scholarly journals Differential Expression of Interferon (IFN) Regulatory Factors and IFN-Stimulated Genes at Early Times after West Nile Virus Infection of Mouse Embryo Fibroblasts

2007 ◽  
Vol 81 (21) ◽  
pp. 12005-12018 ◽  
Author(s):  
Svetlana V. Scherbik ◽  
Bronislava M. Stockman ◽  
Margo A. Brinton
Keyword(s):  
West Nile Virus ◽  
Mouse Embryo ◽  
Stress Responses ◽  
Host Response ◽  
West Nile Virus Infection ◽  
West Nile ◽  
Protein Levels ◽  
Mouse Embryo Fibroblasts ◽  
Primary Mouse ◽  
Embryo Fibroblasts

ABSTRACT Although lineage I West Nile virus (WNV) strain Eg101 induced beta interferon (IFN-β) production as early as 12 h after infection in primary mouse embryo fibroblasts and did not inhibit the JAK-STAT signaling pathway, it was still able to replicate efficiently. To gain insights about possible viral countermeasures used by this virus to suppress the host response, the cell transcriptional profile and the kinetics of IFN regulatory factor (IRF) expression and activation were examined at early times after infection. By 12 h after WNV infection, the majority of the up-regulated genes were ones involved in IFN pathways. However, comparison of IFN-stimulated gene (ISG) expression levels in mock-infected, IFN-treated, and virus-infected cells indicated that WNV infection suppressed the up-regulation of a subset of ISGs, including genes involved in transcriptional regulation, apoptosis, and stress responses, prior to 24 h after infection. Analysis of mRNA and protein levels for representative genes indicated that suppression was at the transcriptional and posttranscriptional levels. Translocation of IRF-3 to the nucleus was observed beginning at 8 h, IRF-7 expression was detected by 12 h, but IRF-1 expression was not detected until 24 h after infection. Virus-induced gene suppression was sufficient to overcome the effect of exogenous IFN pretreatment for 1 h but not for 4 h prior to infection. These data indicate that WNV can selectively counteract the host response at early times after infection by previously unreported mechanisms.

scholarly journals Cellular microRNA-155 Regulates Virus-Induced Inflammatory Response and Protects against Lethal West Nile Virus Infection

Viruses ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 9 ◽  
Author(s):  
Janhavi P. Natekar ◽  
Hussin A. Rothan ◽  
Komal Arora ◽  
Philip G. Strate ◽  
Mukesh Kumar
Keyword(s):  
West Nile Virus ◽  
Critical Role ◽  
West Nile Virus Infection ◽  
High Viral Load ◽  
West Nile ◽  
Gm Csf ◽  
Protein Levels ◽  
Primary Mouse ◽  
Lower Protein ◽  
Human Neuronal Cells

West Nile virus (WNV) is a flavivirus that has disseminated globally as a significant cause of viral encephalitis in humans. MircoRNA-155 (miR-155) regulates various aspects of innate and adaptive immune responses. We previously reported that WNV infection induces upregulation of miR-155 in mice brains. In the current study, we demonstrate the critical role of miR-155 in restricting the pathogenesis of WNV infection in mice. Compared to wild-type (WT) mice, miR-155 knockout mice exhibited significantly higher morbidity and mortality after infection with either a lethal strain, WNV NY99, or a non-lethal strain, WNV Eg101. Increased mortality in miR-155−/− mice was associated with significantly high WNV burden in the serum and brains. Protein levels of interferon (IFN)-α in the serum and brains were higher in miR-155−/− mice. However, miR-155−/− mice exhibited significantly lower protein levels of anti-viral interleukin (IL)-1β, IL-12, IL-6, IL-15, and GM-CSF despite the high viral load. Primary mouse cells lacking miR-155 were more susceptible to infection with WNV compared to cells derived from WT mice. Besides, overexpression of miR-155 in human neuronal cells modulated anti-viral cytokine response and resulted in significantly lower WNV replication. These data collectively indicate that miR-155 restricts WNV production in mouse and human cells and protects against lethal WNV infection in mice.

Two ISREs cooperatively regulate ISG expression in West Nile virus infected IFNAR -/- MEFs.

2021 ◽  
Author(s):  
Dan Cui ◽  
Emilio E. Espínola ◽  
Komal Arora ◽  
Margo A. Brinton
Keyword(s):  
West Nile Virus ◽  
Complex Formation ◽  
Mouse Embryo ◽  
Gene Activation ◽  
West Nile ◽  
Interferon Stimulated Genes ◽  
Mouse Embryo Fibroblasts ◽  
Embryo Fibroblasts

We previously identified a subset of interferon stimulated genes (ISGs) upregulated by West Nile virus (WNV) infection in wildtype mouse embryo fibroblasts (MEFs) after viral proteins had inhibited type 1 interferon (IFN)-mediated JAK-STAT signaling and also in WNV-infected RIG-I -/- , MDA5 -/- , STAT1 -/- , STAT2 -/- , IFNAR -/- , IRF3 -/- , IRF7 -/- , and IRF3/7 -/- MEFs. In this study, ISG upregulation by WNV infection in IFNAR -/- MEFs was confirmed by RNA-seq. ISG upregulation by WNV infection was inhibited in RIG-I -/- /MDA5 -/- MEFs. ISGs were upregulated in IRF1 -/- and IRF5 -/- MEFs but only minimally upregulated in IRF3/5/7 -/- MEFs, suggesting redundant IRF involvement. We previously showed that a single proximal interferon stimulated response element (ISRE) in the Oas1a and Oas1b promoters bound the ISGF3 complex after type 1 IFN treatment. In this study, we used wild-type and mutant promoter luciferase reporter constructs to identify critical regions in the Oas1b and Ifit1 promoters for gene activation in infected IFNAR -/- MEFs. Two ISREs were required in both promoters. Mutation of these ISREs in an Ifit1 promoter DNA probe reduced in vitro complex formation with infected nuclear extracts. An NF-κB inhibitor decreased Ifit1 promoter activity in cells and in vitro complex formation. IRF3 and p50 promoter binding were detected by ChIP for upregulated ISGs with two proximal ISREs. The data indicate that ISREs function cooperatively to upregulate the expression of some ISGs when type 1 IFN-signaling is absent with the binding complex consisting of IRF3, 5, and/or 7 and an NF-κB component(s) as well as other as yet unknown factors. AUTHOR SUMMARY Type 1 IFN signaling in mammalian cells induces formation of the ISGF3 transcription factor complex, which binds to interferon stimulated response elements (ISREs) in the promoters of interferon stimulated genes (ISGs) in the cell nucleus. Flavivirus proteins counteract type 1 IFN signaling by preventing either the formation or nuclear localization of ISGF3. A subset of ISRE-regulated ISGs was still induced in West Nile virus (WNV)-infected mouse embryo fibroblasts (MEFs) indicating that cells have an alternative mechanism for activating these ISGs. In this study, cellular components involved in this ISG upregulation mechanism were identified using gene-knockout MEFs and ChIP and critical promoter regions for gene activation were mapped using reporter assays. The data indicate cooperative function between two ISREs and required binding of IRF3, 5, and/or 7 and an NF-κB component(s). Moreover, type 1 IFN signaling-independent ISG activation requires different additional promoter activation regions than type 1 IFN-dependent activation.

scholarly journals The Host Response to West Nile Virus Infection Limits Viral Spread through the Activation of the Interferon Regulatory Factor 3 Pathway

2004 ◽  
Vol 78 (14) ◽  
pp. 7737-7747 ◽  
Author(s):  
Brenda L. Fredericksen ◽  
Maria Smith ◽  
Michael G. Katze ◽  
Pei-Yong Shi ◽  
Michael Gale
Keyword(s):  
West Nile Virus ◽  
Virus Replication ◽  
Host Response ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
West Nile Virus Infection ◽  
Null Mouse ◽  
Regulatory Factor ◽  
West Nile ◽  
Viral Spread

ABSTRACT Recent outbreaks of West Nile Virus (WNV) have been associated with an increase in morbidity and mortality in humans, birds, and many other species. We have initiated studies to define the molecular mechanisms by which a recent pathogenic isolate of WNV evades the host cell innate antiviral response. Biochemical and microarray analyses demonstrated that WNV induced the expression of beta interferon (IFN-β) and several IFN-stimulated genes late in infection of cultured human cells. The late expression of these antiviral genes was due to the delayed activation of the transcription factor IFN regulatory factor 3 (IRF-3). Despite this host response, WNV was still able to replicate efficiently. The effect of the IRF-3 pathway on WNV replication was assessed by examining virus replication and spread in cultures of wild-type or IRF-3-null mouse embryo fibroblasts. The absence of IRF-3 was marked by a significant increase in plaque size and a sustained production of infectious particles. Although the activation of the IRF-3 pathway was not sufficient to block virus replication, our results suggest that IRF-3 target genes function to constrain WNV infection and limit cell-to-cell virus spread.

WEST NILE VIRUS INFECTION OF PRIMARY MOUSE NEURONAL AND NEUROGLIAL CELLS: THE ROLE OF ASTROCYTES IN CHRONIC INFECTION

2006 ◽  
Vol 75 (4) ◽  
pp. 691-696 ◽  
Author(s):  
JOSE A. P. DINIZ ◽  
HILDA GUZMAN ◽  
VSEVOLOD L. POPOV ◽  
PEDRO F. C. VASCONCELOS ◽  
FANGLING XU ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Virus Infection ◽  
Chronic Infection ◽  
West Nile Virus Infection ◽  
West Nile ◽  
Primary Mouse

Immortalized mouse embryo fibroblasts are resistant to miR-290-induced senescence regardless of p53 status

2011 ◽  
Vol 43 (20) ◽  
pp. 1153-1159
Author(s):  
Milena Rizzo ◽  
Monica Evangelista ◽  
Laura Mariani ◽  
Marcella Simili ◽  
Giuseppe Rainaldi ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Mouse Embryo ◽  
Nih3t3 Cells ◽  
Mouse Embryo Fibroblasts ◽  
Primary Mouse ◽  
Murine Fibroblasts ◽  
P53 Status ◽  
Embryo Fibroblasts ◽  
Potential Tumor Suppressor

The prosenescence role of miR-290 and nocodazole has been documented in primary mouse embryo fibroblasts (MEF), while it is not clear whether immortal murine fibroblasts are still responsive to these senescence inducing stimuli. To establish this point, immortal murine fibroblasts with functional (NIH3T3) or nonfunctional p53 (I-MEF) and low levels of miR-290 were tested for their capability to undergo senescence after exposure to either nocodazole or miR-290. Our results clearly indicate that nocodazole induces senescence only in NIH3T3 cells with a functional p53 but not in I-MEF lacking a functional p53. miR-290 overexpression is unable to address any of the tested immortalized clones toward senescence, regardless of the p53 status, suggesting that the prosenescence role of miR-290 is specific for primary but not for immortal murine fibroblasts. Moreover our findings suggest that the mere downregulation of a potential tumor suppressor miRNA in a given cell type does not necessarily imply that it behaves as a tumor suppressor.

Aryl-hydrocarbon receptor is an inhibitory regulator of lipid synthesis and of commitment to adipogenesis

1998 ◽  
Vol 111 (22) ◽  
pp. 3311-3322 ◽  
Author(s):  
D.L. Alexander ◽  
L.G. Ganem ◽  
P. Fernandez-Salguero ◽  
F. Gonzalez ◽  
C.R. Jefcoate
Keyword(s):  
Cell Cycle ◽  
Aryl Hydrocarbon Receptor ◽  
Mouse Embryo ◽  
Biological Effects ◽  
Ppar Gamma ◽  
Glycerol Phosphate ◽  
Aryl Hydrocarbon ◽  
Mouse Embryo Fibroblasts ◽  
Primary Mouse ◽  
Embryo Fibroblasts

The aryl-hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological effects of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). In mouse embryo fibroblasts, TCDD activates expression of multiple genes, including CYP1B1, the predominant cytochrome P450 expressed in these cells. Here, we analyze constitutive functions of the AhR in primary mouse embryo fibroblasts (MEFs) and spontaneously immortalized MEF cell lines derived from wild-type (WT) C57BL/6 mice and also from congenic mice with a targeted disruption of the AhR gene (AhR-/-). After multiple passages, primary MEFs exhibit spontaneous differentiation, growth cessation and senescence. Eventually, colonies of immortalized MEFs arise to provide clonal lines. The senescent phase occurs much earlier for AhR-/- MEFs, while immortalization is substantially delayed. Comparison of AhR-/- and WT MEFs also indicates that constitutive AhR activity is required for basal expression of CYP1B1 and suppresses lipogenesis in subconfluent cultures. Primary WT and AhR-/- MEFs and the corresponding lines undergo adipogenesis when treated at confluence with the appropriate hormonal inducers. Addition of TCDD before or concurrent with hormonal induction suppressed PPAR gamma mRNA and adipogenesis, as measured by lipid accumulation, glycerol phosphate dehydrogenase activity and stearoyl CoA desaturase type 1 mRNA expression. This effect of TCDD treatment was absent in AhR-/- MEFs, establishing the role of AhR in hormone-induced adipogenesis. Such hormonal activation of confluent MEFs and preadipocytes results in a limited proliferative expansion followed by irreversible growth arrest. TCDD-treated MEFs undergo the mitotic expansion but fail to exit the cell cycle. In AhR-/- MEFs, there is no such effect of TCDD. These findings implicate the AhR as a constitutive inhibitor of triglyceride synthesis, and as an early regulator of adipocyte differentiation. AhR interference with cell-cycle arrest in differentiation may be linked to the increased rate of senescence.

scholarly journals Expression of the small T antigen of Lymphotropic Papovavirus is sufficient to transform primary mouse embryo fibroblasts

Virology ◽  
2016 ◽  
Vol 487 ◽  
pp. 112-120 ◽  
Author(s):  
Tushar Gupta ◽  
Maria Teresa Sáenz Robles ◽  
Rachel M. Schowalter ◽  
Christopher B. Buck ◽  
James M. Pipas
Keyword(s):  
Mouse Embryo ◽  
T Antigen ◽  
Mouse Embryo Fibroblasts ◽  
Primary Mouse ◽  
Embryo Fibroblasts ◽  
Small T Antigen ◽  
Lymphotropic Papovavirus

scholarly journals Induction of Alpha/Beta Interferon by Myxoma Virus Is Selectively Abrogated When Primary Mouse Embryo Fibroblasts Become Immortalized

2009 ◽  
Vol 83 (11) ◽  
pp. 5928-5932 ◽  
Author(s):  
Fuan Wang ◽  
John W. Barrett ◽  
Yiyue Ma ◽  
Gregory A. Dekaban ◽  
Grant McFadden
Keyword(s):  
Virus Infection ◽  
Mouse Embryo ◽  
Myxoma Virus ◽  
Virus Challenge ◽  
Beta Interferon ◽  
Phenotypic Alteration ◽  
Mouse Embryo Fibroblasts ◽  
Primary Mouse ◽  
Alpha Beta ◽  
Embryo Fibroblasts

ABSTRACT Mouse embryo fibroblasts (MEFs) are a widely used cell culture system in life sciences, including virology. Here, we show that although primary MEFs are nonpermissive to myxoma virus replication, the corresponding immortalized MEFs support a highly productive myxoma virus infection. We further demonstrate that this permissive phenotype for myxoma virus in immortalized MEFs is due to the immortalization-associated selective block to the cellular alpha/beta interferon induction machinery involved in responding to myxoma virus challenge. Thus, our report presents a clear example, illustrating that a drastic phenotypic alteration can occur with respect to virus infection between primary cells and their immortalized counterparts.

Fusion of Isolated Mitochondria with Tissue Culture Cells

1972 ◽  
Vol 27 (4) ◽  
pp. 419-423 ◽  
Author(s):  
K. Radsak ◽  
W. Sawicki ◽  
H. Koprowski
Keyword(s):  
Tissue Culture ◽  
Mouse Embryo ◽  
Mouse Tumor ◽  
Resistant Mouse ◽  
Isolated Mitochondria ◽  
Culture Cells ◽  
Mouse Embryo Fibroblasts ◽  
Tissue Culture Cells ◽  
Primary Mouse ◽  
Embryo Fibroblasts

Experiments were undertaken to study the effect of introducing isolated mammalian cell mitochondria into tissue culture cells. The DNA of isolated mitochondria was labeled in vitro with 3H-thymidine. Incorporation of 3H-thymidine into mitochondrial DNA was increased tenfold by the addition of bovine serum albumin and sucrose to the assay.Labeled HeLa cell mitochondria were fused with WI38 (human fibroblast) and I-T-22 (Bromodeoxyuridine resistant mouse cell line) cells in the presence of Sendai virus and autoradiographs were made. The results indicated that isolated mitochondria may have been introduced into the cells by the fusion process.Fusion of mitochondria isolated from mouse tumor cell lines with isogenic primary mouse embryo fibroblasts induced permanent growth of these cells in tissue culture, whereas isolated mitochondria of mouse embryo fibroblasts or allogenic tumor cells did not have this effect.

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