Current Approaches on Viral Infection: Proteomics and Functional Validations

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

Viruses associated with carcinomas and tumors have presented high risks and occupied an important branch of cancer diagnostics. Deciphering virus and host interplays by Co-IP based proteomics could gain tremendous insights into malignant transformations of carcinomas as well as cellular differentiation and proliferations encountered. In addition, studying the effects of those negative mutants could greatly enhance the validity of proteomic data. Adenovirus (Adv) was a DNA virus model suitable for studying tumor oncogenesis, Komorek et al. (2010) successfully utilized TAP based proteomics to identify multiple cellular proteins interacting with the Adv oncoprotein E1A C-terminal region. The forkhead transcription factors, FOXK1/K2, were identified as novel factors specifically bound to E1A. Numerous recombinant adenoviruses carrying distinctive E1A exon 1 and exon 2 mutants were generated in order to map the specific E1A domain interacting with FOXK1/K2 by western blot analysis. One mutant lacking amino acids 224-238 in exon 2 was found to have significantly reduced staining with FOXKI/K2. Functionally, virus bearing this E1A exon 2 mutant was deficient in association with FOXK1/K2, resulting in enhanced cell proliferation and oncogenic transformation. Thus this indicated that the interaction between FOXK1/K2 with exon 2 of E1A could reversely suppress cell proliferation and oncogenic transformation. Human Papillomavirus (HPV) is another DNA virus causing anogenital carcinomas and oropharyngeal squamous cell carcinomas. The viral gene E7 is retained and integrated into the cancer cell chromosomes. Therefore its E7 oncoprotein plays important role in mediating malignant transformation. In one study, in order to find the novel binding partners of the E7 protein, a recombinant E7 from HPV-16 was constructed by tagging S. japonicum GST to its N-terminal, which could be recognized by immobilized glutathione. Human glutathione S-transferase P1-1 (GSTP1) was uniquely identified by MS. The interaction between E7 and GSTP1 was structurally characterized by three-dimensional docking program, which assisted to design a E7 mutant deficient in affinity binding with GSTP1 by subtracting residues Val 55, Phe 57, and Met 84. Although real-time PCR showed similar translation levels of GSTP1 in E7 and mutant group, GSTP1 in HPV E7 expressing cells apparently enhanced cell pro-survival abilities by suppressing Jun N-terminal kinase (JNK) mediated-phosphorylation signaling pathway to induce apoptosis. And siRNA knockdown of GSTP1 in HPV E7 expressing cells could reversely counteract this effect (Mileo et al., 2009) . Viral proteins important for manipulating viral gene expressions could be post-translationally modified, playing essential role as intrinsic functional proteins and cofactors assisting or inhibiting virus infection. Validations with viral PTM-deficient proteins by substituting PTM sites enable us to make a comparative study with the control group, uncovering the impact of these modified proteins at the functional level. For instance, ICP27 is one viral regulatory protein of herpes simplex virus type 1 (HSV-1). It is known to be post-translationally modified by kinases and arginine methyltransferases, and closely involved in viral protein export (Sandri-Goldin and Hibbard, 1996) . ICP27 bears a glycineand arginine-rich (GAR) region within an RGG box, which is characterized as an RNA binding domain mediating viral protein export. Souki et al. empl