scholarly journals Dengue activates mTORC2 signaling to counteract apoptosis and maximize viral replication

2020 ◽  
Author(s):  
Christoph C. Carter ◽  
Jean Paul Olivier ◽  
Alexis Kaushansky ◽  
Fred D. Mast ◽  
John D. Aitchison
Keyword(s):  
Viral Replication ◽  
Cell Survival ◽  
Structural Protein ◽  
Protein Protein Interaction ◽  
Mechanistic Target Of Rapamycin ◽  
Promote Cell Survival ◽  
Important Regulator ◽  
Infected Cells ◽  
Induced Apoptosis

ABSTRACTThe mechanistic target of rapamycin (mTOR) functions in at least two distinct complexes: mTORC1, which regulates cellular anabolic-catabolic homeostasis, and mTORC2, which is an important regulator of cell survival and cytoskeletal maintenance. mTORC1 has been implicated in the pathogenesis of flaviviruses including dengue, where it contributes to the establishment of a pro-viral autophagic state. In contrast, the role of mTORC2 in viral pathogenesis is unknown. In this study, we explore the consequences of a physical protein-protein interaction between dengue non-structural protein 5 (NS5) and host cell mTOR proteins during infection. Using shRNA to differentially target mTORC1 and mTORC2 complexes, we show that mTORC2 is required for optimal dengue replication. Furthermore, we show that mTORC2 is activated during viral replication, and that mTORC2 counteracts virus-induced apoptosis, promoting the survival of infected cells. This work reveals a novel mechanism by which the dengue flavivirus can promote cell survival to maximize viral replication.

scholarly journals Human IFITM3 restricts Chikungunya virus and Mayaro virus infection and is susceptible to virus-mediated counteraction

2020 ◽  
Author(s):  
Sergej Franz ◽  
Thomas Zillinger ◽  
Fabian Pott ◽  
Christiane Schüler ◽  
Sandra Dapa ◽  
...  
Keyword(s):  
Virus Infection ◽  
Influenza A ◽  
Chikungunya Virus ◽  
Protein S ◽  
Human Cells ◽  
Structural Protein ◽  
Infected Cells ◽  
Mayaro Virus ◽  
The Impact

AbstractInterferon-induced transmembrane (IFITM) proteins restrict infection by enveloped viruses through interfering with membrane fusion and virion internalisation. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies remain largely unexplored. Here, we characterized the impact of IFITM proteins and variants on entry and spread of Chikungunya virus (CHIKV) and Mayaro virus (MAYV) in human cells, and provide first evidence for a CHIKV-mediated antagonism of IFITM proteins. IFITM1, 2 and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and during cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in the loss of its antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that has been proposed to associate with severe influenza in humans, restricted CHIKV, MAYV and influenza A virus infection as efficiently as wild-type IFITM3. Finally, all antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several non-structural protein(s) of CHIKV.

scholarly journals Genetic diversity of Collaborative Cross mice controls viral replication, clinical severity and brain pathology induced by Zika virus infection, independently of Oas1b

2019 ◽  
Author(s):  
Caroline Manet ◽  
Etienne Simon-Lorière ◽  
Grégory Jouvion ◽  
David Hardy ◽  
Matthieu Prot ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Viral Load ◽  
Viral Replication ◽  
Clinical Severity ◽  
Brain Pathology ◽  
Zikv Infection ◽  
Infected Cells ◽  
Host Genetic ◽  
Host Genes

ABSTRACTThe explosive spread of Zika virus (ZIKV) has been associated with major variations in severe disease and congenital afflictions among infected populations, suggesting an influence of host genes. We investigated how genome-wide variants could impact susceptibility to ZIKV infection in mice. We first describe that the susceptibility of Ifnar1 knockout mice is largely influenced by their genetic background. We then show that the broad genetic diversity of Collaborative Cross mice, which receptor to type I interferon (IFNAR) was blocked by anti-IFNAR antibody, expressed phenotypes ranging from complete resistance to severe symptoms and death with large variations in the peak and rate of decrease of plasma viral load, in brain viral load, in brain histopathology and in viral replication rate in infected cells. Differences of susceptibility between CC strains were correlated between Zika, Dengue and West Nile viruses. We identified highly susceptible and resistant mouse strains as new models to investigate the mechanisms of human ZIKV disease and other flavivirus infections. Genetic analyses revealed that phenotypic variations are driven by multiple genes with small effects, reflecting the complexity of ZIKV disease susceptibility in human population. Notably, our results rule out a role of the Oas1b gene in the susceptibility to ZIKV. Altogether, this study emphasizes the role of host genes in the pathogeny of ZIKV infection and lays the foundation for further genetic and mechanistic studies.IMPORTANCEIn recent outbreaks, ZIKV has infected millions of people and induced rare but potentially severe complications, including Guillain-Barré syndrome and encephalitis in adults. While several viral sequence variants were proposed to enhance the pathogenicity of ZIKV, the influence of host genetic variants in the clinical heterogeneity remains mostly unexplored. We have addressed this question using a mouse panel which models the genetic diversity of human population and a ZIKV strain from a recent clinical isolate. Through a combination of in vitro and in vivo approaches, we demonstrate that multiple host genetic variants determine viral replication in infected cells, and clinical severity, kinetics of blood viral load and brain pathology in mice. We describe new mouse models expressing high susceptibility or resistance to ZIKV and to other flaviviruses. These models will facilitate the identification and mechanistic characterization of host genes that influence ZIKV pathogenesis.

scholarly journals Transcriptional role of p53 in interferon-mediated antiviral immunity

2008 ◽  
Vol 205 (8) ◽  
pp. 1929-1938 ◽  
Author(s):  
César Muñoz-Fontela ◽  
Salvador Macip ◽  
Luis Martínez-Sobrido ◽  
Lauren Brown ◽  
Joseph Ashour ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Viral Replication ◽  
Viral Infections ◽  
Suppressor Gene ◽  
Central Component ◽  
Type I ◽  
Infected Cells

Tumor suppressor p53 is activated by several stimuli, including DNA damage and oncogenic stress. Previous studies (Takaoka, A., S. Hayakawa, H. Yanai, D. Stoiber, H. Negishi, H. Kikuchi, S. Sasaki, K. Imai, T. Shibue, K. Honda, and T. Taniguchi. 2003. Nature. 424:516–523) have shown that p53 is also induced in response to viral infections as a downstream transcriptional target of type I interferon (IFN) signaling. Moreover, many viruses, including SV40, human papillomavirus, Kaposi's sarcoma herpesvirus, adenoviruses, and even RNA viruses such as polioviruses, have evolved mechanisms designated to abrogate p53 responses. We describe a novel p53 function in the activation of the IFN pathway. We observed that infected mouse and human cells with functional p53 exhibited markedly decreased viral replication early after infection. This early inhibition of viral replication was mediated both in vitro and in vivo by a p53-dependent enhancement of IFN signaling, specifically the induction of genes containing IFN-stimulated response elements. Of note, p53 also contributed to an increase in IFN release from infected cells. We established that this p53-dependent enhancement of IFN signaling is dependent to a great extent on the ability of p53 to activate the transcription of IFN regulatory factor 9, a central component of the IFN-stimulated gene factor 3 complex. Our results demonstrate that p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene.

The Regulation of STAT3 and Its Role in the Adhesion and Migration of Chronic Lymphocytic Leukaemia Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4347-4347
Author(s):  
Sarah Brophy ◽  
Fiona M Quinn ◽  
David O'Brien ◽  
Paul Browne ◽  
Elisabeth A. Vandenberghe ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Adhesion ◽  
Cell Survival ◽  
Lymphocytic Leukaemia ◽  
Serine Phosphorylation ◽  
Pre Treatment ◽  
And Migration ◽  
Induced Apoptosis ◽  
Stat3 Inhibition

Abstract The bone marrow and lymph node microenvironments are important in promoting cell proliferation, survival and protection from drug induced apoptosis in chronic lymphocytic leukaemia (CLL). Chemokine networks, such as the CXCR4/CXCL12 axis, in combination with selectins, such as CD62L and integrins allow the migration of CLL cells to these protective niches. The B-cell receptor (BCR) signalling pathway is the most important pathway involved in micro-environmental crosstalk and CLL cell survival. Further, it has been shown to interact with the signal transducer and activator of transcription 3 (STAT3) signalling pathway. The role of the STAT3 in CLL pathogenesis is unclear; however, it is constitutively phosphorylated on serine residue 727 (serine pSTAT3) in CLL cells. Here, we investigate the role of STAT3 in CLL cell survival and migration, using pharmacological inhibition and siRNA knockdown. Phospho-tyrosine and phospho-serine STAT3 were assessed by flow cytometry and western blotting. Apoptosis was assessed by Annexin V/Propidium Iodide staining by flow cytometry. The expression of cell surface markers involved in cell adhesion and homing was determined by multicolour flow cytometry. Stimulation of the BCR using immunoglobulin F(ab´)2 fragments induced tyrosine phosphorylation of STAT3 in CLL cells with unmutated immunoglobulin (IgVH) genes (n=7) but not mutated IgVH genes (n=5). This induced tyrosine phosphorylation was abrogated by pre-treatment with the Janus kinase (JAK) inhibitor Ruxolitinib and the BCR inhibitors Ibrutinib and Idelalisib (p<0.05, n=5). Gene expression studies using Taqman Assays showed BCR stimulation resulted in an upregulation of STAT3 regulated genes in CLL cells with unmutated IgVH genes. Interestingly, stimulation of BCR resulted in a significant increase in CD62L expression, which was inhibited by pre-treatment with Ibrutinib and Ruxolitinib (p<0.05, n=5). STAT3 inhibition was shown to have a divergent effect on CLL cell survival: In patient samples with >70% positive serine pSTAT3 cells, the STAT3 inhibitor cucurbitacin I induced apoptosis with a concurrent downregulation in serine phosphorylation (n=3); while in patient samples with <70% positive serine pSTAT3 cells, treatment with cucurbitacin I resulted in a decrease in apoptosis and a concurrent increase in serine phosphorylation (n=3). The STAT3 inhibitor S3I-201 had a similar effect but the upstream JAK inhibitor Ruxolitinib had no effect on serine phosphorylation and no effect on the apoptosis of CLL cells. In addition, siRNA mediated STAT3 knockdown and treatment with cucurbitacin I and S31-201, resulted in a significant decrease in CD62L positive CLL cells (p<0.0001, n=29). The role of STAT3 in CLL cell adhesion under shear flow conditions was investigated using a microfluidics system including a neMESYS Low Pressure syringe pump system and Human Umbilical Vein Endothelial Cells (HUVEC) coated biochips. Treatment of CLL cells with cucurbitacin I resulted in a significant decrease in adhesion to endothelial cells (p<0.001, n=4). The effect of STAT3 inhibition on the chemotaxis of CLL cells was investigated using Neuroprobe 96-well ChemoTx plates. Treatment with cucurbitacin I resulted in a significant decrease in CLL cells migrated in response to the chemokine CXCL12 compared to control (p=0.0001, n=8). In addition, treatment of CLL cells with CXCL12 resulted in an increase in serine pSTAT3 that was downregulated by pretreatment with cucurbitacin I. This study has shown: 1. Activation of STAT3 by BCR stimulation occurs in poor prognostic unmutated IgVH genes 2. Serine pSTAT3 has a role in cell survival in response to STAT3 inhibition 3. A role for STAT3 in CLL cell adhesion and migration, in particular in the regulation of the expression of CD62L. In conclusion, this study shows a role for the STAT3 pathway in cell survival and CLL cell-microenvironment crosstalk, suggesting therapeutic potential by interfering with the migration and homing of CLL cells to the lymph node and bone marrow microenvironments. Disclosures No relevant conflicts of interest to declare.

The role of the glucocorticoid receptor (GR) in inhibiting chemotherapy-induced apoptosis in high-grade serous ovarian carcinoma (HGS-OvCa).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 11101-11101
Author(s):  
Erica Michelle Stringer ◽  
Maxwell N. Skor ◽  
Gini F. Fleming ◽  
Suzanne D. Conzen
Keyword(s):  
Ovarian Cancer ◽  
Glucocorticoid Receptor ◽  
Tumor Cell ◽  
Cell Survival ◽  
Cell Lines ◽  
High Grade ◽  
Induced Apoptosis ◽  
Er Alpha ◽  
Ovca Cell

11101 Background: Ovarian cancer is the leading cause of death from gynecologic malignancies. High-grade serous ovarian cancer (HGS-OvCa) is often initially sensitive to platinum-based therapy, but relapse rates remain high. The TCGA recently found that HGS-OvCas have a gene expression and mutational profile similar to that of triple negative breast cancer (TNBC). Previously, our group demonstrated that dexamethasone treatment decreased chemotherapy-induced tumor cell apoptosis in TNBC and HGS-OvCa cell lines. We have also shown that glucocorticoid receptor (GR) activation induces expression of anti-apoptotic genes SGK1 and MKP1/DUSP1 in both HGS-OvCa and TNBC cell lines and in primary human ovarian and TNBC tumors. Methods: We examined glucocorticoid receptor (GR), estrogen receptor (ER), and progesterone receptor (PR) expression in a panel of HGS-OvCa cell lines by Western analysis and qRT-PCR. We also performed apoptosis assays with and without mifepristone, glucocorticoid and/or chemotherapy treatment using IncuCyte live-cell imaging technology in order to measure the effect of GR modulation of chemotherapy sensitivity. Results: HGS-OvCa cell lines (including CAOV3, HeyA8, SKOV3, Monty-1) all had detectable GR expression; HeyA8, SKOV3, and Monty-1 cell lines expressed very low levels of ER-alpha while all other HGS-OvCa cell lines did not express any detectable ER-alpha. Furthermore, none of the HGS-OvCa cell lines tested expressed PR.Apoptosis assays revealed that GR activation significantly inhibited gemcitabine/carboplatin-induced apoptosis in HGS-OvCa cell lines and that mifepristone could reverse this cell survival effect, presumably through GR antagonism. Conclusions: These results suggest that treatment with mifepristone, a GR antagonist, reverses GR-mediated cell survival signaling in HGS-OvCa and increases chemotherapy-induced tumor cell death. To further investigate the role of GR activity in HGS-OvCa, we are currently performing xenograft experiments with chemotherapy +/- mifepristone treatment.

scholarly journals Role of Bcl-2 Family Members in Caspase-Independent Apoptosis during Chlamydia Infection

2002 ◽  
Vol 70 (1) ◽  
pp. 55-61 ◽  
Author(s):  
Jean-Luc Perfettini ◽  
John C. Reed ◽  
Nicole Israël ◽  
Jean-Claude Martinou ◽  
Alice Dautry-Varsat ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Chlamydia Psittaci ◽  
Host Cells ◽  
Chlamydia Infection ◽  
Caspase Inhibitor ◽  
Obligate Intracellular Bacterium ◽  
Obligate Intracellular ◽  
Infected Cells ◽  
Induced Apoptosis

ABSTRACT Infection with an obligate intracellular bacterium, the Chlamydia trachomatis lymphogranuloma venereum (LGV/L2) strain or the guinea pig inclusion conjunctivitis serovar of Chlamydia psittaci, leads to apoptosis of host cells. The apoptosis is not affected by a broad-spectrum caspase inhibitor, and caspase-3 is not activated in infected cells, suggesting that apoptosis mediated by these two strains of Chlamydia is independent of known caspases. Overexpression of the proapoptotic Bcl-2 family member, Bax, was previously shown to induce caspase-independent apoptosis, and we find that Bax is activated and translocates from the cytosol to the mitochondria in C. psittaci-infected cells. C. psittaci-induced apoptosis is inhibited in host cells overexpressing Bax inhibitor-1 and is inhibited through overexpression of Bcl-2, which blocks both caspase-dependent and -independent apoptosis. As Bax and mitochondria are ideally located to sense stress-related metabolic changes emanating from the interior of an infected cell, it is likely that Bax-dependent apoptosis may also be observed in cells infected with other intracellular pathogens.

scholarly journals Human Cytomegalovirus miR-UL70-3p Downregulates the H2O2-Induced Apoptosis by Targeting the Modulator of Apoptosis-1 (MOAP1)

2021 ◽  
Vol 23 (1) ◽  
pp. 18
Author(s):  
Abhishek Pandeya ◽  
Raj Kumar Khalko ◽  
Anup Mishra ◽  
Nishant Singh ◽  
Sukhveer Singh ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Antiviral Responses ◽  
Persistent Infections ◽  
Innate Immune Mechanism ◽  
Infected Cells ◽  
Hcmv Replication ◽  
Induced Apoptosis

Human Cytomegalovirus (HCMV) is a prototypic beta herpesvirus, causing persistent infections in humans. There are medications that are used to treat the symptoms; however, there is no cure yet. Thus, understanding the molecular mechanisms of HCMV replication and its persistence may reveal new prevention strategies. HCMV evasive strategies on the antiviral responses of the human host largely rely on its significant portion of genome. Numerous studies have highlighted the importance of miRNA-mediated regulation of apoptosis, which is an innate immune mechanism that eradicates virus-infected cells. In this study, we explore the antiapoptotic role of hcmv-miR-UL70-3p in HEK293T cells. We establish that hcmv-miR-UL70-3p targets the proapoptotic gene Modulator of Apoptosis-1 (MOAP1) through interaction with its 3’UTR region of mRNA. The ectopic expression of hcmv-miR-UL70-3p mimic significantly downregulates the H2O2-induced apoptosis through the translational repression of MOAP1. Silencing of MOAP1 through siRNA also inhibits the H2O2-induced apoptosis, which further supports the hcmv-miR-UL70-3p mediated antiapoptotic effect by regulating MOAP1 expression. These results uncover a role for hcmv-miR-UL70-3p and its target MOAP1 in regulating apoptosis.

ZEB1 Promotes Chemoresistance to Cisplatin in Ovarian Cancer Cells by Suppressing SLC3A2

Chemotherapy ◽  
2018 ◽  
Vol 63 (5) ◽  
pp. 262-271 ◽  
Author(s):  
Yajie Cui ◽  
Li Qin ◽  
Defu Tian ◽  
Ting Wang ◽  
Lijing Fan ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Viability ◽  
Cancer Cells ◽  
Tumor Development ◽  
Migration And Invasion ◽  
Ovarian Cancer Cells ◽  
Cell Surface Molecule ◽  
Important Regulator ◽  
Induced Apoptosis

Ovarian cancer is one of the deadliest gynecological malignancies in women. Chemoresistance has been a major obstacle for ovarian cancer treatment. Zinc finger E-box-binding homeobox 1 (ZEB1) is an important regulator of tumor development in various types of cancer. Abnormal expression of SLC3A2 (CD98hc), a type 2 transmembrane cell surface molecule, has been described in several cancers. This study was designed to investigate the role of ZEB1 and SLC3A2 in the chemoresistance to cisplatin in ovarian cancer cells. We found that ZEB1 was increased in cisplatin-resistant SKOV3/DPP cells. Downregulation of ZEB1 significantly decreased cell viability in response to cisplatin, increased cis­platin-induced apoptosis, and decreased migration and invasion in the presence of cisplatin. In addition, downregulation of ZEB1 decreased the volume and weight of implanted tumors. SLC3A2 was decreased in cisplatin-resistant SKOV3/DPP cells. Upregulation of SLC3A2 significantly decreased cell viability in response to cisplatin, increased cisplatin-induced apoptosis, and decreased migration and invasion in the presence of cisplatin. Moreover, upregulation of SLC3A2 decreased the volume and weight of implanted tumors. Downregulation of ZEB1 resulted in a significant increase of SLC3A2 expression. Moreover, downregulation of SLC3A2 significantly inhibited ZEB1 knockdown-mediated inhibition of cisplatin-resistance. ZEB1-mediated regulation of SLC3A2 was involved in the chemoresistance to cisplatin in ovarian cancer cells. Overall, we provide new insights into the mechanism of chemoresistance to cisplatin in ovarian cancer cells. ZEB1/SLC3A2 may be promising therapeutic targets for enhancement of the sensitivity of ovarian cancer cells to cisplatin-mediated chemotherapy.

scholarly journals Improved HSC reconstitution and protection from inflammatory stress and chemotherapy in mice lacking granzyme B

2014 ◽  
Vol 211 (5) ◽  
pp. 769-779 ◽  
Author(s):  
Larissa S. Carnevalli ◽  
Roberta Scognamiglio ◽  
Nina Cabezas-Wallscheid ◽  
Susann Rahmig ◽  
Elisa Laurenti ◽  
...  
Keyword(s):  
Granzyme B ◽  
Negative Regulator ◽  
Mitochondrial Activity ◽  
Hematopoietic Stem ◽  
Inflammatory Stress ◽  
Immune Mediated ◽  
Important Regulator ◽  
Response To Stress ◽  
Infected Cells

The serine protease granzyme B (GzmB) is stored in the granules of cytotoxic T and NK cells and facilitates immune-mediated destruction of virus-infected cells. In this study, we use genetic tools to report novel roles for GzmB as an important regulator of hematopoietic stem cell (HSC) function in response to stress. HSCs lacking the GzmB gene show improved bone marrow (BM) reconstitution associated with increased HSC proliferation and mitochondrial activity. In addition, recipients deficient in GzmB support superior engraftment of wild-type HSCs compared with hosts with normal BM niches. Stimulation of mice with lipopolysaccharide strongly induced GzmB protein expression in HSCs, which was mediated by the TLR4–TRIF–p65 NF-κB pathway. This is associated with increased cell death and GzmB secretion into the BM environment, suggesting an extracellular role of GzmB in modulating HSC niches. Moreover, treatment with the chemotherapeutic agent 5-fluorouracil (5-FU) also induces GzmB production in HSCs. In this situation GzmB is not secreted, but instead causes cell-autonomous apoptosis. Accordingly, GzmB-deficient mice are more resistant to serial 5-FU treatments. Collectively, these results identify GzmB as a negative regulator of HSC function that is induced by stress and chemotherapy in both HSCs and their niches. Blockade of GzmB production may help to improve hematopoiesis in various situations of BM stress.

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