title: Novel near-infrared BiFC systems from a bacterial phytochrome for imaging protein interactions and drug evaluation under physiological conditions.
abstract: Monitoring protein-protein interactions (PPIs) in live subjects is critical for understanding these fundamental biological processes. Bimolecular fluorescence complementation (BiFC) provides a good technique for imaging PPIs; however, a BiFC system with a long wavelength remains to be pursued for in vivo imaging. Here, we conducted systematic screening of split reporters from a bacterial phytochrome-based, near-infrared fluorescent protein (iRFP). Several new near-infrared phytochrome BiFC systems were built based on selected split sites including the amino acids residues 97/98, 99/100, 122/123, and 123/124. These new near-infrared BiFC systems from a bacterial phytochrome were verified as powerful tools for imaging PPIs under physiological conditions in live cells and in live mice. The interaction between HIV-1 integrase (IN) and cellular cofactor protein Lens epithelium-derived growth factor (LEDGF/p75) was visualized in live cells using the newly constructed iRFP BiFC system because of its important roles in HIV-1 integration and replication. Because the HIV IN-LEDGF/p75 interaction is an attractive anti-HIV target, drug evaluation assays to inhibit the HIV IN-LEDGF/p75 interaction were also performed using the newly constructed BiFC system. The results showed that compound 6 and carbidopa inhibit the HIV IN-LEDGF/p75 interaction in a dose-dependent manner under physiological conditions in the BiFC assays. This study provides novel near-infrared BiFC systems for imaging protein interactions under physiological conditions and provides guidance for splitting other bacterial phytochrome-like proteins to construct BiFC systems. The study also provides a new method for drug evaluation in live cells based on iRFP BiFC systems and supplies some new information regarding candidate drugs for anti-HIV therapies.
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; Graduate University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Minghai
authors.initials: M
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; Graduate University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Sanying
authors.initials: S
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; National Key Laboratory of Macrobiomolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: zhangxe@ibp.ac.cn.
authors.forename: Xian-En
authors.initials: XE
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China. Electronic address: czq@wh.iov.cn.
authors.forename: Zongqiang
authors.initials: Z
authors.lastname: Cui
mesh_terms: D000818:Animals; D001419:Bacteria / Q000378:metabolism*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D019427:HIV Integrase / Q000378:metabolism; D006801:Humans; D051379:Mice; D010834:Phytochrome / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D019265:Spectroscopy, Near-Infrared / Q000379:methods*
chemical_list: D011506:Proteins; D010834:Phytochrome; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1
keywords: Bacterial phytochrome iRFP; BiFC; Drug evaluation; HIV IN-LEDGF/p75 interaction; Near-infrared