HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase.

The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria.

A flavivirus protein M-derived peptide directly permeabilizes mitochondrial membranes, triggers cell death and reduces human tumor growth in nude mice.

Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events.

The p59 oligoadenylate synthetase-like protein possesses antiviral activity that requires the C-terminal ubiquitin-like domain.

Viral infection of mammalian cells prompts the innate immune system to initiate an antiviral response. The recognition of the virus triggers several antiviral signalling pathways, which among others include the family of 2'-5' oligoadenylate synthetase (OAS) proteins. The p59 protein encoded by the OAS-like (OASL) gene is an atypical member of the OAS family in the sense that it lacks the characteristic 2'-5' oligoadenylate synthetase activity.

Chemosensitization by knockdown of adenine nucleotide translocase-2.

Mitochondrial membrane permeabilization (MMP) is a rate-limiting step of apoptosis, including in anticancer chemotherapy. Adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP on the inner mitochondrial membrane in healthy cells. In addition, ANT can cooperate with Bax to form a lethal pore during apoptosis.

Targeted Vpr-derived peptides reach mitochondria to induce apoptosis of alphaVbeta3-expressing endothelial cells.

The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP.

Upstream control of apoptosis by caspase-2 in serum-deprived primary neurons.

During development as well as in pathological situations, neurons that fail to find appropriate targets or neurotrophic factors undergo cell death. Using primary cortical neurons subjected to acute serum-deprivation (SD), we have examined caspases activation, mitochondrial dysfunction and cell death parameters. Among a panel of metabolic, signaling and caspases inhibitors only those able to interfere with caspase-2 like activity protect primary neurons against SD-induced cell death.

Down-regulation of p53 by double-stranded RNA modulates the antiviral response.

p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells.

Dynamic analysis of apoptosis in primary cortical neurons by fixed- and real-time cytofluorometry.

We describe here a cytofluorometric technology for the characterization of decision, execution, and degradation steps of neuronal apoptosis. Multiparametric flow cytometry was developed and combined to detailed fluorescence microscopy observations to establish the chronology and hierarchy of death-related events: neuron morphological changes, mitochondrial transmembrane potential (DeltaPsi(m)) collapse, caspase-3 and -9 activation, phosphatidyl-serine exposure, nuclear dismantling and final plasma membrane permeabilization. Moreover, we developed a reliable real-time flow cytometric monitoring of DeltaPsi(m) and plasma membrane integrity in response to neurotoxic insults including MPTP treatment.

Real-time flow cytometry analysis of permeability transition in isolated mitochondria.

Mitochondrial membrane permeabilization (MMP) is a key event in necrotic and (intrinsic) apoptotic processes. MMP is controlled by a few major rate-limiting events, one of which is opening of the permeability transition pore (PTP). Here we develop a flow cytometry (FC)-based approach to screen and study inducers and blockers of MMP in isolated mitochondria.

Combined use of radioimagers and radioactive 3'OH DNA nick end labelling to quantify apoptosis in cell lines and tissue sections: applications to virus-induced apoptosis.

DNA fragmentation is a key feature of the degradation phase of apoptosis. In this work we have developed an assay, based on radioimager (beta-IMAGER and micro-IMAGER) quantification of radioactive nick end labelling (RANEL), which is quantitative, rapid and sensitive to study in vitro and in vivo induced apoptosis. To establish the technique, in vitro apoptosis of T cell lines was induced by stimulation of the Fas receptor; cells were labelled using TdT-mediated [alpha-33P] dCTP nick end labelling, after which then radioactivity was quantified using a beta-IMAGER.

Characterization of the gene encoding the 100-kDa form of human 2',5' oligoadenylate synthetase.

The 2'-5' oligoadenylate synthetases (OAS) represent a family of interferon (IFN)-induced proteins implicated in the antiviral action of IFN. When activated by double-stranded (ds) RNA, these proteins polymerize ATP into 2'-5' linked oligomers with the general formula pppA(2'p5'A)n, n greater than or = 1. Three forms of human OAS have been described corresponding to proteins of 40/46, 69/71, and 100 kDa.

Expression of hepatitis C virus proteins interferes with the antiviral action of interferon independently of PKR-mediated control of protein synthesis.

Hepatitis C virus (HCV) of genotype 1 is the most resistant to interferon (IFN) therapy. Here, we have analyzed the response to IFN of the human cell line UHCV-11 engineered to inducibly express the entire HCV genotype 1a polyprotein. IFN-treated, induced UHCV cells were found to better support the growth of encephalomyocarditis virus (EMCV) than IFN-treated, uninduced cells.

The human 2',5'-oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties.

2',5'-Oligoadenylate synthetase (2',5'-OAS) was discovered and characterized as an interferon (IFN)-induced enzyme that in the presence of double-stranded (ds) RNA converts ATP into 2',5'-linked oligomers of adenosine with the general formula pppA(2'p'A)n, n > or = 1. The product is pppG2'p5'G when GTP is used as a substrate. Now, 20 years later, this activity is attributed to several well-characterized, homologous, and IFN-induced proteins in human cells.

The expression of both domains of the 69/71 kDa 2',5' oligoadenylate synthetase generates a catalytically active enzyme and mediates an anti-viral response.

The 2',5' oligoadenylate synthetase (OAS) represents a family of interferon-induced proteins which, when activated by double-stranded (ds) RNA, polymerizes ATP into 2',5'-linked oligomers with the general formula pppA(2'p5'A)n, where n >/= 1. The 69-kDa form of human OAS has two isoforms (p69 and p71) that are identical for their first 683 amino acids and consist of two homologous and adjacent domains, each homologous to the small 40-kDa OAS. Here, we demonstrate that mRNA species specific for the isoforms p69 and p71 are enhanced in interferon-treated cells, with the p69 mRNA being more abundant than that of p71.

The 100-kDa 2',5'-oligoadenylate synthetase catalyzing preferentially the synthesis of dimeric pppA2'p5'A molecules is composed of three homologous domains.

The 2-5A synthetases represent a family of proteins implicated in the mechanism of the antiviral action of interferon. When activated by double-stranded RNA, these proteins polymerize ATP into 2'-5'-linked oligomers with the general formula pppA(2'p5'A)n, n >/= 1. Three forms of human 2-5A synthetases have been described corresponding to proteins of 40/46 (p40/p46), 69/71 (p69/p71), and 100 kDa (p100).

Molecular cloning and characterization of two related and interferon-induced 56-kDa and 30-kDa proteins highly similar to 2'-5' oligoadenylate synthetase.

The 2'-5' oligoadenylate synthetase (OAS) represents a family of interferon-induced proteins which when activated by double-stranded (ds) RNA polymerizes ATP into 2'-5' linked oligomers with the general formula pppA(2'p5'A)n, n> or =1. Three forms of human OASs have been described corresponding to proteins of 40/46, 69/71 and 100 kDa (p40/p46, p69/p71 and p100). Polyclonal antibodies raised against p100 cross reacted with the other forms p40/p46, p69/p71 but also with an interferon-induced 56-kDa protein (p56).

The human 2',5'-oligoadenylate synthetase locus is composed of three distinct genes clustered on chromosome 12q24.2 encoding the 100-, 69-, and 40-kDa forms.

The 2',5'-oligoadenylate synthetases (OAS) represent a family of interferon-induced proteins implicated in the mechanism of the antiviral action of interferon. When activated by double-stranded RNA, these proteins polymerize ATP into 2'-5'-linked oligomers with the general formula pppA(2'p5'A)n, n >/= 1. Three forms of human OAS corresponding to proteins of 40/46, 69/71, and 100 kDa have been described.

Ultrastructural localization of interferon-inducible double-stranded RNA-activated enzymes in human cells.

The protein kinase PKR and the 2',5'-oligoadenylate (2-5A) synthetase are two interferon-induced and double-stranded RNA-activated enzymes which are implicated in the mechanism of action of interferon. Their distribution was undertaken here at the ultrastructural level by the immunogold procedure, following the use of specific monoclonal antibodies directed against PKR and 69- and 100-kDa forms of the 2-5A synthetase. These enzymes were detected as a pool of nonaggregated proteins scattered throughout the cell and as aggregates often associated with electron-dense doughnut-like structures showing a similar aspect whatever their subcellular localization: the cytoplasm, the nuclear envelope, and the nucleus.

69-kDa and 100-kDa isoforms of interferon-induced (2'-5')oligoadenylate synthetase exhibit differential catalytic parameters.

The (2'-5')oligoadenylate synthetase represents a family of interferon-induced proteins which when activated by double-stranded (ds)RNA polymerizes ATP into 2'-5'-linked oligomers with the general formula pppA(2'p5'A)n, where n > 1, which for convenience are referred to as 2-5A. We studied here the influence of pH, dsRNA concentration and time on oligomeric composition of 2-5A synthesized by purified 69-kDa and 100-kDa isoforms of (2'-5')oligo(adenylate) synthetase. In optimal conditions for activity, the 69-kDa form synthesized higher oligomers of 2-5A molecules whereas the 100 kDa form synthesized preferentially dimeric molecules, which are known not to be functional for the activation of RNase L.

Tandem repeat polymers of a critical region of the human interferon-beta promoter exhibit a marked constitutive activity and enhanced responsiveness to transcriptional regulators in transfected HeLa cells.

Multiple copy tandem repeats polymers of an authentic 30-bp region of the human interferon-beta (IFN-beta) promoter between positions-91 to -62 relative to the cap site or the hexanucleotide GAAAGT derived from this region, both acted as strong constitutive regulatory elements in transfected HeLa cells. Such polymers were unresponsive to treatment with IFN-alpha despite their considerable homology with the IFN-responsive elements of other genes but were highly responsive to treatment of HeLa cells with IFN-gamma. Virus induction of HeLa cells transfected with polymers of the 30-bp region linked to a CAT gene increased the activity of the reporter gene 500- to 2,000-fold over baseline levels.