Search

Singapore grouper iridovirus (SGIV) is a lethal grouper virus containing 162 predicted ORFs. Previous proteomic studies led to identification of 73 SGIV structural proteins. Here, SDS-assisted tube-gel digestion and DOC-assisted in-solution digestion coupled with LC-ESI-MS/MS were applied to further profile the SGIV structural proteome. We identified a total of 90 SGIV structural proteins including 24 newly reported proteins. Additionally, several PTMs were identified, including 26 N-terminal acetylated proteins, three phosphorylated proteins, and one myristoylated protein. Importantly, 47 of the proteins that were identified are predicted to contain conserved domains. Our work greatly expands the repertoire of the SGIV structural proteome and provides more insight into the biology of SGIV. [ABSTRACT FROM AUTHOR]

• This study is the first to explore the interactome of SGIV ICP18 using the proximity-dependent BioID method. • A total of 112 cellular proteins including CDK1 is identified as candidate ICP18-interacting proteins. • ICP18 may be involved in modulating various cellular processes such as cell cycle and cell adhesion. Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that is a major threat to grouper aquaculture. The pathogenesis of SGIV is not well understood so far. Previous studies have revealed that ICP18, an immediate early protein encoded by SGIV ORF086R gene, promotes viral replication by regulating cell proliferation and virus assembly. In the present study, the potential functions of ICP18 were further explored by probing into its interactors using a proximity-dependent BioID method. Since our in-house grouper embryonic cells (a natural host cell of SGIV) could not be efficiently transfected with the plasmid DNA, and the grouper genome data for mass spectrometry-based protein identification is not currently available, we chosen a non-permissive cell (HEK293 T) as a substitute for this study. A total of 112 cellular proteins that potentially bind to ICP18 were identified by mass spectrometry analysis. Homology analysis showed that among these identified proteins, 110 candidate ICP18-interactors had homologous proteins in zebrafish (a host of SGIV), and shared high sequence identity. Further analysis revealed that the identified ICP18-interacting proteins modulate various cellular processes such as cell cycle and cell adhesion. In addition, the interaction between ICP18 and its candidate interactor, i.e., cyclin-dependent kinase1 (CDK1), was confirmed using Co-immunoprecipitation (Co-IP) and Pull-down assays. Collectively, our present data provides additional insight into the biological functions of ICP18 during viral infection, which could help in further unraveling the pathogenesis of SGIV. [ABSTRACT FROM AUTHOR]

We report, here, the first proteomics study of a grouper embryonic cell line (GEC) infected by Singapore grouper iridovirus (SGIV). The differential proteomes of GEC with and without viral infection were studied and quantified with iTRAQ labelling followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Forty-nine viral proteins were recognized, of which 11 were identified for the first time. Moreover, 743 host proteins were revealed and classified into 218 unique protein groups. Fourteen host proteins were upregulated and five host proteins were downregulated upon viral infection. The iTRAQ analysis of SGIV infection in GEC provides an insight to viral and host gene products at the protein level. This should facilitate further study and the understanding of virus-host interactions, molecular mechanisms of viral infection and pathogenesis.

Colorectal cancer is the second leading killer cancer worldwide and presently the most common cancer among males in Singapore. The study aimed to detect changes of protein profiles associated with the process of colorectal tumorigenesis to identify specific protein markers for early colorectal cancer detection and diagnosis or as potential therapeutic targets. Seven pairs of colorectal cancer tissues and adjacent normal mucosa were examined by two-dimensional gel electrophoresis at basic pH range (pH 7-10). Intensity changes of 34 spots were detected with statistical significance. 16 of the 34 spots were identified by MALDI-TOF/TOF tandem mass spectrometry. Changes in protein expression levels revealed a significantly enhanced glycolytic pathway (Warburg effect), a decreased gluconeogenesis, a suppressed glucuronic acid pathway, and an impaired tricarboxylic acid cycle. Observed changes in protein abundance were verified by two-dimensional DIGE. These changes reveal an underlying mechanism of colorectal tumorigenesis in which the roles of impaired tricarboxylic acid cycle and the Warburg effect may be critical.

The Singapore grouper iridovirus (SGIV) genome consists of a double-stranded circular DNA of 140,131 base pairs with 162 predicted open reading frames. Our earlier study using peptide mass fingerprints generated from MALDI-TOF MS led to the identification of 26 viral proteins. The present investigation aimed to achieve a more comprehensive and precise identification of the SGIV viral proteome by two workflows: one-dimensional gel electrophoresis (1-DE) separation followed by protein identification by MALDI-TOF/TOF MS/MS (1-DE-MALDI workflow) and shotgun proteomics in which the whole virus was digested by trypsin and the resulting peptides were separated by nano-LC and analyzed by MALDI-TOF/TOF MS/MS (LC-MALDI workflow). In total, 44 viral proteins were identified, 25 of which were reported for the first time. Fourteen proteins were uniquely identified by the 1-DE-MALDI workflow, whereas another 10 proteins were only identified by the LC-MALDI workflow with 20 proteins found by both approaches. Moreover 13 proteins were found to have acetylated N termini. Twenty-three proteins identified contain predicted transmembrane domains, accounting for 52.3% of the total proteins identified. RT-PCR confirmed the transcription products of all the identified viral proteins. A large number of proteins identified by both the 1-DE-MALDI and the LC-MALDI workflows from this study have significantly enhanced the coverage of the SGIV proteome. The SGIV proteome is at present the only iridoviral proteome that has been extensively characterized. Our results should provide further insights into the biology of SGIV and other iridoviruses.