scholarly journals Modulation of GAPDH expression and cellular localization after vaccinia virus infection of human adherent monocytes.

2003 ◽  
Vol 50 (3) ◽  
pp. 667-676 ◽  
Author(s):  
Krystyna W Nahlik ◽  
Anna K Mleczko ◽  
Magdalena K Gawlik ◽  
Hanna B Rokita
Keyword(s):  
Vaccinia Virus ◽  
Cellular Localization ◽  
Nuclear Translocation ◽  
Cell Types ◽  
Glycolytic Enzyme ◽  
Gene Transcript ◽  
Gapdh Expression ◽  
Gapdh Mrna ◽  
Infected Cells ◽  
Novel Target

Vaccinia virus is able to replicate in many cell types and is known to modulate apoptosis in infected cells. In this study, expression of apoptosis-related genes was screened in human adherent monocytes after vaccinia infection using a DNA array. A marked increase of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was found. Increased expression and nuclear translocation of GAPDH have recently been reported to participate in apoptosis of many cell types. To confirm the array results, levels of GAPDH mRNA were estimated by RT-PCR, showing an increase at 4 h p.i. followed by a slight decrease, which correlated with the viral anti-apoptotic E3L gene transcript levels. Subcellular localization of the enzyme in human monocytes was examined by Western blot and immunostaining of the infected cells. Both experiments revealed accumulation of GAPDH in the nucleus at 14 h p.i., which was completely suppressed at 24 h p.i. This might indicate GAPDH as a novel target for vaccinia anti-apoptotic modulation.

scholarly journals Multiple Regulatory Domains of IRF-5 Control Activation, Cellular Localization, and Induction of Chemokines That Mediate Recruitment of T Lymphocytes

2002 ◽  
Vol 22 (16) ◽  
pp. 5721-5740 ◽  
Author(s):  
Betsy J. Barnes ◽  
Merrill J. Kellum ◽  
Ann E. Field ◽  
Paula M. Pitha
Keyword(s):  
Cellular Localization ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Transient Transfection Assay ◽  
Nuclear Localization Signals ◽  
Regulatory Domains ◽  
Inflammatory Protein ◽  
Infected Cells ◽  
Simplex Virus ◽  
And Function

ABSTRACT Transcription factors of the interferon regulatory factor (IRF) family have been identified as critical mediators of early inflammatory gene transcription in infected cells. We recently determined that, besides IRF-3 and IRF-7, IRF-5 serves as a direct transducer of virus-mediated signaling. In contrast to that mediated by the other two IRFs, IRF-5-mediated activation is virus specific. We show that, in addition to Newcastle disease virus (NDV) infection, vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1) infection activates IRF-5, leading to the induction of IFNA gene subtypes that are distinct from subtypes induced by NDV. The IRF-5-mediated stimulation of inflammatory genes is not limited to IFNA since in BJAB/IRF-5-expressing cells IRF-5 stimulates transcription of RANTES, macrophage inflammatory protein 1β, monocyte chemotactic protein 1, interleukin-8, and I-309 genes in a virus-specific manner. By transient- transfection assay, we identified constitutive-activation (amino acids [aa] 410 to 489) and autoinhibitory (aa 490 to 539) domains in the IRF-5 polypeptide. We identified functional nuclear localization signals (NLS) in the amino and carboxyl termini of IRF-5 and showed that both of these NLS are sufficient for nuclear translocation and retention in infected cells. Furthermore, we demonstrated that serine residues 477 and 480 play critical roles in the response to NDV infection. Mutation of these residues from serine to alanine dramatically decreased phosphorylation and resulted in a substantial loss of IRF-5 transactivation in infected cells. Thus, this study defines the regulatory phosphorylation sites that control the activity of IRF-5 in NDV-infected cells and provides further insight into the structure and function of IRF-5. It also shows that the range of IRF-5 immunoregulatory target genes includes members of the cytokine and chemokine superfamilies.

scholarly journals PI3K is critical for the nuclear translocation of IRF-7 and type I IFN production by human plasmacytoid predendritic cells in response to TLR activation

2008 ◽  
Vol 205 (2) ◽  
pp. 315-322 ◽  
Author(s):  
Cristiana Guiducci ◽  
Cristina Ghirelli ◽  
Marie-Annick Marloie-Provost ◽  
Tracy Matray ◽  
Robert L. Coffman ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Cell Types ◽  
Nuclear Factor Κb ◽  
Type I ◽  
Endosomal Trafficking ◽  
Type I Ifn ◽  
Protein Levels ◽  
Proinflammatory Responses ◽  
Novel Target ◽  
Factor Α

Plasmacytoid predendritic cells (pDCs) are the main producers of type I interferon (IFN) in response to Toll-like receptor (TLR) stimulation. Phosphatidylinositol-3 kinase (PI3K) has been shown to be activated by TLR triggering in multiple cell types; however, its role in pDC function is not known. We show that PI3K is activated by TLR stimulation in primary human pDCs and demonstrate, using specific inhibitors, that PI3K is required for type I IFN production by pDCs, both at the transcriptional and protein levels. Importantly, PI3K was not involved in other proinflammatory responses of pDCs, including tumor necrosis factor α and interleukin 6 production and DC differentiation. pDCs preferentially expressed the PI3K δ subunit, which was specifically involved in the control of type I IFN production. Although uptake and endosomal trafficking of TLR ligands were not affected in the presence of PI3K inhibitors, there was a dramatic defect in the nuclear translocation of IFN regulatory factor (IRF) 7, whereas nuclear factor κB activation was preserved. Thus, PI3K selectively controls type I IFN production by regulating IRF-7 nuclear translocation in human pDCs and could serve as a novel target to inhibit pathogenic type I IFN in autoimmune diseases.

scholarly journals Vaccinia Virus Blocks Gamma Interferon Signal Transduction: Viral VH1 Phosphatase Reverses Stat1 Activation

2001 ◽  
Vol 75 (7) ◽  
pp. 3185-3196 ◽  
Author(s):  
Pilar Najarro ◽  
Paula Traktman ◽  
John A. Lewis
Keyword(s):  
Signal Transduction ◽  
Vaccinia Virus ◽  
Okadaic Acid ◽  
Nuclear Translocation ◽  
Gamma Interferon ◽  
Viral Protein Synthesis ◽  
Host Immune Responses ◽  
Infected Cells ◽  
Ifn Γ

ABSTRACT We have analyzed the effects of vaccinia virus (VV) on gamma interferon (IFN-γ) signal transduction. Infection of cells with VV 1 to 2 h prior to treatment with IFN-γ inhibits phosphorylation and nuclear translocation of Stat1 and consequently blocks accumulation of mRNAs normally induced by IFN-γ. While phosphorylation of other proteins in the IFN-γ pathway was not affected, activation of Stat1 by other ligand-receptor systems was also blocked by VV. This block of Stat1 activation was dose dependent, and although viral protein synthesis was not required, entry and uncoating of viral cores appear to be needed to block the accumulation of phosphorylated Stat1. These results suggest that a virion component is responsible for the effect. VV virions contain a phosphatase (VH1) that is sensitive to the phosphatase inhibitor Na3VO4 but not to okadaic acid. Addition of Na3VO4 but not okadaic acid restored normal Stat1 phosphorylation levels in VV-infected cells. Moreover, virions containing reduced levels of VH1 were unable to block the IFN-γ signaling pathway. In vitro studies show that the phosphatase can bind and dephosphorylate Stat1, indicating that this transcription factor can be a substrate for VH1. Our results reveal a novel mechanism by which VV interferes with the onset of host immune responses by blocking the IFN-γ signal cascade through the dephosphorylating activity of the viral phosphatase VH1.

Processing and Characterization of Recombinant von Willebrand Factor Expressed in Different Cell Types Using a Vaccinia Virus Vector

1992 ◽  
Vol 67 (01) ◽  
pp. 154-160 ◽  
Author(s):  
P Meulien ◽  
M Nishino ◽  
C Mazurier ◽  
K Dott ◽  
G Piétu ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Von Willebrand Factor ◽  
Human Fibroblasts ◽  
Active Form ◽  
Cell Types ◽  
Biologically Active ◽  
L Cells ◽  
Von Willebrand ◽  
Different Cell Types ◽  
Willebrand Factor

SummaryThe cloning of the cDNA encoding von Willebrand factor (vWF) has revealed that it is synthesized as a large precursor (pre-pro-vWF) molecule and it is now clear that the prosequence or vWAgll is responsible for the intracellular multimerization of vWF. We have cloned the complete vWF cDNA and expressed it using a recombinant vaccinia virus as vector. We have characterized the structure and function of the recombinant vWF (rvWF) secreted from five different cell types: baby hamster kidney (BHK), Chinese hamster ovary (CHO), human fibroblasts (143B), mouse fibroblasts (L) and primary embryonic chicken cells. Forty-eight hours after infection, the quantity of vWF antigen found in the cell supernatant varied from 3 to 12 U/dl depending on the cell type. By SDS-agarose gel electrophoresis, the percentage of high molecular weight forms of vWF varied from 39 to 49% relative to normal plasma for BHK, CHO, 143B and chicken cells but was less than 10% for L cells. In all cell types, the two anodic subbands of each multimer were missing. The two cathodic subbands were easily detected only in BHK and L cells. By SDS-PAGE of reduced samples, pro-vWF was present in similar quantity to the fully processed vWF subunit in L cells, present in moderate amounts in BHK and CHO and in very low amounts in 143B and chicken cells. rvWF from all cells bound to collagen and to platelets in the presence of ristocetin, the latter showing a high correlation between binding efficiency and degree of multimerization. rvWF from all cells was also shown to bind to purified FVIII and in this case binding appeared to be independent of the degree of multimerization. We conclude that whereas vWF is naturally synthesized only by endothelial cells and megakaryocytes, it can be expressed in a biologically active form from various other cell types.

scholarly journals Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

2001 ◽  
Vol 75 (16) ◽  
pp. 7528-7542 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Recombinant Vaccinia Virus ◽  
Open Reading Frames ◽  
Type I ◽  
Golgi Vesicles ◽  
Infected Cells ◽  
Catalytic Motif ◽  
Green Fluorescent

ABSTRACT The wrapping of intracellular mature vaccinia virions by modifiedtrans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.

Protein kinase C-α and the regulation of diverse cell responses

2017 ◽  
Vol 8 (3-4) ◽  
pp. 143-153 ◽  
Author(s):  
Rishi Kant Singh ◽  
Sanjay Kumar ◽  
Pramod Kumar Gautam ◽  
Munendra Singh Tomar ◽  
Praveen Kumar Verma ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cellular Localization ◽  
Cellular Transformation ◽  
Cell Types ◽  
Adapter Proteins ◽  
Cellular Functions ◽  
Cell Responses ◽  
Kinase C ◽  
Cellular Compartments

AbstractProtein kinase C (PKC) comprises a family of lipid-sensitive enzymes that have been involved in a broad range of cellular functions. PKC-α is a member of classical PKC with ubiquitous expression and different cellular localization. This unique PKC isoform is activated by various signals which evoke lipid hydrolysis, after activation it interacts with various adapter proteins and is localized to specific cellular compartments where it is devised to work. The universal expression and activation by various stimuli make it a perfect player in uncountable cellular functions including differentiation, proliferation, apoptosis, cellular transformation, motility, adhesion and so on. However, these functions are not intrinsic properties of PKC-α, but depend on cell types and conditions. The activities of PKC-α are managed by the various pharmacological activators/inhibitors and antisense oligonucleotides. The aim of this review is to elaborate the structural feature, and provide an insight into the mechanism of PKC-α activation and regulation of its key biological functions in different cellular compartments to develop an effective pharmacological approach to regulate the PKC-α signal array.

scholarly journals Differential Requirement for TANK-binding Kinase-1 in Type I Interferon Responses to Toll-like Receptor Activation and Viral Infection

2004 ◽  
Vol 199 (12) ◽  
pp. 1651-1658 ◽  
Author(s):  
Andrea K. Perry ◽  
Edward K. Chow ◽  
Julia B. Goodnough ◽  
Wen-Chen Yeh ◽  
Genhong Cheng
Keyword(s):  
Viral Infection ◽  
Nuclear Translocation ◽  
Sendai Virus ◽  
Cell Types ◽  
Receptor Activation ◽  
Northern Blotting ◽  
Type I ◽  
Toll Like Receptor ◽  
Embryonic Fibroblasts ◽  
Iκb Kinase

TANK-binding kinase-1 (TBK1) and the inducible IκB kinase (IKK-i) have been shown recently to activate interferon (IFN) regulatory factor-3 (IRF3), the primary transcription factor regulating induction of type I IFNs. Here, we have compared the role and specificity of TBK1 in the type I IFN response to lipopolysaccharide (LPS), polyI:C, and viral challenge by examining IRF3 nuclear translocation, signal transducer and activator of transcription 1 phosphorylation, and induction of IFN-regulated genes. The LPS and polyI:C-induced IFN responses were abolished and delayed, respectively, in macrophages from mice with a targeted disruption of the TBK1 gene. When challenged with Sendai virus, the IFN response was normal in TBK1−/− macrophages, but defective in TBK1−/− embryonic fibroblasts. Although both TBK1 and IKK-i are expressed in macrophages, only TBK1 but not IKK-i was detected in embryonic fibroblasts by Northern blotting analysis. Furthermore, the IFN response in TBK1−/− embryonic fibroblasts can be restored by reconstitution with wild-type IKK-i but not a mutant IKK-i lacking kinase activity. Thus, our studies suggest that TBK1 plays an important role in the Toll-like receptor–mediated IFN response and is redundant with IKK-i in the response of certain cell types to viral infection.

scholarly journals Label-Free Digital Holo-tomographic Microscopy Reveals Virus-Induced Cytopathic Effects in Live Cells

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Artur Yakimovich ◽  
Robert Witte ◽  
Vardan Andriasyan ◽  
Fanny Georgi ◽  
Urs F. Greber
Keyword(s):  
Refractive Index ◽  
Vaccinia Virus ◽  
Virus Type ◽  
Live Cells ◽  
Refractive Index Gradient ◽  
Label Free ◽  
Virus Infections ◽  
Cytopathic Effects ◽  
Infected Cells ◽  
Index Gradient

ABSTRACTCytopathic effects (CPEs) are a hallmark of infections. CPEs are difficult to observe due to phototoxicity from classical light microscopy. We report distinct patterns of virus infections in live cells using digital holo-tomographic microscopy (DHTM). DHTM is label-free and records the phase shift of low-energy light passing through the specimen on a transparent surface with minimal perturbation. DHTM measures the refractive index (RI) and computes the refractive index gradient (RIG), unveiling optical heterogeneity in cells. We find that vaccinia virus (VACV), herpes simplex virus (HSV), and rhinovirus (RV) infections progressively and distinctly increased RIG. VACV infection, but not HSV and RV infections, induced oscillations of cell volume, while all three viruses altered cytoplasmic membrane dynamics and induced apoptotic features akin to those caused by the chemical compound staurosporine. In sum, we introduce DHTM for quantitative label-free microscopy in infection research and uncover virus type-specific changes and CPE in living cells with minimal interference.IMPORTANCEThis study introduces label-free digital holo-tomographic microscopy (DHTM) and refractive index gradient (RIG) measurements of live, virus-infected cells. We use DHTM to describe virus type-specific cytopathic effects, including cyclic volume changes of vaccinia virus infections, and cytoplasmic condensations in herpesvirus and rhinovirus infections, distinct from apoptotic cells. This work shows for the first time that DHTM is suitable to observe virus-infected cells and distinguishes virus type-specific signatures under noninvasive conditions. It provides a basis for future studies, where correlative fluorescence microscopy of cell and virus structures annotate distinct RIG values derived from DHTM.

scholarly journals Vaccinia virus hemagglutinin. A novel member of the immunoglobulin superfamily.

1989 ◽  
Vol 170 (2) ◽  
pp. 571-576 ◽  
Author(s):  
D Y Jin ◽  
Z L Li ◽  
Q Jin ◽  
Y W Hao ◽  
Y D Hou
Keyword(s):  
Amino Acid ◽  
Vaccinia Virus ◽  
Functional Relationship ◽  
Immunoglobulin Superfamily ◽  
Hydrophobic Region ◽  
Acid Glycoprotein ◽  
Ig Superfamily ◽  
Infected Cells ◽  
Membrane Spanning ◽  
Human Cmv

Striking similarities between vaccinia virus hemagglutinin (VVHA) and proteins belonging to the Ig superfamily clearly indicate that VVHA, a 315-amino acid glycoprotein expressed on the surface of the infected cells, is a novel viral protein that can be added to the expanding list of the Ig superfamily. Its deduced amino acid sequence contains one Ig-like domain at the NH2 terminus, followed by two tandem repeating units and a hydrophobic region, suggestive of membrane spanning. The results offer an opportunity for the further study of the probable evolutionary and possible functional relationship between VVHA and other members of the Ig superfamily. Our observation, together with a recent finding that human CMV possibly encodes a protein similar to the MHC class I antigens (13), provides evidence supporting the fact that the viral capture of cellular Ig-related genes is more common than expected in vaccinia and other viruses, and that usage of an Ig-like domain as recognition signals might be extended from higher animals to animal viruses.

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