Loss of IKK subunits limits NF-κB signaling in reovirus infected cells

2019 ◽  
Author(s):  
Andrew J. McNamara ◽  
Pranav Danthi
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Virus Replication ◽  
Nuclear Translocation ◽  
Innate Immune ◽  
Host Cells ◽  
Reovirus Infection ◽  
Cellular Environment ◽  
Ikk Complex ◽  
Infected Cells

ABSTRACTViruses commonly antagonize innate immune pathways that are primarily driven by Nuclear Factor-κB (NF-κB), Interferon Regulatory Factor (IRF) and Signal Transducer and Activator of Transcription (STAT) family of transcription factors. Such a strategy allows viruses to evade immune surveillance and maximize their replication. Using an unbiased RNA-seq based approach to measure gene expression induced by transfected viral genomic RNA (vgRNA) and reovirus infection, we discovered that mammalian reovirus inhibits host cell innate immune signaling. We found that while vgRNA and reovirus infection both induce a similar IRF dependent gene expression program, gene expression driven by the NF-κB family of transcription factors is lower in infected cells. Potent agonists of NF-κB, such as Tumor Necrosis Factor alpha (TNFα) and vgRNA, failed to induce NF-κB dependent gene expression in infected cells. We demonstrate that NF-κB signaling is blocked due to loss of critical members of the Inhibitor of KappaB Kinase (IKK) complex, NF-κB Essential MOdifier (NEMO) and IKKβ. The loss of the IKK complex components prevents nuclear translocation and phosphorylation of NF-κB, thereby preventing gene expression. Our studies demonstrate that reovirus infection selectively blocks NF-κB, likely to counteract its antiviral effects and promote efficient viral replication.IMPORTANCEHost cells mount a response to curb virus replication in infected cells and prevent infection of neighboring, as yet uninfected cells. The NF-κB family of proteins is important for the cell to mediate this response. In this study, we show that in cells infected with mammalian reovirus, NF-κB is inactive. Further, we demonstrate that NF-κB is rendered inactive because virus infection results in reduced levels of upstream intermediaries (called IKKs) that are needed for NF-κB function. Based on previous evidence that active NF-κB limits reovirus infection, we conclude that inactivating NF-κB is a viral strategy to produce a cellular environment that is favorable for virus replication.

scholarly journals Loss of IKK Subunits Limits NF-κB Signaling in Reovirus-Infected Cells

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Andrew J. McNamara ◽  
Pranav Danthi
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Virus Replication ◽  
Nuclear Translocation ◽  
Innate Immune ◽  
Host Cells ◽  
Reovirus Infection ◽  
Cellular Environment ◽  
Ikk Complex ◽  
Infected Cells

ABSTRACT Viruses commonly antagonize innate immune pathways that are primarily driven by nuclear factor kappa B (NF-κB), interferon regulatory factor (IRF), and the signal transducer and activator of transcription proteins (STAT) family of transcription factors. Such a strategy allows viruses to evade immune surveillance and maximize their replication. Using an unbiased transcriptome sequencing (RNA-seq)-based approach to measure gene expression induced by transfected viral genomic RNA (vgRNA) and reovirus infection, we discovered that mammalian reovirus inhibits host cell innate immune signaling. We found that, while vgRNA and reovirus infection both induce a similar IRF-dependent gene expression program, gene expression driven by the NF-κB family of transcription factors is lower in infected cells. Potent agonists of NF-κB such as tumor necrosis factor alpha (TNF-α) and vgRNA failed to induce NF-κB-dependent gene expression in infected cells. We demonstrate that NF-κB signaling is blocked due to loss of critical members of the inhibitor of kappa B kinase (IKK) complex, NF-κB essential modifier (NEMO), and IKKβ. The loss of the IKK complex components prevents nuclear translocation and phosphorylation of NF-κB, thereby preventing gene expression. Our study demonstrates that reovirus infection selectively blocks NF-κB, likely to counteract its antiviral effects and promote efficient viral replication. IMPORTANCE Host cells mount a response to curb virus replication in infected cells and prevent spread of virus to neighboring, as yet uninfected, cells. The NF-κB family of proteins is important for the cell to mediate this response. In this study, we show that in cells infected with mammalian reovirus, NF-κB is inactive. Further, we demonstrate that NF-κB is rendered inactive because virus infection results in reduced levels of upstream intermediaries (called IKKs) that are needed for NF-κB function. Based on previous evidence that active NF-κB limits reovirus infection, we conclude that inactivating NF-κB is a viral strategy to produce a cellular environment that is favorable for virus replication.

scholarly journals “Limiting access to iron decreases infection of Atlantic salmon SHK-1 cells with bacterium Piscirickettsia salmonis”

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Rodrigo Díaz ◽  
José Troncoso ◽  
Eva Jakob ◽  
Stanko Skugor
Keyword(s):  
Gene Expression ◽  
Iron Deficiency ◽  
Atlantic Salmon ◽  
Bacterial Load ◽  
Host Cells ◽  
Immune Gene ◽  
Immune Signaling ◽  
Immune Effector ◽  
Infected Cells ◽  
Piscirickettsia Salmonis

Abstract Background Vertebrate hosts limit the availability of iron to microbial pathogens in order to nutritionally starve the invaders. The impact of iron deficiency induced by the iron chelator deferoxamine mesylate (DFO) was investigated in Atlantic salmon SHK-1 cells infected with the facultative intracellular bacterium Piscirickettsia salmonis. Results Effects of the DFO treatment and P. salmonis on SHK-1 cells were gaged by assessing cytopathic effects, bacterial load and activity, and gene expression profiles of eight immune biomarkers at 4- and 7-days post infection (dpi) in the control group, groups receiving single treatments (DFO or P. salmonis) and their combination. The chelator appears to be well-tolerated by host cells, while it had a negative impact on the number of bacterial cells and associated cytotoxicity. DFO alone had minor effects on gene expression of SHK-1 cells, including an early activation of IL-1β at 4 dpi. In contrast to few moderate changes induced by single treatments (either infection or chelator), most genes had highest upregulation in the infected groups receiving DFO. The mildest induction of hepcidin-1 (antimicrobial peptide precursor and regulator of iron homeostasis) was observed in cells exposed to DFO alone, followed by P. salmonis infected cells while the addition of DFO to infected cells further increased the mRNA abundance of this gene. Transcripts encoding TNF-α (immune signaling) and iNOS (immune effector) showed sustained increase at both time points in this group while cathelicidin-1 (immune effector) and IL-8 (immune signaling) were upregulated at 7 dpi. The stimulation of protective gene responses seen in infected cultures supplemented with DFO coincided with the reduction of bacterial load and activity (judged by the expression of P. salmonis 16S rRNA), and damage to cultured host cells. Conclusion The absence of immune gene activation under normal iron conditions suggests modulation of host responses by P. salmonis. The negative effect of iron deficiency on bacteria likely allowed host cells to respond in a more protective manner to the infection, further decreasing its progression. Presented findings encourage in vivo exploration of iron chelators as a promising strategy against piscirickettsiosis.

Abstract 1772: Cardiomyocyte Damage-Released S100A1 Protein Evokes A Proinflammatory Phenotype In Cardiac Fibroblasts

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
David Rohde ◽  
Melanie Boerries ◽  
Herzog Nicole ◽  
Gang Qiu ◽  
Philipp Ehlermann ◽  
...  
Keyword(s):  
Western Blotting ◽  
Immune Cells ◽  
Nuclear Translocation ◽  
Cardiac Fibroblasts ◽  
Innate Immune ◽  
Host Cells ◽  
Boyden Chamber ◽  
Innate Immune Cells ◽  
Danger Signal

Background: S100A1, a cardiomyocyte specific inotropic calcium sensor protein, is released from infarcted human myocardium in the extracellular environment and circulation, reaching peak serum levels (1–2 μM) 8–9 hours after clinical onset. As growing evidence indicates that S100 proteins can act as pre-existing danger signals triggering the innate immune system into action upon release from injured host cells, we hypothesized that damage-released S100A1 can act as a cardiac danger signal alerting innate immune cells. Methods and Results: Here we report for the first time that necrotic cardiomyocytes release S100A1 protein in vitro, which is exclusively internalized by cardiac fibroblasts (CFs) in a clathrin- and caveolin-independent manner as shown by IF. Internalized S100A1 specifically activated MAPKs/SAPKs (p38, ERK1/2 and JNK) resulting in nuclear translocation of p65 (NF-kB) as assessed by Western blotting, EMSA and IF. In turn, S100A1 triggered an inflammatory gene program in CFs including enhanced expression of adhesion molecules, integrins, chemokines and cytokines including I-CAM, V-CAM, CD11b/18, IL1-alpha, MCP-1, TNF-alpha, SDF-1 among others as obtained by RT-PCR, Western blotting and ELISA. This resulted in enhanced chemoattraction and adhesion of monocytotic and stem cells to S100A1-activated CF as shown by Boyden-chamber and adhesion assays. In line with their proinflammatory transition, S100A1-activated CFs exhibited decreased collagen-1/-3 expression and de-novo collagen production, enhanced collagenolytic MMP-9 abundance and activity and increased levels of the antiangiogenic matricellular factor thrombospondin-2 reflecting extracellular matrix net degradation. Importantly, the immun-modulatory and antifibrotic actions of S100A1 protein in vitro were restricted to CFs, RAGE independent and occurred at concentrations (0.1–1 μM) that were found in patients after AMI. Conclusion: Our in vitro results indicate that S100A1 has the properties of a pre-exisiting endogenous cardiomyocyte danger signal transforming cardiac fibroblasts into immunmodulatory cells that might recruit innate immune cells to the site of cardiac injury and link cardiomyocyte damage to post-MI inflammation.

scholarly journals In vitro integration of an ichnovirus genome segment into the genomic DNA of lepidopteran cells

2007 ◽  
Vol 88 (1) ◽  
pp. 105-113 ◽  
Author(s):  
Daniel Doucet ◽  
Anic Levasseur ◽  
Catherine Béliveau ◽  
Renée Lapointe ◽  
Don Stoltz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virus Replication ◽  
Genomic Dna ◽  
Cultured Cells ◽  
Germ Line ◽  
Choristoneura Fumiferana ◽  
Genome Segment ◽  
Host Cells ◽  
Dsdna Viruses

Polydnaviruses (PDVs) are dsDNA viruses transmitted by ichneumonid and braconid endoparasitoids to their lepidopteran hosts during oviposition. Wasp carriers are asymptomatic and transmit the virus to their progeny through the germ line; replication is confined to the calyx region of the wasp ovary, where the virus accumulates in the fluid bathing the eggs. In the lepidopteran host, however, no virus replication takes place, but PDV gene expression is essential for successful parasitism. Sustained gene expression in the absence of virus replication thus requires that the circular PDV genome segments persist for days within host cells. Available evidence suggests that most genome segments persist as episomes, but recent studies have indicated that some genome segments may undergo integration within lepidopteran genomic DNA, at least in vitro. In the present study, an integrated form of a Tranosema rostrale ichnovirus (TrIV) genome segment was cloned from genomic DNA extracted from infected Choristoneura fumiferana CF-124T cells and junction regions on either side of the viral DNA sequence were sequenced. This is the first proven example of integration of an ichnovirus genome segment in infected lepidopteran cells. Interestingly, circular forms of this genome segment do not appear to persist in these cells; none the less, a gene (TrFrep1) carried by this genome segment displays long-term transcription in infected cultured cells.

scholarly journals Pseudomonas aeruginosa Infection of Airway Epithelial Cells Modulates Expression of Kruppel-Like Factors 2 and 6 via RsmA-Mediated Regulation of Type III Exoenzymes S and Y

2006 ◽  
Vol 74 (10) ◽  
pp. 5893-5902 ◽  
Author(s):  
Eoin P. O'Grady ◽  
Heidi Mulcahy ◽  
Julie O'Callaghan ◽  
Claire Adams ◽  
Fergal O'Gara
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Transcription Factors ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Effector Proteins ◽  
Host Cells ◽  
Airway Epithelial ◽  
Type Iii ◽  
Enhanced Expression

ABSTRACT Pseudomonas aeruginosa is an important opportunistic pathogen which is capable of causing both acute and chronic infections in immunocompromised patients. Successful adaptation of the bacterium to its host environment relies on the ability of the organism to tightly regulate gene expression. RsmA, a small RNA-binding protein, controls the expression of a large number of virulence-related genes in P. aeruginosa, including those encoding the type III secretion system and associated effector proteins, with important consequences for epithelial cell morphology and cytotoxicity. In order to examine the influence of RsmA-regulated functions in the pathogen on gene expression in the host, we compared global expression profiles of airway epithelial cells in response to infection with P. aeruginosa PAO1 and an rsmA mutant. The RsmA-dependent response of host cells was characterized by significant changes in the global transcriptional pattern, including the increased expression of two Kruppel-like factors, KLF2 and KLF6. This increased expression was mediated by specific type III effector proteins. ExoS was required for the enhanced expression of KLF2, whereas both ExoS and ExoY were required for the enhanced expression of KLF6. Neither ExoT nor ExoU influenced the expression of the transcription factors. Additionally, the increased gene expression of KLF2 and KLF6 was associated with ExoS-mediated cytotoxicity. Therefore, this study identifies for the first time the human transcription factors KLF2 and KLF6 as targets of the P. aeruginosa type III exoenzymes S and Y, with potential importance in host cell death.

scholarly journals Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

2015 ◽  
Vol 90 (2) ◽  
pp. 873-886 ◽  
Author(s):  
Jesus A. Silvas ◽  
Vsevolod L. Popov ◽  
Adriana Paulucci-Holthauzen ◽  
Patricia V. Aguilar
Keyword(s):  
Virus Infection ◽  
Extracellular Vesicles ◽  
Virus Replication ◽  
Case Fatality ◽  
Host Protein ◽  
Host Cells ◽  
Limited Information ◽  
Recent Emergence ◽  
Infected Cells ◽  
Cytoplasmic Structures

ABSTRACTSevere fever with thrombocytopenia syndrome (SFTS) virus is a newly recognized member of the genusPhlebovirusin the familyBunyaviridae. The virus was isolated from patients presenting with hemorrhagic manifestations and an initial case fatality rate of 12 to 30% was reported. Due to the recent emergence of this pathogen, there is limited knowledge on the molecular virology of SFTS virus. Recently, we reported that the SFTS virus NSs protein inhibited the activation of the beta interferon (IFN-β) promoter. Furthermore, we also found that SFTS virus NSs relocalizes key components of the IFN response into NSs-induced cytoplasmic structures. Due to the important role these structures play during SFTS virus replication, we conducted live cell imaging studies to gain further insight into the role and trafficking of these cytoplasmic structures during virus infection. We found that some of the SFTS virus NSs-positive cytoplasmic structures were secreted to the extracellular space and endocytosed by neighboring cells. We also found that these secreted structures isolated from NSs-expressing cells and SFTS virus-infected cells were positive for the viral protein NSs and the host protein CD63, a protein associated with extracellular vesicles. Electron microscopy studies also revealed that the isolated CD63-immunoprecipitated extracellular vesicles produced during SFTS virus infection contained virions. The virions harbored within these structures were efficiently delivered to uninfected cells and were able to sustain SFTS virus replication. Altogether, these results suggest that SFTS virus exploits extracellular vesicles to mediate virus receptor-independent transmission to host cells and open the avenue for novel therapeutic strategies against SFTS virus and related pathogens.IMPORTANCESFTS virus is novel bunyavirus associated with hemorrhagic fever illness. Currently, limited information is available about SFTS virus. In the present study, we demonstrated that extracellular vesicles produced by SFTS virus-infected cells harbor infectious virions. We sought to determine whether these “infectious” extracellular vesicles can mediate transmission of the virus and confirmed that the SFTS virions were efficiently transported by these secreted structures into uninfected cells and were able to sustain efficient replication of SFTS virus. These results have significant impact on our understanding of how the novel tick-borne phleboviruses hijack cellular machineries to establish infection and point toward a novel mechanism for virus replication among arthropod-borne viruses.

scholarly journals Dengue virus sustains viability of infected cells by counteracting apoptosis-mediated DNA breakage

2020 ◽  
Author(s):  
Himadri Nath ◽  
Keya Basu ◽  
Abhishek De ◽  
Subhajit Biswas
Keyword(s):  
Dengue Virus ◽  
Virus Replication ◽  
Host Cells ◽  
Severe Dengue ◽  
Dna Breaks ◽  
Dna Breakage ◽  
Major Player ◽  
Infected Cells ◽  
Dna Break ◽  
Cellular Dna

AbstractDengue is the most important arboviral disease inflicting mankind. This mosquito-borne Flavivirus causes mild to severe dengue fever which in some cases leads to life-threatening conditions namely, dengue haemorrhagic fever and dengue shock syndrome. Annual infection is estimated at 390 million globally with 96 million manifesting clinically. So, ≥80% infections are asymptomatic and self-limiting. Dengue virus (DV) non-structural protein 1 (NS1) is a proven virotoxin abundantly present in the victim’s blood. We found that DV-infected or only NS1-expressing cells both can induce Cleaved Caspase3, due to antiviral response of host cells. NS1-transfected cells also showed nuclear damage and significant levels of DNA breaks suggestive of ensuing apoptosis. So, it was established that NS1 alone is capable of causing apoptosis. Surprisingly, despite secreting similar amount of soluble NS1, the DV-infected cells showed intact nuclear morphology and background levels of DNA nicks. These observations suggested that DV downregulates apoptosis of infected cells, which is a viral strategy against host defence. Furthermore, DV-infected cells counteracted Camptothecin-induced apoptotic DNA break. DV-infection was also found to keep the infected cells metabolically more active than only NS1 expressing cells. So, DV bypasses cellular defence against virus i.e. apoptosis by counteracting cellular DNA break and keeps the infected cells metabolically active to support virus replication for longer period which eventually results in high virus titer in circulation. Our findings reveal another level of intricacy involving dengue virus-host interactions and perhaps explain why ≥80% DV infections are asymptomatic/self-limiting despite the presence of NS1 virotoxin in infected cells.Author SummaryNS1, a virotoxin, abundantly present in Dengue patients blood, is a major player behind disease patho-biogenesis including plasma leakage. Despite the presence of NS1 in blood, Dengue is asymptomatic and self-limiting in more than 80% dengue virus (DV) infected people. We investigated this observation and found that plasmid-mediated NS1 expression and secretion in cells are sufficient to cause programmed cell death (apoptosis) and associated cellular DNA breakage. However, cells infected with dengue virus and secreting equivalent amounts of NS1 didn’t exhibit apoptotic DNA breakage. Consequently, DV-infected cells showed better survival than cells in which only NS1 was transiently expressed by transfection with expression plasmid. We also found that DV can even prevent chemical induced apoptotic DNA damage in infected host cells. So, DV bypasses host antiviral defence i.e. apoptosis by counteracting cellular DNA breakages and keeps the infected cells metabolically active to prolong virus replication.

Targeting cap-dependent translation to inhibit Chikungunya virus replication: selectivity of p38 MAPK inhibitors to virus-infected cells due to autophagy-mediated down regulation of phospho-ERK

2021 ◽  
Vol 102 (7) ◽  
Author(s):  
Prashant Mudaliar ◽  
Parvanendhu Pradeep ◽  
Rachy Abraham ◽  
Easwaran Sreekumar
Keyword(s):  
Protein Expression ◽  
P38 Mapk ◽  
Virus Replication ◽  
Chikungunya Virus ◽  
Host Cells ◽  
Hek293 Cells ◽  
Viral Titre ◽  
Mtor Inhibition ◽  
Infected Cells ◽  
Eif2 Alpha

The 5′ capped, message-sense RNA genome of Chikungunya virus (CHIKV) utilizes the host cell machinery for translation. Translation is regulated by eIF2 alpha at the initiation phase and by eIF4F at cap recognition. Translational suppression by eIF2 alpha phosphorylation occurs as an early event in many alphavirus infections. We observe that in CHIKV-infected HEK293 cells, this occurs as a late event, by which time the viral replication has reached an exponential phase, implying its minimal role in virus restriction. The regulation by eIF4F is mediated through the PI3K-Akt-mTOR, p38 MAPK and RAS-RAF-MEK-ERK pathways. A kinetic analysis revealed that CHIKV infection did not modulate AKT phosphorylation, but caused a significant reduction in p38 MAPK phosphorylation. It caused degradation of phospho-ERK 1/2 by increased autophagy, leaving the PI3K-Akt-mTOR and p38 MAPK pathways for pharmacological targeting. mTOR inhibition resulted in moderate reduction in viral titre, but had no effect on CHIKV E2 protein expression, indicating a minimal role of the mTOR complex in virus replication. Inhibition of p38 MAPK using SB202190 caused a significant reduction in viral titre and CHIKV E2 and nsP3 protein expression. Furthermore, inhibiting the two pathways together did not offer any synergism, indicating that inhibiting the p38 MAPK pathway alone is sufficient to cause restriction of CHIKV replication. Meanwhile, in uninfected cells the fully functional RAS-RAF-MEK-ERK pathway can circumvent the effect of p38 MAPK inhibition on cap-dependent translation. Thus, our results show that host-directed antiviral strategies targeting cellular p38 MAPK are worth exploring against Chikungunya as they could be selective against CHIKV-infected cells with minimal effects on uninfected host cells.

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