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2.
J Med Virol ; 84(4): 555-61, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22337293

ABSTRACT

The high incidence of progressive multifocal leukoencephalopathy (PML) among individuals with acquired immunodeficiency syndrome (AIDS) is similar to the incidence of other immunocompromised diseases. The pathogenic JC virus (JCV) with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease in the brains of immunocompromised patients. In a previous study, Tat protein, encoded by human immunodeficiency virus type 1 (HIV-1), markedly enhanced the expression of a reporter gene under control of the JCV late promoter. In order to examine the enhancement of JCV replication by Tat protein, the neuroblastoma cell line IMR-32 was used because it enables IMR-32-adapted JCV. The extent of JCV replication in IMR-32 cells treated with Tat protein was significantly higher than that in untreated IMR-32 cells. The enhancement of JCV propagation by Tat protein was also examined using IMR-32-derived JCV producing (JCI) cells which continuously produce JCV. Treatment of JCI cells with Tat protein led to a significant increase in the titers of progeny viruses. It has also been shown that Tat protein leads to a decrease in the expression of purine-rich element binding protein α (Purα) as an important mediator of JCV replication in IMR-32 cells. Thus, it is probable that Tat protein enhances JCV replication in IMR-32 cells via the down-regulation of Purα expression and cell proliferation. To our knowledge, this is the first report that exogenous Tat protein enhances the replication of JCV efficiently in neuroblastoma cell lines.


Subject(s)
JC Virus/growth & development , Neurons/virology , Virus Replication , tat Gene Products, Human Immunodeficiency Virus/metabolism , Cell Line, Tumor , Humans , Virus Cultivation , tat Gene Products, Human Immunodeficiency Virus/genetics
3.
Microbiol Immunol ; 54(12): 758-62, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21223365

ABSTRACT

The high incidence of progressive multifocal leukoencephalopathy (PML) in AIDS patients compared with many other immunosuppressive diseases suggests that HIV-1 infection is strictly related to the activation of JC virus (JCV) propagation. In this report, propagation of PML-type JCV in COS-7-derived cell lines stably expressing HIV-1 Tat (COS-tat cells) has been examined. In COS-tat cells, production of viral particles and replication of genomic DNA were markedly increased compared to COS-7 cells, as judged by HA and real-time PCR analyses. These results demonstrate that COS-tat cells provide a useful model system for studying HIV-1 Tat-mediated propagation of PML-type JCV.


Subject(s)
HIV-1/physiology , JC Virus/growth & development , tat Gene Products, Human Immunodeficiency Virus/physiology , Animals , COS Cells , Chlorocebus aethiops
4.
Microbiol Immunol ; 53(11): 621-8, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19903262

ABSTRACT

Pathogenic JCV with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease, in the brains of immunocompromised patients. On the other hand, archetype JCV persistently infecting the kidney is thought to be converted to PML-type virus during JCV replication in the infected host under immunosuppressed conditions. In addition, Tat protein, encoded by HIV-1, markedly enhances the expression of a reporter gene under control of the JCV late promoter. In order to examine the influence of Tat on JCV propagation, we used kidney-derived COS-7 cells, which only permit archetype JCV, and established COS-tat cells, which express HIV-1 Tat stably. We found that the extent of archetype JCV propagation in COS-tat cells is significantly greater than in COS-7 cells. On the other hand, COS-7 cells express SV40 T antigen, which is a strong stimulator of archetype JCV replication. The expression of SV40 T antigen was enhanced by HIV-1 Tat slightly according to real-time RT-PCR, this was not closely related to JCV replication in COS-tat cells. The efficiency of JCV propagation depended on the extent of expression of functional Tat. To our knowledge, this is the first report of increased production of archetype JCV in a culture system using cell lines stably expressing HIV-1 Tat. We propose here that COS-tat cells are a useful tool for studying the role of Tat in archetype JCV replication in the development of PML.


Subject(s)
COS Cells/virology , HIV-1/physiology , JC Virus/physiology , Virus Cultivation/methods , Virus Replication/physiology , tat Gene Products, Human Immunodeficiency Virus/physiology , Animals , Antigens, Polyomavirus Transforming/biosynthesis , Chlorocebus aethiops , DNA Replication , DNA, Viral/genetics , DNA, Viral/metabolism , Genome, Viral , Humans , Reverse Transcriptase Polymerase Chain Reaction , Viral Proteins/genetics , Viral Proteins/metabolism
6.
Med Mol Morphol ; 41(4): 204-10, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19107610

ABSTRACT

A few studies of the morphogenesis of human herpesvirus (HHV) 6 type A and B (HHV-6A, -6B) have been performed using neurogenic, lymphoid, or epithelial cells. When human MT-4 T-lymphotropic virus type I (HTLV-I)-producing lymphoid cells were coinfected with HHV-6B in vitro, viral-specific proteins were clearly detected. We therefore attempted to detect virus particles at the ultrastructural level, focusing on the morphogenesis of such particles. Ultrastructurally, HHV-6B virus particles could be observed in the nuclei, cytoplasm, and extracellular spaces of MT-4 cells, in addition to extracellular HTLV-I particles of C type. In the nuclei, dense-cored or doughnut-shaped viral capsids were found, as well as peculiar tubular rods. When budding to perinuclear spaces, these intranuclear capsids exhibited a thin tegument on their surfaces. Distinct teguments were found in the intracytoplasmic particles, which budded into cytoplasmic vacuoles during the process of maturation. The mature particles were detected in the extracellular spaces and the intracytoplasmic vacuoles, with a distinct tegument and surface spikes. An electron-dense layer in the outer part of the tegument was found in some mature particles located in the extracellular space, but no such layer was detected in mature particles in intracytoplasmic vacuoles. No annulate lamellae, but intranuclear tubular rods, were found in the cytoplasm of MT-4 cells. These observations indicate that HHV-6B in MT-4 cells is similar to HHV-6A in fine structure, but differs from HHV-7 and HHV-8 in ultrastructural characteristics. Further comparisons of HHV-6B with HHV-6A, HHV-7, and HHV-8 are needed with regard to functional activity.


Subject(s)
Herpesvirus 6, Human , Human T-lymphotropic virus 1/physiology , Lymphocytes/virology , Morphogenesis , Cell Line , Herpesvirus 6, Human/physiology , Herpesvirus 6, Human/ultrastructure , Humans , Lymphocytes/cytology , Lymphocytes/metabolism
9.
Blood ; 110(1): 323-33, 2007 Jul 01.
Article in English | MEDLINE | ID: mdl-17360941

ABSTRACT

Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer, including acute myeloid leukemia (AML), and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML, we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins, including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells, and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis, and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles.


Subject(s)
Leukemia, Megakaryoblastic, Acute/genetics , Oncogene Proteins, Fusion/physiology , RNA-Binding Proteins/genetics , Receptor, Macrophage Colony-Stimulating Factor/genetics , Animals , Cell Line , Cell Line, Tumor , Cell Proliferation , Cell Survival , Chromosomes, Human, Pair 3 , Chromosomes, Human, Pair 5 , Humans , Leukemia, Megakaryoblastic, Acute/etiology , Mice , Neoplasm Transplantation , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/isolation & purification , Protein-Tyrosine Kinases/isolation & purification , Receptor, Macrophage Colony-Stimulating Factor/isolation & purification , Sequence Analysis, DNA , Translocation, Genetic , Transplantation, Heterologous
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