title: Delaying Photobleaching of a Light-Switch Complex for Real-Time Imaging of Single Viral Particle Uncoating.
abstract: Photobleaching is a major obstacle in the real-time imaging of biological events, particularly at the single-molecule/particle level. Here, we report a strategy to delay photobleaching of a light-switch complex, [Ru(phen)2dppx]2+, by insertion of a six-cysteine peptide into virus particles. The six-cysteine peptide was inserted into viral protein R of HIV-1 and assembled into infectious HIV-1 viral particles, where it effectively delayed the photobleaching of the [Ru(phen)2dppx]2+complex used to label viral genomic RNAs. This delay in photobleaching allowed for a monofluorescent assay to be constructed for the real-time monitoring of viral uncoating, a poorly understood process. This novel strategy to delay photobleaching in infectious viral particles provides a powerful method to analyze viral uncoating at the single-particle level in real time.
authors.affiliation: Beijing Key Lab of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology , Beijing 100029, People's Republic of China.|State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, People's Republic of China.
authors.forename: Yingxin
authors.initials: Y
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, People's Republic of China.
authors.forename: Zongqiang
authors.initials: Z
authors.lastname: Cui
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine, Ministry of Education, College of Chemistry and Sciences, Wuhan University , Wuhan, Hubei 430072, People's Republic of China.
authors.forename: Zhike
authors.initials: Z
authors.lastname: He
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071, People's Republic of 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, People's Republic of 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, People's Republic of China.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.
authors.forename: Xian-En
authors.initials: XE
authors.lastname: Zhang
authors.affiliation: Beijing Key Lab of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology , Beijing 100029, People's Republic of China.
authors.forename: Tianwei
authors.initials: T
authors.lastname: Tan
mesh_terms: D056831:Coordination Complexes / Q000737:chemistry / Q000528:radiation effects*; D003545:Cysteine / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry / Q000528:radiation effects*; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D010455:Peptides / Q000235:genetics; D038761:Photobleaching; D012367:RNA, Viral / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012428:Ruthenium / Q000737:chemistry; D057646:Virus Uncoating; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000235:genetics
chemical_list: D056831:Coordination Complexes; D005456:Fluorescent Dyes; D010455:Peptides; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; D012428:Ruthenium; D003545:Cysteine