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ABSTRACT: Rift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus infecting a wide range of vertebrate hosts. Of particular interest is the nonstructural NSs protein, which forms large filamentous fibril bundles in the nucleus. Past studies have shown NSs to be a multifaceted protein important for virulence through modulation of the interferon response as well acting as a general inhibitor of transcription. Here we investigated the regulation of the DNA damage signaling cascades by RVFV infection and found virally inducted phosphorylation of the classical DNA damage signaling proteins, ataxia-telangiectasia mutated (ATM) (Ser-1981), Chk.2 (Thr-68), H2A.X (Ser-139), and p53 (Ser-15). In contrast, ataxia-telangiectasia mutated and Rad3-related kinase (ATR) (Ser-428) phosphorylation was decreased following RVFV infection. Importantly, both the attenuated vaccine strain MP12 and the fully virulent strain ZH548 showed strong parallels in their up-regulation of the ATM arm of the DNA damage response and in the down-regulation of the ATR pathway. The increase in DNA damage signaling proteins did not result from gross DNA damage as no increase in DNA damage was observed following infection. Rather the DNA damage signaling was found to be dependent on the viral protein NSs, as an NSs mutant virus was not found to induce the equivalent signaling pathways. RVFV MP12-infected cells also displayed an S phase arrest that was found to be dependent on NSs expression. Use of ATM and Chk.2 inhibitors resulted in a marked decrease in S phase arrest as well as viral production. These results indicate that RVFV NSs induces DNA damage signaling pathways that are beneficial for viral replication.
Journal of Biological Chemistry 03/2012; 287(10):7399-410. · 4.77 Impact Factor
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Dana Austin,
Alan Baer,
Lindsay Lundberg,
Nazly Shafagati,
Annalise Schoonmaker,
Aarthi Narayanan, Taissia Popova,
Jean Jacques Panthier,
Fatah Kashanchi,
Charles Bailey,
Kylene Kehn-Hall
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ABSTRACT: Rift Valley fever virus (RVFV) is an emerging viral zoonosis that is responsible for devastating outbreaks among livestock and is capable of causing potentially fatal disease in humans. Studies have shown that upon infection, certain viruses have the capability of utilizing particular cellular signaling pathways to propagate viral infection. Activation of p53 is important for the DNA damage signaling cascade, initiation of apoptosis, cell cycle arrest and transcriptional regulation of multiple genes. The current study focuses on the role of p53 signaling in RVFV infection and viral replication. These results show an up-regulation of p53 phosphorylation at several serine sites after RVFV MP-12 infection that is highly dependent on the viral protein NSs. qRT-PCR data showed a transcriptional up-regulation of several p53 targeted genes involved in cell cycle and apoptosis regulation following RVFV infection. Cell viability assays demonstrate that loss of p53 results in less RVFV induced cell death. Furthermore, decreased viral titers in p53 null cells indicate that RVFV utilizes p53 to enhance viral production. Collectively, these experiments indicate that the p53 signaling pathway is utilized during RVFV infection to induce cell death and increase viral production.
PLoS ONE 01/2012; 7(5):e36327. · 4.09 Impact Factor
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ABSTRACT: Rift Valley fever (RVF) is a zoonotic disease caused by Rift Valley fever virus (RVFV). RVFV is a category A pathogen that belongs to the genus Phlebovirus, family Bunyaviridae. Understanding early host events to an infectious exposure to RVFV will be of significant use in the development of effective therapeutics that not only control pathogen multiplication, but also contribute to cell survival. In this study, we have carried out infections of human cells with a vaccine strain (MP12) and virulent strain (ZH501) of RVFV and determined host responses to viral infection. We demonstrate that the cellular antioxidant enzyme superoxide dismutase 1 (SOD1) displays altered abundances at early time points following exposure to the virus. We show that the enzyme is down regulated in cases of both a virulent (ZH501) and a vaccine strain (MP12) exposure. Our data demonstrates that the down regulation of SOD1 is likely to be due to post transcriptional processes and may be related to up regulation of TNFα following infection. We also provide evidence for extensive oxidative stress in the MP12 infected cells. Concomitantly, there is an increase in the activation of the p38 MAPK stress response, which our earlier published study demonstrated to be an essential cell survival strategy. Our data suggests that the viral anti-apoptotic protein NSm may play a role in the regulation of the cellular p38 MAPK response. Alterations in the host protein SOD1 following RVFV infection appears to be an early event that occurs in multiple cell types. Activation of the cellular stress response p38 MAPK pathway can be observed in all cell types tested. Our data implies that maintaining oxidative homeostasis in the infected cells may play an important role in improving survival of infected cells.
PLoS ONE 01/2011; 6(5):e20354. · 4.09 Impact Factor
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ABSTRACT: The effect of anthrax infection on phosphoprotein signaling was studied in human small airway lung epithelial cells exposed to B. anthracis spores of the plasmidless dSterne strain in comparison with the Sterne strain containing the toxigenic plasmid (pXO1). The differential regulation of phosphorylation was found in the mitogen-activated protein kinase cascade (ERK, p38, and P90RSK), the PI3K cascade (AKT, GSK-3alpha/beta), and downstream in the case of the proapoptotic BAD and the transcription factor STAT3. Both strains stimulate phosphorylation of CREB and inhibit phosphorylation of 4E-BP1 required for activation of cap-dependent translation. Downregulation of the survival AKT phosphorylation by the Sterne strain inhibits the process of Ca(2+)-dependent homophilic interaction of E-cadherin (EC) upon formation or repair of cell-cell contacts. Both lethal and edema toxins produced by the Sterne strain inhibit the AKT phosphorylation induced during the EC-mediated signaling. Activity of ERK1/2 and p38 inhibitors indicates that inhibition of AKT phosphorylation takes place through the ERK1/2-PI3K crosstalk. In Sterne spore-challenged mice, a specific inhibitor of PI3K/AKT, wortmannin, accelerates the lethal outcome, and reduction of AKT phosphorylation in the circulating blood cells coincides with the death of animals. We conclude that the PI3K/AKT pathway controlling the integrity of epithelium plays an important survival role in anthrax infection.
FEMS Immunology & Medical Microbiology 05/2009; 56(2):129-42. · 2.44 Impact Factor
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ABSTRACT: Bacillus anthracis secretes several virulence factors targeting different host organs and cell types during inhalational anthrax infection. The bacterial expression of a key virulence factor, lethal toxin (LeTx) is closely tied to another factor, edema toxin (EdTx). Both are transcribed on the same virulence plasmid (pXO1) and both have been the subject of much individual study. Their combined effect during virulent anthrax likely modulates both the global transcriptional and the phenotypic response of macrophages and phagocytes. In fact, responses brought about by the toxins may be different than each of their individual effects.
Here we report the transcriptional and apoptotic responses of the macrophage-like phagocytic cell line THP-1 exposed to B. anthracis Sterne (pXO1+) spores, and B. anthracis Delta Sterne (pXO1-) spores. These cells are resistant to LeTx-induced cytolysis, a phenotype seen in macrophages from several mouse strains which are sensitive to toxigenic anthrax infection. Our results indicate that the pXO1-containing strain induces higher pro-inflammatory transcriptional responses during the first 4 hours of interaction with bacterium, evident in the upregulation of several genes relevant to Nf-kappaB, phosphatases, prostaglandins, and TNF-alpha, along with decreases in expression levels of genes for mitochondrial components. Both bacterial strains induce apoptosis, but in the toxigenic strain-challenged cells, apoptosis is delayed.
This delay in apoptosis occurs despite the much higher level of TNF-alpha secretion induced by the toxigenic-strain challenge. Interestingly, CFLAR, an important apoptotic inhibitor which blocks apoptosis induced by large amounts of extracellular TNF-alpha, is upregulated significantly during toxigenic-strain infection, but not at all during non-toxigenic-strain infection, indicating that it may play a role in blocking or delaying TNF-alpha-mediated apoptosis. The suppression of apoptosis by the toxigenic anthrax strain is consistent with the notion that apoptosis itself may represent a protective host cell response.
BMC Immunology 12/2008; 9:67. · 2.53 Impact Factor
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ABSTRACT: Bacterial products such as cell walls (CW) and peptidoglycan (PGN) are known to activate macrophages and NK cells during microbial infections. In this report, we demonstrated that whole CW and PGN of four Gram-positive bacteria are capable of enhancing the anti-poxviral activity of murine macrophage RAW 264.7 cells. Among the major Bacillus alcalophilus CW components, PGN contributes the most to antiviral activity and induces remarkably higher levels of IFN-alpha. Anti-IFN-alpha/beta antibody, but not anti-IFN-gamma, anti-IFN-gamma receptor, or anti-IL-12, reversed the PGN-induced inhibition of vaccinia virus replication and reduced nitric oxide (NO) production. Our data thus suggest that PGN induce antiviral activity through IFN-alpha and to a lesser extent, through NO production.
FEMS Immunology & Medical Microbiology 11/2004; 42(2):197-204. · 2.44 Impact Factor
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ABSTRACT: Lethal toxin (LeTx) plays a central role in anthrax pathogenesis, however a cytotoxicity of LeTx has been difficult to demonstrate in vitro. No cytolytic effect has been reported for human cells, in contrast to murine cell lines, indicating that cell lysis can not be considered as a marker of LeTx activity. We have recently shown that murine macrophage-like RAW 264.7 cells treated with LeTx or infected with anthrax spores underwent changes typical of apoptotic death. Here we demonstrate that cells from human peripheral blood display a proapoptotic behavior similar to murine cells. TUNEL assay detected a nucleosomal degradation typical of apoptosis in peripheral blood mononuclear cells (PBMC) treated with LeTx. Membrane staining with apoptotic dyes was detected in macrophages derived from monocytes in presence of LeTx. The toxin inhibited production of proinflammatory cytokines in PBMC stimulated with a preparation of Bacillus anthracis cell wall. Infection of PBMC with anthrax spores led to the appearance of a large population of cells stained positively for apoptosis, with a reduced capacity to eliminate spores and vegetative bacteria. The aminopeptidase inhibitor, bestatin, capable of protecting cells from LeTx, restored a bactericidal activity of infected cells. These findings may be explained by LeTx expression within phagocytes and support an important role of LeTx as an early intracellular virulence factor contributing to bacterial dissemination and disease progression.
FEBS Letters 10/2002; 527(1-3):211-5. · 3.54 Impact Factor
