scholarly journals Members of the AP-1 Family, c-Jun and c-Fos, Functionally Interact with JC Virus Early Regulatory Protein Large T Antigen

2003 ◽  
Vol 77 (9) ◽  
pp. 5241-5252 ◽  
Author(s):  
Joanne Kim ◽  
Stefanie Woolridge ◽  
Renato Biffi ◽  
Elisa Borghi ◽  
Adam Lassak ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Jc Virus ◽  
Regulatory Protein ◽  
T Antigen ◽  
Immediate Early ◽  
Human Polyomavirus ◽  
Viral Gene ◽  
Large T Antigen ◽  
Functional Interaction ◽  
Transcription And Replication

ABSTRACT The activating protein 1 (AP-1) family of regulatory proteins is characterized as immediate-early inducible transcription factors which were shown to be activated by a variety of stress-related stimuli and to be involved in numerous biological processes, including cellular and viral gene expression, cell proliferation, differentiation, and tumorigenesis. We have recently demonstrated the involvement of the AP-1 family members c-Jun and c-Fos in transcriptional regulation of the human polyomavirus, JC virus (JCV), genome. Here, we further examined their role in JCV gene regulation and replication through their physical and functional interaction with JCV early regulatory protein large T antigen (T-Ag). Transfection and replication studies indicated that c-Jun and c-Fos can significantly diminish T-Ag-mediated JCV gene transcription and replication. Affinity chromatography and coimmunoprecipitation assays demonstrated that c-Jun and T-Ag physically interact with each other. Results from band shift assays showed that the binding efficiency of c-Jun to the AP-1 site was reduced in the presence of T-Ag. In addition, we have mapped, through the use of a series of deletion mutants, the regions of these proteins which are important for their interaction. While the c-Jun interaction domain of T-Ag is localized to the middle portion of the protein, the T-Ag interacting domain of c-Jun maps to its basic-DNA binding region. Results of transient-transfection assays with various c-Jun mutants and T-Ag expression constructs further confirm the specificity of the functional interaction between c-Jun and T-Ag. Taken together, these data demonstrate that immediate-early inducible transcription factors c-Jun and c-Fos physically and functionally interact with JCV major early regulatory protein large T-Ag and that this interaction modulates JCV transcription and replication in glial cells.

scholarly journals Physical and Functional Interaction between the Y-Box Binding Protein YB-1 and Human Polyomavirus JC Virus Large T Antigen

1999 ◽  
Vol 73 (12) ◽  
pp. 10146-10157 ◽  
Author(s):  
Mahmut Safak ◽  
Gary L. Gallia ◽  
Sameer A. Ansari ◽  
Kamel Khalili
Keyword(s):  
Rna Binding ◽  
Jc Virus ◽  
Binding Protein ◽  
Regulatory Protein ◽  
T Antigen ◽  
Human Polyomavirus ◽  
Gene Promoters ◽  
Large T Antigen ◽  
Functional Interaction ◽  
Polyomavirus Jc

ABSTRACT Y-box binding protein YB-1 is a member of a family of DNA and RNA binding proteins which have been shown to affect gene expression at both the transcriptional and translational levels. We have previously shown that YB-1 modulates transcription from the promoters of the ubiquitous human polyomavirus JC virus (JCV). Here we investigate the physical and functional interplay between YB-1 and the viral regulatory protein large T antigen (T-antigen), using JCV as a model system. Results of mobility band shift assays demonstrated that the efficiency of binding of YB-1 to a 23-bp single-stranded viral target sequence was significantly increased when T-antigen was included in the binding reaction mixture. Affinity chromatography and coimmunoprecipitation assays demonstrated that YB-1 and T-antigen physically interact with each other. Additionally, results of transcription studies demonstrated that these two proteins interact functionally on the JCV early and late gene promoters. Whereas ectopic expression of YB-1 and T-antigen results in synergistic transactivation of the viral late promoter, YB-1 alleviates T-antigen-mediated transcriptional suppression of the viral early promoter activity. Furthermore, we have localized, through the use of a series of deletion mutants, the sequences of these proteins which are important for their interaction. The T-antigen-interacting region of YB-1 is located in the cold shock domain of YB-1 and its immediate flanking sequences, and the YB-1-interacting domain of T-antigen maps to the carboxy-terminal half of T-antigen. Results of transient transfection assays with various YB-1 mutants and T-antigen expression constructs confirm the specificity of the functional interaction between YB-1 and T-antigen. Taken together, these data demonstrate that the cellular factor YB-1 and the viral regulatory protein T-antigen interact both physically and functionally and that this interaction modulates transcription from the JCV promoters.

scholarly journals JC virus promoter/enhancers contain TATA box-associated Spi-B-binding sites that support early viral gene expression in primary astrocytes

2012 ◽  
Vol 93 (3) ◽  
pp. 651-661 ◽  
Author(s):  
Leslie J. Marshall ◽  
Lisa D. Moore ◽  
Matthew M. Mirsky ◽  
Eugene O. Major
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Jc Virus ◽  
Viral Gene Expression ◽  
Antigen Expression ◽  
T Antigen ◽  
Viral Gene ◽  
Large T Antigen ◽  
Viral Promoter ◽  
The Brain

JC virus (JCV) is the aetiological agent of the demyelinating disease progressive multifocal leukoencephalopathy, an AIDS defining illness and serious complication of mAb therapies. Initial infection probably occurs in childhood. In the working model of dissemination, virus persists in the kidney and lymphoid tissues until immune suppression/modulation causes reactivation and trafficking to the brain where JCV replicates in oligodendrocytes. JCV infection is regulated through binding of host factors such as Spi-B to, and sequence variation in the non-coding control region (NCCR). Although NCCR sequences differ between sites of persistence and pathogenesis, evidence suggests that the virus that initiates infection in the brain disseminates via B-cells derived from latently infected haematopoietic precursors in the bone marrow. Spi-B binds adjacent to TATA boxes in the promoter/enhancer of the PML-associated JCV Mad-1 and Mad-4 viruses but not the non-pathogenic, kidney-associated archetype. The Spi-B-binding site of Mad-1/Mad-4 differs from that of archetype by a single nucleotide, AAAAGGGAAGGGA to AAAAGGGAAGGTA. Point mutation of the Mad-1 Spi-B site reduced early viral protein large T-antigen expression by up to fourfold. Strikingly, the reverse mutation in the archetype NCCR increased large T-antigen expression by 10-fold. Interestingly, Spi-B protein binds the NCCR sequence flanking the viral promoter/enhancer, but these sites are not essential for early viral gene expression. The effect of mutating Spi-B-binding sites within the JCV promoter/enhancer on early viral gene expression strongly suggests a role for Spi-B binding to the viral promoter/enhancer in the activation of early viral gene expression.

scholarly journals Complete Nucleotide Sequence of Polyomavirus SA12

2005 ◽  
Vol 79 (20) ◽  
pp. 13094-13104 ◽  
Author(s):  
Paul Cantalupo ◽  
Adrienne Doering ◽  
Christopher S. Sullivan ◽  
Achintya Pal ◽  
K. W. C. Peden ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Complete Nucleotide Sequence ◽  
Jc Virus ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Bk Virus ◽  
Human Polyomavirus ◽  
Close Relative ◽  
Large T Antigen ◽  
A Genome

ABSTRACT The Polyomaviridae have small icosahedral virions that contain a genome of approximately 5,000 bp of circular double-stranded DNA. Polyomaviruses infect hosts ranging from humans to birds, and some members of this family induce tumors in test animals or in their natural hosts. We report the complete nucleotide sequence of simian agent 12 (SA12), whose natural host is thought to be Papio ursinus, the chacma baboon. The 5,230-bp genome has a genetic organization typical of polyomaviruses. Sequences encoding large T antigen, small t antigen, agnoprotein, and the viral capsid proteins VP1, VP2, and VP3 are present in the expected locations. We show that, like its close relative simian virus 40 (SV40), SA12 expresses microRNAs that are encoded by the late DNA strand overlapping the 3′ end of large T antigen coding sequences. Based on sequence comparisons, SA12 is most closely related to BK virus (BKV), a human polyomavirus. We have developed a real-time PCR test that distinguishes SA12 from BKV and the other closely related polyomaviruses JC virus and SV40. The close relationship between SA12 and BKV raises the possibility that these viruses circulate between human and baboon hosts.

scholarly journals Interaction of JC Virus Agno Protein with T Antigen Modulates Transcription and Replication of the Viral Genome in Glial Cells

2001 ◽  
Vol 75 (3) ◽  
pp. 1476-1486 ◽  
Author(s):  
Mahmut Safak ◽  
Robert Barrucco ◽  
Armine Darbinyan ◽  
Yuki Okada ◽  
Kazuo Nagashima ◽  
...  
Keyword(s):  
Dna Replication ◽  
Glial Cells ◽  
Viral Genome ◽  
Jc Virus ◽  
T Antigen ◽  
Lytic Cycle ◽  
Viral Gene ◽  
Deletion Mutants ◽  
Amino Terminal ◽  
Transcription And Replication

ABSTRACT In addition to encoding the structural and regulatory proteins, many viruses encode auxiliary proteins, some of which have been shown to play important roles in lytic and latent states of the viruses. The human neurotropic JC virus (JCV) genome encodes an auxiliary protein called Agno whose function remains unknown. Here, we investigated the functional role of JCV Agno protein on transcription and replication of the viral genome in glial cells. Results from transfection of human glial cells showed that Agno protein suppresses both T-antigen-mediated transcription of the viral late gene promoter and T-antigen-induced replication of viral DNA. Affinity chromatography and coimmunoprecipitation assays demonstrated that the Agno protein and T antigen physically interact with each other. Through the use of a series of deletion mutants, we demonstrated that the T-antigen-interacting region of Agno protein is localized to its amino-terminal half and the Agno-interacting domain of T antigen maps to its central portion. Furthermore, utilizing various Agno deletion mutants in functional studies, we confirmed the importance of the Agno-T antigen interaction in the observed down-modulation of T antigen function upon viral gene transcription and DNA replication by Agno protein. Taken together these data suggest that the Agno protein of JCV, which is produced late during the late phase of the lytic cycle, can physically and functionally interact with the viral early protein, T antigen, and downregulate viral gene expression and DNA replication. The importance of these observations in the lytic cycle of JCV is discussed.

scholarly journals Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Elvis T. Ajuh ◽  
Zongsong Wu ◽  
Emma Kraus ◽  
Fabian H. Weissbach ◽  
Tobias Bethge ◽  
...  
Keyword(s):  
Gene Expression ◽  
Host Cell ◽  
Binding Sites ◽  
T Antigen ◽  
Hek293 Cells ◽  
Human Polyomavirus ◽  
Gene Region ◽  
Viral Gene ◽  
Cell Tropism ◽  
Large T Antigen

ABSTRACTHuman polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman'sr, 0.625;P< 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTag-dependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific.IMPORTANCEHPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets.

scholarly journals Growth of immortal simian virus 40 tsA-transformed human fibroblasts is temperature dependent.

1989 ◽  
Vol 9 (7) ◽  
pp. 3093-3096 ◽  
Author(s):  
R L Radna ◽  
Y Caton ◽  
K K Jha ◽  
P Kaplan ◽  
G Li ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Cellular Aging ◽  
Cellular Growth ◽  
Viral Gene ◽  
Large T Antigen ◽  
Temperature Dependent

Simian virus 40 (SV40)-mediated transformation of human fibroblasts offers an experimental system for studying both carcinogenesis and cellular aging, since such transformants show the typical features of altered cellular growth but still have a limited life span in culture and undergo senescence. We have previously demonstrated (D. S. Neufeld, S. Ripley, A. Henderson, and H. L. Ozer, Mol. Cell. Biol. 7:2794-2802, 1987) that transformants generated with origin-defective mutants of SV40 show an increased frequency of overcoming senescence and becoming immortal. To clarify further the role of large T antigen, we have generated immortalized transformants by using origin-defective mutants of SV40 encoding a heat-labile large T antigen (tsA58 transformants). At a temperature permissive for large-T-antigen function (35 degrees C), the cell line AR5 had properties resembling those of cell lines transformed with wild-type SV40. However, the AR5 cells were unable to proliferate or form colonies at temperatures restrictive for large-T-antigen function (39 degrees C), demonstrating a continuous need for large T antigen even in immortalized human fibroblasts. Such immortal temperature-dependent transformants should be useful cell lines for the identification of other cellular or viral gene products that induce cell proliferation in human cells.

Polyomavirus-plasmid recombinants capable of replicating have an enhanced transforming potential

1983 ◽  
Vol 3 (9) ◽  
pp. 1670-1674
Author(s):  
W J Muller ◽  
M A Naujokas ◽  
J A Hassell
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
T Antigen ◽  
Viral Gene ◽  
Large T Antigen ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Transfected Cells ◽  
Cis Acting ◽  
Viral Gene Product

The frequency of transformation of rodent fibroblasts by polyomavirus is enhanced by a viral gene product, large T-antigen. However, this effect of large T-antigen cannot be demonstrated with pBR322-cloned viral DNA. Recently, it was discovered that pBR322 contains cis-acting sequences inhibitory to DNA replication in mammalian cells. Because polyomavirus large T-antigen is required for viral DNA replication, we examined the possibility that our inability to demonstrate a requirement for large T-antigen in transformation with pBR322-cloned viral DNA was due to the failure of the chimeric DNA to replicate in the transfected cells. To this end we constructed polyomavirus recombinant molecules with a plasmid (pML-2) that lacks these "poison" sequences and measured their capacity to transform cells. Here we report that recombinant plasmids capable of replicating in the transfected cells transform these cells at frequencies approximately sixfold greater than their replication-defective counterparts.

scholarly journals Biochemical Characterization and Localization of JC Virus Large T Antigen Phosphorylation Domains

Virology ◽  
1995 ◽  
Vol 212 (2) ◽  
pp. 295-308 ◽  
Author(s):  
Jennifer J. Swenson ◽  
Richard J. Frisque
Keyword(s):  
Biochemical Characterization ◽  
Jc Virus ◽  
T Antigen ◽  
Large T Antigen

scholarly journals High-Throughput Cell-Based Screen for Chemicals That Inhibit Infection by Simian Virus 40 and Human Polyomaviruses

2009 ◽  
Vol 83 (11) ◽  
pp. 5630-5639 ◽  
Author(s):  
Edward C. Goodwin ◽  
Walter J. Atwood ◽  
Daniel DiMaio
Keyword(s):  
High Throughput ◽  
Ellagic Acid ◽  
Viral Vector ◽  
Jc Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Early Gene ◽  
Large T Antigen ◽  
Human Polyomaviruses

ABSTRACT We developed a high-throughput, cell-based screen to identify chemicals that inhibit infection by the primate polyomaviruses. The screen is based on the detection of compounds that inhibit the ability of a replication-defective simian virus 40 (SV40)-based viral vector to cause growth arrest in HeLa cells by repressing the expression of the endogenous human papillomavirus E7 oncogene in these cells. We identified two compounds, ellagic acid and spiperone, that suppressed the ability of the SV40 recombinant virus to inhibit cellular DNA synthesis. These compounds caused a marked reduction of the ability of wild-type SV40 to productively infect permissive monkey cells, even when the compounds were added several hours after infection. The fraction of cells expressing SV40 large T antigen and the levels of T antigen mRNA were reduced in infected human and monkey cells treated with ellagic acid and spiperone, suggesting that these compounds block a step in the virus life cycle prior to SV40 early gene expression. Ellagic acid and spiperone also inhibited large T antigen expression by BK virus and JC virus, two important, pathogenic human polyomaviruses.

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