scholarly journals Genome Packaging Sense Is Controlled by the Efficiency of the Nick Site in the Right-End Replication Origin of Parvoviruses Minute Virus of Mice and LuIII

2005 ◽  
Vol 79 (4) ◽  
pp. 2287-2300 ◽  
Author(s):  
Susan F. Cotmore ◽  
Peter Tattersall
Keyword(s):  
Replication Origin ◽  
Close Relative ◽  
Transfer Model ◽  
Minute Virus Of Mice ◽  
Positive Sense ◽  
Minute Virus ◽  
Nick Site ◽  
The Right

ABSTRACT The parvovirus minute virus of mice (MVM) packages predominantly negative-sense single strands, while its close relative LuIII encapsidates strands of both polarities with equal efficiency. Using genomic chimeras and mutagenesis, we show that the ability to package positive strands maps not, as originally postulated, to divergent untranslated regions downstream of the capsid gene but to the viral hairpins and predominantly to the nick site of OriR, the right-end replication origin. In MVM, the sequence of this site is 5′-CTAT▾TCA-3′, while in LuIII a two-base insertion (underlined) changes it to 5′-CTAT AT ▾TCA-3′. Matched LuIII genomes differing only at this position (designated LuIII and LuΔ2) packaged 47 and <8% positive-sense strands, respectively. OriR sequences from these viruses were both able to support NS1-mediated nicking in vitro, but initiation efficiency was consistently two- to threefold higher for LuΔ2 derivatives, suggesting that LuIII's ability to package positive strands is determined by a suboptimal right-end origin rather than by strand-specific packaging sequences. These observations support a mathematical “kinetic hairpin transfer” model, previously described by Chen and colleagues (K. C. Chen, J. J. Tyson, M. Lederman, E. R. Stout, and R. C. Bates, J. Mol. Biol. 208:283-296, 1989), that postulates that preferential excision of particular strands is solely responsible for packaging specificity. By analyzing replicative-form (RF) DNA generated in vivo during LuIII and LuΔ2 infections, we extend this model, showing that positive-sense strands do accumulate in LuΔ2 infections as part of duplex RF DNA, but these do not support packaging. However, replication is biphasic, so that accumulation of positive-sense strands is ultimately suppressed, probably because the onset of packaging removes newly displaced single strands from the replicating pool.

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 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 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 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.

scholarly journals Severe Leukopenia and Dysregulated Erythropoiesis in SCID Mice Persistently Infected with the Parvovirus Minute Virus of Mice

1999 ◽  
Vol 73 (3) ◽  
pp. 1774-1784 ◽  
Author(s):  
José C. Segovia ◽  
Jesús M. Gallego ◽  
Juan A. Bueren ◽  
José M. Almendral
Keyword(s):  
Fold Increase ◽  
Scid Mice ◽  
Productive Infection ◽  
Small Subset ◽  
Compensatory Mechanisms ◽  
Minute Virus Of Mice ◽  
Absolute Increase ◽  
Minute Virus

ABSTRACT Parvovirus minute virus of mice strain i (MVMi) infects committed granulocyte-macrophage CFU and erythroid burst-forming unit (CFU-GM and BFU-E, respectively) and pluripotent (CFU-S) mouse hematopoietic progenitors in vitro. To study the effects of MVMi infection on mouse hemopoiesis in the absence of a specific immune response, adult SCID mice were inoculated by the natural intranasal route of infection and monitored for hematopoietic and viral multiplication parameters. Infected animals developed a very severe viral-dose-dependent leukopenia by 30 days postinfection (d.p.i.) that led to death within 100 days, even though the number of circulating platelets and erythrocytes remained unaltered throughout the disease. In the bone marrow of every lethally inoculated mouse, a deep suppression of CFU-GM and BFU-E clonogenic progenitors occurring during the 20- to 35-d.p.i. interval corresponded with the maximal MVMi production, as determined by the accumulation of virus DNA replicative intermediates and the yield of infectious virus. Viral productive infection was limited to a small subset of primitive cells expressing the major replicative viral antigen (NS-1 protein), the numbers of which declined with the disease. However, the infection induced a sharp and lasting unbalance of the marrow hemopoiesis, denoted by a marked depletion of granulomacrophagic cells (GR-1+ and MAC-1+) concomitant with a twofold absolute increase in erythroid cells (TER-119+). A stimulated definitive erythropoiesis in the infected mice was further evidenced by a 12-fold increase per femur of recognizable proerythroblasts, a quantitative apoptosis confined to uninfected TER-119+ cells, as well as by a 4-fold elevation in the number of circulating reticulocytes. Therefore, MVMi targets and suppresses primitive hemopoietic progenitors leading to a very severe leukopenia, but compensatory mechanisms are mounted specifically by the erythroid lineage that maintain an effective erythropoiesis. The results show that infection of SCID mice with the parvovirus MVMi causes a novel dysregulation of murine hemopoiesis in vivo.

In vitro and in vivo susceptibility of mouse megakaryocytic progenitors to strain i of parvovirus minute virus of mice

2001 ◽  
Vol 29 (11) ◽  
pp. 1303-1309 ◽  
Author(s):  
Marı́a L Lamana ◽  
Beatriz Albella ◽  
Juan A Bueren ◽  
Jose C Segovia
Keyword(s):  
Minute Virus Of Mice ◽  
Minute Virus

scholarly journals In VivoExamination of Mouse APOBEC3- and Human APOBEC3A- and APOBEC3G-Mediated Restriction of Parvovirus and Herpesvirus Infection in Mouse Models

2016 ◽  
Vol 90 (17) ◽  
pp. 8005-8012 ◽  
Author(s):  
Yuki Nakaya ◽  
Spyridon Stavrou ◽  
Kristin Blouch ◽  
Peter Tattersall ◽  
Susan R. Ross
Keyword(s):  
Mouse Models ◽  
Restriction Factors ◽  
Herpes Simplex Virus 1 ◽  
Minute Virus Of Mice ◽  
Minute Virus ◽  
Apobec3 Proteins ◽  
Simplex Virus ◽  
Cytidine Deamination

ABSTRACTAPOBEC3 knockout and human APOBEC3A and -3G transgenic mice were tested for their ability to be infected by the herpesviruses herpes simplex virus 1 and murine herpesvirus 68 and the parvovirus minute virus of mice (MVM). Knockout, APOBEC3A and APOBEC3G transgenic, and wild-type mice were equally infected by the herpesviruses, while APOBEC3A but not mouse APOBEC3 conferred resistance to MVM. No viruses showed evidence of cytidine deamination by mouse or human APOBEC3s. These data suggest thatin vitrostudies implicating APOBEC3 proteins in virus resistance may not reflect their rolein vivo.IMPORTANCEIt is well established that APOBEC3 proteins in different species are a critical component of the host antiretroviral defense. Whether these proteins also function to inhibit other viruses is not clear. There have been a number ofin vitrostudies suggesting that different APOBEC3 proteins restrict herpesviruses and parvoviruses, among others, but whether they also workin vivohas not been demonstrated. Our studies looking at the role of mouse and human APOBEC3 proteins in transgenic and knockout mouse models of viral infection suggest that these restriction factors are not broadly antiviral and demonstrate the importance of testing their activityin vivo.

scholarly journals Structural Determinants of Tissue Tropism and In Vivo Pathogenicity for the Parvovirus Minute Virus of Mice

2005 ◽  
Vol 79 (17) ◽  
pp. 10931-10943 ◽  
Author(s):  
Maria Kontou ◽  
Lakshmanan Govindasamy ◽  
Hyun-Joo Nam ◽  
Nathan Bryant ◽  
Antonio L. Llamas-Saiz ◽  
...  
Keyword(s):  
Host Cells ◽  
Structural Determinants ◽  
Threefold Axis ◽  
Minute Virus Of Mice ◽  
Axis Ii ◽  
Host Interactions ◽  
Minute Virus ◽  
Forward Mutation

ABSTRACT Two strains of the parvovirus minute virus of mice (MVM), the immunosuppressive (MVMi) and the prototype (MVMp) strains, display disparate in vitro tropism and in vivo pathogenicity. We report the crystal structures of MVMp virus-like particles (MVMpb) and native wild-type (wt) empty capsids (MVMpe), determined and refined to 3.25 and 3.75 Å resolution, respectively, and their comparison to the structure of MVMi, also refined to 3.5 Å resolution in this study. A comparison of the MVMpb and MVMpe capsids showed their structures to be the same, providing structural verification that some heterologously expressed parvovirus capsids are indistinguishable from wt capsids produced in host cells. The structures of MVMi and MVMp capsids were almost identical, but local surface conformational differences clustered from symmetry-related capsid proteins at three specific domains: (i) the icosahedral fivefold axis, (ii) the “shoulder” of the protrusion at the icosahedral threefold axis, and (iii) the area surrounding the depression at the icosahedral twofold axis. The latter two domains contain important determinants of MVM in vitro tropism (residues 317 and 321) and forward mutation residues (residues 399, 460, 553, and 558) conferring fibrotropism on MVMi. Furthermore, these structural differences between the MVM strains colocalize with tropism and pathogenicity determinants mapped for other autonomous parvovirus capsids, highlighting the importance of common parvovirus capsid regions in the control of virus-host interactions.

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