Abstract17Correspondence should be addressed to G.K. (e-mail: E-mail: kroemer@igr.fr). 16 These authors contributed equally to this paper. AUTHOR CONTRIBUTIONS E.T., M.C.M., L.G. and I.V. conducted experiments, prepared figures and analysed data; M.D.-M., M.D.'A., A.C., E.M., C.Z., F.H., U.N., C.S., P.P., J.M.V, R.C., F.M., P.P.B, G.S., G.P., K.B., N.T., P.C. and F.C. performed experiments; E.T. and G.K. planned the project; G.K. supervised the project and wrote the manuscript.Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2009 May 4. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptMultiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that knockout, knockdown or pharmacological inhibition of p53 can induce autophagy in human, mouse and nematode cells. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53 -/-cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.Autophagy (`self-eating') is an important eukaryotic response to cellular stress. During autophagy, portions of the cytosol and cytoplasmic organelles are sequestered within characteristic double-or multi-membraned autophagosomes and delivered to lysosomes for bulk degradation. By promoting catabolic reactions, autophagy generates new metabolic substrates that meet the bioenergetic needs of cells and allows for adaptive protein synthesis. Autophagy also constitutes a homeostatic `clean-up' process to rid cells of intracellular parasites, damaged organelles and potentially toxic, aggregate-prone proteins. Finally, autophagy has been viewed as a self-destructive process in which stressed cells succumb to the so-called autophagic cell death 1 .Autophagy is essential for the long-term survival of mammalian cells and a partial reduction in the autophagic capacity may constitute an oncogenic event. At least one of the phylogenetically conserved autophagy genes, atg6/beclin 1, is frequently inactivated at one locus in human cancers, and mouse studies have confirmed that beclin 1 is a haploinsufficient tumour suppressor 2 . There are two non-exclusive hypotheses to explain how inhibition of autoph...

There is now abundant evidence to substantiate an important role of hepatitis C virus (HCV) core protein in cellular gene expression as well as in the viral cycle. Thus the subcellular localization of this protein has important implications. However, several studies have shown controversial results: the HCV core has been, indeed, described as cytoplasmic or nuclear depending on the size of the protein or on the genotype analyzed. We have studied the localization of the HCV core protein in two different cell lines, one nonhepatic (CHO) and the other hepatic (HepG2). Double immunof luorescence staining using a nuclear membrane marker and confocal analysis showed the core protein pattern to be cytoplasmic and globular. This pattern is not cell cycleregulated. Electron microscopy analysis revealed the nature of the globular staining observed in immunof luorescence. The HCV core protein accumulated at the surface of lipid droplets that were also the unique morphological feature of nonhepatic core transfected cells. The lipid droplets were isolated by sequential ultracentrifugation on the basis of their density; biochemical analysis revealed a prevalence of triglycerides. In addition the core protein colocalized with apolipoprotein AII at the surface of the lipid droplets as revealed by confocal microscopy. Moreover analysis of liver biopsies from chronically HCV-infected chimpanzees revealed that HCV core is cytoplasmic and localized on the endoplasmic reticulum and on lipid droplets. These results clearly define the subcellular localization of the HCV core protein and suggest a relationship between the expression of the HCV core protein and cellular lipid metabolism.The hepatitis C virus (HCV), the major causative agent of non-A͞non-B hepatitis (1), is a positive-stranded RNA virus of about 10 kb evolutionary related to pestivirus and flavivirus (2, 3). The HCV ORF is flanked by a 341-bp long 5Ј untranslated region and a 3Ј untranslated region with a poly(U) or a poly(A) tail (3, 4) and encodes for a precursor polyprotein of about 3000 aa that is then cleaved into structural and nonstructural proteins (5, 6).A major characteristic of HCV infection is the extremely high (up to 80%) risk of chronicity; in addition, chronic infection can lead to liver cirrhosis and liver cancer (7,8). An important issue regarding the pathogenesis of HCV-associated liver lesions is to determine whether or not HCV proteins might have a direct effect on cellular phenotype as suggested by some recent works (9, 10). In this view, a regulative effect by HCV core protein, one of the structural proteins of the virus, has been shown by transfection both on hepatitis B viral genome expression and replication (11) and on expression of different cellular genes such as c-myc oncogene (12) or genes encoding for interferon ␤ in a human cell line (13).The observations reported above would suggest that the HCV core protein could have not only a packaging function in the cytoplasm, but also a regulatory role on cell functions. Precise informatio...

Activated human T lymphocytes exposed to apoptotic stimuli targeting mitochondria (i.e. staurosporine), enter an early, caspase-independent phase of commitment to apoptosis characterized by cell shrinkage and peripheral chromatin condensation. We show that during this phase, AIF is selectively released from the intermembrane space of mitochondria, and that Bax undergo conformational change, relocation to mitochondria, and insertion into the outer mitochondrial membrane, in a Bid-independent manner. We analyzed the subcellular distribution of cathepsins (Cat) B, D, and L, in a search for caspase-independent factors responsible for Bax activation and AIF release. All were translocated from lysosomes to the cytosol, in correlation with limited destabilization of the lysosomes and release of lysosomal molecules in a size selective manner. However, only inhibition of Cat D activity by pepstatin A inhibited the early apoptotic events and delayed cell death, even in the presence of bafilomycin A 1 , an inhibitor of vacuolar type H ؉ -ATPase, which inhibits acidification in lysosomes. Small interfering RNA-mediated gene silencing was used to inactivate Cat D, Bax, and AIF gene expression. This allowed us to define a novel sequence of events in which Cat D triggers Bax activation, Bax induces the selective release of mitochondrial AIF, and the latter is responsible for the early apoptotic phenotype.

Human Endogenous Retroviruses are expected to be the remnants of ancestral infections of primates by active retroviruses that have thereafter been transmitted in a Mendelian fashion. Here, we derived in silico the sequence of the putative ancestral "progenitor" element of one of the most recently amplified family-the HERV-K family-and constructed it. This element, Phoenix, produces viral particles that disclose all of the structural and functional properties of a bona-fide retrovirus, can infect mammalian, including human, cells, and integrate with the exact signature of the presently found endogenous HERV-K progeny. We also show that this element amplifies via an extracellular pathway involving reinfection, at variance with the non-LTR-retrotransposons (LINEs, SINEs) or LTR-retrotransposons, thus recapitulating ex vivo the molecular events responsible for its dissemination in the host genomes. We also show that in vitro recombinations among present-day human HERV-K (also known as ERVK) loci can similarly generate functional HERV-K elements, indicating that human cells still have the potential to produce infectious retroviruses.[Supplemental material is available online at www.genome.org.]Nearly 8% of the human genome is composed of sequences of retroviral origin. Most of them are degenerate, either due to recombination between the two provirus LTRs or to mutations interrupting the retroviral ORFs. The env gene seems to be best conserved, with 18 intact genes retaining a full coding capacity in the human genome (Benit et al. 2001;de Parseval et al. 2003;Villesen et al. 2004), possibly because of its potential role in human physiology. The HERV-K(HML2) family of endogenous retroviruses is an exception to this general rule, since some copies still contain complete ORFs for the other retroviral genes (Löwer et al. 1996; for review, see Bannert and Kurth 2004). This family includes the most recently amplified endogenous retroviruses, most of which have integrated into the genome <5 million years ago, with a few insertions showing polymorphism within the human population (Steinhuber et al. 1995;Medstrand and Mager 1998;Barbulescu et al. 1999;Turner et al. 2001;Hughes and Coffin 2004;Belshaw et al. 2005). Some of these recently integrated proviruses are responsible for the synthesis of retroviral particles that can be observed in teratocarcinoma and melanoma-derived cell lines Löwer et al. 1993;Bieda et al. 2001;Muster et al. 2003;Buscher et al. 2005), and possibly in human placenta (Kalter et al. 1973;Dirksen and Levy 1977;Wilkinson et al. 1994). Because of this "activity," the HERV-K(HML2) family has been the subject of numerous studies in the past years, with the description of alleles with nearly intact proviruses and complete coding capacity (Mayer et al. 1999;Reus et al. 2001;Turner et al. 2001). Despite these efforts, no functional provirus able to produce infectious particles has yet been described. Even if several loci containing complete ORFs have been identified, and in spite of the availability of the comp...

In response to stress, cells start transcriptional and transcription-independent programs that can lead to adaptation or death. Here, we show that multiple inducers of autophagy, including nutrient depletion, trigger the activation of the IKK (IjB kinase) complex that is best known for its essential role in the activation of the transcription factor NF-jB by stress. Constitutively active IKK subunits stimulated autophagy and transduced multiple signals that operate in starvation-induced autophagy, including the phosphorylation of AMPK and JNK1. Genetic inhibition of the nuclear translocation of NF-jB or ablation of the p65/RelA NF-jB subunit failed to suppress IKK-induced autophagy, indicating that IKK can promote the autophagic pathway in an NF-jB-independent manner. In murine and human cells, knockout and/or knockdown of IKK subunits (but not that of p65) prevented the induction of autophagy in response to multiple stimuli. Moreover, the knockout of IKK-b suppressed the activation of autophagy by food deprivation or rapamycin injections in vivo, in mice. Altogether, these results indicate that IKK has a cardinal role in the stimulation of autophagy by physiological and pharmacological stimuli.

The reduction of intracellular 1,4,5-inositol trisphosphate (IP 3 ) levels stimulates autophagy, whereas the enhancement of IP 3 levels inhibits autophagy induced by nutrient depletion. Here, we show that knockdown of the IP 3 receptor (IP 3 R) with small interfering RNAs and pharmacological IP 3 R blockade is a strong stimulus for the induction of autophagy. The IP 3 R is known to reside in the membranes of the endoplasmic reticulum (ER) as well as within ER-mitochondrial contact sites, and IP 3 R blockade triggered the autophagy of both ER and mitochondria, as exactly observed in starvation-induced autophagy. ER stressors such as tunicamycin and thapsigargin also induced autophagy of ER and, to less extent, of mitochondria. Autophagy triggered by starvation or IP 3 R blockade was inhibited by Bcl-2 and Bcl-X L specifically targeted to ER but not Bcl-2 or Bcl-X L proteins targeted to mitochondria. In contrast, ER stress-induced autophagy was not inhibited by Bcl-2 and Bcl-X L . Autophagy promoted by IP 3 R inhibition could not be attributed to a modulation of steady-state Ca 2 þ levels in the ER or in the cytosol, yet involved the obligate contribution of Beclin-1, autophagy-related gene (Atg)5, Atg10, Atg12 and hVps34. Altogether, these results strongly suggest that IP 3 R exerts a major role in the physiological control of autophagy. The first step of macroautophagy consists in the gradual envelopment of cytoplasmic material (cytosol and/or organelles) in the phagophore, a cistern that finally sequesters cytoplasmic material in autophagosomes (also called autophagic vacuoles (AVs)) lined by two membranes. Autophagosomes then undergo a progressive maturation by fusion with endosomes and/or lysosomes. This latter step creates autolysosomes in which the inner membrane as well as the luminal content of the AVs is degraded by lysosomal enzymes. The process of autophagy is controlled by a series of evolutionary conserved genes, the atg genes, whose products are essential for specific steps of the autophagic process. 1,2One of the strongest triggers of autophagy is nutrient stress. 3,4 In response to starvation, cells degrade nonessential components thereby generating nutrients for meeting the cell's energetic demand as well as for vital biosynthetic reactions. In such circumstances, when autophagy is an adaptation, inhibition of autophagy has a negative impact on cell survival. For example, mice lacking the atg5 gene survive without any major developmental defect until birth, yet succumb to the stressful neonatal period unless puppies are force-fed with milk within the first hours after birth.5 Similarly, human cell lines cultured in nutrient-free media mount a cytoprotective autophagic response. Suppression of autophagy by chemical inhibitors or knockdown of essential genes thus sensitize cells to starvation-induced cell death. 6,7Autophagy inhibition also sensitizes cells to the depletion of obligatory growth (or survival) factors resulting in a decrease of nutrient import through the plasma membrane. For example...

Rabies virus infection induces the formation of cytoplasmic inclusion bodies that resemble Negri bodies found in the cytoplasm of some infected nerve cells. We have studied the morphogenesis and the role of these Negri body-like structures (NBLs) during viral infection. The results indicate that these spherical structures (one or two per cell in the initial stage of infection), composed of the viral N and P proteins, grow during the virus cycle before appearing as smaller structures at late stages of infection. We have shown that the microtubule network is not necessary for the formation of these inclusion bodies but is involved in their dynamics. In contrast, the actin network does not play any detectable role in these processes. These inclusion bodies contain Hsp70 and ubiquitinylated proteins, but they are not misfolded protein aggregates. NBLs, in fact, appear to be functional structures involved in the viral life cycle. Specifically, using in situ fluorescent hybridization techniques, we show that all viral RNAs (genome, antigenome, and every mRNA) are located inside the inclusion bodies. Significantly, short-term RNA labeling in the presence of BrUTP strongly suggests that the NBLs are the sites where viral transcription and replication take place.

We describe the spatial organization of the two NEAT1 noncoding (nc)RNAs required for the integrity of the paraspeckle nuclear bodies. The central sequences of the long transcript are internal when its extremities and the short isoform are peripheral, indicating how RNA can contribute to the architecture of nuclear bodies.