Current Approaches on Viral Infection: Proteomics and Functional Validations

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

oyed Co-IP approach to purify ICP27 in virus infected cells and performed independent trypsin, pepsin, and thermolysin digestion to comprehensively map the sequence of ICP27 by MALDI TOF/TOF MS (Souki et al., 2009) . As a result, this combined protease digestion method was able to detect peptides covering of 90% of the ICP27 sequence, including the major arginine methylation sites, e.g., arginines 138, 148, and 150 within the RGG box. To functionally study the arginine methylation associated with virus infection, site-directed mutagenesis was used to construct point mutation (arginine to lysine) in ICP27, which was inserted in a plasmid and co-transfected with viral DNA into cells. The R150K mutant exhibited the most delayed virus maturations as well as smallest plaque size. Also, microarray assay revealed that both viral gene expression and replication were reduced in the mutants compared to the wild type, suggesting that the role of arginine methylation of ICP27 is closely associated with virus trafficking, assembly, as well as virus genome transcriptions. Similarly, phosphoprotein (P) of parainfluenza virus 5 (PIV5) was heavily phosphorylated and it was responsible for viral RNA synthesis upon infection (Sun et al., 2011b) . IP was carried out to bait the viral P protein by using immobilized anti-V5-conjugated agarose, followed by SDS-PAGE gel and LC-MS/MS analysis. T286 of the P protein was found to be phosphorylated and it was further substituted by alanine, aspartic acid, or glutamic acid to study the effects of P mutants to viral infection. Compared to the wide type, the virus carrying the mutant P protein T286A was observed to have a slower growth rate as well as delayed viral mRNA synthesis at the transcription level. This work demonstrated that phosphorylation of the PIV5 P protein is important for virus replication and formation; however the detailed mechanism of key kinases involved in signaling pathways remains elusive. In another study, Duellman et al. (2009) used immobilized metalaffinity chromatography (IMAC) to enrich the phosphorylated peptides of EBV nuclear antigen 1 (EBNA1) followed by nano LC-MS/MS analysis, which resulted in identification of 10 phosphosites on EBNA1. In the subsequent validation works, all the 10 phosphosites were mutated to alanine by PCR mutagenesis and constructs were amplified in stable 293T cells after transfection. The phosphorylation of EBNA1 was essential to virus transcriptions as its phosphorylation-deficient mutant revealed a reduction on transcription activities.