives. Assignments were made from a combination of 1D and 2D spectra and include stereochemical 1H assignments for the C-22 and C-23 protons of 5 alpha-cholesta-8,24-dien-3 beta-ol and other delta 24 sterols with a C8 side chain. At the temperature and concentration range described, chemical shifts were generally reproducible to +/- 0.01 ppm in 13C spectra and +/- 0.001 ppm in 1H spectra. Except for some overlapped or strongly coupled 1H resonances, chemical shifts are given to these precisions, which are an order of magnitude better than for most data given previously. Full 1H NMR data have been reported previously for only three of the 46 compounds, and 13C data were unavailable for many, including the previously undescribed cholesta-5,8(14)-dien-3 beta-ol. An extensive set of 1H-1H coupling constants for the unsaturated sterols indicated considerable conformational diversity, which was confirmed by molecular modeling. The conformational diversity together with other factors led to a complex pattern of 13C substituent-induced chemical shifts (SCS) that appeared to elude any simple empirical correlations with structure. By contrast, the 1H SCS correlated reasonably well with simple structural features. The high precision of the SCS revealed small but measurable effects of a double bond on 1H resonances up to 12 bonds away. Also discussed are the utility and limitations of NMR for the identification of unsaturated sterols, estimation of purity, and analysis of mixtures, with an emphasis on special problems encountered at a microgram level.

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.

In recent years, the process of selective autophagy has received much attention with respect to the clearance of protein aggregates, damaged mitochondria and bacteria. However, until recently, there have been virtually no studies on the selective autophagy of viruses, although they are perhaps one of the most ubiquitous unwanted constituents in human cells. Recently, we have shown that the ability of neuronal Atg5 to protect against lethal Sindbis virus central nervous system (CNS) infection in mice is associated with impaired viral capsid clearance, increased p62 accumulation and increased neuronal cell death. In vitro, we showed that p62 interacts with the Sindbis capsid protein and targets it for degradation in autophagosomes. Herein, we review these findings and broadly speculate about potential roles of selective viral autophagy in the regulation of host immunity and viral pathogenesis.

Hepatitis C virus (HCV) is a major human pathogen that infects 170 million people. A hallmark of HCV is its ability to establish persistent infections reflecting the evasion of host immunity and interference with alpha/beta-IFN innate immune defenses. We demonstrate that disruption of retinoic acid-inducible gene I (RIG-I) signaling by the viral NS3/4A protease contributes to the ability of HCV to control innate antiviral defenses. RIG-I was essential for virus or HCV RNA-induced signaling to the IFN-beta promoter in human hepatoma cells. This signaling was disrupted by the protease activity of NS3/4A, which ablates RIG-I signaling of downstream IFN regulatory factor 3 and NF-kappaB activation, attenuating expression of host antiviral defense genes and interrupting an IFN amplification loop that otherwise suppresses HCV replication. Treatment of cells with an active site inhibitor of the NS3/4A protease relieved this suppression and restored intracellular antiviral defenses. Thus, NS3/4A control of RIG-I supports HCV persistence by preventing IFN regulatory factor 3 and NF-kappaB activation. Our results demonstrate that these processes are amenable to restoration through pharmacologic inhibition of viral protease function.

Hepatitis C virus (HCV) RNA replication depends on viral protein association with intracellular membranes, but the influence of membrane composition on viral replication is unclear. We report that HCV RNA replication and assembly of the viral replication complex require geranylgeranylation of one or more host proteins. In cultured hepatoma cells, HCV RNA replication was disrupted by treatment with lovastatin, an inhibitor of 3-hydroxy-3-methyglutaryl CoA reductase, or with an inhibitor of protein geranylgeranyl transferase I, each of which induced the dissolution of the HCV replication complex. Viral replication was not affected by treatment of cells with an inhibitor of farnesyl transferase. When added to lovastatin-treated cells, geranylgeraniol, but not farnesol, restored replication complex assembly and viral replication. Inasmuch as the HCV genome does not encode a canonical geranylgeranylated protein, the data suggest the involvement of a geranylgeranylated host protein in HCV replication. Inhibition of its geranylgeranylation affords a therapeutic strategy for treatment of HCV infection.

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.

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.

The use of the intra-aortic balloon pump (IABP) in cardiac surgical patients has become accepted treatment. The purposes of this study were (1) to determine the frequency of use of the balloon, (2) to re-evaluate the indications for the IABP, and (3) to assess the hemodynamic effects of the balloon during surgery. In the past 24 months, the IABP was used in 63 of 1,738 (3.62%) adult cardiac surgical patients. Eight patients required the IABP prior to surgery due to complicated acute myocardial infarctions. In 13 patients, the IABP was used in the operating room before bypass for complicated infarctions or severe left ventricular dysfunction. It was not considered necessary before bypass in patients with left main coronary artery disease, moderately depressed left ventricular function, or unstable angina. In addition, 42 patients required the IABP to discontinue cardiopulmonary bypass. Detailed hemodynamic measurements were made in 11 patients. The IABP decreased systolic blood pressure, left and right ventricular filling pressures, and peripheral resistance, while it increased diastolic and mean arterial pressures, stroke work, cardiac output, and the endocardial viability ratio. The intra-aortic balloon was shown to be life-saving in certain patients. However, it should only be used selectively for specific indications. Careful surgical and anesthetic management with good monitoring can be used in many patients instead of the balloon.

Propranolol therapy has been implicated as a cause of myocardial depression and increased morbidity and mortality in patients undergoing coronary artery surgery. The authors reviewed 169 consecutive patients undergoing cardiac surgery, of whom 143 had been taking propranolol, with regard to preoperative administration of propranolol and intraoperative or postoperative complications. Patients taking propranolol until 24 hours before surgery showed no increased incidence of hypotension or bradycardia before cardiopulmonary bypass. Hypotension after bypass was no more common in patients off propranolol 12 to 48 hours than in patients who either discontinued the drug over 48 hours before operation or had never taken the drug. Myocardial contractility as measured by systolic time intervals was normal 24 to 48 hours after stopping propranolol therapy. Five patients had preoperative myocardial infarctions within 48 hours of discontinuing the drug. The operative mortality rate was 4 percent in patients taking propranolol within 48 hours of surgery and 6 percent in all other patients. Seven risk factors, other than propranolol, were identified in those patients requiring inotropic support. The authors conclude that propranolol can be given safely within 24 to 48 hours of coronary artery surgery provided the patient is a satisfactory candidate for myocardial revascularization.

Patients who had undergone aorto-coronary bypass grafts (ACBG) were assessed for the incidence of cardiac complications in the postoperative period following subsequent non-cardiac surgery. One hundred and twenty-one patients had 13 complications (11 per cent). A significantly higher risk of cardiac complications (27 per cent) was found in patients undergoing non-cardiac procedures in the first month after ACBG. This remained higher (17 per cent) until the sixth month following ACBG. Significant factors which increased the risk of cardiac complications in the postoperative period included preoperative congestive heart failure (33 per cent), cardiac risk index score classification of III or IV (37 per cent), surgery on major vessels, and surgery necessitated because of a complication of the ACBG itself (17 per cent). No correlation was found between cardiac complication rates and recurrent angina, hypertension, the use of beta-blockers or digoxin, or anaesthetic technique. It is suggested that all but emergency surgery should be postponed in the first month following ACBG, and elective surgery be delayed for up to six months.