Abstract
The clinical manifestations of West Nile virus (WNV), a member of the Flavivirus family, include febrile illness, sporadic encephalitis, and paralysis. The capsid (Cp) of WNV is thought to participate in these processes by inducing apoptosis through mitochondrial dysfunction and activation of caspase-9 and caspase-3. To further identify the molecular mechanism of the WNV capsid protein (WNVCp), yeast two-hybrid assays were employed using WNV-Cp as bait. Jab1, the fifth subunit of the COP9 signalosome, was subsequently identified as a molecule that interacts with WNVCp. Immunoprecipitation and glutathione S-transferase pulldown assays confirmed that direct interaction could occur between WNVCp and Jab1. Immunofluorescence microscopy demonstrated that the overexpressed WNVCp, which localized to the nucleolus, was translocated to the cytoplasm upon its co-expression with Jab1. When treated with leptomycin B, Jab1-facilitated nuclear exclusion of WNVCp was prevented, which indicated that the CRM1 complex is required for Jab1-facilitated nuclear export of WNVCp. Moreover, Jab1 promoted the degradation of WNVCp in a proteasome-dependent way. Consistent with this, WNVCp-mediated cell cycle arrest at the G(2) phase in H1299 was prevented by exogenous Jab1. Finally, an analysis of WNVCp deletion mutants indicated that the first 15 amino acids were required for interaction with Jab1. Furthermore, the double-point mutant of the WNVCp, P5A/P8A, was incapable of binding to Jab1. These results indicate that Jab1 has a potential protective effect against pathogenic WNVCp and might provide a novel target site for the treatment of disease caused by WNV.
Highlights
West Nile virus (WNV) contains a single positive-sense RNA genome of ϳ11 kb in length; this genome encodes polyprotein
The double-point mutant of the WNV capsid protein (WNVCp), P5A/P8A, was incapable of binding to Jab1. These results indicate that Jab1 has a potential protective effect against pathogenic WNVCp and might provide a novel target site for the treatment of disease caused by WNV
The detailed mechanism involved in this process has not yet been defined clearly, several reports have suggested that molecules derived from the WNV genome are responsible for apoptosis
Summary
Plasmids—pGBK-T7-WNVCp, a bait plasmid for yeast two-hybrid screening, was created by cloning an EcoRI-XhoI fragment from pcDNA3-His-WNVCp into pGBK-T7 (Clontech). pGADT7-Jab was generated by subcloning an EcoRI-XhoI fragment from pGEX-4T-1-Jab The cells were transfected with 200 nM of siRNA by using Oligofectamine according to the instructions of the manufacturer (Invitrogen). Whole cell extracts were incubated with the target antibody for 2 h at 4 °C. H1299 (p53-null lung carcinoma) cells, mostly localized to the nucleolus This was confirmed by its colocalization with nucleolin, the nucleolus protein (Fig. 2A). On the other hand, when WNVCp was cotransfected with Jab, ϳ45% of cells displayed WNVCp that had translocated to the cytoplasm (Fig. 2B, panels 5– 8, and graph). After washing three indicated that Jab directly interacted with WNVCp, which times with PBS, the fixed cells were incubated with RNase A Stained cells were detected by The N-terminal Domain of WNVCp Is Responsible for Its flow cytometry analysis (BD Biosciences). Data were analyzed Interaction with Jab1—we analyzed the WNVCp domain, using CellQuest Pro software (BD Biosciences)
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