Current Approaches on Viral Infection: Proteomics and Functional Validations

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

What is more, some host proteins could also balance their distinctive PTM sites, and therefore manipulate cellular immune responses upon virus infections. Retinoic acid-inducible gene I (RIG-I) protein is one cytosolic receptor sensitive to viral RNAs. And this recognition could further undergo ubiquitination at Lys72, which enables to induce the innate immune responses, e.g., type-I IFNs, to inhibit the viral replication. Whereas under the normal condition, some kinases could phosphorylate RIG-I and thereby inhibit ubiquitination and downstream antiviral signal transduction. To map the phosphorylation sites on RIG-I, Gack et al. constructed the GST fusion vector inserted with the RIG-I PCR product, which was expressed in HEK293T cells (Gack et al., 2010) . By employing GST pull-down and LC-MS/MS analysis, three phosphorylation sites in the N-terminal caspase recruitment domains (CARDs) of RIG-I were identified. It was hypothesized that phosphorylation at T170 suppresses ubiquitination at Lys172. The T170E mutant was generated to explore this hypothesis under normal circumstances, the phosphorylation level of RIG-I was markedly reduced after virus infection whereas its ubiquitination level was increased in a time-dependent manner. However, the T170E mutant lacked binding ability with tripartite motif protein 25 (TRIM25), which could induce ubiquitination and IFN signal transduction. Furthermore, Maharaj et al. (2012) successfully identified two upstream kinases, PKC -α and C-β that are responsible for phosphorylation of the RIG-I protein. Double knockdown of PKC-α/β by shRNA or siRNA revealed remarkably decreased phosphorylation levels, resulting in increased susceptibility of cells to virus infection.