Viral proteomics: The emerging cutting-edge of virus research

Zhou, ShengTao; Liu, Rui; Zhao, Xia; Huang, CanHua; Wei, YuQuan

Currently, protein isolation by 2DE coupled with MS-based analysis of differentially expressed spots, is the most widely utilized among a wide spectrum of protein-expression analysis technologies, primarily because of its simplicity and wide applicability. Viruses studied by this approach comprise HBV, EBV, SARS-CoV, HCV, and HIV-1. In 2008, Tong et al. [48] successfully utilized 2-DE and MS/MS analysis to compare and identify differentially expressed proteins between an HBV-producing cell line, HepG2.2.15, and its parental cell line, HepG2, to determine the effects of HBV replication on host cell-protein expression. Sixty-two spots were positively identified by MS/MS analysis. Cluster and metabolic/signaling pathway analysis divided the proteins into three groups: retinol metabolism pathway, calcium ion-binding proteins, and proteins associated with protein degradation pathways. This study provided novel insights into the mechanisms underlying HBV-host cell interactions and the development of HCC [11] . In another study, 2D-GE-MALDI-TOF-MS was applied to clinical HBV-associated hepatocellular carcinoma. One hundred protein spots, corresponding to 80 distinct gene products, were identified. Among these 100 proteins, proliferating cell antigen and stathmin 1 were further corroborated by Western blotting. In an attempt to profile EBV-induced cellular proteome alterations, EBV transformed B-lymphoblastoid cell lines (LCLs) were used as a model system to identify the proteins essential for the im- In the Y2H method, two plasmids including a bait-encoding protein fused to the C-terminus of a transcription factor BD (binding domain) and a prey-encoding protein fused to an AD (activation domain) are constructed. Subsequently, the plasmids are introduced into a suitable yeast strain, and if the two proteins physically interact with one another, the BD and AD are brought together to reconstitute an active transcription factor. This active transcription factor binds to upstream specific activation sequences in the reporter gene promoter to activate expression. By measuring the expression of a reporter gene, the interaction between the proteins can be examined. In the TAP immunoprecipitation method, a fusion tag comprising two sites, one protein A IgG-binding domain and one CBP tag, separated by a spacer containing the specific recognition site for the TEV protease, is expressed in the target cell. The purification steps for interacting proteins are summarized in the right picture. AD, activation domain; BD, DNA-binding domain; CBP, calmodulin-binding peptide; TAP, tandem affinity purification; TEV, tobacco etch virus. mortalization process. By surveying the differential protein expression profile between pre-and post-immortal cells, this study ultimately identified 20 proteins from 32 spots. Upregulated proteins included proliferating cell nuclear antigen, nucleoside diphosphate kinase-A, and ubiquitin C-terminal hydrolase. Downregulated proteins included ubiquitin ligase N and stathmin [49] . Coiras et al. [50] examined the impact of the HIV-I tat protein on Jurkat T cells using 2-D DIGE followed by MS analysis. This study noted downregulation of several cytoskeletal proteins, such as actin, β-tubulin, and annexin II, as well as gelsolin, cofilin, and the Rac/Rho-GDI complex. This expression pattern change could account for the survival of long-term reservoirs of HIV-infected CD4 + T cells responsible for continuous viral production.