Identification of DDX1 as a JC virus transcriptional control region-binding protein.
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 purifi-cation 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.
Sunden
Yuji
Y
Laboratory of Comparative Pathology, Hokkaido University School of Veterinary Medicine, Sapporo, Hokkaido, Japan.
Semba
Shingo
S
Suzuki
Tadaki
T
Okada
Yuki
Y
Orba
Yasuko
Y
Nagashima
Kazuo
K
Umemura
Takashi
T
Sawa
Hirofumi
H
D045744:Cell Line, Tumor; D039241:Cleavage Stimulation Factor / Q000235:genetics / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007577:JC Virus / Q000235:genetics* / Q000302:isolation & purification; D009447:Neuroblastoma / Q000378:metabolism / Q000821:virology; D027601:Polyomavirus Infections / Q000235:genetics / Q000378:metabolism / Q000821:virology; D011485:Protein Binding; D014412:Tumor Virus Infections / Q000235:genetics / Q000378:metabolism / Q000821:virology
D039241:Cleavage Stimulation Factor; C086252:DDX1 protein, human; D053487:DEAD-box RNA Helicases