scholarly journals Identification of TAZ as a Binding Partner of the Polyomavirus T Antigens

2004 ◽  
Vol 78 (22) ◽  
pp. 12657-12664 ◽  
Author(s):  
Yu Tian ◽  
Dawei Li ◽  
Jean Dahl ◽  
John You ◽  
Thomas Benjamin
Keyword(s):  
Dna Replication ◽  
Host Range ◽  
T Antigen ◽  
Binding Motif ◽  
Wild Type ◽  
T Antigens ◽  
Ww Domain ◽  
Binding Partner ◽  
N Terminus ◽  
The Common

ABSTRACT A polyomavirus mutant isolated by the tumor host range selection procedure (19) has a three-amino-acid deletion (Δ2-4) in the common N terminus of the T antigens. To search for a cellular protein bound by wild-type but not the mutant T antigen(s), a yeast two-hybrid screen of a mouse embryo cDNA library was carried out with a bait of wild-type small T antigen (sT) fused N terminally to the DNA-binding domain of Gal4. TAZ, a transcriptional coactivator with a WW domain and PDZ-binding motif (17), was identified as a binding partner. TAZ bound in vivo to all three T antigens with different apparent affinities estimated as 1:7:100 (large T antigen [lT]:middle T antigen [mT]:sT). The Δ2-4 mutant T antigens showed no detectable binding. The sT and mT of the host range transformation-defective (hr-t) mutant NG59 with an alteration in the common sT/mT region (179 D→NI) and a normal N terminus also failed to bind TAZ, while the unaltered lT bound but with reduced affinity compared to that seen in a wild-type virus infection. The WW domain but not the PDZ-binding motif of TAZ was essential for T antigen binding. The Δ2-4 mutant was defective in viral DNA replication. Forced overexpression of TAZ blocked wild-type DNA replication in a manner dependent on the binding site for the polyomavirus enhancer-binding protein 2α. Wild-type polyomavirus T antigens effectively block transactivation by TAZ. The functional significance of TAZ interactions with polyomavirus T antigens is discussed.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3694-3704
Author(s):  
C Prives ◽  
Y Murakami ◽  
F G Kern ◽  
W Folk ◽  
C Basilico ◽  
...  
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Early Gene ◽  
Wild Type ◽  
The Core ◽  
Cell Extracts ◽  
Sequence Requirements

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

scholarly journals Kilham Polyomavirus: Activation of Gene Expression and DNA Replication in Mouse Fibroblast Cells by an Enhancer Substitution

2001 ◽  
Vol 75 (21) ◽  
pp. 10015-10023 ◽  
Author(s):  
Shouting Zhang ◽  
Göran Magnusson
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
T Antigen ◽  
Mouse Fibroblast ◽  
Fibroblast Cells ◽  
Large T Antigen ◽  
Wild Type ◽  
3T3 Cells ◽  
Late Promoter ◽  
Substitution Mutant

ABSTRACT The Kilham strain of polyomavirus (KV) infects vascular endothelial cells in vivo (J. E. Greenlee, Infect. Immun. 26:705–713, 1979), but no permissive cell type for growth of the virus in vitro has been identified. The failure of KV DNA to replicate in mouse fibroblast cells after transfection suggested that viral gene expression had narrow cell specificity. A KV substitution mutant having a part of the regulatory region of KV DNA replaced with a segment of the polyomavirus transcriptional enhancer was constructed. The substitution mutant was able to replicate in transfected 3T3 cells, and the newly replicated viral DNA associated with protein to form particles with the density of virions in CsCl equilibrium gradients. However, these particles were noninfectious when tested on 3T3 cells, suggesting that absorption or uptake of virus particles was defective for these cells. Analysis of early and late promoter activities by luciferase reporter gene expression showed that the enhancer substitution had a moderate positive effect on early gene expression and a large effect on the expression of the late genes. KV large T antigen inhibited the activities of both the wild-type and the substitution mutant early promoter, whereas only the mutant late promoter was activated under the same conditions. A comparison of the KV and polyomavirus large T antigens showed that they were not interchangeable in the initiation of KV and polyomavirus DNA synthesis. Furthermore, the wild-type KV origin of DNA replication was less active than the mutant structure in the presence of saturating amounts of KV large T antigen. Together, our data demonstrate several differences between the two types of large T antigen in their interactions with cellular proteins.

scholarly journals A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling

2016 ◽  
Vol 91 (2) ◽  
Author(s):  
Deborah Denis ◽  
Cecile Rouleau ◽  
Brian S. Schaffhausen
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
T Antigen ◽  
Pi3 Kinase ◽  
Binding Motif ◽  
Wild Type ◽  
Pi3k Activity ◽  
Murine Polyomavirus ◽  
Middle T Antigen

ABSTRACT Middle T antigen (MT), the principal oncoprotein of murine polyomavirus, transforms by association with cellular proteins. Protein phosphatase 2A (PP2A), YAP, Src family tyrosine kinases, Shc, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLCγ1) have all been implicated in MT transformation. Mutant dl1015, with deletion of residues 338 to 347 in the C-terminal region, has been an enigma, because the basis for its transformation defect has not been apparent. This work probes the dl1015 region of MT. Because the region is proline rich, the hypothesis that it targets Src homology domain 3 (SH3) domains was tested, but mutation of the putative SH3 binding motif did not affect transformation. During this work, two point mutants, W348R and E349K, were identified as transformation defective. Extensive analysis of the E349K mutant is described here. Similar to wild-type MT, the E349K mutant associates with PP2A, YAP, tyrosine kinases, Shc, PI3 kinase, and PLCγ1. The E349K mutant was examined to determine the mechanism for its transformation defect. Assays of cell localization and membrane targeting showed no obvious difference in localization. Src association was normal as assayed by in vitro kinase and MT phosphopeptide mapping. Shc activation was confirmed by its tyrosine phosphorylation. Association of type 1 PI3K with MT was demonstrated by coimmunoprecipitation, showing both PI3K subunits and in vitro activity. Nonetheless, expression of the mutants failed to lead to the activation of two known downstream targets of PI3K, Akt and Rac-1. Strikingly, despite normal association of the E349K mutant with PI3K, cells expressing the mutant failed to elevate phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in mutant-expressing cells. These results indicate a novel unsuspected aspect to PI3K control. IMPORTANCE The gene coding for middle T antigen (MT) is the murine polyomavirus oncogene most responsible for tumor formation. Its study has a history of uncovering novel aspects of mammalian cell regulation. The importance of PI3K activity and tyrosine phosphorylation are two examples of insights coming from MT. This study describes new mutants unable to transform like the wild type that point to novel regulation of PI3K signaling. Previous mutants were defective in PI3K because they failed to bind the enzyme and bring the activity to the membrane. These mutants recruit PI3K activity like the wild type, but fail to elevate the cellular level of PIP3, the product used to signal downstream of PI3K. As a result, they fail to activate either Akt or Rac1, explaining the transformation defect.

Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A

1992 ◽  
Vol 12 (11) ◽  
pp. 4883-4895
Author(s):  
D M Virshup ◽  
A A Russo ◽  
T J Kelly
Keyword(s):  
Dna Replication ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Insertion Mutation ◽  
Wild Type ◽  
Phosphatase 2A ◽  
Half Site

The catalytic subunit of protein phosphatase 2A (PP2Ac) stimulates the initiation of replication of simian virus 40 DNA in vitro by dephosphorylating T antigen at specific phosphoserine residues (K. H. Scheidtmann, D. M. Virshup, and T. J. Kelly, J. Virol. 65:2098-2101, 1991). To better define the biochemical mechanism responsible for this stimulation, we investigated the effect of PP2Ac on the interaction of T antigen with wild-type and mutant origins of replication. Analysis of the binding of T antigen to the wild-type origin as a function of protein concentration revealed that binding occurs in two relatively discrete steps: the assembly of a T-antigen hexamer on one half-site of the origin, followed by the assembly of the second hexamer on the other half-site. The major effect of PP2Ac was to stimulate binding of the second hexamer, so that the binding reaction became much more cooperative. This observation suggests that dephosphorylation of T antigen by PP2Ac primarily affects interactions between the two hexamers bound to the origin. Pretreatment with PP2Ac increased the ability of the bound T antigen to unwind the origin of replication but had no effect on the intrinsic helicase activity of the protein. Thus, dephosphorylation of PP2Ac appears to increase the efficiency of the initial opening of the origin by T antigen. An insertion mutation at the dyad axis in the simian virus 40 origin, which altered the structural relationship of the two halves of the origin, abolished the effect of the phosphatase on the cooperativity of binding and completely prevented origin unwinding. These findings suggest that the ability of T antigen to open the viral origin of DNA replication is critically dependent on the appropriate functional interactions between T-antigen hexamers and that these interactions are regulated by the phosphorylation state of the viral initiator protein.

scholarly journals Immortalization of rat embryo fibroblasts by mutant polyomavirus large T antigens deficient in DNA binding.

1986 ◽  
Vol 6 (12) ◽  
pp. 4344-4352 ◽  
Author(s):  
A Cowie ◽  
J de Villiers ◽  
R Kamen
Keyword(s):  
Dna Binding ◽  
T Antigen ◽  
Wild Type ◽  
T Antigens ◽  
Rat Embryo ◽  
Viral Origin ◽  
Selection For ◽  
Embryo Fibroblasts ◽  
Neomycin Resistance

We have identified a putative DNA-binding domain in polyomavirus large T antigen. Mutations introduced into the gene between amino acids 290 and 310 resulted in proteins that no longer bound to the high-affinity binding sites on the polyomavirus genome, showed no detectable nonspecific DNA binding, and were not able to initiate DNA replication from the viral origin. These mutant T antigen genes were introduced into rat embryo fibroblasts together with the neomycin resistance gene to allow selection for growth in the presence of G418. All the mutations tested facilitated the establishment of these cells in long-term culture at an efficiency indistinguishable from that of the wild-type protein.

scholarly journals Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans

2007 ◽  
Vol 104 (18) ◽  
pp. 7546-7551 ◽  
Author(s):  
Shaoman Yin ◽  
Nancy Pham ◽  
Shuiliang Yu ◽  
Chaoyang Li ◽  
Poki Wong ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Prion Proteins ◽  
Prion Diseases ◽  
Point Mutations ◽  
Binding Activity ◽  
Binding Motif ◽  
Wild Type ◽  
Pathogenic Mutations ◽  
N Terminus ◽  
Ligand Binding Activity

Mutation in the prion gene PRNP accounts for 10–15% of human prion diseases. However, little is known about the mechanisms by which mutant prion proteins (PrPs) cause disease. Here we investigated the effects of 10 different pathogenic mutations on the conformation and ligand-binding activity of recombinant human PrP (rPrP). We found that mutant rPrPs react more strongly with N terminus-specific antibodies, indicative of a more exposed N terminus. The N terminus of PrP contains a glycosaminoglycan (GAG)-binding motif. Binding of GAG is important in prion disease. Accordingly, all mutant rPrPs bind more GAG, and GAG promotes the aggregation of mutant rPrPs more efficiently than wild-type recombinant normal cellular PrP (rPrPC). Furthermore, point mutations in PRNP also cause conformational changes in the region between residues 109 and 136, resulting in the exposure of a second, normally buried, GAG-binding motif. Importantly, brain-derived PrP from transgenic mice, which express a pathogenic mutant with nine extra octapeptide repeats, also binds more strongly to GAG than wild-type PrPC. Thus, several rPrPs with distinct pathogenic mutations have common conformational changes, which enhance binding to GAG. These changes may contribute to the pathogenesis of inherited prion diseases.

scholarly journals The Ubiquitin-Specific Protease Usp7, a Novel Merkel Cell Polyomavirus Large T-Antigen Interaction Partner, Modulates Viral DNA Replication

2019 ◽  
Vol 94 (5) ◽  
Author(s):  
Manja Czech-Sioli ◽  
Svenja Siebels ◽  
Sonja Radau ◽  
René P. Zahedi ◽  
Claudia Schmidt ◽  
...  
Keyword(s):  
Dna Replication ◽  
Viral Replication ◽  
T Antigen ◽  
Origin Of Replication ◽  
Merkel Cell ◽  
Large T Antigen ◽  
T Antigens ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Cellular Factors

ABSTRACT Merkel cell polyomavirus (MCPyV) is the major cause for Merkel cell carcinoma (MCC), a rare but highly aggressive skin cancer predominantly found in elderly and immunosuppressed patients. The early viral gene products large T-antigen (LT) and small T-antigen (sT) are important for efficient viral DNA replication, and both contribute to transformation processes. These functions are executed mainly through interactions with host factors. Here, we identify the cellular ubiquitin-specific processing protease 7 (Usp7) as a new interaction partner of the MCPyV LT. Using glutathione S-transferase pulldown experiments, we show that MCPyV LT directly binds to Usp7 and that N- as well as C-terminal regions of LT bind to the TRAF (tumor necrosis factor receptor-associated) domain of Usp7. We demonstrate that endogenous Usp7 coprecipitates with MCPyV T-antigens and relocalizes to viral DNA replication centers in cells actively replicating MCPyV genomes. We show that Usp7 does not alter ubiquitination levels of the T-antigens; however, Usp7 binding increases the binding affinity of LT to the origin of replication, thereby negatively regulating viral DNA replication. Together, these data identify Usp7 as a restriction factor of MCPyV replication. In contrast to other DNA viruses, Usp7 does not affect MCPyV gene expression via its ubiquitination activity but influences MCPyV DNA replication solely via a novel mechanism that modulates binding of LT to viral DNA. IMPORTANCE MCPyV is the only human polyomavirus that is associated with cancer; the majority of Merkel cell cancers have a viral etiology. While much emphasis was placed on investigations to understand the transformation process by MCPyV oncoproteins and cellular factors, we have only limited knowledge of cellular factors participating in the MCPyV life cycle. Here, we describe Usp7, a cellular deubiquitination enzyme, as a new factor involved in MCPyV replication. Usp7 is known in the context of large DNA tumor viruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma herpesvirus, to restrict viral replication. Similar to EBV, where Usp7 binding to EBNA1 increases EBNA1 binding affinity to viral DNA, we find MCPyV LT binding to the origin of replication to be increased in the presence of Usp7, resulting in restriction of viral DNA replication. However, Usp7-induced restriction of MCPyV replication is independent of its enzymatic activity, thereby constituting a novel mechanism of Usp7-induced restriction of viral replication.

scholarly journals Two Regions of Simian Virus 40 T Antigen Determine Cooperativity of Double-Hexamer Assembly on the Viral Origin of DNA Replication and Promote Hexamer Interactions during Bidirectional Origin DNA Unwinding

1999 ◽  
Vol 73 (3) ◽  
pp. 2201-2211 ◽  
Author(s):  
Klaus Weisshart ◽  
Poonam Taneja ◽  
Andreas Jenne ◽  
Utz Herbig ◽  
Daniel T. Simmons ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Binding Domain ◽  
Dna Unwinding ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Viral Origin

ABSTRACT Phosphorylation of simian virus 40 large tumor (T) antigen on threonine 124 is essential for viral DNA replication. A mutant T antigen (T124A), in which this threonine was replaced by alanine, has helicase activity, assembles double hexamers on viral-origin DNA, and locally distorts the origin DNA structure, but it cannot catalyze origin DNA unwinding. A class of T-antigen mutants with single-amino-acid substitutions in the DNA binding domain (class 4) has remarkably similar properties, although these proteins are phosphorylated on threonine 124, as we show here. By comparing the DNA binding properties of the T124A and class 4 mutant proteins with those of the wild type, we demonstrate that mutant double hexamers bind to viral origin DNA with reduced cooperativity. We report that T124A T-antigen subunits impair the ability of double hexamers containing the wild-type protein to unwind viral origin DNA, suggesting that interactions between hexamers are also required for unwinding. Moreover, the T124A and class 4 mutant T antigens display dominant-negative inhibition of the viral DNA replication activity of the wild-type protein. We propose that interactions between hexamers, mediated through the DNA binding domain and the N-terminal phosphorylated region of T antigen, play a role in double-hexamer assembly and origin DNA unwinding. We speculate that one surface of the DNA binding domain in each subunit of one hexamer may form a docking site that can interact with each subunit in the other hexamer, either directly with the N-terminal phosphorylated region or with another region that is regulated by phosphorylation.

scholarly journals Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A.

1992 ◽  
Vol 12 (11) ◽  
pp. 4883-4895 ◽  
Author(s):  
D M Virshup ◽  
A A Russo ◽  
T J Kelly
Keyword(s):  
Dna Replication ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Insertion Mutation ◽  
Wild Type ◽  
Phosphatase 2A ◽  
Half Site

The catalytic subunit of protein phosphatase 2A (PP2Ac) stimulates the initiation of replication of simian virus 40 DNA in vitro by dephosphorylating T antigen at specific phosphoserine residues (K. H. Scheidtmann, D. M. Virshup, and T. J. Kelly, J. Virol. 65:2098-2101, 1991). To better define the biochemical mechanism responsible for this stimulation, we investigated the effect of PP2Ac on the interaction of T antigen with wild-type and mutant origins of replication. Analysis of the binding of T antigen to the wild-type origin as a function of protein concentration revealed that binding occurs in two relatively discrete steps: the assembly of a T-antigen hexamer on one half-site of the origin, followed by the assembly of the second hexamer on the other half-site. The major effect of PP2Ac was to stimulate binding of the second hexamer, so that the binding reaction became much more cooperative. This observation suggests that dephosphorylation of T antigen by PP2Ac primarily affects interactions between the two hexamers bound to the origin. Pretreatment with PP2Ac increased the ability of the bound T antigen to unwind the origin of replication but had no effect on the intrinsic helicase activity of the protein. Thus, dephosphorylation of PP2Ac appears to increase the efficiency of the initial opening of the origin by T antigen. An insertion mutation at the dyad axis in the simian virus 40 origin, which altered the structural relationship of the two halves of the origin, abolished the effect of the phosphatase on the cooperativity of binding and completely prevented origin unwinding. These findings suggest that the ability of T antigen to open the viral origin of DNA replication is critically dependent on the appropriate functional interactions between T-antigen hexamers and that these interactions are regulated by the phosphorylation state of the viral initiator protein.

scholarly journals The Carboxy Terminus of Simian Virus 40 Large T Antigen Is Required To Disrupt the Yeast Cell Cycle

2002 ◽  
Vol 76 (9) ◽  
pp. 4621-4624 ◽  
Author(s):  
Sheara W. Fewell ◽  
Dena M. Markle ◽  
Jeffrey L. Brodsky
Keyword(s):  
Cell Cycle ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Yeast Cells ◽  
Wild Type ◽  
T Antigens ◽  
Normal Morphology ◽  
Cell Cycle Disruption ◽  
Carboxy Terminal

ABSTRACT Wild-type and J domain mutant simian virus 40 large T antigens alter the cell cycle and bud morphology of Saccharomyces cerevisiae. In contrast, yeast cells expressing mutant T antigen lacking the carboxy-terminal 150 aa exhibit normal morphology, indicating that this region of T antigen is required for cell cycle disruption.

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