Current Approaches on Viral Infection: Proteomics and Functional Validations

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

Construction of plasmid cloning vector encoding gene targets and its subsequent transfection into cells, or bacteria, could lead to the co-expression of endogenous and exogenous proteins. Therefore, this target-based cloning enables us to additionally induce the over-expression of one protein or heterogeneously express proteins encoded by genes of an alternative microorganism, respectively. In recent years, numerous pull-down based proteomic techniques have been widely applied to study PPIs. Specifically, TAP based proteomic approaches facilitate the characterization of interacting partners of the highlighted viral protein, which could be exogenously expressed in host cells by cloning specific construct. And Most TAP based or pull-down proteomics were initial designs followed by proteomics rather than functional validation studies to proteomic data. In one study, in order to discover the binding partners of HCVc fusion protein, Lee et al. (2011) designed a vector construct by inserting the HCVc gene fragment into the pMSCV-BCP vector, this enabled them to express a biotinylated (B-tag) bait protein with CBP/protein A tags. The interacting protein complex of the exogenous intact HCVc protein could be identified by LC-MS/MS and confirmed by confocal imaging microscopy. In another study, Jorba et al. (2008) constructed plasmids encoding influenza A viral polymerase genes fused with the TAP tag, and used MALDI TOF MS to find the binding partners of this heterotrimeric polymerase complex. Human HEK293T cells were transfected with these plasmids expressing viral polymerase proteins fused with the TAP tag. Most successfully identified host proteins were nuclear proteins involved in cellular RNA synthesis, modification, and nuclear trafficking. To further validate the proteomic data, one abundant host factor, KIAA0136 (NXP2), was designed to be over-expressed from the plasmid pcDNA3HA-NXP2 by fusing the gene with the HA tag. And subsequent immunofluorescence imaging assay supported mass spectrometric data and confirmed the co-localization of this tagged-NXP2 with viral RNPs.