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

Poliovirus is a single-stranded, plus-polarity RNA virus. Upon infection ofhuman cells, the input RNA is translated in the cytoplasm into a long polyprotein,which is initially processed into three precursor polyproteins (P1,P2,P3). Furtherprocessing of these precursors by virally encoded proteases gives rise to manyimportant mature proteins. Some viral proteins, host proteins and RNA virus composethe viral replication complex, which guides viral RNA synthesis rapidly andefficiently. Among them, proteins 3AB, VPg and 3D, which are the shear productsfrom P3 precursor, play important effect in the formation and function of viralreplication complex. 3B (VPg) is covalent linkage to 5' genome RNA and can initiateRNA replication; 3D is a RNA polymerase and can elongate nascent RNA chains.While 3AB is a membrane-bound protein and can help viral replication complexlocated in cell membrane.Research related to the protein interactions in viral replication complex is ofgreat importance for illuminating the mechanism of viral replication complex andunderstanding the virus-host interaction. So far, there are many methods for studyingthe protein interactions, such as: yeast two-hybrid, co-immunoprecipitation, SPR andso on. However, these methods can't achieve protein interactions in situ, in vivo anddo real-time study. Fluorescence resonance energy transfer (FRET) is a new methodfor researching protein interaction in living cells and can visualize protein interactionsnondestructively in vivo. In our study, we visualized protein interactions in poliovirusRNA replication complex in living cells using FRET for the first time.In this thesis we mainly study the location and interactions of proteins 3AB, VPg, and 3D, which are key proteins in viral replication complex, in living cells. FRETanalysis were done with laser scanning confocal microscope 24 hours later aftertransfecting the recombinant plasmids separately or corporately into Vero cells. Singleplasmid transfection experiments indicated that protein 3D and VPg could beobserved distributing throughout the cell, while 3AB localized only in the cytoplasmand accumulated in small, irregularly sized granules. Two plasmids co-transfectionexperiments showed that 3AB-VPg could interact in living cell and the interactionregion is centralized in the cytoplasm region with the mean value of FRET efficiency8.3±0.6% after 44 groups analysises, 3AB-3D could interact in living cell and theinteraction region is centralized in the cytoplasm region with the mean value of FRETefficiency 6.1±0.8% after 46 groups analysises; and VPg-3D could also interact inliving cell but the interaction region is distributed throughout the cell with the meanvalue of FRET efficiency 6.8±0.5% after 51 groups analysis. From the above results,we could see that when co-transfections involving 3AB, the interaction region was inthe cytoplasm region, where also the viral replication complex buds. Besides that, wefound there was no change in 3AB-VPg, 3AB-3D interactions after poliovirusinfection. But viral infection enhanced interaction of VPg and 3D with the mean valueof FRET efficiency rising from 6.8±0.5% (n=51) to 11.1±1.1% (n=43).By studying the location and interactions of protein 3AB,VPg and 3D, we provedthat these proteins play important functions in the formation of viral replicationcomplex. This study also provides important data for illustrating virus replicationmechanism and understanding the formation and effect mechanism of viral replicationcomplex.