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Identification of DDX1 as a JC Virus Transcriptional Control Region‐Binding Protein
Author(s) -
Sunden Yuji,
Semba Shingo,
Suzuki Tadaki,
Okada Yuki,
Orba Yasuko,
Nagashima Kazuo,
Umemura Takashi,
Sawa Hirofumi
Publication year - 2007
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.2007.tb03915.x
Subject(s) - biology , chromatin immunoprecipitation , immunoprecipitation , microbiology and biotechnology , t cell receptor , rna binding protein , electrophoretic mobility shift assay , binding protein , transcription factor , nuclear protein , complementary dna , dna binding protein , rna , cell culture , t cell , gene expression , biochemistry , gene , genetics , promoter , immune system
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.