Current Approaches on Viral Infection: Proteomics and Functional Validations

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

Co-localization has been widely applied to virology studies in order to visualize the co-existence of target proteins in different sub-cellular distributions. In proteomic studies, the confocal imaging technique could further visualize virus-host interactions at the protein level and thus enhance proteomic data from another aspect. In one research, Lee et al. (2011) employed TAP based pull-down assay combined with 1D LC-MS/MS to investigate the essential interaction partners of hepatitis C virus core (HCVc) protein. Huh7 cells were transiently transfected with a plasmid construct bearing exogenous HCVc and TAP components, which were designed to express the biotinylated (B-tag) bait protein with calmodulin-binding peptide (CBP)/protein A tags. This TAP based proteomic approach enabled the authors to identify 36 candidates. Three highest-ranking proteins, hnRNPH1, NF45, and C14orf166, were selected for further validations. These three proteins were also specifically identified and validated as binding partners of HCVc in another affinity pull-down system, which is based on streptavidin-Dynabeads instead of IgG-Dynabeads. Confocal imaging analysis was performed to visualize these interacting partners. In a mock group, hnRNPH1 was found to be localized in the nucleus whereas NF45 and C14orf166 were evenly distributed in both the cytoplasm and the nucleus. In HCVc expressed Huh7 cells, co-staining of HCVc with these three proteins were predominantly localized in the nuclear region, suggesting that HCVc was transferred to nucleus from cytoplasm to interact with its binding partners. In another example, an EBV-encoded protein, latent membrane protein 1 (LMP1), was known to be able to interact with cellular prenylated Rab acceptor 1 (PRA1). This association mainly occurred in Golgi apparatus visualized by immunofluorescence imaging and it was highly involved in LMP1 mediated intracellular trafficking and NF-kB signaling pathways in nasopharyngeal carcinoma (NPC) cells (Liu et al., 2006) . To further clarify the role of PRA1 in EBV infected NPC cells, siRNA knockdown of PRA1 was performed to generate PRA1-knockdown NPC clones, which were analyzed by the isobaric mass tags (iTRAQ) labeling approach coupled with 2D LC-MS/MS (Liu et al., 2011) . Seventy proteins were found to be significantly up-regulated whereas 20 were downregulated. The significantly up-regulated proteins (e.g., LAMC2, ITGA6, ITGB4, FABP5, CAV1, and TIP47) responsible for lipid homeostasis and cell migration were selected and analyzed by immunofluorescence imaging in PRA1-knockdown NPC cells. In consistent with the proteomic data, spatial distributions of ITGA6, ITGB4, and CAV1 to perinuclear regions were observed with their elevated fluorescence staining patterns.