Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to generate reagents to study the murine type I interferon (IFN) system, recombinant murine IFN-alpha1 (rMuIFN-alpha1) protein was expressed in the methylotropic yeast Pichia pastoris. rMuIFN-alpha1 with a phosphate acceptor site engineered at the C-terminus (rMuIFN-alpha1P) to enable radiolabeling by gamma(32)P-ATP and cAMP-dependent protein kinase was also generated. Proteins of 20, 25 (MuIFN-alpha1) and 25.5 (MuIFN-alpha1P), kDa were detected in the yeast growth medium, had type I IFN activity, and were recognized by antimurine L929 cell IFN antibodies. The MuIFN-alpha1 proteins produced in P. pastoris were a mixture of glycosylated and unglycosylated forms, with sugars of approximately 5 kDa added via N-linked glycosylation. The recombinant proteins were highly purified using a single RP-HPLC elution step, and their authenticity was confirmed by amino-terminal amino acid sequencing. The MuIFN-alpha1 and MuIFN-alpha1P protein preparations had specific antiviral activities of 1.3 x 10(7) and 4.7 x 10(6) IU/mg protein, respectively. MuIFN-alpha1P could be radiolabeled to a high specific radioactivity (0.6-2 x 10(8) cpm/microg protein) with gamma(32)P-ATP and cAMP-dependent protein kinase without significantly altering its biologic activity or electrophoretic properties. Binding experiments on COS-7 cells transiently transfected with MuIFNAR-2 and IFNAR-2 demonstrated specific and dose-dependent binding of gamma(32)P-ATP-MuIFN-alpha1P to cell surface type I IFN receptors.
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PMID:Generation and characterization of recombinant unmodified and phosphorylatable murine IFN-alpha1 in the methylotropic yeast Pichia pastoris. 1451 61

Successful host defense against viral infections relies on early production of type I interferon (IFN) and subsequent activation of a cellular cytotoxic response. The acute IFN and inflammatory response against virus infections is mediated by cellular pattern-recognition receptors (PRRs) that recognize specific molecular structures on viral particles or products of viral replication. Toll-like receptors (TLRs) constitute a class of membrane-bound PRRs capable of detecting microbial infections. While TLR2 and TLR4, which were first identified to recognize Gram-positive and Gram-negative bacteria, respectively, sense specific viral proteins on the cell surface, TLRs 3, 7, 8, and 9 serve as receptors for viral nucleic acids in endosomic compartments. In addition to TLRs, cells express cytoplasmic PRRs such as the RNA helicase retinoic acid inducible gene I and the kinase double-stranded RNA-activated protein kinase R, both of which sense dsRNA, a characteristic signature of viral replication, and initiate a protective cellular response. Here we review the recent progress in our understanding of PRRs and viral infections and discuss the molecular and cellular responses evoked by virus-activated PRRs. Finally, we look into what is currently known about the role of PRRs in viral infections in vivo.
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PMID:Reading the viral signature by Toll-like receptors and other pattern recognition receptors. 1563 78

We have previously shown that the leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) blocks cap-dependent mRNA translation and that a genetically engineered FMDV lacking the leader proteinase coding region (A12-LLV2) is attenuated in cell culture and susceptible animals. The attenuated phenotype apparently is a consequence of the inability of A12-LLV2 to block the expression of type I interferon (IFN-alpha/beta) protein, resulting in IFN-induced inhibition of FMDV replication. Here we show that in addition to preventing IFN-alpha/beta protein synthesis, L(pro) reduces the level of immediate-early induction of IFN-beta mRNA and IFN-stimulated gene products such as double-stranded RNA-dependent protein kinase R (PKR), 2',5'-oligoadenylate synthetase, and Mx1 mRNAs in swine cells. Down-regulation of cellular PKR by RNA interference did not affect wild-type virus yield but resulted in a higher yield of A12-LLV2, indicating a direct role of PKR in controlling FMDV replication in the natural host. The observation that L(pro) controls the transcription of genes involved in innate immunity reveals a novel role of this protein in antagonizing the cellular response to viral infection.
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PMID:The leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mRNA and blocks the host innate immune response. 1643 46

The interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase PKR plays a key role in interferon-mediated host defense against viral infection, and is implicated in cellular transformation and apoptosis. We have isolated a cDNA of simian PKR encoding a product with 83% amino acid identity to the human homolog and showed that PKR expression is significantly attenuated in the Vero E6 African green monkey kidney cells devoid of type I interferon genes. A variant form of PKR lacking the exon 12 in the kinase domain is produced in these cells, presumably from an alternatively spliced transcript. Unlike wild type PKR, the variant protein named PKR-DE12 is incapable of auto-phosphorylation and phosphorylation of eIF2-a, indicating that the kinase sub-domains III and IV embedded in exon 12 are indispensable for catalytic function. PKR-DE12 had no dominant negative effect but was weakly phosphorylated in trans by wild type PKR.
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PMID:Attenuated expression of interferon-induced protein kinase PKR in a simian cell devoid of type I interferons. 1651 43

In 129 mice, infection with the nairovirus Dugbe virus (DUGV) was lethal following intracerebral but not intraperitoneal inoculation. Following both routes of inoculation, immunostaining of tissue sections demonstrated virus-positive cells in the brain, indicating that DUGV is neuroinvasive in mice. Many brain areas were affected and neurones were the main cell type infected. Infected cells showed punctate accumulations of viral nucleoprotein in the cytoplasm, indicative of virus replication sites. Immunostaining for activated caspase 3 demonstrated no evidence of apoptosis. The type I interferon (IFN) system plays a significant role in defence against DUGV, as 129 IFN-alpha/beta R(-/-) mice died rapidly following both intraperitoneal and intracerebral inoculations. Studies were undertaken to determine whether the IFN-inducible proteins, protein kinase R (PKR) and MxA, were important for protection; neither PKR nor constitutively expressed human MxA played significant roles.
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PMID:Pathogenesis of Dugbe virus infection in wild-type and interferon-deficient mice. 1676 Apr 3

Dendritic cells (DCs) play a role in anti-viral immunity by providing early innate protection against viral replication and by presenting antigen to T cells for initiation of the adaptive immune response. Studies show the adaptive response to porcine reproductive and respiratory syndrome virus (PRRSV) is ineffective for complete viral elimination. Other studies describe the kinetics of the adaptive response to PRRSV, but have not investigated the early response by DCs. We hypothesize that there is an aberrant activation of DCs early in PRRSV infection; consequently, the adaptive response is triggered inadequately. The current study characterized a subtype of porcine lung DCs (L-DCs) and investigated the ability of PRRSV to infect and replicate in L-DCs and monocyte-derived DCs (MDDCs). Furthermore, the type I interferon anti-viral response to PRRSV with and without the addition of recombinant porcine IFN-alpha (rpIFN-alpha), an important cytokine that signals for anti-viral mediator activation, was analysed. Results show that PRRSV replicated in MDDCs but not L-DCs, providing evidence that these cells have followed distinct differentiation pathways. Although both cell types responded to PRRSV with an induction of IFN-beta mRNA, the magnitude and duration of the response differed between cell types. The addition of rpIFN-alpha was protective in MDDCs, and mRNA synthesis of Mx (myxovirus resistant) and PKR (double-stranded RNA dependent protein kinase) was observed in both cell types after rpIFN-alpha addition. Overall, PRRSV replicated in MDDCs but not L-DCs, and rpIFN-alpha was required for the transcription of protective anti-viral mediators. DC response to PRRSV was limited to IFN-beta transcription, which may be inadequate in triggering the adaptive immune response.
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PMID:Differential type I interferon activation and susceptibility of dendritic cell populations to porcine arterivirus. 1711 72

Non-structural protein NS1 of influenza A virus counteracts the host immune response by blocking the synthesis of type I interferon (IFN). As deletion of the complete NS1 gene has to date been reported only in the human H1N1 strain A/PR/8/34, it remained unclear whether NS1 is a non-essential virulence factor in other influenza A virus strains as well. In this report, the properties of NS1-deficient mutants derived from strain SC35M (H7N7) are described. A mutant of SC35M that completely lacks the NS1 gene was an excellent inducer of IFN in mammalian and avian cells in culture and, consequently, was able to multiply efficiently only in cell lines with defects in the type I IFN system. Virus mutants carrying C-terminally truncated versions of NS1 were less powerful inducers of IFN and were attenuated less strongly in human A549 cells. Although attenuated in wild-type mice, these mutants remained highly pathogenic for mice lacking the IFN-regulated antiviral factor Mx1. In contrast, the NS1-deficient SC35M mutant was completely non-pathogenic for wild-type mice, but remained pathogenic for mice lacking Mx1 and double-stranded RNA-activated protein kinase (PKR). Wild-type SC35M, but not the NS1-deficient mutant virus, was able to replicate in the upper respiratory tract of birds, but neither virus induced severe disease in adult chickens. Altogether, this study supports the view that NS1 represents a non-essential virulence factor of different influenza A viruses.
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PMID:Properties of H7N7 influenza A virus strain SC35M lacking interferon antagonist NS1 in mice and chickens. 1741 66

Cells carry a variety of molecules, referred to as pathogen recognition receptors (PRRs), which are able to sense invading pathogens. Interaction of PRRs with viral compounds instigates a signaling pathway(s), resulting in the activation of genes, including those for type I interferon (IFN), which are critical for an effective antiviral response. Here we demonstrate that the double-stranded RNA (dsRNA)-dependent protein kinase PKR, which has been shown to function as a PRR in cells treated with the dsRNA mimetic poly(I:C), serves as a PRR in West Nile virus (WNV)-infected cells. Evidence for PKR's role as a PRR was obtained from both human and murine cells. Using mouse embryonic fibroblasts (MEFs), we demonstrated that PKR gene knockout, posttranscriptional gene silencing of PKR mRNA using small interfering RNA (siRNA), and chemical inhibition of PKR function all interfered with IFN synthesis following WNV infection. In three different human cell lines, siRNA knockdown and chemical inhibition of PKR blocked WNV-induced IFN synthesis. Using the same approaches, we demonstrated that PKR was not necessary for Sendai virus-induced IFN synthesis, suggesting that PKR is particularly important for recognition of WNV infection. Taken together, our data suggest that PKR could serve as a PRR for recognition of WNV infection.
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PMID:West Nile virus-induced interferon production is mediated by the double-stranded RNA-dependent protein kinase PKR. 1768 61

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus strain undergoing clinical evaluation as a replication-deficient vaccine vector against various infections and tumor diseases. To analyze the basis of its high immunogenicity, we investigated the mechanism of how MVA induces type I interferon (IFN) responses. MVA stimulation of bone marrow-derived dendritic cells (DC) showed that plasmacytoid DC were main alpha IFN (IFN-alpha) producers that were triggered independently of productive infection, viral replication, or intermediate and late viral gene expression. Increased IFN-alpha levels were induced upon treatment with mildly UV-irradiated MVA, suggesting that a virus-encoded immune modulator(s) interfered with the host cytokine response. Mice devoid of Toll-like receptor 9 (TLR9), the receptor for double-stranded DNA, mounted normal IFN-alpha responses upon MVA treatment. Furthermore, mice devoid of the adaptors of TLR signaling MyD88 and TRIF and mice deficient in protein kinase R (PKR) showed IFN-alpha responses that were only slightly reduced compared to those of wild-type mice. MVA-induced IFN-alpha responses were critically dependent on autocrine/paracrine triggering of the IFN-alpha/beta receptor and were independent of IFN-beta, thus involving "one-half" of a positive-feedback loop. In conclusion, MVA-mediated type I IFN secretion was primarily triggered by non-TLR molecules, was independent of virus propagation, and critically involved IFN feedback stimulation. These data provide the basis to further improve MVA as a vaccine vector.
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PMID:Modified vaccinia virus Ankara induces Toll-like receptor-independent type I interferon responses. 1785 54

The effect of viral hemorrhagic septicemia virus (VHSV) was studied on the established rainbow trout (Oncorhynchus mykiss) monocyte/macrophage-like cell line RTS11. The virus was not able to complete its replication cycle as infectious viral particles were not released from the cells. However, in RTS11, the virus was capable of producing mRNA from at least its N and G genes. At the protein level, only N protein was detected 2 days post-infection, whereas a faint band corresponding to the G protein was also observed after 5 days post-infection. These results suggest an interruption of viral protein translation at some point. The expression of N mRNA was significantly inhibited in cells pre-treated with Poly I:C, but not affected by 2-aminopurine (2-AP), an inhibitor of the dsRNA-dependent protein kinase (PKR), thus indicating that PKR has no effect on mRNA expression directly. However, when cells were preincubated with Poly I:C in the presence of 2-AP, the levels of N mRNA were restored suggesting that Poly I:C can limit viral transcription through an antiviral mechanism dependent of PKR. The effect of VHSV on the expression of transcripts for different immune genes was determined, but significant induction was found only for genes related to the type I interferon (IFN) response, such as IFN-1 and -2 and the three Mx isoforms. Heat-inactivated virus failed to induce IFN-1 and -2, suggesting that early events in the VHSV life cycle were necessary for the type I IFN response. Poly I:C alone also induced transcripts for the antiviral Mx proteins. Prior exposure of RTS11 to VHSV did not prevent Poly I:C from inducing transcripts for Mx1, Mx2 and Mx3. Perhaps the failure of VHSV to disable antiviral mechanisms in RTS11 accounts for the aborted infections.
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PMID:Effects of viral hemorrhagic septicemia virus (VHSV) on the rainbow trout (Oncorhynchus mykiss) monocyte cell line RTS-11. 1792 55


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