Current Approaches on Viral Infection: Proteomics and Functional Validations

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

Although MS based proteomics has high throughput for identification of the altered proteome upon virus infections, it is unable to characterize the dynamic changes of enzymes, which could be induced by distinctive modifications, proteolytic processing, and alternative regulatory proteins. ABPP is a common functional technique designed to measure the dynamic changes of enzyme activities during virus infections (Cravatt et al., 2008; Frontiers in Microbiology | Virology 2012). The activity-based probes (ABPs) consist of two essential components: a warhead reactive group and a reporter tag, which are designed to covalently target the active site of enzymes and for purification or visualization, respectively. In addition, different levels of enzyme activity induced by viral or host proteins upon virus infections could be studied by comparative ABPP. A nondirected ABPP probe, PS4, was used to profile different levels of enzyme activity related to HCV replications in Huh7 cells (Singaravelu et al., 2010) . Nine host candidates such as HSPA8, protein disulfide isomerase A5, and nuclear distribution gene C homolog were identified by MS based on comparative ABPP analysis. What is more, a FP-rhodamine ABPP probe was used to examine the host serine hydrolases required for HCV replication . After 2D gel, protein spots recognized by the FP-rhodamine ABPP probes were visualized by fluorescence prior to LC/MS/MS analysis. Carboxyl-esterase 1 (CES1) was identified as a differentially active enzyme involved in regulating triglycerides and cholesterol. siRNA knockdown of CES1 further resulted in reduced HCV replication levels and over-expression of CES1 benefited virus replications. To explore different activities of ubiquitin specific proteases under different pathological conditions, Ovaa et al. used HA-tagged Ub-derived active-site-directed probes to comparatively study the enzyme activities in healthy, virus infected, and tumor-derived cells. The altered USPs were purified and followed by LC/MS/MS analysis, resulting in identification of a list of up-regulated USPs in different stages of cellular differentiation (Ovaa et al., 2004) . Furthermore, ABPP could also serve as a novel tool for discovering selective drugs or inhibitors. For instance, tetrahydroquinoline oxocarbazate was characterized as a blocker against SARS coronavirus and Ebola pseudotype virus by inhibiting cathepsin L, a member of human lysosomal cysteine proteases (Shah et al., 2010) . Therefore, this breakthrough technology demonstrated its advantages by functionally identifying enzyme activities associated with a wide range of diseases, as well as filling up the gap that are unreachable to MS based proteomics.