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 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 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 Biochemical Activities of Minute Virus of Mice Nonstructural Protein NS1 Are Modulated In Vitro by the Phosphorylation State of the Polypeptide

1998 ◽  
Vol 72 (10) ◽  
pp. 8002-8012 ◽  
Author(s):  
Jürg P. F. Nüesch ◽  
Romuald Corbau ◽  
Peter Tattersall ◽  
Jean Rommelaere
Keyword(s):  
Posttranslational Modifications ◽  
Nonstructural Protein ◽  
Virus Production ◽  
Progeny Virus ◽  
Helicase Activity ◽  
Minute Virus Of Mice ◽  
Target Dna ◽  
Minute Virus ◽  
Cytotoxic Stress

ABSTRACT NS1, the 83-kDa major nonstructural protein of minute virus of mice (MVM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as late during infection. NS1 is the initiator protein for viral DNA replication. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities; trans regulates viral and cellular promoters; and exerts cytotoxic stress on the host cell. To investigate whether these multiple activities of NS1 depend on posttranslational modifications, in particular phosphorylation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residues with calf intestine alkaline phosphatase, and compared the biochemical activities of the purified un(der)phosphorylated (NS1O) and the native (NS1P) polypeptides. Biochemical analyses of replicative functions of NS1O revealed a severe reduction of intrinsic helicase activity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA]2–3 was enhanced compared to that of NS1P. In the presence of endogenous protein kinases found in replication extracts, NS1O showed all functions necessary for resolution and replication of the 3′ dimer bridge, indicating reactivation of NS1O by rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1O was incubated with protein kinase C.

The multiple lives of DEAD-box RNA helicase DP103/DDX20/Gemin3

2018 ◽  
Vol 46 (2) ◽  
pp. 329-341 ◽  
Author(s):  
Frank Curmi ◽  
Ruben J. Cauchi
Keyword(s):  
Rna Binding ◽  
Muscular Atrophy ◽  
Essential Gene ◽  
Rna Helicase ◽  
Survival Motor Neuron ◽  
In Vivo Studies ◽  
Critical Function ◽  
Dead Box ◽  
Small Nuclear Ribonucleoprotein

Gemin3, also known as DDX20 or DP103, is a DEAD-box RNA helicase which is involved in more than one cellular process. Though RNA unwinding has been determined in vitro, it is surprisingly not required for all of its activities in cellular metabolism. Gemin3 is an essential gene, present in Amoeba and Metazoa. The highly conserved N-terminus hosts the helicase core, formed of the helicase- and DEAD-domains, which, based on crystal structure determination, have key roles in RNA binding. The C-terminus of Gemin3 is highly divergent between species and serves as the interaction site for several accessory factors that could recruit Gemin3 to its target substrates and/or modulate its function. This review article focuses on the known roles of Gemin3, first as a core member of the survival motor neuron (SMN) complex, in small nuclear ribonucleoprotein biogenesis. Although mechanistic details are lacking, a critical function for Gemin3 in this pathway is supported by numerous in vitro and in vivo studies. Gene expression activities of Gemin3 are next underscored, mainly messenger ribonucleoprotein trafficking, gene silencing via microRNA processing, and transcriptional regulation. The involvement of Gemin3 in abnormal cell signal transduction pathways involving p53 and NF-κB is also highlighted. Finally, the clinical implications of Gemin3 deregulation are discussed including links to spinal muscular atrophy, poliomyelitis, amyotrophic lateral sclerosis, and cancer. Impressive progress made over the past two decades since the discovery of Gemin3 bodes well for further work that refines the mechanism(s) underpinning its multiple activities.

scholarly journals The Methylosome, a 20S Complex Containing JBP1 and pICln, Produces Dimethylarginine-Modified Sm Proteins

2001 ◽  
Vol 21 (24) ◽  
pp. 8289-8300 ◽  
Author(s):  
Westley J. Friesen ◽  
Sergey Paushkin ◽  
Anastasia Wyce ◽  
Severine Massenet ◽  
G. Scott Pesiridis ◽  
...  
Keyword(s):  
Muscular Atrophy ◽  
Ring Structure ◽  
Survival Motor Neuron ◽  
Sm Proteins ◽  
Particle Assembly ◽  
Interacting Protein ◽  
Smn Complex ◽  
Snrnp Core

ABSTRACT snRNPs, integral components of the pre-mRNA splicing machinery, consist of seven Sm proteins which assemble in the cytoplasm as a ring structure on the snRNAs U1, U2, U4, and U5. The survival motor neuron (SMN) protein, the spinal muscular atrophy disease gene product, is crucial for snRNP core particle assembly in vivo. SMN binds preferentially and directly to the symmetrical dimethylarginine (sDMA)-modified arginine- and glycine-rich (RG-rich) domains of SmD1 and SmD3. We found that the unmodified, but not the sDMA-modified, RG domains of SmD1 and SmD3 associate with a 20S methyltransferase complex, termed the methylosome, that contains the methyltransferase JBP1 and a JBP1-interacting protein, pICln. JBP1 binds SmD1 and SmD3 via their RG domains, while pICln binds the Sm domains. JBP1 produces sDMAs in the RG domain-containing Sm proteins. We further demonstrate the existence of a 6S complex that contains pICln, SmD1, and SmD3 but not JBP1. SmD3 from the methylosome, but not that from the 6S complex, can be transferred to the SMN complex in vitro. Together with previous results, these data indicate that methylation of Sm proteins by the methylosome directs Sm proteins to the SMN complex for assembly into snRNP core particles and suggest that the methylosome can regulate snRNP assembly.

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 Repurposing inhibitors for SARS-CoV2 main protease

2020 ◽  
Author(s):  
Kumar Sharp
Keyword(s):  
Viral Replication ◽  
Active Sites ◽  
Drug Repurposing ◽  
Chemotherapeutic Drugs ◽  
Short Term ◽  
Main Protease ◽  
Potential Binding ◽  
Approved Drugs ◽  
Fda Approved Drugs

Abstract SARS-CoV2 main protease is important for viral replication and one of the most potential targets for drug development in this current pandemic. Drug repurposing is a promising field to provide potential short-term acceptable therapy for management of coronavirus till a specific anti-viral for coronavirus is developed. In-silico drug repurposing screening is the current fastest way to repurpose drugs by targeting active sites in fraction of seconds. In this study, SARS-CoV2 main protease is being targeted by 1050 FDA-approved drugs to inhibit its activity thereby interfering with viral replication. Chemotherapeutic drugs and anti-retroviral drugs have shown potential binding as inhibitor. In-vitro and clinical trials required to establish final fact.

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