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 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.

Effect of Daunorubicin and Adriamycin on Nucleic ACID Synthesis of Serum Stimulated Mouse Embryo Fibroblasts

1977 ◽  
Vol 63 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Rosanna Supino ◽  
Anna M. Casazza ◽  
Aurelio Di Marco
Keyword(s):  
Dna Synthesis ◽  
Mouse Embryo ◽  
Rna Synthesis ◽  
Calf Serum ◽  
Acid Synthesis ◽  
Anthracycline Antibiotics ◽  
Simple Method ◽  
Mouse Embryo Fibroblasts ◽  
Embryo Fibroblasts

This paper reports the effects of daunorubicin and adriamycin on DNA and RNA synthesis of in vitro cultured mouse embryo fibroblasts (MEF) stimulated by fetal calf serum (FCS). The addition of FCS to quiescent MEF cultures brings about a wave of RNA synthesis, followed by DNA synthesis which starts between 8 and 12 h after change of medium and proceed for up to 24 h. These cells are therefore partially synchronized. The level of DNA synthesis depends on the amount of FCS added. Daunorubicin and adriamycin are almost equally effective in inhibiting DNA synthesis, as well as cell proliferation, which takes place later. Adriamycin is more active than daunorubicin on RNA synthesis. In cultures treated for an 8 h period starting at different times after FCS addition, the highest DNA synthesis inhibition is achieved by treatment during the first 8 h, when DNA synthesis has not yet started. The cellular uptake of daunorubicin is constantly higher than that of adriamycin, in any experimental condition tested. The results show that FCS-stimulated MEF can provide a simple method for studying the effects of anthracycline antibiotics on partially synchronized cells.

scholarly journals Continuous replication of Friend virus complex (spleen focus-forming virus-lymphatic leukemia-inducing virus) in mouse embryo fibroblasts. Retention of leukemogenicity and loss of immunosuppressive properties.

1975 ◽  
Vol 142 (4) ◽  
pp. 936-948 ◽  
Author(s):  
R J Eckner
Keyword(s):  
Mouse Embryo ◽  
Helper Virus ◽  
Productive Infection ◽  
Friend Virus ◽  
Focus Formation ◽  
Lymphatic Leukemia ◽  
Mouse Embryo Fibroblasts ◽  
Embryo Fibroblasts

Exposure of NIH Swiss mouse embryo fibroblasts (MEF) to infectious Friend virus (FV) complex [containing defective spleen focus-forming virus (SFFV) and endogenous NB-tropic leukemia-inducing helper virus (LLV-F)] resulted in the productive infection of these cells by both SFFV and LLV-F. Stocks of SFFV derived after extensive growth in this Swiss MEF cell culture system are fully leukemogenic in adult mice for the induction of erythroleukemia and spleen foci. In addition, in vitro-derived LLV-F, when isolated free of SFFV, is fully leukemogenic for the induction of lymphatic leukemia when inoculated into susceptible newborn BALB/c mice. The host range of in vitro-derived FV complex (i.e., FV-TC) for focus formation in vivo is NB-tropic. Unlike in vivo-derived FV complex, FV-TC does not suppress the responsiveness of murine thymocytes to concanavalin A (Con A) in vitro. Rather, FV-TC acts as a mitogen to nonspecifically stimulate the proliferation of BALB/c thymocytes. The mitogenicity of in vitro-derived FV complex is directly associated with the presence of type-C virus particles, is a heat-labile and UV-sensitive property of the virus, and may be primarily due to LLV since equivalent amounts of LLV with or without SFFV present are equally mitogenic. One in vivo passage of FV-TC resulted in the total loss of this mitogenic property with the reappearance of full immunosuppressive properties. This result demonstrates a clear association between in vivo growth of FV and its ability to suppress mouse thymocytes, and suggests that FV complex (SFFV-LLV) is not inherently immunosuppressive for these cells. While the mechanism of this interconversion between immunostimulatory and fully suppressive virus is unknown, both virus markers appear to be dependent upon the presence of infectious FV.

The reproductive potential of normal mouse embryo fibroblasts during culture in vitro

1984 ◽  
Vol 66 (1) ◽  
pp. 401-409
Author(s):  
C. Karatza ◽  
S. Shall
Keyword(s):  
Mouse Embryo ◽  
Colony Size ◽  
Reproductive Potential ◽  
Cell Strain ◽  
Growth Fraction ◽  
Growth Behaviour ◽  
Direct Estimate ◽  
Mouse Embryo Fibroblasts ◽  
Embryo Fibroblasts

A direct estimate of the reproductive potential of mouse embryo fibroblasts through their entire lifespan has been made using the mini-clone technique, which permits the direct observation of the growth fraction in a bulk population by inspection of the growth behaviour of individual cells. We have measured the colony size on each island that contained one or two cells at the beginning and the fraction of islands which, starting from one or two cells failed to divide even once. We observed that even in a young culture there are individual cells that can only reproduce two or three times. With each succeeding passage the distribution of colony sizes shifts to a greater proportion of small colonies. The median colony size decreases with each passage. Furthermore, the fraction of non-dividers directly observed increases smoothly with time; the fraction of non-dividers is quite small at the first passage but increases steadily to reach 0.6 at the last passage, after about 30 generations. These smooth changes in the growth behaviour of this cell strain are accurately described by the mortalization theory of Shall and Stein, in which the single parameter gamma, describes the change in reproductive potential over the entire lifespan. The parameter gamma describes the rate at which the doubling time of the culture increases; it is the number of generations at which half of the newborn cells are themselves reproductively sterile. Our present data provide an estimate of gamma for this cell strain equal to 21.2 generations, which compares well with a previous estimate of 20.3 generations.

Effect of Syngeneic and Allogeneic Lymphocytes on L-cells and Mouse Embryo Fibroblasts in vitro

Nature ◽  
1967 ◽  
Vol 214 (5094) ◽  
pp. 1229-1230 ◽  
Author(s):  
INESSA YU. CHERNYAKHOVSKAYA ◽  
ZAIRA G. KADAGGIDZE ◽  
GEORGE J. SVET-MOLDAVSKY
Keyword(s):  
Mouse Embryo ◽  
L Cells ◽  
Mouse Embryo Fibroblasts ◽  
Embryo Fibroblasts ◽  
Allogeneic Lymphocytes

Regulation of Id1 Protein Expression in Mouse Embryo Fibroblasts by the Type 1 Insulin-like Growth Factor Receptor

2002 ◽  
Vol 277 (1) ◽  
pp. 107-118 ◽  
Author(s):  
Barbara Belletti ◽  
Robert Drakas ◽  
Andrea Morrione ◽  
Xiao Tu ◽  
Marco Prisco ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Expression ◽  
Mouse Embryo ◽  
Growth Factor Receptor ◽  
Insulin Like Growth Factor ◽  
Mouse Embryo Fibroblasts ◽  
Embryo Fibroblasts

scholarly journals 5′-AMP-Activated Protein Kinase (AMPK) Is Induced by Low-Oxygen and Glucose Deprivation Conditions Found in Solid-Tumor Microenvironments

2006 ◽  
Vol 26 (14) ◽  
pp. 5336-5347 ◽  
Author(s):  
Keith R. Laderoute ◽  
Khalid Amin ◽  
Joy M. Calaoagan ◽  
Merrill Knapp ◽  
Theresamai Le ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mouse Embryo ◽  
Cellular Response ◽  
Null Mouse ◽  
Low Oxygen ◽  
Tumor Microenvironments ◽  
Mouse Embryo Fibroblasts ◽  
Embryo Fibroblasts ◽  
Amp Activated Protein Kinase

ABSTRACT Low oxygen gradients (hypoxia and anoxia) are important determinants of pathological conditions under which the tissue blood supply is deficient or defective, such as in solid tumors. We have been investigating the relationship between the activation of hypoxia-inducible factor 1 (HIF-1), the primary transcriptional regulator of the mammalian response to hypoxia, and 5′-AMP-activated protein kinase (AMPK), another regulatory system important for controlling cellular energy metabolism. In the present study, we used mouse embryo fibroblasts nullizygous for HIF-1α or AMPK expression to show that AMPK is rapidly activated in vitro by both physiological and pathophysiological low-oxygen conditions, independently of HIF-1 activity. These findings imply that HIF-1 and AMPK are components of a concerted cellular response to maintain energy homeostasis in low-oxygen or ischemic-tissue microenvironments. Finally, we used transformed derivatives of wild-type and HIF-1α- or AMPKα-null mouse embryo fibroblasts to determine whether AMPK is activated in vivo. We obtained evidence that AMPK is activated in authentic hypoxic tumor microenvironments and that this activity overlaps with regions of hypoxia detected by a chemical probe. We also showed that AMPK is important for the growth of this tumor model.

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