Visualization of the Npro protein in living cells using biarsenically labeling tetracysteine-tagged classical swine fever virus

• Published in: Virus research 2014-08, Vol.189, p.67-74
• Main Authors: Li, Yongfeng, Shen, Liang, Li, Chao, Huang, Junhua, Zhao, Bibo, Sun, Yuan, Li, Su, Luo, Yuzi, Qiu, Hua-Ji
• Format: Article
• Language: English
• Subjects: Animals; Cell Line; Cell Nucleus - chemistry; Classical swine fever virus; Classical swine fever virus - enzymology; Cytoplasm - chemistry; Endopeptidases - analysis; Npro protein; Optical Imaging - methods; Real-time fluorescence imaging; Swine; Tetracysteine tag; Viral Proteins - analysis
• ISSN: 0168-1702; 1872-7492
• DOI: 10.1016/j.virusres.2014.04.018

•We generated CSFV mutants bearing the tetracysteine tag in the Npro protein.•Biarsenical labeling resulted in fluorescent Npro protein in CSFV-infected cells.•Labeled Npro was localized in the cytoplasm at 27 hpi and in the nucleus at 48 hpi.•Real-time Npro nuclear import and export from 36.5 to 37 hpi was visualized.•Npro protein transported into the nucleus by passive diffusion. Real-time fluorescence imaging of viral proteins in living cells is a valuable means to study virus–host interactions, and tetracysteine (TC)-biarsenical technology has been used in several viruses but not in classical swine fever virus (CSFV). Here, we generated CSFV mutants vSMTC385 or vSMTC412 bearing the small TC tag (CCPGCC) in the N-terminal region of the Npro protein. The mutants showed growth characteristics indistinguishable from that of the wild-type virus, and retained similar Npro subcellular localization to that of the parent virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in the fluorescent Npro protein in the context of virus infection. Finally, we showed that Npro was localized in the cytoplasm of CSFV-infected cells at 27h post-infection (hpi) and present in the nucleus at 48 hpi, and the nuclear import and export was clearly observed from 36.5 to 37 hpi. Interestingly, our results demonstrated that Npro transported across the nuclear pores by passive diffusion, which might be prevented by exogenous interferon regulatory factor 3 interacting with Npro. Taken together, biarsenical labeling allows real-time visualization of the nucleus import and export of the fluorescent Npro protein in CSFV-infected living cells.