scholarly journals Interaction between Parvovirus NS2 Protein and Nuclear Export Factor Crm1 Is Important for Viral Egress from the Nucleus of Murine Cells

2002 ◽  
Vol 76 (7) ◽  
pp. 3257-3266 ◽  
Author(s):  
Cathy L. Miller ◽  
David J. Pintel
Keyword(s):  
Viral Infection ◽  
Nuclear Export ◽  
Null Mutant ◽  
Wild Type ◽  
Single Stranded Dna ◽  
Minute Virus Of Mice ◽  
Export Pathway ◽  
Viral Egress ◽  
Late Step ◽  
Minute Virus

ABSTRACT A mutation that disrupts the interaction between the NS2 protein of minute virus of mice and the nuclear export factor Crm1 results in a block to egress of mutant-generated full virions from the nucleus of infected murine cells. These mutants produce wild-type levels of monomer and dimer replicative DNA forms but are impaired in their ability to generate progeny single-stranded DNA in restrictive murine cells in the first round of infection. The NS2-Crm1 interaction mutant can be distinguished phenotypically from an NS2-null mutant and reveals a role for the Crm1-mediated export pathway at a late step in viral infection.

scholarly journals Nuclear Export Factor CRM1 Interacts with Nonstructural Proteins NS2 from Parvovirus Minute Virus of Mice

1999 ◽  
Vol 73 (9) ◽  
pp. 7769-7779 ◽  
Author(s):  
Ursula Bodendorf ◽  
Celina Cziepluch ◽  
Jean-Claude Jauniaux ◽  
Jean Rommelaere ◽  
Nathalie Salomé
Keyword(s):  
Nuclear Export ◽  
Protein A ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Leptomycin B ◽  
Minute Virus Of Mice ◽  
Export Pathway ◽  
Exact Function ◽  
Infected Cells ◽  
Minute Virus

ABSTRACT The nonstructural NS2 proteins of autonomous parvoviruses are known to act in a host cell-dependent manner and to play a role in viral DNA replication, efficient translation of viral mRNA, and/or encapsidation. Their exact function during the parvovirus life cycle remains, however, still obscure. We report here the characterization of the interaction with the NS2 proteins from the parvovirus minute virus of mice (MVM) and rat as well as mouse homologues of the human CRM1 protein, a member of the importin-beta family recently identified as an essential nuclear export factor. Using the two-hybrid system, we could detect the interaction between the carboxy-terminal region of rat CRM1 and each of the three isoforms of NS2 (P [or major], Y [or minor], and L [or rare]). NS2 proteins were further shown to interact with the full-length CRM1 by coimmunoprecipitation experiments using extracts from both mouse and rat cell lines. Our data show that CRM1 preferentially binds to the nonphosphorylated isoforms of NS2. Moreover, we observed that the treatment of MVM-infected cells with leptomycin B, a drug that specifically inhibits the CRM1-dependent nuclear export pathway, leads to a drastic accumulation of NS2 proteins in the nucleus. Both NS2 interaction with CRM1 and nuclear accumulation upon leptomycin B treatment strongly suggest that these nonstructural viral proteins are actively exported out of the nuclei of infected cells via a CRM1-mediated nuclear export pathway.

scholarly journals Cloning and Sequencing of Defective Particles Derived from the Autonomous Parvovirus Minute Virus of Mice for the Construction of Vectors with Minimal cis-Acting Sequences

2001 ◽  
Vol 75 (3) ◽  
pp. 1284-1293 ◽  
Author(s):  
Nathalie Clément ◽  
Bernard Avalosse ◽  
Karim El Bakkouri ◽  
Thierry Velu ◽  
Annick Brandenburger
Keyword(s):  
Virus Production ◽  
Dna Amplification ◽  
Wild Type ◽  
Nonstructural Proteins ◽  
Helper Plasmid ◽  
Minute Virus Of Mice ◽  
Coding Sequences ◽  
Cis Acting ◽  
Minute Virus ◽  
Helper Plasmids

ABSTRACT The production of wild-type-free stocks of recombinant parvovirus minute virus of mice [MVM(p)] is difficult due to the presence of homologous sequences in vector and helper genomes that cannot easily be eliminated from the overlapping coding sequences. We have therefore cloned and sequenced spontaneously occurring defective particles of MVM(p) with very small genomes to identify the minimalcis-acting sequences required for DNA amplification and virus production. One of them has lost all capsid-coding sequences but is still able to replicate in permissive cells when nonstructural proteins are provided in trans by a helper plasmid. Vectors derived from this particle produce stocks with no detectable wild-type MVM after cotransfection with new, matched, helper plasmids that present no homology downstream from the transgene.

scholarly journals Replication of Minute Virus of Mice DNA Is Critically Dependent on Accumulated Levels of NS2

2005 ◽  
Vol 79 (19) ◽  
pp. 12375-12381 ◽  
Author(s):  
Eun-Young Choi ◽  
Ann E. Newman ◽  
Lisa Burger ◽  
David Pintel
Keyword(s):  
Splice Site ◽  
Nuclear Export ◽  
Single Nucleotide ◽  
Minute Virus Of Mice ◽  
U2 Snrna ◽  
Large Intron ◽  
Murine Fibroblasts ◽  
Minute Virus ◽  
Nuclear Export Protein ◽  
Export Protein

ABSTRACT Following transfection of murine fibroblasts, the lymphotropic strain of minute virus of mice (MVMi) does not efficiently produce progeny single-strand DNA (ssDNA). However, changing a single nucleotide in the MVMi 3′ splice site to that found in the fibrotropic strain MVMp enabled full DNA replication and production of ssDNA. This change enhanced excision of the large intron and the production of NS2, likely by improving interaction, in fibroblasts with the branch point-binding U2 snRNA. One function of NS2 involves interaction with the nuclear export protein Crm1. The defect in production of MVMi ssDNA in fibroblasts can also be overcome by introducing a mutation in MVMi NS2 that enhances its interaction with Crm1. Although MVMi contains a 3′ splice site that performs poorly in fibroblasts, MVMi generated at least as much R2 and NS2 in murine lymphocytes as did MVMp in fibroblasts. Therefore, it appears that MVMp has acquired a mutation that improves the excision of the large intron, as it adapted to fibroblasts to accommodate the need for NS2 for replication in these cells, and that the ratio of NS1 to NS2 may play a larger role in the host range of MVM than previously appreciated.

CRM1 Mediates Nuclear Export of Nonstructural Protein 2 from Parvovirus Minute Virus of Mice

1999 ◽  
Vol 264 (1) ◽  
pp. 144-150 ◽  
Author(s):  
Takayuki Ohshima ◽  
Toshihiro Nakajima ◽  
Takayuki Oishi ◽  
Naoko Imamoto ◽  
Yoshihiro Yoneda ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nonstructural Protein ◽  
Minute Virus Of Mice ◽  
Nonstructural Protein 2 ◽  
Minute Virus

scholarly journals Nuclear Export Through Nuclear Envelope Remodeling inSaccharomyces cerevisiae

2017 ◽  
Author(s):  
Baojin Ding ◽  
Anne M. Mirza ◽  
James Ashley ◽  
Vivian Budnik ◽  
Mary Munson
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Budding Yeast ◽  
Mrna Export ◽  
Low Frequency ◽  
Nuclear Pore ◽  
Wild Type ◽  
Export Pathway ◽  
Wild Type Yeast

ABSTRACTIn eukaryotes, subsets of exported mRNAs are organized into large ribonucleoprotein (megaRNP) granules. How megaRNPs exit the nucleus is unclear, as their diameters are much larger than the nuclear pore complex (NPC) central channel. We previously identified a non-canonical nuclear export mechanism inDrosophila(Speese et al.,Cell2012) and mammals (Ding et al., in preparation), in which megaRNPs exit the nucleus by budding across nuclear envelope (NE) membranes. Here, we present evidence for a similar pathway in the nucleus of the budding yeast S.cerevisiae, which contain morphologically similar granules bearing mRNAs. Wild-type yeast displayed these granules at very low frequency, but this frequency was dramatically increased when the non-essential NPC protein Nup116 was deleted. These granules were not artifacts of defective NPCs; a mutation in the exportinXPO1(CRM1), in which NPCs are normal, induced similar megaRNP upregulation. We hypothesize that a non-canonical nuclear export pathway, analogous to those observed inDrosophilaand in mammalian cells, exists in yeast, and that this pathway is upregulated for use when NPCs or nuclear export are impaired.SUMMARYDing et al., describe a non-canonical mRNA export pathway in budding yeast similar to that observed inDrosophila. This pathway appears upregulated when the NPC is impaired, nuclear envelope integrity is disrupted, or the export factor Xpo1 (CRM1) is defective.

scholarly journals Minute virus of mice small non-structural protein NS2 localizes within, but is not required for the formation of, Smn-associated autonomous parvovirus-associated replication bodies

2005 ◽  
Vol 86 (4) ◽  
pp. 1009-1014 ◽  
Author(s):  
Philip J. Young ◽  
Ann Newman ◽  
Klaus T. Jensen ◽  
Lisa R. Burger ◽  
David J. Pintel ◽  
...  
Keyword(s):  
Virus Replication ◽  
Survival Motor Neuron ◽  
Structural Proteins ◽  
Temporal Delay ◽  
Structural Protein ◽  
Wild Type ◽  
Minute Virus Of Mice ◽  
Minute Virus ◽  
Nuclear Structures

The non-structural proteins NS1 and NS2 of the parvovirus minute virus of mice (MVM) are required for efficient virus replication. It has previously been shown that NS1 and NS2 interact and colocalize with the survival motor neuron (Smn) gene product in novel nuclear structures that are formed late in infection, termed Smn-associated APAR (autonomous parvovirus-associated replication) bodies (SAABs). It is not clear what molecular viral intermediate(s) contribute to SAAB formation. The current results address the role of NS2 in SAAB formation. In highly synchronized wild-type MVM infection of murine A92L cells, NS2 colocalizes with Smn and other SAAB constituents. An MVM mutant that does not produce NS2 still generates SAABS, albeit with a temporal delay. The lag in SAAB formation seen in the absence of NS2 is probably related to the temporal delay in virus replication, suggesting that, whilst NS2 is required for efficient viral infection, it is dispensable for SAAB formation.

scholarly journals Late Maturation Steps Preceding Selective Nuclear Export and Egress of Progeny Parvovirus

2016 ◽  
Vol 90 (11) ◽  
pp. 5462-5474 ◽  
Author(s):  
Raphael Wolfisberg ◽  
Christoph Kempf ◽  
Carlos Ros
Keyword(s):  
Nuclear Export ◽  
Mutational Analysis ◽  
Cell Lysis ◽  
Exchange Chromatography ◽  
Active Process ◽  
Surface Charges ◽  
Minute Virus Of Mice ◽  
Passive Release ◽  
Minute Virus ◽  
Late Maturation

ABSTRACTAlthough the mechanism is not well understood, growing evidence indicates that the nonenveloped parvovirus minute virus of mice (MVM) may actively egress before passive release through cell lysis. We have dissected the late maturation steps of the intranuclear progeny with the aims of confirming the existence of active prelytic egress and identifying critical capsid rearrangements required to initiate the process. By performing anion-exchange chromatography (AEX), we separated intranuclear progeny particles by their net surface charges. Apart from empty capsids (EC), two distinct populations of full capsids (FC) arose in the nuclei of infected cells. The earliest population of FC to appear was infectious but, like EC, could not be actively exported from the nucleus. Further maturation of this early population, involving the phosphorylation of surface residues, gave rise to a second, late population with nuclear export potential. While capsid surface phosphorylation was strictly associated with nuclear export capacity, mutational analysis revealed that the phosphoserine-rich N terminus of VP2 (N-VP2) was dispensable, although it contributed to passive release. The reverse situation was observed for the incoming particles, which were dephosphorylated in the endosomes. Our results confirm the existence of active prelytic egress and reveal a late phosphorylation event occurring in the nucleus as a selective factor for initiating the process.IMPORTANCEIn general, the process of egress of enveloped viruses is active and involves host cell membranes. However, the release of nonenveloped viruses seems to rely more on cell lysis. At least for some nonenveloped viruses, an active process before passive release by cell lysis has been reported, although the underlying mechanism remains poorly understood. By using the nonenveloped model parvovirus minute virus of mice, we could confirm the existence of an active process of nuclear export and further characterize the associated capsid maturation steps. Following DNA packaging in the nucleus, capsids required further modifications, involving the phosphorylation of surface residues, to acquire nuclear export potential. Inversely, those surface residues were dephosphorylated on entering capsids. These spatially controlled phosphorylation-dephosphorylation events concurred with the nuclear export-import potential required to complete the infectious cycle.

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