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Updated: Jul 7, 2026
Imaging Protein-protein Interactions in vivo
Published on: October 10, 2010
Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy
Na Li1, Zong-Qiang Cui, Ji-Kai Wen
1State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, 44#, Xiao Hong Shan, Wuchang, Wuhan 430071, China.
Abstract:
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.
Insights
Poliovirus replication proteins 3AB, VPg, and 3D interact within viral replication complexes. Live cell imaging revealed poliovirus infection enhances these protein interactions, crucial for RNA replication.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Poliovirus RNA replication occurs in a complex machinery involving specific viral proteins.
- Proteins 3AB, VPg (3B), and 3D from the poliovirus genome are essential for replication complex formation and function.
Purpose of the Study:
- To visualize and quantify protein interactions within the poliovirus RNA replication complex in living cells.
- To investigate the impact of poliovirus infection on these protein interactions.
Main Methods:
- Utilized acceptor photobleaching protocol for fluorescence resonance energy transfer (FRET) imaging in living cells.
- Quantified FRET efficiencies to determine interaction strengths between 3AB, VPg, and 3D proteins.
Main Results:
- Demonstrated interactions between 3AB-3D, VPg-3D, and 3AB-VPg with measured FRET efficiencies.
- Observed significantly enhanced interactions of VPg-3D and 3AB-3D upon poliovirus infection.
Conclusions:
- Live cell FRET analysis provides a method to study real-time protein interactions in the poliovirus replication complex.
- Poliovirus infection dynamically modulates protein interactions essential for viral RNA replication.
