Current Approaches on Viral Infection: Proteomics and Functional Validations

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

In another study associated with dengue virus infected human endothelial cells,Alix (apoptosis-linked gene-2-interacting protein X) was identified as an up-regulated protein by 2D gel coupled with Q-TOF MS/MS (Pattanakitsakul et al., 2010) . Alix was known to play an important role in viral protein transport from endosome to cytosol for viral replication purpose. And double immunofluorescence staining assay confirmed the co-localization of Alix with the late endosome marker, lysobisphosphatidic acid (LBPA), suggesting that interaction of Alix with late endosome could assist the viral nucleocapsid export to the cytoplasm. Also, cells with and without anti-LBPA pretreatment were infected with dengue virus and then measured in different time post infection; immunofluorescence staining by targeting DENV envelop protein revealed delayed virus replication when pre-treated with anti-LBPA, suggesting that the inhibition of endosomal protein could impair functions of Alix to assist viral replication. To explore the possible mechanism underlying vascular leakage upon dengue infection, a similar proteomic approach was utilized to quantitatively analyze significantly regulated proteins (Kanlaya et al., 2009) . β-actin was identified to be greatly up-regulated. However, subsequent western blot analysis revealed that the expression level of β-actin was decreased. Furthermore, by applying immunofluorescence imaging, dengue infected cells showed remarkably decreased expression of actin networks and proteins associated with adherens junction, intercellular adhesion, and transendothelial migration. These observed alternations of actin networks and endothelial integrity could shed a light on vascular leakage upon virus infection. Although β-actin was noted as an up-regulated protein in one particular spot by 2D Gel proteomic approach, it was possible that the overall expression level of this abundant protein was decreased, which was consistent with the western blot and immunofluorescence imaging assays. This observation also reflects the necessity of functional validations to proteomic data, which may include false-positive identification or biased quantification results. Meanwhile, due to the limitations of the 2D gel based proteomic approach (Bunai and Yamane, 2005) , its accuracy needs to be improved. Besides www.frontiersin.org β-actin, hnRNPs were also found to be up-regulated upon virus infection, and IP based proteomics identified vimentin as the binding partner of hnRNPs (Kanlaya et al., 2010b) . The subsequent co-localization study confirmed the co-existence of vimentin, hnRNPs, and dengue NS1 in perinuclear regions, suggesting their roles associated with assembling in perinucleus upon dengue virus infection. The advances of immunofluorescence staining in visualizing the sub-cellular localizations of specific proteins and their interactions ensure that it remains a vital technique for functional proteomic studies.