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. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with DeltaPsi(m) loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.

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. We decided to investigate the putative antiviral activity of p59 by ectopically expressing this protein in Vero cells and then infecting these cells with virus. We demonstrate that OASL has an antiviral effect against the single-stranded RNA virus picornavirus, encephalomyocarditis virus, but not against a large DNA virus, herpes simplex virus 1. Importantly, this antiviral activity was lost in a truncated version of p59 lacking the ubiquitin-like C-terminal domain of p59. Taken together our results indicate that p59 is indeed an antiviral protein that works through a novel mechanism distinct from other OAS proteins.

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. Humans possess four distinct ANT isoforms, encoded by four genes, whose transcription depends on the cell type, developmental stage, cell proliferation, and hormone status. Here, we show that the ANT2 gene is up-regulated in several hormone-dependent cancers. Knockdown of ANT2 by RNA interference induced no major changes in the aspect of the mitochondrial network or cell cycle but provoked minor increase in mitochondrial transmembrane potential and reactive oxygen species level and reduced intracellular ATP concentration without affecting glycolysis. At expression and functional levels, ANT2 depletion was not compensated by other ANT isoforms. Most importantly, ANT2, but not ANT1, silencing facilitated MMP induction by lonidamine, a mitochondrion-targeted antitumor compound already used in clinical studies for breast, ovarian, glioma, and lung cancer as well as prostate adenoma. The combination of ANT2 knockdown with lonidamine induced apoptosis irrespective of the Bcl-2 status. These data identify ANT2 as an endogenous inhibitor of MMP and suggest that its selective inhibition could constitute a promising strategy of chemosensitization.

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. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G(1) arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G(1) arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication.

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. Fixed-time and real-time FC permits to co-evaluate and order modifications of mitochondrial size, structure and inner membrane (IM) electrochemical potential (DeltaPsi(m)) during MMP. Calcium, a major PTP opener, and alamethicin, a PTP-independent MMP inducer, trigger significant mitochondrial forward scatter (FSC) increase and side scatter (SSC) decrease, correlating with spectrophotometrically detected swelling. FC-based fluorescence detection of the DeltaPsi(m)-sensitive cationic lipophilic dye JC-1 permits to detect DeltaPsi(m) variations induced by PTP openers or specific inducers of inner MMP such as carbonylcyanide m-chlorophenylhydrazone (mClCCP). These simple, highly sensitive and quantitative FC-based methods will be pertinent to evaluate compounds for their ability to control MMP.