The Experts below are selected from a list of 1425 Experts worldwide ranked by ideXlab platform
Felix N Toka - One of the best experts on this subject based on the ideXlab platform.
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mitochondria related gene expression profiles in murine fibroblasts and macrophages during later stages of Ectromelia Virus infection in vitro
Acta Virologica, 2020Co-Authors: Lidia Szulcdąbrowska, Malgorzata Gierynska, Zbigniew Wyzewski, Karolina P Gregorczykzboroch, Justyna Struzik, Joanna Szczepanowska, Felix N Toka, Z Nowakżyczynska, Marek NiemialtowskiAbstract:Mitochondria are multitasking organelles that play a central role in energy production, survival and primary host defense against viral infections. Therefore, Viruses target mitochondria dynamics and functions to benefit their replication and morphogenetic processes. We endeavor to understand the role of mitochondria during infection of Ectromelia Virus (ECTV), hence our investigations on mitochondria-related genes in non-immune (L929 fibroblasts) and immune (RAW 264.7 macrophages) cells. Our results show that during later stages of infection, ECTV significantly decreases the expression of mitochondria-related genes regulating many aspects of mitochondrial physiology and functions, including mitochondrial transport, small molecule transport, membrane polarization and potential, targeting proteins to mitochondria, inner membrane translocation, and apoptosis. Such down-regulation is cell-specific, since macrophages exhibited a more profound down-regulation of mitochondria-related genes compared to infected L929 fibroblasts. Only L929 cells exhibited up-regulation of two important genes responsible for oxidative phosphorylation and subsequent ATP production: Slc25a23 and Slc25a31. Changes in the expression of mitochondria-related genes are accompanied by altered mitochondria morphology and distribution in both types of cells. In depth Ingenuity Pathway Analysis (IPA) identified the "Sirtuin Signaling Pathway" as the most significant top canonical pathway associated with ECTV infection in both analyzed cell types. Taken together, down-regulation of mitochondria-related genes observed especially in macrophages indicates dysfunctional mitochondria, possibly contributing to energy collapse and induction of intrinsic pathway of apoptosis. Meanwhile, alteration of the expression of several mitochondria-related genes in fibroblasts without apoptosis induction may represent poxviral strategy to control cellular energy metabolism for efficient replication. Keywords: Ectromelia Virus; mitochondria; fibroblasts; macrophages.
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mitochondrial heat shock response induced by Ectromelia Virus is accompanied by reduced apoptotic potential in murine l929 fibroblasts
Archivum Immunologiae Et Therapiae Experimentalis, 2019Co-Authors: Zbigniew Wyzewski, Karolina P Gregorczykzboroch, Matylda B Mielcarska, Magdalena Bossowskanowicka, Justyna Struzik, Joanna Szczepanowska, Felix N Toka, Marek Niemialtowski, Lidia SzulcdąbrowskaAbstract:PoxViruses utilize multiple strategies to prevent activation of extrinsic and intrinsic apoptotic pathways for successful replication. Mitochondrial heat shock proteins (mtHsps), especially Hsp60 and its cofactor Hsp10, are engaged in apoptosis regulation; however, until now, the influence of poxViruses on mtHsps has never been studied. We used highly infectious Moscow strain of Ectromelia Virus (ECTV) to investigate the mitochondrial heat shock response and apoptotic potential in permissive L929 fibroblasts. Our results show that ECTV-infected cells exhibit mostly mitochondrial localization of Hsp60 and Hsp10, and show overexpression of both proteins during later stages of infection. ECTV infection has only moderate effect on the electron transport chain subunit expression. Moreover, increase of mtHsp amounts is accompanied by lack of apoptosis, and confirmed by reduced level of pro-apoptotic Bax protein and elevated levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Taken together, we show a positive relationship between increased levels of Hsp60 and Hsp10 and decreased apoptotic potential of L929 fibroblasts, and further hypothesize that Hsp60 and/or its cofactor play important roles in maintaining protein homeostasis in mitochondria for promotion of cell survival allowing efficient replication of ECTV.
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Ectromelia Virus suppresses expression of cathepsins and cystatins in conventional dendritic cells to efficiently execute the replication process
BMC Microbiology, 2019Co-Authors: Magdalena Bossowskanowicka, Malgorzata Gierynska, Karolina P Gregorczykzboroch, Matylda B Mielcarska, Justyna Struzik, Felix N Toka, Marta Romaniewicz, Monika M Kaczmarek, Marta GrodzikAbstract:Cathepsins are a group of endosomal proteases present in many cells including dendritic cells (DCs). The activity of cathepsins is regulated by their endogenous inhibitors – cystatins. Cathepsins are crucial to antigen processing during viral and bacterial infections, and as such are a prerequisite to antigen presentation in the context of major histocompatibility complex class I and II molecules. Due to the involvement of DCs in both innate and adaptive immune responses, and the quest to understand the impact of poxVirus infection on host cells, we investigated the influence of Ectromelia Virus (ECTV) infection on cathepsin and cystatin levels in murine conventional DCs (cDCs). ECTV is a poxVirus that has evolved many mechanisms to avoid host immune response and is able to replicate productively in DCs. Our results showed that ECTV-infection of JAWS II DCs and primary murine GM-CSF-derived bone marrow cells down-regulated both mRNA and protein of cathepsin B, L and S, and cystatin B and C, particularly during the later stages of infection. Moreover, the activity of cathepsin B, L and S was confirmed to be diminished especially at later stages of infection in JAWS II cells. Consequently, ECTV-infected DCs had diminished ability to endocytose and process a soluble antigen. Close examination of cellular protein distribution showed that beginning from early stages of infection, the remnants of cathepsin L and cystatin B co-localized and partially co-localized with viral replication centers (viral factories), respectively. Moreover, viral yield increased in cDCs treated with siRNA against cathepsin B, L or S and subsequently infected with ECTV. Taken together, our results indicate that infection of cDCs with ECTV suppresses cathepsins and cystatins, and alters their cellular distribution which impairs the cDC function. We propose this as an additional viral strategy to escape immune responses, enabling the Virus to replicate effectively in infected cells.
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Ectromelia Virus induces tubulin cytoskeletal rearrangement in immune cells accompanied by a loss of the microtubule organizing center and increased α tubulin acetylation
Archives of Virology, 2019Co-Authors: Lidia Szulcdąbrowska, Ada Schollenberger, Karolina P Gregorczykzboroch, Justyna Struzik, Felix N Toka, Mateusz Palusinski, Malgorzata GierynskaAbstract:Ectromelia Virus (ECTV) is an orthopoxVirus that productively replicates in dendritic cells (DCs), but its influence on the microtubule (MT) cytoskeleton in DCs is not known. Here, we show that ECTV infection of primary murine granulocyte-macrophage colony stimulating factor-derived bone marrow cells (GM-BM) downregulates numerous genes engaged in MT cytoskeleton organization and dynamics. In infected cells, the MT cytoskeleton undergoes dramatic rearrangement and relaxation, accompanied by disappearance of the microtubule organizing centre (MTOC) and increased acetylation and stabilization of MTs, which are exploited by progeny virions for intracellular transport. This indicates a strong ability of ECTV to subvert the MT cytoskeleton of highly specialized immune cells.
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Ectromelia Virus suppresses expression of cathepsins and cystatins in conventional dendritic cells to efficiently execute the replication process
BMC, 2019Co-Authors: Magdalena Bossowska-nowicka, Matylda B Mielcarska, Justyna Struzik, Felix N Toka, Marta Romaniewicz, Monika M Kaczmarek, Marta Grodzik, Małgorzata M. Gieryńska, Karolina P. Gregorczyk-zboroch, Lidia Szulc-dąbrowskaAbstract:Abstract Background Cathepsins are a group of endosomal proteases present in many cells including dendritic cells (DCs). The activity of cathepsins is regulated by their endogenous inhibitors – cystatins. Cathepsins are crucial to antigen processing during viral and bacterial infections, and as such are a prerequisite to antigen presentation in the context of major histocompatibility complex class I and II molecules. Due to the involvement of DCs in both innate and adaptive immune responses, and the quest to understand the impact of poxVirus infection on host cells, we investigated the influence of Ectromelia Virus (ECTV) infection on cathepsin and cystatin levels in murine conventional DCs (cDCs). ECTV is a poxVirus that has evolved many mechanisms to avoid host immune response and is able to replicate productively in DCs. Results Our results showed that ECTV-infection of JAWS II DCs and primary murine GM-CSF-derived bone marrow cells down-regulated both mRNA and protein of cathepsin B, L and S, and cystatin B and C, particularly during the later stages of infection. Moreover, the activity of cathepsin B, L and S was confirmed to be diminished especially at later stages of infection in JAWS II cells. Consequently, ECTV-infected DCs had diminished ability to endocytose and process a soluble antigen. Close examination of cellular protein distribution showed that beginning from early stages of infection, the remnants of cathepsin L and cystatin B co-localized and partially co-localized with viral replication centers (viral factories), respectively. Moreover, viral yield increased in cDCs treated with siRNA against cathepsin B, L or S and subsequently infected with ECTV. Conclusions Taken together, our results indicate that infection of cDCs with ECTV suppresses cathepsins and cystatins, and alters their cellular distribution which impairs the cDC function. We propose this as an additional viral strategy to escape immune responses, enabling the Virus to replicate effectively in infected cells
Justyna Struzik - One of the best experts on this subject based on the ideXlab platform.
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first insight into the modulation of noncanonical nf κb signaling components by poxViruses in established immune derived cell lines an in vitro model of Ectromelia Virus infection
Pathogenetics, 2020Co-Authors: Justyna Struzik, Matylda B Mielcarska, Magdalena Bossowskanowicka, Lidia Szulcdąbrowska, Michal Koper, Malgorzata GierynskaAbstract:Dendritic cells (DCs) and macrophages are the first line of antiviral immunity. Viral pathogens exploit these cell populations for their efficient replication and dissemination via the modulation of intracellular signaling pathways. Disruption of the noncanonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling has frequently been observed in lymphoid cells upon infection with oncogenic Viruses. However, several nononcogenic Viruses have been shown to manipulate the noncanonical NF-κB signaling in different cell types. This study demonstrates the modulating effect of Ectromelia Virus (ECTV) on the components of the noncanonical NF-κB signaling pathway in established murine cell lines: JAWS II DCs and RAW 264.7 macrophages. ECTV affected the activation of TRAF2, cIAP1, RelB, and p100 upon cell treatment with both canonical and noncanonical NF-κB stimuli and thus impeded DNA binding by RelB and p52. ECTV also inhibited the expression of numerous genes related to the noncanonical NF-κB pathway and RelB-dependent gene expression in the cells treated with canonical and noncanonical NF-κB activators. Thus, our data strongly suggest that ECTV influenced the noncanonical NF-κB signaling components in the in vitro models. These findings provide new insights into the noncanonical NF-κB signaling components and their manipulation by poxViruses in vitro.
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mitochondria related gene expression profiles in murine fibroblasts and macrophages during later stages of Ectromelia Virus infection in vitro
Acta Virologica, 2020Co-Authors: Lidia Szulcdąbrowska, Malgorzata Gierynska, Zbigniew Wyzewski, Karolina P Gregorczykzboroch, Justyna Struzik, Joanna Szczepanowska, Felix N Toka, Z Nowakżyczynska, Marek NiemialtowskiAbstract:Mitochondria are multitasking organelles that play a central role in energy production, survival and primary host defense against viral infections. Therefore, Viruses target mitochondria dynamics and functions to benefit their replication and morphogenetic processes. We endeavor to understand the role of mitochondria during infection of Ectromelia Virus (ECTV), hence our investigations on mitochondria-related genes in non-immune (L929 fibroblasts) and immune (RAW 264.7 macrophages) cells. Our results show that during later stages of infection, ECTV significantly decreases the expression of mitochondria-related genes regulating many aspects of mitochondrial physiology and functions, including mitochondrial transport, small molecule transport, membrane polarization and potential, targeting proteins to mitochondria, inner membrane translocation, and apoptosis. Such down-regulation is cell-specific, since macrophages exhibited a more profound down-regulation of mitochondria-related genes compared to infected L929 fibroblasts. Only L929 cells exhibited up-regulation of two important genes responsible for oxidative phosphorylation and subsequent ATP production: Slc25a23 and Slc25a31. Changes in the expression of mitochondria-related genes are accompanied by altered mitochondria morphology and distribution in both types of cells. In depth Ingenuity Pathway Analysis (IPA) identified the "Sirtuin Signaling Pathway" as the most significant top canonical pathway associated with ECTV infection in both analyzed cell types. Taken together, down-regulation of mitochondria-related genes observed especially in macrophages indicates dysfunctional mitochondria, possibly contributing to energy collapse and induction of intrinsic pathway of apoptosis. Meanwhile, alteration of the expression of several mitochondria-related genes in fibroblasts without apoptosis induction may represent poxviral strategy to control cellular energy metabolism for efficient replication. Keywords: Ectromelia Virus; mitochondria; fibroblasts; macrophages.
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mitochondrial heat shock response induced by Ectromelia Virus is accompanied by reduced apoptotic potential in murine l929 fibroblasts
Archivum Immunologiae Et Therapiae Experimentalis, 2019Co-Authors: Zbigniew Wyzewski, Karolina P Gregorczykzboroch, Matylda B Mielcarska, Magdalena Bossowskanowicka, Justyna Struzik, Joanna Szczepanowska, Felix N Toka, Marek Niemialtowski, Lidia SzulcdąbrowskaAbstract:PoxViruses utilize multiple strategies to prevent activation of extrinsic and intrinsic apoptotic pathways for successful replication. Mitochondrial heat shock proteins (mtHsps), especially Hsp60 and its cofactor Hsp10, are engaged in apoptosis regulation; however, until now, the influence of poxViruses on mtHsps has never been studied. We used highly infectious Moscow strain of Ectromelia Virus (ECTV) to investigate the mitochondrial heat shock response and apoptotic potential in permissive L929 fibroblasts. Our results show that ECTV-infected cells exhibit mostly mitochondrial localization of Hsp60 and Hsp10, and show overexpression of both proteins during later stages of infection. ECTV infection has only moderate effect on the electron transport chain subunit expression. Moreover, increase of mtHsp amounts is accompanied by lack of apoptosis, and confirmed by reduced level of pro-apoptotic Bax protein and elevated levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Taken together, we show a positive relationship between increased levels of Hsp60 and Hsp10 and decreased apoptotic potential of L929 fibroblasts, and further hypothesize that Hsp60 and/or its cofactor play important roles in maintaining protein homeostasis in mitochondria for promotion of cell survival allowing efficient replication of ECTV.
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Ectromelia Virus suppresses expression of cathepsins and cystatins in conventional dendritic cells to efficiently execute the replication process
BMC Microbiology, 2019Co-Authors: Magdalena Bossowskanowicka, Malgorzata Gierynska, Karolina P Gregorczykzboroch, Matylda B Mielcarska, Justyna Struzik, Felix N Toka, Marta Romaniewicz, Monika M Kaczmarek, Marta GrodzikAbstract:Cathepsins are a group of endosomal proteases present in many cells including dendritic cells (DCs). The activity of cathepsins is regulated by their endogenous inhibitors – cystatins. Cathepsins are crucial to antigen processing during viral and bacterial infections, and as such are a prerequisite to antigen presentation in the context of major histocompatibility complex class I and II molecules. Due to the involvement of DCs in both innate and adaptive immune responses, and the quest to understand the impact of poxVirus infection on host cells, we investigated the influence of Ectromelia Virus (ECTV) infection on cathepsin and cystatin levels in murine conventional DCs (cDCs). ECTV is a poxVirus that has evolved many mechanisms to avoid host immune response and is able to replicate productively in DCs. Our results showed that ECTV-infection of JAWS II DCs and primary murine GM-CSF-derived bone marrow cells down-regulated both mRNA and protein of cathepsin B, L and S, and cystatin B and C, particularly during the later stages of infection. Moreover, the activity of cathepsin B, L and S was confirmed to be diminished especially at later stages of infection in JAWS II cells. Consequently, ECTV-infected DCs had diminished ability to endocytose and process a soluble antigen. Close examination of cellular protein distribution showed that beginning from early stages of infection, the remnants of cathepsin L and cystatin B co-localized and partially co-localized with viral replication centers (viral factories), respectively. Moreover, viral yield increased in cDCs treated with siRNA against cathepsin B, L or S and subsequently infected with ECTV. Taken together, our results indicate that infection of cDCs with ECTV suppresses cathepsins and cystatins, and alters their cellular distribution which impairs the cDC function. We propose this as an additional viral strategy to escape immune responses, enabling the Virus to replicate effectively in infected cells.
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Ectromelia Virus induces tubulin cytoskeletal rearrangement in immune cells accompanied by a loss of the microtubule organizing center and increased α tubulin acetylation
Archives of Virology, 2019Co-Authors: Lidia Szulcdąbrowska, Ada Schollenberger, Karolina P Gregorczykzboroch, Justyna Struzik, Felix N Toka, Mateusz Palusinski, Malgorzata GierynskaAbstract:Ectromelia Virus (ECTV) is an orthopoxVirus that productively replicates in dendritic cells (DCs), but its influence on the microtubule (MT) cytoskeleton in DCs is not known. Here, we show that ECTV infection of primary murine granulocyte-macrophage colony stimulating factor-derived bone marrow cells (GM-BM) downregulates numerous genes engaged in MT cytoskeleton organization and dynamics. In infected cells, the MT cytoskeleton undergoes dramatic rearrangement and relaxation, accompanied by disappearance of the microtubule organizing centre (MTOC) and increased acetylation and stabilization of MTs, which are exploited by progeny virions for intracellular transport. This indicates a strong ability of ECTV to subvert the MT cytoskeleton of highly specialized immune cells.
Michele Barry - One of the best experts on this subject based on the ideXlab platform.
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Ectromelia Virus encodes an anti-apoptotic protein that regulates cell death.
Virology, 2014Co-Authors: Ninad Mehta, John M. Taylor, Douglas Quilty, Michele BarryAbstract:Apoptosis serves as a powerful defense against damaged or pathogen-infected cells. Since apoptosis is an effective defense against viral infection, many Viruses including poxViruses, encode proteins to prevent or delay apoptosis. Here we show that Ectromelia Virus, the causative agent of mousepox encodes an anti-apoptotic protein EVM025. Here we demonstrate that expression of functional EVM025 is crucial to prevent apoptosis triggered by Virus infection and staurosporine. We demonstrate that the expression of EVM025 prevents the conformational activation of the pro-apoptotic proteins Bak and Bax, allowing the maintenance of mitochondrial membrane integrity upon infection with ECTV. Additionally, EVM025 interacted with intracellular Bak. We were able to demonstrate that EVM025 ability to inhibit Bax activation is a function of its ability to inhibit the activity of an upstream BH3 only protein Bim. Collectively, our data indicates that EVM025 inhibits apoptosis by sequestering Bak and inhibiting the activity of Bak and Bax.
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Ectromelia Virus encodes a family of ankyrin f box proteins that regulate nfκb
Virology, 2014Co-Authors: Kristin Burles, Nicholas Van Buuren, Michele BarryAbstract:A notable feature of poxViruses is their ability to inhibit the antiviral response, including the nuclear factor kappa B (NFκB) pathway. NFκB is a transcription factor that is sequestered in the cytoplasm until cell stimulation, and relies on the SCF (Skp1, culllin-1, F-box) ubiquitin ligase to target its inhibitor, IκBα, for degradation. IκBα is recruited to the SCF by the F-box domain-containing protein βTrCP. Here, we show that Ectromelia Virus, the causative agent of mousepox, encodes four F-box-containing proteins, EVM002, EVM005, EVM154, and EVM165, all of which contain Ankyrin (Ank) domains. The Ank/F-box proteins inhibit NFκB nuclear translocation, and this inhibition is dependent on the F-box domain. We also demonstrate that EVM002, EVM005, EVM154, and EVM165 prevent IκBα degradation, suggesting that they target the SCF. This study identifies a new mechanism by which Ectromelia Virus inhibits NFκB.
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Ectromelia Virus encodes a family of Ankyrin/F-box proteins that regulate NFκB
Virology, 2014Co-Authors: Kristin Burles, Nicholas Van Buuren, Michele BarryAbstract:A notable feature of poxViruses is their ability to inhibit the antiviral response, including the nuclear factor kappa B (NFκB) pathway. NFκB is a transcription factor that is sequestered in the cytoplasm until cell stimulation, and relies on the SCF (Skp1, culllin-1, F-box) ubiquitin ligase to target its inhibitor, IκBα, for degradation. IκBα is recruited to the SCF by the F-box domain-containing protein βTrCP. Here, we show that Ectromelia Virus, the causative agent of mousepox, encodes four F-box-containing proteins, EVM002, EVM005, EVM154, and EVM165, all of which contain Ankyrin (Ank) domains. The Ank/F-box proteins inhibit NFκB nuclear translocation, and this inhibition is dependent on the F-box domain. We also demonstrate that EVM002, EVM005, EVM154, and EVM165 prevent IκBα degradation, suggesting that they target the SCF. This study identifies a new mechanism by which Ectromelia Virus inhibits NFκB.
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EVM005: an Ectromelia-encoded protein with dual roles in NF-κB inhibition and virulence.
PLoS pathogens, 2014Co-Authors: Nicholas Van Buuren, R. Mark L. Buller, Scott Parker, Jill Schriewer, Kristin Burles, Ninad Mehta, Michele BarryAbstract:PoxViruses contain large dsDNA genomes encoding numerous open reading frames that manipulate cellular signalling pathways and interfere with the host immune response. The NF-κB signalling cascade is an important mediator of innate immunity and inflammation, and is tightly regulated by ubiquitination at several key points. A critical step in NF-κB activation is the ubiquitination and degradation of the inhibitor of kappaB (IκBα), by the cellular SCFβ-TRCP ubiquitin ligase complex. We show here that upon stimulation with TNFα or IL-1β, OrthopoxVirus-infected cells displayed an accumulation of phosphorylated IκBα, indicating that NF-κB activation was inhibited during poxVirus infection. Ectromelia Virus is the causative agent of lethal mousepox, a natural disease that is fatal in mice. Previously, we identified a family of four Ectromelia Virus genes (EVM002, EVM005, EVM154 and EVM165) that contain N-terminal ankyrin repeats and C-terminal F-box domains that interact with the cellular SCF ubiquitin ligase complex. Since degradation of IκBα is catalyzed by the SCFβ-TRCP ubiquitin ligase, we investigated the role of the Ectromelia Virus ankyrin/F-box protein, EVM005, in the regulation of NF-κB. Expression of Flag-EVM005 inhibited both TNFα- and IL-1β-stimulated IκBα degradation and p65 nuclear translocation. Inhibition of the NF-κB pathway by EVM005 was dependent on the F-box domain, and interaction with the SCF complex. Additionally, Ectromelia Virus devoid of EVM005 was shown to inhibit NF-κB activation, despite lacking the EVM005 open reading frame. Finally, Ectromelia Virus devoid of EVM005 was attenuated in both A/NCR and C57BL/6 mouse models, indicating that EVM005 is required for virulence and immune regulation in vivo.
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Ectromelia Virus encodes a btb kelch protein evm150 that inhibits nf κb signaling
Journal of Virology, 2014Co-Authors: Qian Wang, Brianne Couturier, Kristin Burles, Crystal M H Randall, Joanna L Shisler, Michele BarryAbstract:The NF-κB signaling pathway plays a critical role in inflammation and innate immunity. Consequently, many Viruses have evolved strategies to inhibit NF-κB in order to facilitate replication and evasion of the host immune response. Recently, we determined that Ectromelia Virus, the causative agent of mousepox, contains a family of four BTB/kelch proteins that interact with cullin-3-based ubiquitin ligases. We demonstrate here that expression of EVM150, one of the four BTB/kelch proteins, inhibited NF-κB activation induced by tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). Although EVM150 inhibited NF-κB p65 nuclear translocation, IκBα degradation was observed, indicating that EVM150 functioned downstream of IκBα degradation. Significantly, expression of the BTB-only domain of EVM150 blocked NF-κB activation, demonstrating that EVM150 functioned independently of the kelch domain and its role as an adapter for cullin-3-based ubiquitin ligases. Furthermore, cullin-3 knockdown by small interfering RNA demonstrated that cullin-3-based ubiquitin ligases are dispensable for TNF-α-induced NF-κB activation. Interestingly, nuclear translocation of IRF3 and STAT1 still occurred in the presence of EVM150, indicating that EVM150 prevented NF-κB nuclear translocation specifically. In addition to identifying EVM150 as an inhibitor of the NF-κB pathway, this study provides new insights into the role of BTB/kelch proteins during Virus infection. IMPORTANCE With the exception of virulence studies, little work has been done to determine the role of poxviral BTB/kelch proteins during infection. This study, for the first time, has identified a mechanism for the Ectromelia Virus BTB/kelch protein EVM150. Here, we show that EVM150 is a novel inhibitor of the cellular NF-κB pathway, an important component of the antiviral response. This study adds EVM150 to the growing list of NF-κB inhibitors in poxViruses and provides new insights into the role of BTB/kelch proteins during Virus infection.
Lidia Szulcdąbrowska - One of the best experts on this subject based on the ideXlab platform.
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first insight into the modulation of noncanonical nf κb signaling components by poxViruses in established immune derived cell lines an in vitro model of Ectromelia Virus infection
Pathogenetics, 2020Co-Authors: Justyna Struzik, Matylda B Mielcarska, Magdalena Bossowskanowicka, Lidia Szulcdąbrowska, Michal Koper, Malgorzata GierynskaAbstract:Dendritic cells (DCs) and macrophages are the first line of antiviral immunity. Viral pathogens exploit these cell populations for their efficient replication and dissemination via the modulation of intracellular signaling pathways. Disruption of the noncanonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling has frequently been observed in lymphoid cells upon infection with oncogenic Viruses. However, several nononcogenic Viruses have been shown to manipulate the noncanonical NF-κB signaling in different cell types. This study demonstrates the modulating effect of Ectromelia Virus (ECTV) on the components of the noncanonical NF-κB signaling pathway in established murine cell lines: JAWS II DCs and RAW 264.7 macrophages. ECTV affected the activation of TRAF2, cIAP1, RelB, and p100 upon cell treatment with both canonical and noncanonical NF-κB stimuli and thus impeded DNA binding by RelB and p52. ECTV also inhibited the expression of numerous genes related to the noncanonical NF-κB pathway and RelB-dependent gene expression in the cells treated with canonical and noncanonical NF-κB activators. Thus, our data strongly suggest that ECTV influenced the noncanonical NF-κB signaling components in the in vitro models. These findings provide new insights into the noncanonical NF-κB signaling components and their manipulation by poxViruses in vitro.
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mitochondria related gene expression profiles in murine fibroblasts and macrophages during later stages of Ectromelia Virus infection in vitro
Acta Virologica, 2020Co-Authors: Lidia Szulcdąbrowska, Malgorzata Gierynska, Zbigniew Wyzewski, Karolina P Gregorczykzboroch, Justyna Struzik, Joanna Szczepanowska, Felix N Toka, Z Nowakżyczynska, Marek NiemialtowskiAbstract:Mitochondria are multitasking organelles that play a central role in energy production, survival and primary host defense against viral infections. Therefore, Viruses target mitochondria dynamics and functions to benefit their replication and morphogenetic processes. We endeavor to understand the role of mitochondria during infection of Ectromelia Virus (ECTV), hence our investigations on mitochondria-related genes in non-immune (L929 fibroblasts) and immune (RAW 264.7 macrophages) cells. Our results show that during later stages of infection, ECTV significantly decreases the expression of mitochondria-related genes regulating many aspects of mitochondrial physiology and functions, including mitochondrial transport, small molecule transport, membrane polarization and potential, targeting proteins to mitochondria, inner membrane translocation, and apoptosis. Such down-regulation is cell-specific, since macrophages exhibited a more profound down-regulation of mitochondria-related genes compared to infected L929 fibroblasts. Only L929 cells exhibited up-regulation of two important genes responsible for oxidative phosphorylation and subsequent ATP production: Slc25a23 and Slc25a31. Changes in the expression of mitochondria-related genes are accompanied by altered mitochondria morphology and distribution in both types of cells. In depth Ingenuity Pathway Analysis (IPA) identified the "Sirtuin Signaling Pathway" as the most significant top canonical pathway associated with ECTV infection in both analyzed cell types. Taken together, down-regulation of mitochondria-related genes observed especially in macrophages indicates dysfunctional mitochondria, possibly contributing to energy collapse and induction of intrinsic pathway of apoptosis. Meanwhile, alteration of the expression of several mitochondria-related genes in fibroblasts without apoptosis induction may represent poxviral strategy to control cellular energy metabolism for efficient replication. Keywords: Ectromelia Virus; mitochondria; fibroblasts; macrophages.
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mitochondrial heat shock response induced by Ectromelia Virus is accompanied by reduced apoptotic potential in murine l929 fibroblasts
Archivum Immunologiae Et Therapiae Experimentalis, 2019Co-Authors: Zbigniew Wyzewski, Karolina P Gregorczykzboroch, Matylda B Mielcarska, Magdalena Bossowskanowicka, Justyna Struzik, Joanna Szczepanowska, Felix N Toka, Marek Niemialtowski, Lidia SzulcdąbrowskaAbstract:PoxViruses utilize multiple strategies to prevent activation of extrinsic and intrinsic apoptotic pathways for successful replication. Mitochondrial heat shock proteins (mtHsps), especially Hsp60 and its cofactor Hsp10, are engaged in apoptosis regulation; however, until now, the influence of poxViruses on mtHsps has never been studied. We used highly infectious Moscow strain of Ectromelia Virus (ECTV) to investigate the mitochondrial heat shock response and apoptotic potential in permissive L929 fibroblasts. Our results show that ECTV-infected cells exhibit mostly mitochondrial localization of Hsp60 and Hsp10, and show overexpression of both proteins during later stages of infection. ECTV infection has only moderate effect on the electron transport chain subunit expression. Moreover, increase of mtHsp amounts is accompanied by lack of apoptosis, and confirmed by reduced level of pro-apoptotic Bax protein and elevated levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Taken together, we show a positive relationship between increased levels of Hsp60 and Hsp10 and decreased apoptotic potential of L929 fibroblasts, and further hypothesize that Hsp60 and/or its cofactor play important roles in maintaining protein homeostasis in mitochondria for promotion of cell survival allowing efficient replication of ECTV.
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Ectromelia Virus induces tubulin cytoskeletal rearrangement in immune cells accompanied by a loss of the microtubule organizing center and increased α tubulin acetylation
Archives of Virology, 2019Co-Authors: Lidia Szulcdąbrowska, Ada Schollenberger, Karolina P Gregorczykzboroch, Justyna Struzik, Felix N Toka, Mateusz Palusinski, Malgorzata GierynskaAbstract:Ectromelia Virus (ECTV) is an orthopoxVirus that productively replicates in dendritic cells (DCs), but its influence on the microtubule (MT) cytoskeleton in DCs is not known. Here, we show that ECTV infection of primary murine granulocyte-macrophage colony stimulating factor-derived bone marrow cells (GM-BM) downregulates numerous genes engaged in MT cytoskeleton organization and dynamics. In infected cells, the MT cytoskeleton undergoes dramatic rearrangement and relaxation, accompanied by disappearance of the microtubule organizing centre (MTOC) and increased acetylation and stabilization of MTs, which are exploited by progeny virions for intracellular transport. This indicates a strong ability of ECTV to subvert the MT cytoskeleton of highly specialized immune cells.
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long actin based cellular protrusions as novel evidence of the cytopathic effect induced in immune cells infected by the Ectromelia Virus
Central European Journal of Immunology, 2018Co-Authors: Lidia Szulcdąbrowska, Zbigniew Wyzewski, Karolina P Gregorczykzboroch, Justyna Struzik, Felix N Toka, Agnieszka Ostrowska, Malgorzata GierynskaAbstract:The aim of the study was to evaluate the influence of Ectromelia Virus (ECTV) infection on actin cytoskeleton rearrangement in immune cells, such as macrophages and dendritic cells (DCs). Using scanning electron and fluorescence microscopy analysis we observed the presence of long actin-based cellular extensions, formed by both types of immune cells at later stages of infection with ECTV. Such extensions contained straight tubulin filaments and numerous punctuate mitochondria. Moreover, these long cellular projections extended to a certain length and formed convex structures termed "cytoplasmic packets". These structures contained numerous viral particles and presumably were sites of progeny virions' release via budding. Further, discrete mitochondria and separated tubulin filaments that formed a scaffold for accumulated mitochondria were visible within cytoplasmic packets. ECTV-induced long actin-based protrusions resemble "cytoplasmic corridors" and probably participate in Virus dissemination. Our data demonstrate the incredible capacity for adaptation of ECTV to its natural host immune cells, in which it can survive, replicate and induce effective mechanisms for viral spread and dissemination.
Marek Niemialtowski - One of the best experts on this subject based on the ideXlab platform.
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mitochondria related gene expression profiles in murine fibroblasts and macrophages during later stages of Ectromelia Virus infection in vitro
Acta Virologica, 2020Co-Authors: Lidia Szulcdąbrowska, Malgorzata Gierynska, Zbigniew Wyzewski, Karolina P Gregorczykzboroch, Justyna Struzik, Joanna Szczepanowska, Felix N Toka, Z Nowakżyczynska, Marek NiemialtowskiAbstract:Mitochondria are multitasking organelles that play a central role in energy production, survival and primary host defense against viral infections. Therefore, Viruses target mitochondria dynamics and functions to benefit their replication and morphogenetic processes. We endeavor to understand the role of mitochondria during infection of Ectromelia Virus (ECTV), hence our investigations on mitochondria-related genes in non-immune (L929 fibroblasts) and immune (RAW 264.7 macrophages) cells. Our results show that during later stages of infection, ECTV significantly decreases the expression of mitochondria-related genes regulating many aspects of mitochondrial physiology and functions, including mitochondrial transport, small molecule transport, membrane polarization and potential, targeting proteins to mitochondria, inner membrane translocation, and apoptosis. Such down-regulation is cell-specific, since macrophages exhibited a more profound down-regulation of mitochondria-related genes compared to infected L929 fibroblasts. Only L929 cells exhibited up-regulation of two important genes responsible for oxidative phosphorylation and subsequent ATP production: Slc25a23 and Slc25a31. Changes in the expression of mitochondria-related genes are accompanied by altered mitochondria morphology and distribution in both types of cells. In depth Ingenuity Pathway Analysis (IPA) identified the "Sirtuin Signaling Pathway" as the most significant top canonical pathway associated with ECTV infection in both analyzed cell types. Taken together, down-regulation of mitochondria-related genes observed especially in macrophages indicates dysfunctional mitochondria, possibly contributing to energy collapse and induction of intrinsic pathway of apoptosis. Meanwhile, alteration of the expression of several mitochondria-related genes in fibroblasts without apoptosis induction may represent poxviral strategy to control cellular energy metabolism for efficient replication. Keywords: Ectromelia Virus; mitochondria; fibroblasts; macrophages.
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mitochondrial heat shock response induced by Ectromelia Virus is accompanied by reduced apoptotic potential in murine l929 fibroblasts
Archivum Immunologiae Et Therapiae Experimentalis, 2019Co-Authors: Zbigniew Wyzewski, Karolina P Gregorczykzboroch, Matylda B Mielcarska, Magdalena Bossowskanowicka, Justyna Struzik, Joanna Szczepanowska, Felix N Toka, Marek Niemialtowski, Lidia SzulcdąbrowskaAbstract:PoxViruses utilize multiple strategies to prevent activation of extrinsic and intrinsic apoptotic pathways for successful replication. Mitochondrial heat shock proteins (mtHsps), especially Hsp60 and its cofactor Hsp10, are engaged in apoptosis regulation; however, until now, the influence of poxViruses on mtHsps has never been studied. We used highly infectious Moscow strain of Ectromelia Virus (ECTV) to investigate the mitochondrial heat shock response and apoptotic potential in permissive L929 fibroblasts. Our results show that ECTV-infected cells exhibit mostly mitochondrial localization of Hsp60 and Hsp10, and show overexpression of both proteins during later stages of infection. ECTV infection has only moderate effect on the electron transport chain subunit expression. Moreover, increase of mtHsp amounts is accompanied by lack of apoptosis, and confirmed by reduced level of pro-apoptotic Bax protein and elevated levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Taken together, we show a positive relationship between increased levels of Hsp60 and Hsp10 and decreased apoptotic potential of L929 fibroblasts, and further hypothesize that Hsp60 and/or its cofactor play important roles in maintaining protein homeostasis in mitochondria for promotion of cell survival allowing efficient replication of ECTV.
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modulation of proinflammatory nf κb signaling by Ectromelia Virus in raw 264 7 murine macrophages
Archives of Virology, 2015Co-Authors: Justyna Struzik, Lidia Szulcdąbrowska, Anna Winnicka, Diana Papiernik, Marek NiemialtowskiAbstract:Macrophages are antigen-presenting cells (APCs) that play a crucial role in the innate immune response and may be involved in both clearance and spread of Viruses. Stimulation of macrophages via Toll-like receptors (TLRs) results in activation of nuclear factor κB (NF-κB) and synthesis of proinflammatory cytokines. In this work, we show modulation of proinflammatory NF-κB signaling by a member of the family Poxviridae, genus OrthopoxVirus – Ectromelia Virus (ECTV) – in RAW 264.7 murine macrophages. ECTV interfered with p65 NF-κB nuclear translocation induced by TLR ligands such as lipopolysaccharide (LPS) (TLR4), polyinosinic-polycytidylic acid (poly(I:C)) (TLR3) and diacylated lipopeptide Pam2CSK4 (TLR2/6). We observed that ECTV modulates phosphorylation of Ser32 of inhibitor of κB (IκBα) and Ser536 of p65. Interference of ECTV with TLR signaling pathways implied that proinflammatory cytokine synthesis was inhibited. Our studies provide new insights into the strategies of proinflammatory signaling modulation by orthopoxViruses during their replication cycle in immune cells. Understanding important immune interactions between viral pathogens and APCs might contribute to the identification of drug targets and the development of vaccines.
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changes in the mitochondrial network during Ectromelia Virus infection of permissive l929 cells
Acta Biochimica Polonica, 2014Co-Authors: Karolina P Gregorczyk, Zbigniew Wyzewski, Justyna Struzik, Lidia Szulcdąbrowska, Marek NiemialtowskiAbstract:Mitochondria are extremely important organelles in the life of a cell. Recent studies indicate that mitochondria also play a fundamental role in the cellular innate immune mechanisms against viral infections. Moreover, mitochondria are able to alter their shape continuously through fusion and fission. These tightly regulated processes are activated or inhibited under physiological or pathological (e.g. viral infection) conditions to help restore homeostasis. However, many types of Viruses, such as orthopoxViruses, have developed various strategies to evade the mitochondrial-mediated antiviral innate immune responses. Moreover, orthopoxViruses exploit the mitochondria for their survival. Such viral activity has been reported during vaccinia Virus (VACV) infection. Our study shows that the Moscow strain of Ectromelia Virus (ECTV-MOS), an orthopoxVirus, alters the mitochondrial network in permissive L929 cells. Upon infection, the branching structure of the mitochondrial network collapses and becomes disorganized followed by destruction of mitochondrial tubules during the late stage of infection. Small, discrete mitochondria co-localize with progeny virions, close to the cell membrane. Furthermore, clustering of mitochondria is observed around viral factories, particularly between the nucleus and viroplasm. Our findings suggest that ECTV-MOS modulates mitochondrial cellular distribution during later stages of the replication cycle, probably enabling viral replication and/or assembly as well as transport of progeny virions inside the cell. However, this requires further investigation.
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quantitative immunophenotypic analysis of antigen presenting cells involved in Ectromelia Virus antigen presentation in balb c and c57bl 6 mice
Pathogens and Disease, 2013Co-Authors: Lidia Szulcdąbrowska, Malgorzata Gierynska, Anna Winnicka, Anna Boratynskajasinska, Lech Martyniszyn, Marek NiemialtowskiAbstract:During mousepox in resistant (C57BL/6) or susceptible (BALB/c) strains of mice, stimulation of Th1 or Th2 cytokine immune response, respectively, is observed. Because mechanisms of different polarization of T cells remain elusive, in this study, we quantitatively assessed the phenotype of antigen-presenting cells (APCs) involved in Ectromelia Virus (ECTV) antigen presentation and cluster formation with effector cells in secondary lymphoid organs of BALB/c and C57BL/6 mice. We showed that both strains of mice display similar dynamics and kinetics of viral antigen presentation by CD11c+, CD11b+, and CD19+ cells. CD11c+ and CD11b+ cells highly participated in viral antigen presentation during all stages of mousepox, whereas CD19+ cells presented viral peptides later in infection. The main population of dendritic cells (DCs) engaged in ECTV antigen presentation and cell junction formation with effector cells was a population of myeloid CD11b+ DCs (mDCs). We suggest that, on the one hand, ECTV may differentially affect the functions of APCs depending on the strain of mice. On the other hand, we suggest that some types of APCs, such as mDCs or other DCs subsets, have different abilities to direct the shape of immune response depending on the host resistance to mousepox.
