scholarly journals Inhibition of transcription-regulating properties of nonstructural protein 1 (NS1) of parvovirus minute virus of mice by a dominant-negative mutant form of NS1

2001 ◽  
Vol 82 (8) ◽  
pp. 1929-1934 ◽  
Author(s):  
Laurent Deleu ◽  
Aurora Pujol ◽  
Jürg P. F. Nüesch ◽  
Jean Rommelaere
Keyword(s):  
Nonstructural Protein ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mutant Form ◽  
Viral Dna ◽  
Minute Virus Of Mice ◽  
Viral Dna Replication ◽  
Nonstructural Protein 1 ◽  
Minute Virus ◽  
Negative Mutant

Nonstructural protein 1 (NS1) of minute virus of mice is involved in viral DNA replication, transcriptional regulation and cytotoxic action in the host cell. Viral DNA replication is dependent on the ability of NS1 to form homo-oligomers. To investigate whether oligomerization is required for NS1 transcriptional activities, a functionally impaired mutant derivative of NS1 that was able to interact with the wild-type (wt) protein and inhibit its activity in a dominant-negative manner was designed. This mutant provided evidence that transactivation of the parvoviral P38 promoter and transinhibition of a heterologous promoter by NS1 were both affected by the co-expression of the wt and the dominant-negative mutant form of NS1. These results indicate that additional functions of NS1, involved in promoter regulation, require oligomer formation.

scholarly journals Regulation of Minute Virus of Mice NS1 Replicative Functions by Atypical PKCλ In Vivo

2003 ◽  
Vol 77 (1) ◽  
pp. 433-442 ◽  
Author(s):  
Jürg P. F. Nüesch ◽  
Sylvie Lachmann ◽  
Romuald Corbau ◽  
Jean Rommelaere
Keyword(s):  
Dna Replication ◽  
Dominant Negative ◽  
Progeny Virus ◽  
Ns1 Protein ◽  
Viral Dna ◽  
Minute Virus Of Mice ◽  
Viral Dna Replication ◽  
Minute Virus

ABSTRACT Minute virus of mice NS1 protein is a multifunctional phosphoprotein endowed with a variety of enzymatic and regulatory activities necessary for progeny virus particle production. To regulate all of its different functions in the course of a viral infection, NS1 has been proposed to be modulated by posttranslational modifications, in particular, phosphorylation. Indeed, it was shown that the NS1 phosphorylation pattern is altered during the infectious cycle and that the biochemical profile of the protein is dependent on the phosphorylation state of the polypeptide. Moreover, in vitro approaches have identified members of the protein kinase C (PKC) family, in particular, atypical PKC, as regulators of viral DNA replication through the phosphorylation of NS1 residues T435 and S473, thereby activating the protein for DNA unwinding activities. In order to substantiate these findings in vivo, we produced NS1 in the presence of a dominant-negative PKCλ mutant and characterized the purified protein in vitro. The NS1 protein produced under these conditions was found to be only partially phosphorylated and as a consequence to be deficient for viral DNA replication. However, it could be rescued for this viral function by treatment with recombinant activated PKCλ. Our data clearly demonstrate that NS1 is a target for PKCλ phosphorylation in vivo and that this modification is essential for the helicase activity of the viral polypeptide. In addition, the phosphorylation of NS1 at residues T435 and S473 appeared to occur mainly in the nucleus, providing further evidence for the involvement of PKCλ which, unlike PKCζ, accumulates in the nuclear compartment of infected cells.

scholarly journals Novel PKCη Is Required To Activate Replicative Functions of the Major Nonstructural Protein NS1 of Minute Virus of Mice

2003 ◽  
Vol 77 (14) ◽  
pp. 8048-8060 ◽  
Author(s):  
Sylvie Lachmann ◽  
Jean Rommeleare ◽  
Jürg P. F. Nüesch
Keyword(s):  
Protein Kinase ◽  
Nonstructural Protein ◽  
Brdu Incorporation ◽  
Dominant Negative Mutant ◽  
Rolling Circle Replication ◽  
Minute Virus Of Mice ◽  
Rolling Circle ◽  
Minute Virus

ABSTRACT The multifunctional protein NS1 of minute virus of mice (MVMp) is posttranslationally modified and at least in part regulated by phosphorylation. The atypical lambda isoform of protein kinase C (PKCλ) phosphorylates residues T435 and S473 in vitro and in vivo, leading directly to an activation of NS1 helicase function, but it is insufficient to activate NS1 for rolling circle replication. The present study identifies an additional cellular protein kinase phosphorylating and regulating NS1 activities. We show in vitro that the recombinant novel PKCη phosphorylates NS1 and in consequence is able to activate the viral polypeptide in concert with PKCλ for rolling circle replication. Moreover, this role of PKCη was confirmed in vivo. We thereby created stably transfected A9 mouse fibroblasts, a typical MVMp-permissive host cell line with Flag-tagged constitutively active or inactive PKCη mutants, in order to alter the activity of the NS1 regulating kinase. Indeed, tryptic phosphopeptide analyses of metabolically 32P-labeled NS1 expressed in the presence of a dominant-negative mutant, PKCηDN, showed a lack of distinct NS1 phosphorylation events. This correlates with impaired synthesis of viral DNA replication intermediates, as detected by Southern blotting at the level of the whole cell population and by BrdU incorporation at the single-cell level. Remarkably, MVM infection triggers an accumulation of endogenous PKCη in the nuclear periphery, suggesting that besides being a target for PKCη, parvovirus infections may also affect the regulation of this NS1 regulating kinase. Altogether, our results underline the tight interconnection between PKC-mediated signaling and the parvoviral life cycle.

Abstract 11985: C1q / tnf-Related Protein 9 Protects Against Acute Myocardial Injury

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Takahiro Kambara ◽  
Rei Shibata ◽  
Koji Ohashi ◽  
Yuusuke Joki ◽  
Satoko Hayakawa ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Inflammatory Cytokines ◽  
Ischemia Reperfusion ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mutant Form ◽  
Related Protein ◽  
Increased Risk ◽  
Tnf Α ◽  
Negative Mutant

Background: Obesity-related disorders are associated with an increased risk for cardiovascular disease. C1q/TNF-related protein (CTRP) 9 is an anti-diabetic adipokine that is downregulated in obese mice. Recently, we have reported that CTRP9 prevents adverse vascular remodeling in response to injury. Here, we investigated the effect of CTRP9 on acute cardiac injury with loss-of-function genetic manipulations. Methods and Results: CTRP9-deficient (CTRP9-KO) mice at the age of 12 weeks were indistinguishable from Wild-type (WT) mice under basal conditions. CTRP9-KO mice showed increased myocardial infarct size and elevated expression of inflammatory cytokines including TNF-α and IL-6 in ischemic heart following ischemia-reperfusion compared with WT mice. CTRP9-KO mice also had exacerbated contractile left ventricle (LV) dysfunction and increased myocardial expression of inflammatory cytokines following intraperitoneal injection of lipopolysaccharide (LPS) compared with WT mice. Conversely, systemic delivery of an adenoviral vector expressing CTRP9 (Ad-CTRP9) attenuated inflammatory responses to myocardial ischemia-reperfusion or LPS administration in WT mice. In cultured cardiac myocytes, treatment with CTRP9 protein suppressed LPS-stimulated expression of TNF-α and IL-6 with an accompanying reduction of NFkB phosphorylation. Treatment of cardiac myocytes with CTRP9 enhanced AMPK phosphorylation, and transduction with dominant-negative mutant form of AMPK reversed the suppressive effect of CTRP9 on TNF-α and IL-6 expression. Furthermore, systemic administration of Ad-CTRP9 improved LPS-induced cardiac dysfunction in WT mice but not in muscle-specific transgenic mice expressing dominant-negative mutant form of AMPK. Conclusions: These findings suggest that CTRP9 protects against acute cardiac damage in response to pathological stimuli by suppressing inflammatory reactions through AMPK-dependent mechanisms.

Small molecule inhibitors of the c-Jun N-terminal kinase (JNK) possess antiviral activity against highly pathogenic avian and human pandemic influenza A viruses

2012 ◽  
Vol 393 (6) ◽  
pp. 525-534 ◽  
Author(s):  
Wolfgang Nacken ◽  
Christina Ehrhardt ◽  
Stephan Ludwig
Keyword(s):  
Influenza A ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Dominant Negative ◽  
Lung Epithelial Cells ◽  
Viral Rna ◽  
Dominant Negative Mutant ◽  
Highly Pathogenic ◽  
Nonstructural Protein 1 ◽  
Jnk Inhibitors

Abstract C-Jun N-terminal kinases (JNK) are activated in course of many viral infections. Here we analyzed the activity of JNK inhibitors on influenza A virus (IAV) amplification. Human lung epithelial cells were infected with either the highly pathogenic avian virus strain A/FPV/Bratislava/79 (H7N7) or the pandemic swine-origin influenza virus A/Hamburg/4/09 (H1N1v). The application of the JNK inhibitors SP600125 and AS601245 reduced IAV amplification by suppressing viral protein and RNA synthesis. Although AS601245 appeared to generally block the transcription of newly introduced genes, SP600125 specifically affected viral RNA synthesis. Overexpression of a dominant negative mutant of SEK/MKK4 and siRNA-mediated suppression of JNK2 expression confirmed that specific manipulation of the JNK pathway attenuates virus propagation. An IAV minigenome replication assay revealed that SP600125 did not directly affect the activity of the viral RNA polymerase complex but seems to suppress an anti-influenza nonstructural protein 1-mediated virus supportive function. Finally, when H7N7- or H1N1v-infected mice were treated with SP600125, the viral load is reduced in lungs of treated compared with untreated mice. Our data suggest that this class of ATP competitive inhibitors once optimized for antiviral action potentially represent novel drugs for antiviral intervention.

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