Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy transfer (FRET).
Poliovirus RNA replication is directed by a replication complex on the rosette-like arrangement of membranous vesicles. Proteins derived from the p3 region of the polioviral genome, such as 3D, 3AB, and 3B (VPg), play key roles in the formation and function of the replication complex. In the present study, by using an acceptor photobleaching protocol for fluorescence resonance energy transfer (FRET) imaging, we visualized the interactions of 3D, 3AB, and VPg in living cells. The interaction of 3AB-VPg was determined by live cell FRET analysis. Quantitative analyses showed that the FRET efficiencies of 3AB-3D, VPg-3D, and 3AB-VPg were 3.9+/-0.4% (n=36), 4.5+/-0.4% (n=39), and 8.3+/-0.6% (n=44), respectively, in the cell cytoplasm where viral replication complexes are formed and function. Poliovirus infection enhanced the protein interactions of VPg-3D and 3AB-3D, with FRET efficiencies in the virus-infected cells of 10.7+/-1.1% (n=39) and 9.0+/-0.9% (n=37), respectively. This method of live cell analysis of protein interactions in the poliovirus RNA replication complex lays the foundation for further understanding of the real-time process of poliovirus RNA replication.
Li
Na
N
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, 44#, Xiao Hong Shan, Wuchang, Wuhan 430071, China.
Cui
Zong-Qiang
ZQ
Wen
Ji-Kai
JK
Zhang
Zhi-Ping
ZP
Wei
Hong-Ping
HP
Zhou
Ya-Feng
YF
Zhang
Xian-En
XE
D000818:Animals; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017955:Poliovirus / Q000502:physiology* / Q000648:ultrastructure; D025941:Protein Interaction Mapping / Q000379:methods*; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014709:Vero Cells; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins