Current Approaches on Viral Infection: Proteomics and Functional Validations

Zheng, Jie; Tan, Boon Huan; Sugrue, Richard; Tang, Kai

Immunofluorescence staining assay is a powerful tool which combines the utility of specific fluorescent probes, advanced confocal microscopy, and digital image analysis. It has been frequently used for analyzing distinctive biological samples, such as neurons (Zinchuk and Grossenbacher-Zinchuk, 2009 ), plant cells (French et al., 2008) , virus infections, and so on. Co-localization refers to the co-existence of multiple fluorescent probes generated through different fluorochromes, resulting in overlapped images. For instance, in a same specimen, two antigens are visualized by their respective fluorescence-labeled secondary antibodies in microscopy (one is red while the other is green), and a third yellow staining image emerges when these two antigens co-localize, suggesting the interactions of these two macromolecules or particular sub-cellular localizations where this co-existence belongs. However, there also exist some limitations of this technique (Smallcombe, 2001) . For example, fluorescence bleed-through and tissue autofluorescence could take place between two fluorochromes, thus resulting in increased non-specific background noises. These obstacles could be overcome by careful sample preparation and appropriate optimization of image acquisition (Smallcombe, 2001; Zinchuk et al., 2007) . In addition, the bimolecular fluorescence complementation (BiFC) assay could investigate PPIs in live cells and organisms (Kerppola, 2006 (Kerppola, , 2008 . Two fluorescent protein fragments are fused to target proteins that interact, and these fragments would refold to produce fluorescence upon target protein association. BiFC has been utilized to study the virus-host interaction between HSV-1 regulatory protein ICP27 and one cellular protein, TAP/NXF1 (Hernandez and Sandri-Goldin, 2010 ). Yet few works were present as a subsequent method to validate proteomics data related to virus infections.