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 N(pro) protein. The mutants showed growth characteristics indistinguishable from that of the wild-type virus, and retained similar N(pro) subcellular localization to that of the parent virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in the fluorescent N(pro) protein in the context of virus infection. Finally, we showed that N(pro) was localized in the cytoplasm of CSFV-infected cells at 27 h 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 N(pro) transported across the nuclear pores by passive diffusion, which might be prevented by exogenous interferon regulatory factor 3 interacting with N(pro). Taken together, biarsenical labeling allows real-time visualization of the nucleus import and export of the fluorescent N(pro) protein in CSFV-infected living cells.
Keywords: Classical swine fever virus; N(pro) protein; Real-time fluorescence imaging; Tetracysteine tag.
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