Characterization and application of polyclonal antibodies that specifically recognize JC virus large T antigen

Sunden, Yuji; Suzuki, Tadaki; Orba, Yasuko; Umemura, Takashi; Asamoto, Makoto; Nagashima, Kazuo; Tanaka, Shinya

doi:10.1007/s00401-005-0025-9

Cited by 15 publications

(13 citation statements)

References 42 publications

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“…Recombination was confirmed by sequencing the plasmid DNA. pCXN2-Flag-JCV-TAg was used as a plasmid expressing Flag-tagged JCV TAg (58). Mutagenesis on pCXN2-Flag-JCV-TAg was performed in a manner similar to that described for the JCV Mad1 genome.…”

Section: Methodsmentioning

confidence: 99%

Deep-Sequence Identification and Role in Virus Replication of a JC Virus Quasispecies in Patients with Progressive Multifocal Leukoencephalopathy

Takahashi

Sekizuka

Fukumoto

et al. 2017

J Virol

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JC virus (JCV) is a DNA virus causing progressive multifocal leukoencephalopathy (PML) in immunodeficient patients. In the present study, 22 genetic quasispecies with more than 1.5% variant frequency were detected in JCV genomes from six clinical samples of PML by next-generation sequencing. A mutation from A to C at nucleotide (nt) 3495 in JCV Mad1 resulting in a V-to-G amino acid substitution at amino acid (aa) position 392 of the large T antigen (TAg) was identified in all six cases of PML at 3% to 19% variant frequencies. Transfection of JCV Mad1 DNA possessing the V392G substitution in TAg into IMR-32 and human embryonic kidney 293 (HEK293) cells resulted in dramatically decreased production of JCV-encoded proteins. The virus DNA copy number was also reduced in supernatants of the mutant virus-transfected cells. Transfection of the IMR-32 and HEK293 cells with a virus genome containing a revertant mutation recovered viral production and protein expression. Cotransfection with equal amounts of wild-type genome and mutated JCV genome did not reduce the expression of viral proteins or viral replication, suggesting that the mutation did not have any dominantnegative function. Finally, immunohistochemistry demonstrated that TAg was expressed in all six pathological samples in which the quasispecies were detected. In conclusion, the V392G amino acid substitution in TAg identified frequently in PML lesions has a function in suppressing JCV replication, but the frequency of the mutation was restricted and its role in PML lesions was limited.IMPORTANCE DNA viruses generally have lower mutation frequency than RNA viruses, and the detection of quasispecies in JCV has rarely been reported. In the present study, a next-generation sequencer identified a JCV quasispecies with an amino acid substitution in the T antigen in patients with PML. In vitro studies showed that the mutation strongly repressed the expression of JC viral proteins and reduced the viral replication. However, because the frequency of the mutation was low in each case, the total expression of virus proteins was sustained in vivo. Thus, JC virus replicates in PML lesions in the presence of a mutant virus which is able to repress virus replication.

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Section: Methodsmentioning

confidence: 99%

Deep-Sequence Identification and Role in Virus Replication of a JC Virus Quasispecies in Patients with Progressive Multifocal Leukoencephalopathy

Takahashi

Sekizuka

Fukumoto

et al. 2017

J Virol

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“…Gene expression vectors and luciferase reporter vectors: Complementary DNA (cDNA) of JCV TAg was subcloned into the mammalian expression vector pCXN2-Flag, and pCXN2-Flag-JCV-TAg was constructed (31). We amplified the coding sequence (CDS) of MeCP2 from the plasmid, pcd-MP4, which encoded the CDS of MeCP2 in the pcDNAI (32), and the insert was cloned into the XhoI and NotI sites of pCMV-Flag (Sigma), designated as pCMV-Flag-MeCP2.…”

Section: Methodsmentioning

confidence: 99%

Relationship between Methyl CpG Binding Protein 2 and JC Viral Proteins

et al. 2013

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SUMMARY: JC virus (JCV) is a causative agent of progressive multifocal leukoencephalopathy (PML). Methyl CpG binding protein 2 (MeCP2) is a transcriptional control nuclear protein that is abundantly expressed in neurons. We previously observed that the MeCP2 protein is expressed in JCV large T antigen (TAg)-expressing glial cells in PML brains. To investigate the relationship between MeCP2 and JCV TAg, we examined the promoter activity and mRNA and protein expression levels of MeCP2 in JCV TAg-expressing cells. We found that JCV TAg enhances the promoter activity of MeCP2, but does not enhance the mRNA and protein levels of MeCP2. These results suggest that post-transcriptional mechanisms may play a role in MeCP2 expression.

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“…Beads were collected using a magnetic separation stand and washed with binding buffer. Bound proteins were subjected to SDS-PAGE and immunoblotting using anti-CstF or anti-DDX antibodies, as described previously (16,20). For competition experiments, a tenfold excess of nonbiotinylated probe was added to the binding reaction mixture.…”

Section: Methodsmentioning

confidence: 99%

Identification of DDX1 as a JC Virus Transcriptional Control Region‐Binding Protein

Sunden

Semba

Suzuki

et al. 2007

Microbiology and Immunology

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To investigate the mechanism behind JC virus (JCV) cell specificity we performed electrophoretic mobility shift assays (EMSA) using probes derived from the JCV transcriptional control region (JCV‐TCR). Using nuclear extracts from the JCV‐susceptible neuroblastoma cell line IMR‐32, EMSA revealed a 670 kDa JCV‐TCR‐binding protein complex designated as #3‐bp. This complex could not be detected in nuclear extracts from non‐susceptible cell lines. Using column chromatographic purification and microsequencing, we identified cleavage stimulation factor (CstF) as a component of #3‐bp. However, as CstF is present in many cell types, we speculated that the IMR‐32‐specific component(s) of #3‐bp bind CstF. We performed a yeast two‐hybrid assay using CstF‐77 as the bait against a HeLa cDNA‐subtracted IMR‐32 cDNA library. This analysis detected binding between CstF‐77 and the RNA helicase DDX1. Subsequently, biotinylated DNA affinity precipitation and chromatin immunoprecipitation assays also confirmed that DDX1 binds specifically to JCV‐TCR. Our findings indicate that an association between DDX1 and the JCV‐TCR may play a significant role in JCV infection in IMR‐32 cells.

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Characterization and application of polyclonal antibodies that specifically recognize JC virus large T antigen

Cited by 15 publications

References 42 publications

Deep-Sequence Identification and Role in Virus Replication of a JC Virus Quasispecies in Patients with Progressive Multifocal Leukoencephalopathy

Deep-Sequence Identification and Role in Virus Replication of a JC Virus Quasispecies in Patients with Progressive Multifocal Leukoencephalopathy

Relationship between Methyl CpG Binding Protein 2 and JC Viral Proteins

Identification of DDX1 as a JC Virus Transcriptional Control Region‐Binding Protein

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