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Live Cell Imaging the Protein Interactions in Poliovirus RNA Replication Complex Using Fluorescence Resonance Energy Transfer (FRET)

Author: LiNa
Tutor: ZhangZhiPing
School: Wuhan Institute of Virology
Course: Biochemistry and Molecular Biology
Keywords: Living cells Poliovirus Replication complex Fluorescence resonance energy transfer Protein interactions Protein 3AB, VPg, 3D
CLC: R373
Type: Master's thesis
Year: 2007
Downloads: 115
Quote: 0
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Abstract


Poliovirus single-stranded RNA virus. When the viral genomic RNA into the host cytoplasm, can be directly translated to a macromolecule poly-peptide, and is hydrolyzed rapidly processed into secondary precursor P1, P2 and P3, and then three sub precursors were further fractionated shear into a variety of end products of protein has an important function. The number of viral protein and host proteins, and the viral RNA together form the viral replication complex, rapidly and efficiently guide the synthesis of viral nucleic acid. Wherein P3 shear product 3AB, 3B (VPg), 3D has an important role in the composition and function of the composite body of the replication of the virus in exercise: 3B (VPG) covalently bound to RNA nascent chain 5 'end of the starting RNA synthesis initiating priming effect; 3D is extended the nascent chain of the RNA polymerase; the 3AB membrane-bound proteins, viral replication in the composite body is positioned at the cell membrane. Interaction between the composite key protein in vivo study poliovirus replication, to clarify the mechanism of action of the viral replication complex to reveal the mechanism of viral replication, and understand the interaction between virus and host cell having a significance. So far, it has been the development of many protein interaction studies: the yeast two-hybrid, co-immunoprecipitation, SPR, etc.. However, these methods can not achieve protein interactions in situ, in vivo, real-time research. Fluorescence resonance energy transfer technology is a new technology to study protein interactions in living cells, in vivo, in situ, nondestructive visualization of protein - protein interactions. In this study, using fluorescence resonance energy transfer in living cells for the first time visualization of the interaction between the poliovirus replication complex key proteins in the body. In this thesis, the poliovirus replication complex three key proteins in vivo 3AB, VPg position of 3D in living cells and interactions. The use of laser confocal microscopy and FRET analysis of samples. Single transfection experiments show: 3AB protein mainly located in the cytoplasmic region, and are gathered into small irregular particles; VPg and 3D protein mainly evenly distributed in the whole cell. Protein co-transfection experiments: 3AB-VPg this histone interactions in living cells, and the role of regional concentration in the cytoplasm, the analysis showed that the FRET efficiency average of 8.3 ± 0.6% (n = 44); 3AB- 3D this group of proteins in living cells can interact and effect region is concentrated in the cytoplasm, the analysis showed the average FRET efficiency was 6.1 ± 0.8% (n = 46); VPg-3D of this group of proteins in living cells observed a significant interaction effect region is dispersed in the whole cells, and is not particularly concentrated area, and analysis results show that the average FRET efficiency was 6.8 ± 0.5% (n = 51). Seen The 3AB protein involved in the interaction region the two groups are concentrated in the cytoplasmic region of the viral replication complex position similar. Further, by poliovirus infection experiments, we found that the virus infection on the 3AB-VPg, no significant effect on the interaction between these two groups of proteins 3AB-3D, but enhanced protein VPg and 3D interaction between, so that FRET average efficiency from 6.8 ± 0.5% (n = 51) increased to 11.1 ± 1.1% (n = 43). With fluorescent protein labeling and fluorescence resonance energy transfer technology, this thesis the poliovirus replication complex three key proteins in vivo 3AB, VPg, 3D in living cells positioning and interaction. Proteins in living cells 3AB, VPg, 3D interaction studies proved that they are important in the formation of the poliovirus replication complex, for an in-depth understanding of the formation and mechanism of action of the viral replication complex, to clarify the mechanism of viral replication the important data.

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus)
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