scholarly journals Intron Definition Is Required for Excision of the Minute Virus of Mice Small Intron and Definition of the Upstream Exon

1998 ◽  
Vol 72 (3) ◽  
pp. 1834-1843 ◽  
Author(s):  
Donald D. Haut ◽  
D. J. Pintel
Keyword(s):  
Alternative Splicing ◽  
Consensus Sequence ◽  
Critical Role ◽  
Viral Mrnas ◽  
Minute Virus Of Mice ◽  
Natural Context ◽  
Minute Virus ◽  
Definition Of ◽  
Intron Definition ◽  
Upstream Exon

ABSTRACT Alternative splicing of pre-mRNAs plays a critical role in maximizing the coding capacity of the small parvovirus genome. The small-intron region of minute virus of mice (MVM) pre-mRNAs undergoes an unusual pattern of overlapping alternative splicing—using two donors (D1 and D2) and two acceptors (A1 and A2) within a region of 120 nucleotides—that determines the steady-state ratios of the various viral mRNAs. In this report, we show that the determinants that govern excision of the small intron are complex and are also required for efficient definition of the upstream exon. For the MVM small intron in its natural context, the two donors appear to compete for the splicing machinery: the position of D1 favors its usage, while the primary sequence of D2 must be more like the consensus sequence than is D1 to be used efficiently. We have genetically defined the branch points that are used for generation of the major and minor spliced forms and show that recognition of components of the small-intron acceptors is likely to be the dominant determinant in alternative small-intron excision. We have also identified a G-rich intronic enhancer sequence within the small intron that is essential for splicing of the minor form (D2 to A2) but not the major form (D1 to A1) of MVM mRNAs and is required for efficient definition of the upstream NS2-specific exon. In its natural context, the small intron appears to be excised by a mechanism consistent with intron definition. When the MVM small intron is expanded, various parameters of its excision are altered, indicating that critical cis-acting signals are context dependent. Relative use of the donors and acceptors is altered, and the upstream NS2-specific exon is no longer efficiently defined. The fact that definition of the upstream NS2-specific exon can be achieved by the MVM small intron in its natural context, but not when it is expanded, suggests that the multiple determinants that govern definition and excision of the small intron are required, in concert, for upstream exon definition. Our data are consistent with a model in which alternative splicing of the MVM P4-generated pre-mRNAs is governed by a hybrid of intron- and exon-defining mechanisms.

scholarly journals Hypoxic-response elements in the oncolytic parvovirus Minute virus of mice do not allow for increased vector production at low oxygen concentration

2006 ◽  
Vol 87 (5) ◽  
pp. 1197-1201 ◽  
Author(s):  
Charlotte Servais ◽  
Perrine Caillet-Fauquet ◽  
Marie-Louise Draps ◽  
Thierry Velu ◽  
Yvan de Launoit ◽  
...  
Keyword(s):  
Cell Metabolism ◽  
Consensus Sequence ◽  
High Titre ◽  
Host Cells ◽  
Low Oxygen ◽  
Minute Virus Of Mice ◽  
Minute Virus ◽  
Responsive Element ◽  
Low Oxygen Concentration

Vectors derived from the autonomous parvovirus Minute virus of mice, MVM(p), are promising tools for the gene therapy of cancer. The validation of their in vivo anti-tumour effect is, however, hampered by the difficulty to produce high-titre stocks. In an attempt to increase vector titres, host cells were subjected to low oxygen tension (hypoxia). It has been shown that a number of viruses are produced at higher titres under these conditions. This is the case, among others, for another member of the family Parvoviridae, the erythrovirus B19 virus. Hypoxia stabilizes a hypoxia-inducible transcription factor (HIF-1α) that interacts with a ‘hypoxia-responsive element’ (HRE), the consensus sequence of which (A/GCGTG) is present in the B19 and MVM promoters. Whilst the native P4 promoter was induced weakly in hypoxia, vector production was reduced dramatically, and adding HRE elements to the P4 promoter of the vector did not alleviate this reduction. Hypoxia has many effects on cell metabolism. Therefore, even if the P4 promoter is activated, the cellular factors that are required for the completion of the parvoviral life cycle may not be expressed.

scholarly journals A Supraphysiological Nuclear Export Signal Is Required for Parvovirus Nuclear Export

2008 ◽  
Vol 19 (6) ◽  
pp. 2544-2552 ◽  
Author(s):  
Dieuwke Engelsma ◽  
Noelia Valle ◽  
Alexander Fish ◽  
Nathalie Salomé ◽  
José M. Almendral ◽  
...  
Keyword(s):  
Infected Mouse ◽  
Nuclear Export ◽  
Critical Role ◽  
Nuclear Export Signal ◽  
Hydrophobic Residue ◽  
Minute Virus Of Mice ◽  
Mouse Fibroblasts ◽  
Endogenous Protein ◽  
Minute Virus ◽  
Export Signal

CRM1 exports proteins that carry a short leucine-rich peptide signal, the nuclear export signal (NES), from the nucleus. Regular NESs must have low affinity for CRM1 to function optimally. We previously generated artificial NESs with higher affinities for CRM1, termed supraphysiological NESs. Here we identify a supraphysiological NES in an endogenous protein, the NS2 protein of parvovirus Minute Virus of Mice (MVM). NS2 interacts with CRM1 without the requirement of RanGTP, whereas addition of RanGTP renders the complex highly stable. Mutation of a single hydrophobic residue that inactivates regular NESs lowers the affinity of the NS2 NES for CRM1 from supraphysiological to regular. Mutant MVM harboring this regular NES is compromised in viral nuclear export and productivity. In virus-infected mouse fibroblasts we observe colocalization of NS2, CRM1 and mature virions, which is dependent on the supraphysiological NS2 NES. We conclude that supraphysiological NESs exist in nature and that the supraphysiological NS2 NES has a critical role in active nuclear export of mature MVM particles before cell lysis.

scholarly journals Binding of CCCTC-Binding Factor (CTCF) to the Minute Virus of Mice Genome Is Important for Proper Processing of Viral P4-Generated Pre-mRNAs

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1368
Author(s):  
Maria Boftsi ◽  
Kinjal Majumder ◽  
Lisa R. Burger ◽  
David J. Pintel
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Genome Organization ◽  
Dna Binding Protein ◽  
Minute Virus Of Mice ◽  
Cell Extracts ◽  
Binding Factor ◽  
Minute Virus ◽  
Definition Of ◽  
Novel Exon ◽  
Affect Processing

Specific chromatin immunoprecipitation of salt-fractionated infected cell extracts has demonstrated that the CCCTC-binding factor (CTCF), a highly conserved, 11-zinc-finger DNA-binding protein with known roles in cellular and viral genome organization and gene expression, specifically binds the genome of Minute Virus of Mice (MVM). Mutations that diminish binding of CTCF to MVM affect processing of the P4-generated pre-mRNAs. These RNAs are spliced less efficiently to generate the R1 mRNA, and definition of the NS2-specific exon upstream of the small intron is reduced, leading to relatively less R2 and the generation of a novel exon-skipped product. These results suggest a model in which CTCF is required for proper engagement of the spliceosome at the MVM small intron and for the first steps of processing of the P4-generated pre-mRNA.

scholarly journals Evolution to Pathogenicity of the Parvovirus Minute Virus of Mice in Immunodeficient Mice Involves Genetic Heterogeneity at the Capsid Domain That Determines Tropism

2007 ◽  
Vol 82 (3) ◽  
pp. 1195-1203 ◽  
Author(s):  
Alberto López-Bueno ◽  
José C. Segovia ◽  
Juan A. Bueren ◽  
M. Gerard O'Sullivan ◽  
Feng Wang ◽  
...  
Keyword(s):  
Genetic Heterogeneity ◽  
Evolutionary Dynamics ◽  
Consensus Sequence ◽  
Plaque Assay ◽  
Scid Mice ◽  
Virus Infections ◽  
Minute Virus Of Mice ◽  
Immunodeficient Mice ◽  
Minute Virus ◽  
High Level

ABSTRACT Very little is known about the role that evolutionary dynamics plays in diseases caused by mammalian DNA viruses. To address this issue in a natural host model, we compared the pathogenesis and genetics of the attenuated fibrotropic and the virulent lymphohematotropic strains of the parvovirus minute virus of mice (MVM), and of two invasive fibrotropic MVM (MVMp) variants carrying the I362S or K368R change in the VP2 major capsid protein, in the infection of severe combined immunodeficient (SCID) mice. By 14 to 18 weeks after oronasal inoculation, the I362S and K368R viruses caused lethal leukopenia characterized by tissue damage and inclusion bodies in hemopoietic organs, a pattern of disease found by 7 weeks postinfection with the lymphohematotropic MVM (MVMi) strain. The MVMp populations emerging in leukopenic mice showed consensus sequence changes in the MVMi genotype at residues G321E and A551V of VP2 in the I362S virus infections or A551V and V575A changes in the K368R virus infections, as well as a high level of genetic heterogeneity within a capsid domain at the twofold depression where these residues lay. Amino acids forming this capsid domain are important MVM tropism determinants, as exemplified by the switch in MVMi host range toward mouse fibroblasts conferred by coordinated changes of some of these residues and by the essential character of glutamate at residue 321 for maintaining MVMi tropism toward primary hemopoietic precursors. The few viruses within the spectrum of mutants from mice that maintained the respective parental 321G and 575V residues were infectious in a plaque assay, whereas the viruses with the main consensus sequences exhibited low levels of fitness in culture. Consistent with this finding, a recombinant MVMp virus carrying the consensus sequence mutations arising in the K368R virus background in mice failed to initiate infection in cell lines of different tissue origins, even though it caused rapid-course lethal leukopenia in SCID mice. The parental consensus genotype prevailed during leukopenia development, but plaque-forming viruses with the reversion of the 575A residue to valine emerged in affected organs. The disease caused by the DNA virus in mice, therefore, involves the generation of heterogeneous viral populations that may cooperatively interact for the hemopoietic syndrome. The evolutionary changes delineate a sector of the surface of the capsid that determines tropism and that surrounds the sialic acid receptor binding domain.

scholarly journals The NS2 Proteins of Parvovirus Minute Virus of Mice Are Required for Efficient Nuclear Egress of Progeny Virions in Mouse Cells

2002 ◽  
Vol 76 (20) ◽  
pp. 10307-10319 ◽  
Author(s):  
Virginie Eichwald ◽  
Laurent Daeffler ◽  
Michèle Klein ◽  
Jean Rommelaere ◽  
Nathalie Salomé
Keyword(s):  
Nuclear Export ◽  
Critical Role ◽  
Nuclear Export Signal ◽  
Wild Type Virus ◽  
Nuclear Retention ◽  
Minute Virus Of Mice ◽  
Genomic Clones ◽  
Minute Virus ◽  
Mouse Cells ◽  
Nuclear Export Receptor

ABSTRACT The small nonstructural NS2 proteins of parvovirus minute virus of mice (MVMp) were previously shown to interact with the nuclear export receptor Crm1. We report here the analysis of two MVM mutant genomic clones generating NS2 proteins that are unable to interact with Crm1 as a result of amino acid substitutions within their nuclear export signal (NES) sequences. Upon transfection of human and mouse cells, the MVM-NES21 and MVM-NES22 mutant genomic clones were proficient in synthesis of the four virus-encoded proteins. While the MVM-NES22 clone was further able to produce infectious mutant virions, no virus could be recovered from cells transfected with the MVM-NES21 clone. Whereas the defect of MVM-NES21 appeared to be complex, the phenotype of MVM-NES22 could be traced back to a novel distinct NS2 function. Infection of mouse cells with the MVM-NES22 mutant led to stronger nuclear retention not only of the NS2 proteins but also of infectious progeny MVM particles. This nuclear sequestration correlated with a severe delay in the release of mutant virions in the medium and with prolonged survival of the infected cell populations compared with wild-type virus-treated cultures. This defect could explain, at least in part, the small size of the plaques generated by the MVM-NES22 mutant when assayed on mouse indicator cells. Altogether, our data indicate that the interaction of MVMp NS2 proteins with the nuclear export receptor Crm1 plays a critical role at a late stage of the parvovirus life cycle involved in release of progeny viruses.

A precise map of splice junctions in the mRNAs of minute virus of mice, an autonomous parvovirus.

1986 ◽  
Vol 59 (3) ◽  
pp. 564-573 ◽  
Author(s):  
C V Jongeneel ◽  
R Sahli ◽  
G K McMaster ◽  
B Hirt
Keyword(s):  
Minute Virus Of Mice ◽  
Minute Virus ◽  
Splice Junctions
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