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.

scholarly journals Ezrin-Radixin-Moesin Family Proteins Are Involved in Parvovirus Replication and Spreading

2009 ◽  
Vol 83 (11) ◽  
pp. 5854-5863 ◽  
Author(s):  
Jürg P. F. Nüesch ◽  
Séverine Bär ◽  
Sylvie Lachmann ◽  
Jean Rommelaere
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nonstructural Protein ◽  
Virus Propagation ◽  
Minute Virus Of Mice ◽  
Erm Proteins ◽  
Kinase C ◽  
Phosphorylation Pattern ◽  
Minute Virus

ABSTRACT The propagation of autonomous parvoviruses is strongly dependent on the phosphorylation of the major nonstructural protein NS1 by members of the protein kinase C (PKC) family. Minute virus of mice (MVM) replication is accompanied by changes in the overall phosphorylation pattern of NS1, which is newly modified at consensus PKC sites. These changes result, at least in part, from the ability of MVM to modulate the PDK-1/PKC pathway, leading to activation and redistribution of both PDK-1 and PKCη. We show that proteins of the ezrin-radixin-moesin (ERM) family are essential for virus propagation and spreading through their functions as adaptors for PKCη. MVM infection led to redistribution of radixin and moesin in the cell, resulting in increased colocalization of these proteins with PKCη. Radixin was found to control the PKCη-driven phosphorylation of NS1 and newly synthesized capsids in vivo. Conversely, radixin phosphorylation and activation were driven by the NS1/CKIIα complex. Altogether, these data argue for ERM proteins being both targets and modulators of parvovirus infection.

scholarly journals Minute Virus of Mice Small Nonstructural Protein NS2 Interacts and Colocalizes with the Smn Protein

2002 ◽  
Vol 76 (12) ◽  
pp. 6364-6369 ◽  
Author(s):  
Philip J. Young ◽  
Klaus T. Jensen ◽  
Lisa R. Burger ◽  
David J. Pintel ◽  
Christian L. Lorson
Keyword(s):  
Nonstructural Protein ◽  
Survival Motor Neuron ◽  
Minute Virus Of Mice ◽  
Survival Motor Neuron Protein ◽  
Motor Neuron Protein ◽  
Minute Virus ◽  
Smn Protein ◽  
Efficient Viral Replication

ABSTRACT The small nonstructural protein NS2 of the minute virus of mice (MVM) is required for efficient viral replication, although its mode of action is unclear. Here we demonstrate that NS2 and survival motor neuron protein (Smn) interact in vitro and in vivo. NS2 and Smn also colocalize in infected nuclei at late times following MVM infection.

scholarly journals Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA]2 motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication

2002 ◽  
Vol 83 (7) ◽  
pp. 1659-1664 ◽  
Author(s):  
Kurt Willwand ◽  
Adela Moroianu ◽  
Rita Hörlein ◽  
Wolfgang Stremmel ◽  
Jean Rommelaere
Keyword(s):  
Dna Replication ◽  
Structural Transition ◽  
Conformational Transition ◽  
Nonstructural Protein ◽  
Specific Interaction ◽  
Sequence Motifs ◽  
Minute Virus Of Mice ◽  
Minute Virus ◽  
The Right

The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA]2, from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA]2 sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.

scholarly journals Recruitment of Single-Stranded Recombinant Adeno-Associated Virus Vector Genomes and Intermolecular Recombination Are Responsible for Stable Transduction of Liver In Vivo

2000 ◽  
Vol 74 (20) ◽  
pp. 9451-9463 ◽  
Author(s):  
Hiroyuki Nakai ◽  
Theresa A. Storm ◽  
Mark A. Kay
Keyword(s):  
Rolling Circle Replication ◽  
Chromosomal Dna ◽  
Adeno Associated Virus ◽  
Rolling Circle ◽  
Steady State Levels ◽  
Slow Rise ◽  
Mouse Hepatocytes ◽  
Enzyme Recognition

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.

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 Host Cell Specificity of Minute Virus of Mice in the Developing Mouse Embryo

2004 ◽  
Vol 78 (17) ◽  
pp. 9474-9486 ◽  
Author(s):  
Refael Itah ◽  
Jacov Tal ◽  
Claytus Davis
Keyword(s):  
Host Cell ◽  
Mouse Embryo ◽  
Cell Types ◽  
Late Gestation ◽  
Productive Infection ◽  
Minute Virus Of Mice ◽  
Minute Virus ◽  
Overlapping Sets

ABSTRACT Productive infection by the murine autonomous parvovirus minute virus of mice (MVM) depends on a dividing cell population and its differentiation state. We have extended the in vivo analysis of the MVM host cell type range into the developing embryo by in utero inoculation followed by further gestation. The fibrotropic p strain (MVMp) and the lymphotropic i strain (MVMi) did not productively infect the early mouse embryo but were able to infect overlapping sets of cell types in the mid- or late-gestation embryo. Both MVMp and MVMi infected developing bone primordia, notochord, central nervous system, and dorsal root ganglia. MVMp exhibited extensive infection in fibroblasts, in the epithelia of lung and developing nose, and, to a lesser extent, in the gut. MVMi also infected endothelium. The data indicated that the host ranges of the two MVM strains consist of overlapping sets of cell types that are broader than previously known from neonate and in vitro infection experiments. The correlation between MVM host cell types and the cell types that activate the transgenic P4 promoter is consistent with the hypothesis that activation of the incoming viral P4 promoter by the host cell is one of the host range determinants of MVM.

scholarly journals Minute Virus of Mice NS1 Interacts with the SMN Protein, and They Colocalize in Novel Nuclear Bodies Induced by Parvovirus Infection

2002 ◽  
Vol 76 (8) ◽  
pp. 3892-3904 ◽  
Author(s):  
Philip J. Young ◽  
Klaus T. Jensen ◽  
Lisa R. Burger ◽  
David J. Pintel ◽  
Christian L. Lorson
Keyword(s):  
Viral Replication ◽  
Active Sites ◽  
Muscular Atrophy ◽  
Nonstructural Protein ◽  
Survival Motor Neuron ◽  
Nuclear Bodies ◽  
Cajal Bodies ◽  
Minute Virus Of Mice ◽  
Minute Virus

ABSTRACT The human survival motor neuron (SMN) gene is the spinal muscular atrophy-determining gene, and a knockout of the murine Smn gene results in preembryonic lethality. Here we show that SMN can directly interact in vitro and in vivo with the large nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVM), a protein essential for viral replication and a potent transcriptional activator. Typically, SMN localizes within nuclear Cajal bodies and diffusely in the cytoplasm. Following transient NS1expression, SMN and NS1 colocalize within Cajal bodies. At early time points following parvovirus infection, NS1 fails to colocalize with SMN within Cajal bodies; however, during the course of MVM infection, dramatic nuclear alterations occur. Formerly distinct nuclear bodies such as Cajal bodies, promyelocytic leukemia gene product (PML) oncogenic domains (PODs), speckles, and autonomous parvovirus-associated replication (APAR) bodies are seen aggregating at later points in infection. These newly formed large nuclear bodies (termed SMN-associated APAR bodies) are active sites of viral replication and viral capsid assembly. These results highlight the transient nature of nuclear bodies and their contents and identify a novel nuclear body formed during infection. Furthermore, simple transient expression of the viral nonstructural proteins is insufficient to induce this nuclear reorganization, suggesting that this event is induced specifically by a step in the viral infection process.

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 The block to transcription elongation at the minute virus of mice attenuator is regulated by cellular elongation factors.

1991 ◽  
Vol 11 (7) ◽  
pp. 3515-3521 ◽  
Author(s):  
A Krauskopf ◽  
E Bengal ◽  
Y Aloni
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Cell Extract ◽  
Elongation Factors ◽  
Minute Virus Of Mice ◽  
Whole Cell ◽  
Minute Virus ◽  
Whole Cell Extract

We have previously reported that both in vivo and in vitro, RNA polymerase II pauses or prematurely terminates transcription at a specific attenuation site located 142 to 147 nucleotides downstream from the P4 promoter of minute virus of mice (MVM). In this report, we show that an in vitro block to transcription elongation in HeLa whole-cell extract occurs at elevated KCl concentrations (0.2 to 1.5 M) but not at the standard KCl concentration (50 mM). Briefly initiated transcription complexes, devoid of dissociated elongation factors by passage through a Sephacryl S-1000 column at 0.3 M KCl, were allowed to elongate the briefly initiated nascent RNA, and a block to transcription elongation at the attenuation site was observed independently of the KCl concentration at the time of elongation. Moreover, the block to elongation was overcome by the addition, during elongation, to the column of purified complexes of whole-cell extract from EA cells but not from MVM-infected EA cells or HeLa cells. The general transcription factors IIF and IIX were also shown to alleviate this block to transcription elongation. On the basis of these results, we suggest that the block to elongation at the MVM attenuation site observed late in MVM infection results, at least in part, from the inactivation of the general transcription elongation factors.

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