M. Safak - Academia.edu (original) (raw)
Papers by M. Safak
Journal of Virological Methods, 2009
JC virus (JCV) is a human neurotropic polyomavirus whose replication in the central nervous syste... more JC virus (JCV) is a human neurotropic polyomavirus whose replication in the central nervous system induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV particles have been detected primarily in oligodendrocytes and astrocytes of the brains of patients with PML and in the laboratory its propagation is limited to primary cultures of human fetal glial cells. In this short communication, the development of a new cell culture system is described through the fusion of primary human fetal astrocytes with the human glioblastoma cell line, U-87MG. The new hybrid cell line obtained from this fusion has the capacity to support efficiently expression of JCV and replication of viral DNA in vitro up to 16 passages. This cell line can serve as a reliable culture system to study the biology of JCV host cell interaction, determine the mechanisms involved in cell type specific replication of JCV, and provide a convenient cell culture system for high throughput screening of anti-viral agents.
Journal of virology, 1996
The transcription control region of the archetype strain of the human polyomavirus JC virus (JCV(... more The transcription control region of the archetype strain of the human polyomavirus JC virus (JCV(Cy)), unlike its neurotropic counterpart (JCV(Mad-1)), contains only one copy of the 98-bp enhancer/promoter repeat with the 23-bp and the 66-bp insertion blocks. Early studies by us and others have indicated that the structural organization of JCV(Mad-1) is critical for glial cell-specific transcription of the viral genome. In addition, the kappa B regulatory motif found in the JCV(Mad-1) genome, which also exists in JCV(Cy), confers inducibility to the JCV(Mad-1) early and late promoters in response to extracellular stimuli. In this study, we have investigated the regulatory role of the 23- and the 66-bp blocks and their functional relationship to the kappa B motif in stimulating transcription of the Cy early and late promoters in glial cells. We demonstrate that mutations in the kappa B motif reduce the basal activity of the Cy early promoter and decrease the levels of its induction b...
Molecular and Cellular Biology
Cross communication between regulatory proteins is an important event in the control of eukaryoti... more Cross communication between regulatory proteins is an important event in the control of eukaryotic gene transcription. Here we have examined the structural and functional interaction between two cellular regulatory proteins, YB-1 and Purα, on the 23-bp sequence element derived from the enhancer-promoter of the human polyomavirus JCV. YB-1 and Purα are single-stranded DNA binding proteins which recognize C/T- and GC/GA-rich sequences, respectively. Results from band shift studies demonstrated that while both proteins interact directly with their DNA target sequences within the 23-bp motif, each protein can regulate the association of the other one with the DNA. Affinity chromatography and coimmunoprecipitation provide evidence for a direct interaction between Purα and YB-1 in the absence of the DNA sequence. Ectopic expression of YB-1 and Purα in glial cells synergistically stimulated viral promoter activity via the 23-bp sequence element. Results from mutational studies revealed tha...
Journal of cellular biochemistry, 2017
Agnoprotein is an important regulatory protein of the human polyoma JC virus (JCV) and plays crit... more Agnoprotein is an important regulatory protein of the human polyoma JC virus (JCV) and plays critical roles during the viral replication cycle. It forms highly stable dimers and oligomers through its Leu/Ile/Phe-rich domain, which is important for the stability and function of the protein. We recently resolved the partial 3D structure of this protein by NMR using a synthetic peptide encompassing amino acids Thr17 to Gln52, where the Leu/Ile/Phe- rich region was found to adopt a major alpha-helix conformation spanning amino acids 23-39. Here, we report the resolution of the 3D structure of full-length JCV agnoprotein by NMR, which not only confirmed the existence of the previously reported major α-helix domain at the same position but also revealed the presence of an additional minor α-helix region spanning amino acid residues Leu6 to lys13. The remaining regions of the protein adopt an intrinsically unstructured conformation. J. Cell. Biochem. 118: 3268-3280, 2017. © 2017 Wiley Peri...
Journal of Cellular Physiology, 2016
Agnoprotein is an important regulatory protein of polyomaviruses, including JCV, BKV, and SV40. I... more Agnoprotein is an important regulatory protein of polyomaviruses, including JCV, BKV, and SV40. In the absence of its expression, these viruses are unable to sustain their productive life cycle. It is a highly basic phosphoprotein that localizes mostly to the perinuclear area of infected cells, although a small amount of the protein is also found in nucleus. Much has been learned about the structure and function of this important regulatory protein in recent years. It forms highly stable dimers/oligomers in vitro and in vivo through its Leu/Ile/Phe-rich domain. Structural NMR studies revealed that this domain adopts an alpha-helix conformation and plays a critical role in the stability of the protein. It associates with cellular proteins, including YB-1, p53, Ku70, FEZ1, HP1α, PP2A, AP-3, PCNA, and α-SNAP; and viral proteins, including small t antigen, large T antigen, HIV-1 Tat, and JCV VP1; and significantly contributes the viral transcription and replication. This review summarizes the recent advances in the structural and functional properties of this important regulatory protein.
Journal of General Virology, 1999
Transcriptional regulation of the human immunodeficiency virus type 1 (HIV-1) genome is mediated ... more Transcriptional regulation of the human immunodeficiency virus type 1 (HIV-1) genome is mediated by viral and cellular factors. TAR, an unusual RNA regulatory element with a stem–bulge–loop structure at the 5′ ends of all nascent viral transcripts is critical for HIV-1 transcription. TAR is the target for Tat, a viral transcription factor encoded early in the HIV-1 life-cycle and essential for gene expression. Evidence demonstrating the interaction of a cellular ssDNA/RNA binding protein, YB-1, with TAR through a region which is important for Tat interaction is presented. Interestingly, results from protein–protein interaction studies revealed that YB-1 can also form a complex with Tat. Results from mapping experiments suggest that while the region spanning aa 125–203 within YB-1 is essential for its association with TAR, a truncated YB-1 spanning aa 1–125 can weakly bind to Tat. Functionally, overexpression of full-length YB-1 enhanced Tat-induced activation of the HIV-1 minimal pr...
Encyclopedia of Virology, 2008
Both JC virus (JCV) and BK virus (BKV) were first isolated in 1971 from the brain tissue of a pat... more Both JC virus (JCV) and BK virus (BKV) were first isolated in 1971 from the brain tissue of a patient suffering from progressive multifocal encephalopathy (PML) and from the urine of a kidney allograft recipient with chronic pyelonephritis respectively. Both viruses are members of the polyomavirus family and are non-enveloped DNA viruses with icosahedral capsids containing a small, circular, double-stranded DNA genome. JCV is the etiologic agent of PML, whereas BKV is involved in polyomavirus-associated nephropathy (PVAN) in patients receiving renal transplants. The genomic organization of both viruses is very similar to that of simian vacuolating virus 40 (SV40) in that regulatory and coding regions make up of the genome of each virus. The regulatory region of each virus consists of the origin of DNA replication and promoter/enhancer elements and is responsible for the regulation of viral early and late gene expression. Both viral genomes are oncogenic in experimental animals and can transform cells in tissue culture system. In addition, both genomes have been detected in a variety of human tumors. Despite the extensive structural and genomic similarities, JCV and BKV display a distinct biology with respect to the infection of the specific tissues in human host. There has been a rapid expansion in the number of the additional human polyomaviruses in recent years, due to new discoveries, bringing the present number to 13. Those include merkel cell carcinoma-associated polyomavirus (MCPyV) and Trichodysplasia Spinulosa-associated polyomavirus (TSPyV) Karolinska Institute polyomavirus (KIPyV), Washington University polyomavirus (WUPyV), human polyomavirus-6 (HPyV6), human polyomavirus-7 (HPyV7), human polyomavirus-9 (HPyV9), human polyomavirus-10 (HPyV10) Malawi human polyomavirus (MWPyV), Mexico X human polyomavirus (MXPyV) and human polyomavirus-12 (HPyV12). Among those recently discovered human polyomaviruses, only MCPyV and TSPyV appear to be associated a human disease; the rest has yet to be definitively linked to with a particular human disease. In this review, a particular attention was devoted to the description of the biology of JCV, BKV, MCPyV and TSPyV.
Journal of Virology, 2014
Reference Module in Biomedical Sciences, 2014
Virology, 2003
Expression of several cytokines involved in signal transduction such as TGF-1 and the inflammato... more Expression of several cytokines involved in signal transduction such as TGF-1 and the inflammatory chemokines including MCP-1 is elevated during the course of AIDS progression. The enhancement of these cellular proteins in astrocytic cells is mediated, at least in part, by HIV-1 Tat protein. Here, we investigate the possible regulation of MCP-1 transcription by Tat and the Smad family of transcription factors whose activities are induced by the TGF-1 pathway. Results from transfection studies revealed that Smad-3 stimulates basal and Tat-mediated transcription of MCP-1 in human astrocytic cells. Smad-4, on the other hand, had no effect on the basal activity of the MCP-1 promoter, but showed the ability to decrease both Smad-3 and Tat-induced transcription of the MCP promoter. Results from protein-binding studies revealed the ability of both Smad-3 and Smad-4 to associate with the region of Tat spanning residues 1-40. Examination of the transcriptional activity of the various domains of Smad including MH1, at the N-terminus, and MH2, at the C-terminus of the protein indicated that neither MH1 or MH2 alone positively cooperate with Tat in modulating MCP-1 transcription. However, ectopic expression of MH1 and, more notably, MH2 severely suppressed transcriptional activation of MCP-1 by Tat in astrocytic cells. Binding studies revealed that similar to the full-length Smad protein, both MH1 and MH2 associate with Tat protein and that the residues between 1 and 40 of Tat are important for their interaction. These observations reveal a novel mechanism for Tat-mediated transcriptional activation via TGF signaling pathway and provide evidence for regulation of MCP-1 gene transcription by this signaling pathway in human astrocytic cells.
Journal of Virology, 2003
The activating protein 1 (AP-1) family of regulatory proteins is characterized as immediate-early... more 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 fr...
Journal of Virology, 2009
Polyomaviruses are a growing family of small DNA viruses with a narrow tropism for both the host ... more Polyomaviruses are a growing family of small DNA viruses with a narrow tropism for both the host species and the cell type in which they productively replicate. Species host range may be constrained by requirements for precise molecular interactions between the viral T antigen, host replication proteins, including DNA polymerase, and the viral origin of replication, which are required for viral DNA replication. Cell type specificity involves, at least in part, transcription factors that are necessary for viral gene expression and restricted in their tissue distribution. In the case of the human polyomaviruses, BK virus (BKV) replication occurs in the tubular epithelial cells of the kidney, causing nephropathy in kidney allograft recipients, while JC virus (JCV) replication occurs in the glial cells of the central nervous system, where it causes progressive multifocal leukoencephalopathy. Three new human polyomaviruses have recently been discovered: MCV was found in Merkel cell carci...
Journal of Virology, 2003
The activating transcription factor 1 (AP-1) family of proteins consists of a large number of ind... more The activating transcription factor 1 (AP-1) family of proteins consists of a large number of inducible factors that are implicated in many biological processes, including cellular and viral gene expression, cell proliferation, differentiation, and tumorigenesis. Here, we investigated the role of the AP-1 family members c-Jun and c-Fos in transcriptional regulation of the JC virus (JCV) promoter in glial cells. DNA binding studies demonstrated the specific association of c-Jun with its DNA sequences corresponding to the AP-1 site within the JCV promoter. Functional analysis of the promoter showed that ectopic expression of c-Jun and c-Fos results in an additive activation of the JCV early and late promoters. Further functional assays indicated that the JCV AP-1 binding site is sufficient to confer responsiveness to both c-Jun/c-Fos- and UV-induced activation when transposed to a heterologous promoter. Analysis of c-Jun expression during the viral infection cycle by Western blotting ...
Journal of Virology, 2006
Many eukaryotic and viral regulatory proteins are known to undergo posttranslational modification... more Many eukaryotic and viral regulatory proteins are known to undergo posttranslational modifications including phosphorylation, which plays a critical role in many aspects of cell function. Previous studies from our and other laboratories indicated that the JC virus (JCV) late regulatory protein, agnoprotein, plays an important role in the JCV life cycle. Agnoprotein contains several potential phosphorylation sites, including Ser7, Ser11, and Thr21, which are potential targets for the serine/threonine-specific protein kinase C (PKC). In this study, we investigated the functional significance of these phosphorylation sites for the activity of agnoprotein. In vitro and in vivo kinase assays demonstrated that agnoprotein is a target for phosphorylation by PKC. In addition, each of the PKC phosphorylation sites was mutated to Ala singly and in combination, and the effects of these mutations on the JCV life cycle were analyzed. Although the expression of each mutant agnoprotein was detecta...
Journal of Virology, 2002
Human polyomavirus JC virus (JCV) is a causative agent of progressive multifocal leukoencephalopa... more Human polyomavirus JC virus (JCV) is a causative agent of progressive multifocal leukoencephalopathy which results from lytic infection of glial cells. Although significant progress has been made in understanding the regulation of JCV gene transcription, the mechanism(s) underlying the viral lytic cycle remains largely unknown. We recently reported that the JCV late auxiliary Agnoprotein may have a regulatory role in JCV gene transcription and replication. Here, we investigated its regulatory function in viral gene transcription through its physical and functional interaction with YB-1, a cellular transcription factor which contributes to JCV gene expression in glial cells. Time course studies revealed that Agnoprotein is first detected at day 3 postinfection and that its level increased during the late stage of the infection cycle. Agnoprotein is mainly localized to the cytoplasmic compartment of the infected cell, with high concentrations found in the perinuclear region. While the...
Journal of Neurovirology, 2004
Journal of Neurovirology, 2004
Journal of Biological Chemistry, 1998
Cross communication between regulatory proteins is an important event in the control of eukaryoti... more Cross communication between regulatory proteins is an important event in the control of eukaryotic gene transcription. Here we have examined the structural and functional interaction between two cellular regulatory proteins, YB-1 and Pur␣, on the 23-bp sequence element derived from the enhancer-promoter of the human polyomavirus JCV. YB-1 and Pur␣ are single-stranded DNA binding proteins which recognize C/T-and GC/GA-rich sequences, respectively. Results from band shift studies demonstrated that while both proteins interact directly with their DNA target sequences within the 23-bp motif, each protein can regulate the association of the other one with the DNA. Affinity chromatography and coimmunoprecipitation provide evidence for a direct interaction between Pur␣ and YB-1 in the absence of the DNA sequence. Ectopic expression of YB-1 and Pur␣ in glial cells synergistically stimulated viral promoter activity via the 23-bp sequence element. Results from mutational studies revealed that residues between amino acids 75 and 203 of YB-1 and between amino acids 85 and 215 of Pur␣ are important for the interaction between these two proteins. Functional studies with glial cells indicated that the region within Pur␣ which mediates its association with YB-1 and binding to the 23-bp sequence is important for the observed activation of the JCV promoter by the Pur␣ and YB-1 proteins. The results of this study suggest that the cooperative interaction between YB-1 and Pur␣ mediates the synergistic activation of the human polyomavirus JCV genome by these cellular proteins. The importance of these findings for cellular and viral genes which are regulated by Pur␣ and YB-1 is discussed.
Journal of Virological Methods, 2009
JC virus (JCV) is a human neurotropic polyomavirus whose replication in the central nervous syste... more JC virus (JCV) is a human neurotropic polyomavirus whose replication in the central nervous system induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV particles have been detected primarily in oligodendrocytes and astrocytes of the brains of patients with PML and in the laboratory its propagation is limited to primary cultures of human fetal glial cells. In this short communication, the development of a new cell culture system is described through the fusion of primary human fetal astrocytes with the human glioblastoma cell line, U-87MG. The new hybrid cell line obtained from this fusion has the capacity to support efficiently expression of JCV and replication of viral DNA in vitro up to 16 passages. This cell line can serve as a reliable culture system to study the biology of JCV host cell interaction, determine the mechanisms involved in cell type specific replication of JCV, and provide a convenient cell culture system for high throughput screening of anti-viral agents.
Journal of virology, 1996
The transcription control region of the archetype strain of the human polyomavirus JC virus (JCV(... more The transcription control region of the archetype strain of the human polyomavirus JC virus (JCV(Cy)), unlike its neurotropic counterpart (JCV(Mad-1)), contains only one copy of the 98-bp enhancer/promoter repeat with the 23-bp and the 66-bp insertion blocks. Early studies by us and others have indicated that the structural organization of JCV(Mad-1) is critical for glial cell-specific transcription of the viral genome. In addition, the kappa B regulatory motif found in the JCV(Mad-1) genome, which also exists in JCV(Cy), confers inducibility to the JCV(Mad-1) early and late promoters in response to extracellular stimuli. In this study, we have investigated the regulatory role of the 23- and the 66-bp blocks and their functional relationship to the kappa B motif in stimulating transcription of the Cy early and late promoters in glial cells. We demonstrate that mutations in the kappa B motif reduce the basal activity of the Cy early promoter and decrease the levels of its induction b...
Molecular and Cellular Biology
Cross communication between regulatory proteins is an important event in the control of eukaryoti... more Cross communication between regulatory proteins is an important event in the control of eukaryotic gene transcription. Here we have examined the structural and functional interaction between two cellular regulatory proteins, YB-1 and Purα, on the 23-bp sequence element derived from the enhancer-promoter of the human polyomavirus JCV. YB-1 and Purα are single-stranded DNA binding proteins which recognize C/T- and GC/GA-rich sequences, respectively. Results from band shift studies demonstrated that while both proteins interact directly with their DNA target sequences within the 23-bp motif, each protein can regulate the association of the other one with the DNA. Affinity chromatography and coimmunoprecipitation provide evidence for a direct interaction between Purα and YB-1 in the absence of the DNA sequence. Ectopic expression of YB-1 and Purα in glial cells synergistically stimulated viral promoter activity via the 23-bp sequence element. Results from mutational studies revealed tha...
Journal of cellular biochemistry, 2017
Agnoprotein is an important regulatory protein of the human polyoma JC virus (JCV) and plays crit... more Agnoprotein is an important regulatory protein of the human polyoma JC virus (JCV) and plays critical roles during the viral replication cycle. It forms highly stable dimers and oligomers through its Leu/Ile/Phe-rich domain, which is important for the stability and function of the protein. We recently resolved the partial 3D structure of this protein by NMR using a synthetic peptide encompassing amino acids Thr17 to Gln52, where the Leu/Ile/Phe- rich region was found to adopt a major alpha-helix conformation spanning amino acids 23-39. Here, we report the resolution of the 3D structure of full-length JCV agnoprotein by NMR, which not only confirmed the existence of the previously reported major α-helix domain at the same position but also revealed the presence of an additional minor α-helix region spanning amino acid residues Leu6 to lys13. The remaining regions of the protein adopt an intrinsically unstructured conformation. J. Cell. Biochem. 118: 3268-3280, 2017. © 2017 Wiley Peri...
Journal of Cellular Physiology, 2016
Agnoprotein is an important regulatory protein of polyomaviruses, including JCV, BKV, and SV40. I... more Agnoprotein is an important regulatory protein of polyomaviruses, including JCV, BKV, and SV40. In the absence of its expression, these viruses are unable to sustain their productive life cycle. It is a highly basic phosphoprotein that localizes mostly to the perinuclear area of infected cells, although a small amount of the protein is also found in nucleus. Much has been learned about the structure and function of this important regulatory protein in recent years. It forms highly stable dimers/oligomers in vitro and in vivo through its Leu/Ile/Phe-rich domain. Structural NMR studies revealed that this domain adopts an alpha-helix conformation and plays a critical role in the stability of the protein. It associates with cellular proteins, including YB-1, p53, Ku70, FEZ1, HP1α, PP2A, AP-3, PCNA, and α-SNAP; and viral proteins, including small t antigen, large T antigen, HIV-1 Tat, and JCV VP1; and significantly contributes the viral transcription and replication. This review summarizes the recent advances in the structural and functional properties of this important regulatory protein.
Journal of General Virology, 1999
Transcriptional regulation of the human immunodeficiency virus type 1 (HIV-1) genome is mediated ... more Transcriptional regulation of the human immunodeficiency virus type 1 (HIV-1) genome is mediated by viral and cellular factors. TAR, an unusual RNA regulatory element with a stem–bulge–loop structure at the 5′ ends of all nascent viral transcripts is critical for HIV-1 transcription. TAR is the target for Tat, a viral transcription factor encoded early in the HIV-1 life-cycle and essential for gene expression. Evidence demonstrating the interaction of a cellular ssDNA/RNA binding protein, YB-1, with TAR through a region which is important for Tat interaction is presented. Interestingly, results from protein–protein interaction studies revealed that YB-1 can also form a complex with Tat. Results from mapping experiments suggest that while the region spanning aa 125–203 within YB-1 is essential for its association with TAR, a truncated YB-1 spanning aa 1–125 can weakly bind to Tat. Functionally, overexpression of full-length YB-1 enhanced Tat-induced activation of the HIV-1 minimal pr...
Encyclopedia of Virology, 2008
Both JC virus (JCV) and BK virus (BKV) were first isolated in 1971 from the brain tissue of a pat... more Both JC virus (JCV) and BK virus (BKV) were first isolated in 1971 from the brain tissue of a patient suffering from progressive multifocal encephalopathy (PML) and from the urine of a kidney allograft recipient with chronic pyelonephritis respectively. Both viruses are members of the polyomavirus family and are non-enveloped DNA viruses with icosahedral capsids containing a small, circular, double-stranded DNA genome. JCV is the etiologic agent of PML, whereas BKV is involved in polyomavirus-associated nephropathy (PVAN) in patients receiving renal transplants. The genomic organization of both viruses is very similar to that of simian vacuolating virus 40 (SV40) in that regulatory and coding regions make up of the genome of each virus. The regulatory region of each virus consists of the origin of DNA replication and promoter/enhancer elements and is responsible for the regulation of viral early and late gene expression. Both viral genomes are oncogenic in experimental animals and can transform cells in tissue culture system. In addition, both genomes have been detected in a variety of human tumors. Despite the extensive structural and genomic similarities, JCV and BKV display a distinct biology with respect to the infection of the specific tissues in human host. There has been a rapid expansion in the number of the additional human polyomaviruses in recent years, due to new discoveries, bringing the present number to 13. Those include merkel cell carcinoma-associated polyomavirus (MCPyV) and Trichodysplasia Spinulosa-associated polyomavirus (TSPyV) Karolinska Institute polyomavirus (KIPyV), Washington University polyomavirus (WUPyV), human polyomavirus-6 (HPyV6), human polyomavirus-7 (HPyV7), human polyomavirus-9 (HPyV9), human polyomavirus-10 (HPyV10) Malawi human polyomavirus (MWPyV), Mexico X human polyomavirus (MXPyV) and human polyomavirus-12 (HPyV12). Among those recently discovered human polyomaviruses, only MCPyV and TSPyV appear to be associated a human disease; the rest has yet to be definitively linked to with a particular human disease. In this review, a particular attention was devoted to the description of the biology of JCV, BKV, MCPyV and TSPyV.
Journal of Virology, 2014
Reference Module in Biomedical Sciences, 2014
Virology, 2003
Expression of several cytokines involved in signal transduction such as TGF-1 and the inflammato... more Expression of several cytokines involved in signal transduction such as TGF-1 and the inflammatory chemokines including MCP-1 is elevated during the course of AIDS progression. The enhancement of these cellular proteins in astrocytic cells is mediated, at least in part, by HIV-1 Tat protein. Here, we investigate the possible regulation of MCP-1 transcription by Tat and the Smad family of transcription factors whose activities are induced by the TGF-1 pathway. Results from transfection studies revealed that Smad-3 stimulates basal and Tat-mediated transcription of MCP-1 in human astrocytic cells. Smad-4, on the other hand, had no effect on the basal activity of the MCP-1 promoter, but showed the ability to decrease both Smad-3 and Tat-induced transcription of the MCP promoter. Results from protein-binding studies revealed the ability of both Smad-3 and Smad-4 to associate with the region of Tat spanning residues 1-40. Examination of the transcriptional activity of the various domains of Smad including MH1, at the N-terminus, and MH2, at the C-terminus of the protein indicated that neither MH1 or MH2 alone positively cooperate with Tat in modulating MCP-1 transcription. However, ectopic expression of MH1 and, more notably, MH2 severely suppressed transcriptional activation of MCP-1 by Tat in astrocytic cells. Binding studies revealed that similar to the full-length Smad protein, both MH1 and MH2 associate with Tat protein and that the residues between 1 and 40 of Tat are important for their interaction. These observations reveal a novel mechanism for Tat-mediated transcriptional activation via TGF signaling pathway and provide evidence for regulation of MCP-1 gene transcription by this signaling pathway in human astrocytic cells.
Journal of Virology, 2003
The activating protein 1 (AP-1) family of regulatory proteins is characterized as immediate-early... more 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 fr...
Journal of Virology, 2009
Polyomaviruses are a growing family of small DNA viruses with a narrow tropism for both the host ... more Polyomaviruses are a growing family of small DNA viruses with a narrow tropism for both the host species and the cell type in which they productively replicate. Species host range may be constrained by requirements for precise molecular interactions between the viral T antigen, host replication proteins, including DNA polymerase, and the viral origin of replication, which are required for viral DNA replication. Cell type specificity involves, at least in part, transcription factors that are necessary for viral gene expression and restricted in their tissue distribution. In the case of the human polyomaviruses, BK virus (BKV) replication occurs in the tubular epithelial cells of the kidney, causing nephropathy in kidney allograft recipients, while JC virus (JCV) replication occurs in the glial cells of the central nervous system, where it causes progressive multifocal leukoencephalopathy. Three new human polyomaviruses have recently been discovered: MCV was found in Merkel cell carci...
Journal of Virology, 2003
The activating transcription factor 1 (AP-1) family of proteins consists of a large number of ind... more The activating transcription factor 1 (AP-1) family of proteins consists of a large number of inducible factors that are implicated in many biological processes, including cellular and viral gene expression, cell proliferation, differentiation, and tumorigenesis. Here, we investigated the role of the AP-1 family members c-Jun and c-Fos in transcriptional regulation of the JC virus (JCV) promoter in glial cells. DNA binding studies demonstrated the specific association of c-Jun with its DNA sequences corresponding to the AP-1 site within the JCV promoter. Functional analysis of the promoter showed that ectopic expression of c-Jun and c-Fos results in an additive activation of the JCV early and late promoters. Further functional assays indicated that the JCV AP-1 binding site is sufficient to confer responsiveness to both c-Jun/c-Fos- and UV-induced activation when transposed to a heterologous promoter. Analysis of c-Jun expression during the viral infection cycle by Western blotting ...
Journal of Virology, 2006
Many eukaryotic and viral regulatory proteins are known to undergo posttranslational modification... more Many eukaryotic and viral regulatory proteins are known to undergo posttranslational modifications including phosphorylation, which plays a critical role in many aspects of cell function. Previous studies from our and other laboratories indicated that the JC virus (JCV) late regulatory protein, agnoprotein, plays an important role in the JCV life cycle. Agnoprotein contains several potential phosphorylation sites, including Ser7, Ser11, and Thr21, which are potential targets for the serine/threonine-specific protein kinase C (PKC). In this study, we investigated the functional significance of these phosphorylation sites for the activity of agnoprotein. In vitro and in vivo kinase assays demonstrated that agnoprotein is a target for phosphorylation by PKC. In addition, each of the PKC phosphorylation sites was mutated to Ala singly and in combination, and the effects of these mutations on the JCV life cycle were analyzed. Although the expression of each mutant agnoprotein was detecta...
Journal of Virology, 2002
Human polyomavirus JC virus (JCV) is a causative agent of progressive multifocal leukoencephalopa... more Human polyomavirus JC virus (JCV) is a causative agent of progressive multifocal leukoencephalopathy which results from lytic infection of glial cells. Although significant progress has been made in understanding the regulation of JCV gene transcription, the mechanism(s) underlying the viral lytic cycle remains largely unknown. We recently reported that the JCV late auxiliary Agnoprotein may have a regulatory role in JCV gene transcription and replication. Here, we investigated its regulatory function in viral gene transcription through its physical and functional interaction with YB-1, a cellular transcription factor which contributes to JCV gene expression in glial cells. Time course studies revealed that Agnoprotein is first detected at day 3 postinfection and that its level increased during the late stage of the infection cycle. Agnoprotein is mainly localized to the cytoplasmic compartment of the infected cell, with high concentrations found in the perinuclear region. While the...
Journal of Neurovirology, 2004
Journal of Neurovirology, 2004
Journal of Biological Chemistry, 1998
Cross communication between regulatory proteins is an important event in the control of eukaryoti... more Cross communication between regulatory proteins is an important event in the control of eukaryotic gene transcription. Here we have examined the structural and functional interaction between two cellular regulatory proteins, YB-1 and Pur␣, on the 23-bp sequence element derived from the enhancer-promoter of the human polyomavirus JCV. YB-1 and Pur␣ are single-stranded DNA binding proteins which recognize C/T-and GC/GA-rich sequences, respectively. Results from band shift studies demonstrated that while both proteins interact directly with their DNA target sequences within the 23-bp motif, each protein can regulate the association of the other one with the DNA. Affinity chromatography and coimmunoprecipitation provide evidence for a direct interaction between Pur␣ and YB-1 in the absence of the DNA sequence. Ectopic expression of YB-1 and Pur␣ in glial cells synergistically stimulated viral promoter activity via the 23-bp sequence element. Results from mutational studies revealed that residues between amino acids 75 and 203 of YB-1 and between amino acids 85 and 215 of Pur␣ are important for the interaction between these two proteins. Functional studies with glial cells indicated that the region within Pur␣ which mediates its association with YB-1 and binding to the 23-bp sequence is important for the observed activation of the JCV promoter by the Pur␣ and YB-1 proteins. The results of this study suggest that the cooperative interaction between YB-1 and Pur␣ mediates the synergistic activation of the human polyomavirus JCV genome by these cellular proteins. The importance of these findings for cellular and viral genes which are regulated by Pur␣ and YB-1 is discussed.