Joao T Marques

Publications of Joao T Marques

• Viral apoptosis is induced by IRF-3-mediated activation of Bax.

  Authors: Saurabh Chattopadhyay, Joao T Marques, Michifumi Yamashita, Kristi L Peters, Kevin Smith, Avanti Desai, Bryan R G Williams, Ganes C Sen

  The EMBO journal. 04/2010; 29(10):1762-73.

  Upon infection with many RNA viruses, the cytoplasmic retinoic acid inducible gene-I (RIG-I) pathway activates the latent transcription factor IRF-3, causing its nuclear translocation and theUpon infection with many RNA viruses, the cytoplasmic retinoic acid inducible gene-I (RIG-I) pathway activates the latent transcription factor IRF-3, causing its nuclear translocation and the induction of many antiviral genes, including those encoding interferons. Here, we report a novel and distinct activity of IRF-3, in virus-infected cells, that induces apoptosis. Using genetically defective mouse and human cell lines, we demonstrated that, although both pathways required the presence of RIG-I, IPS1, TRAF3 and TBK1, only the apoptotic pathway required the presence of TRAF2 and TRAF6 in addition. More importantly, transcriptionally inactive IRF-3 mutants, such as the one missing its DNA-binding domain, could efficiently mediate apoptosis. Apoptosis was triggered by the direct interaction of IRF-3, through a newly identified BH3 domain, with the pro-apoptotic protein Bax, their co-translocation to the mitochondria and the resulting activation of the mitochondrial apoptotic pathway. Thus, IRF-3 is a dual-action cytoplasmic protein that, upon activation, translocates to the nucleus or to the mitochondrion and triggers two complementary antiviral responses of the infected cell.

• The role of PACT in mediating gene induction, PKR activation, and apoptosis in response to diverse stimuli.

  Authors: Joao T Marques, Christine L White, Gregory A Peters, Bryan R G Williams, Ganes C Sen

  Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 09/2008; 28(8):469-76.

  PACT, the protein activator of the double-stranded (ds)RNA-activated protein kinase (PKR) has been shown to strongly interact with and activate PKR in cultured cells and in vitro. To further analyzePACT, the protein activator of the double-stranded (ds)RNA-activated protein kinase (PKR) has been shown to strongly interact with and activate PKR in cultured cells and in vitro. To further analyze the functions of PACT we have recently generated PACT knockout (KO) mice and described several developmental defects that are absent in PKR KO mice. Importantly, PACT has been previously suggested to be involved in different signaling pathways that include endoplasmic reticulum stress, serum deprivation, growth factor withdrawal, viral infection, and cytokine responses. In this study, we have analyzed the contribution of PACT to these pathways using cells derived from wildtype (WT) and PACT KO mice. Notably, we have been unable to detect any significant differences in the responses to stress stimuli comparing WT and PACT KO cells, although we have been able to validate the specific interaction between PACT and PKR. Taken together, our results reinforce the importance of genetic loss of function analysis to infer protein function.

• Determinants of cytokine induction by small interfering RNA in human peripheral blood mononuclear cells.

  Authors: Maryam Zamanian-Daryoush, Joao T Marques, Michael P Gantier, Mark A Behlke, Matthias John, Patricia Rayman, James Finke, Bryan R G Williams

  Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 05/2008; 28(4):221-33.

  Synthetic small interfering RNAs (siRNAs) can trigger a strong innate immune response in mammalian cells. This nonspecific side effect may hinder the application of siRNAs as tools in gene silencing.Synthetic small interfering RNAs (siRNAs) can trigger a strong innate immune response in mammalian cells. This nonspecific side effect may hinder the application of siRNAs as tools in gene silencing. Chemically synthesized siRNAs, including traditional 19-mers with 2-nt 3' overhangs, longer duplexes with blunt or 3' overhangs, and asymmetric duplexes with a blunt end and a 2-nt 3' overhang, can evoke strong dose-dependent interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) release in human peripheral blood mononuclear cells (PBMCs). This response is independent of retinoic acid-inducible gene I but may involve endosomal toll-like receptors (TLRs). The immunostimulatory effect of the siRNAs is directly related to either or both of the strands of the duplex in a sequence-dependent manner. However, although some single-stranded RNAs and siRNAs potently evoked both IFN-alpha and TNF-alpha induction, these responses were not always coupled. In accordance with this, specific chemical modifications differentially altered cytokine production, suggesting recruitment of different TLRs in a sequence-dependent manner.

• A call to arms: coevolution of animal viruses and host innate immune responses.

  Authors: Joao T Marques, Richard W Carthew

  Trends in genetics : TIG. 08/2007; 23(7):359-64.

  Virus infection is generally disadvantageous to the host and strongly selects for host antiviral mechanisms. Therefore, viruses must develop counter-mechanisms to guarantee their survival. This armsVirus infection is generally disadvantageous to the host and strongly selects for host antiviral mechanisms. Therefore, viruses must develop counter-mechanisms to guarantee their survival. This arms race between pathogen and host leads to positive selection for both cellular antiviral mechanisms and viral inhibitors of such mechanisms. Here, we characterize this arms race in the context of the RNA interference (RNAi) pathway, which is used as an innate immune response against viral infection by animals. We review how RNAi is used as an antiviral strategy and the mechanisms that viruses have evolved to suppress the RNAi response.

• Light controllable siRNAs regulate gene suppression and phenotypes in cells.

  Authors: Quan N Nguyen, Rajesh V Chavli, Joao T Marques, Peter G Conrad, Die Wang, Weihai He, Barbara E Belisle, Aiguo Zhang, Larry M Pastor, Frank R Witney, May Morris, Frederic Heitz, Gilles Divita, Bryan R G Williams, Gary K McMaster

  Biochimica et biophysica acta. 04/2006; 1758(3):394-403.

  Small interfering RNA (siRNA) is widely recognized as a powerful tool for targeted gene silencing. However, siRNA gene silencing occurs during transfection, limiting its use is in kinetic studies,Small interfering RNA (siRNA) is widely recognized as a powerful tool for targeted gene silencing. However, siRNA gene silencing occurs during transfection, limiting its use is in kinetic studies, deciphering toxic and off-target effects and phenotypic assays requiring temporal, and/or spatial regulation. We developed a novel controllable siRNA (csiRNA) that is activated by light. A single photo removable group is coupled during oligonucleotide synthesis to the 5' end of the antisense strand of the siRNA, which blocks the siRNA's activity. A low dose of light activates the siRNA, independent of transfection resulting in knock down of specific target mRNAs and proteins (GAPDH, p53, survivin, hNuf2) without stimulating non-specific effects such as regulated protein kinase PKR and induction of the interferon response. We demonstrate survivin and hNuf2 csiRNAs temporally knockdown their mRNAs causing multinucleation and cell death by mitotic arrest, respectively. Furthermore, we demonstrate a dose-dependent light regulation of hNuf2 csiRNA activity and resulting phenotype. The light controllable siRNAs are introduced into cells using commercially available reagents including the MPG peptide based delivery system. The csiRNAs are comparable to standard siRNAs in their transfection efficiency and potency of gene silencing. This technology should be of interest for phenotypic assays such as cell survival, cell cycle regulation, and cell development.

• Efficient suppression of secretory clusterin levels by polymer-siRNA nanocomplexes enhances ionizing radiation lethality in human MCF-7 breast cancer cells in vitro.

  Authors: Damon Sutton, Saejeong Kim, Xintao Shuai, Konstantin Leskov, Joao T Marques, Bryan R G Williams, David A Boothman, Jinming Gao

  International journal of nanomedicine. 02/2006; 1(2):155-62.

  Small interfering RNA molecules (siRNA) hold great promise to specifically target cytoprotective factors to enhance cancer therapy. Like antisense RNA strategies, however, the use of siRNA is limitedSmall interfering RNA molecules (siRNA) hold great promise to specifically target cytoprotective factors to enhance cancer therapy. Like antisense RNA strategies, however, the use of siRNA is limited because of in vivo instability. As a first step to overcome delivery issues, a series of graft copolymers of polyethylene glycol and polyethylenimine (PEI-g-PEG) were synthesized and investigated as nontoxic carriers for delivery of siRNA targeting the signaling peptide of secretory clusterin (sCLU), a prosurvival factor that protects cells from ionizing radiation (IR) injury, as well as chemotherapeutic agents. Three copolymers with different PEG grafting densities were tested for their abilities to bind and form nanocomplexes with siRNA. A copolymer composed of 10 PEG grafts (2 kDa each) per PEI polymer (2k10 copolymer) gave the highest binding affinity to siRNA by ethidium bromide exclusion assays, and had the smallest nanocomplex size (115 +/- 13 nm diameter). In human breast cancer MCF-7 cells, 2k10-siRNA-sCLU nanocomplexes suppressed both basal as well as IR-induced sCLU protein expression, which led to an over 3-fold increase in IR-induced lethality over 2k10-siRNA scrambled controls. In summary, this study demonstrates the proof-of-principle in using nanoparticle-mediated delivery of specific siRNAs to enhance the lethality of IR exposure in vitro, opening the door for siRNA-mediated knockdown of specific cytoprotective factors, such as DNA repair, anti-apoptotic, free radical scavenging, and many other proteins.

• Activation of the mammalian immune system by siRNAs.

  Authors: Joao T Marques, Bryan R G Williams

  Nature biotechnology. 12/2005; 23(11):1399-405.

  Inhibition of gene expression through RNA interference (RNAi) is emerging as a powerful experimental tool for gene function and target validation studies. The potential uses of this technology seemInhibition of gene expression through RNA interference (RNAi) is emerging as a powerful experimental tool for gene function and target validation studies. The potential uses of this technology seem unlimited, extending to the prevention and therapy of human diseases. However, recent work demonstrating that there are unanticipated, different nonspecific effects associated with the use of small interfering RNAs in mammals has raised concerns about the safe use of RNAi in vivo. These nonspecific effects include activation of the immune system, potentially harming the individual. The application of screening assays for nonspecific activation of both innate and acquired immunity will be necessary for further development of RNAi as a therapeutic tool.

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

  Authors: Joao T Marques, Dominique Rebouillat, Chilakamarti V Ramana, Junko Murakami, Jason E Hill, Andrei Gudkov, Robert H Silverman, George R Stark, Bryan R G Williams

  Journal of virology. 10/2005; 79(17):11105-14.

  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 activatedp53 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.

• A call to arms: coevolution of animal viruses and host innate immune responses

  Authors: Joao T. Marques, Richard W. Carthew

  Trends in Genetics.

  Virus infection is generally disadvantageous to the host and strongly selects for host antiviral mechanisms. Therefore, viruses must develop counter-mechanisms to guarantee their survival. This armsVirus infection is generally disadvantageous to the host and strongly selects for host antiviral mechanisms. Therefore, viruses must develop counter-mechanisms to guarantee their survival. This arms race between pathogen and host leads to positive selection for both cellular antiviral mechanisms and viral inhibitors of such mechanisms. Here, we characterize this arms race in the context of the RNA interference (RNAi) pathway, which is used as an innate immune response against viral infection by animals. We review how RNAi is used as an antiviral strategy and the mechanisms that viruses have evolved to suppress the RNAi response.