Construction and Application of a Protein Interaction Map for White Spot Syndrome Virus (WSSV) |
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Authors: | Pakkakul Sangsuriya Jiun-Yan Huang Yu-Fei Chu Kornsunee Phiwsaiya Pimlapas Leekitcharoenphon Watcharachai Meemetta Saengchan Senapin Wei-Pang Huang Boonsirm Withyachumnarnkul Timothy W Flegel Chu-Fang Lo |
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Institution: | 3. Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand;;4. Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand;;5. Institute of Zoology, National Taiwan University, Taipei, Taiwan, Republic of China;;6. National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, 12120, Thailand;;12. Department of Anatomy, Faculty of Science, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand;;9. Institute of Bioinformatics and Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan, Republic of China |
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Abstract: | White spot syndrome virus (WSSV) is currently the most serious global threat for cultured shrimp production. Although its large, double-stranded DNA genome has been completely characterized, most putative protein functions remain obscure. To provide more informative knowledge about this virus, a proteomic-scale network of WSSV-WSSV protein interactions was carried out using a comprehensive yeast two-hybrid analysis. An array of yeast transformants containing each WSSV open reading frame fused with GAL4 DNA binding domain and GAL4 activation domain was constructed yielding 187 bait and 182 prey constructs, respectively. On screening of ∼28,000 pairwise combinations, 710 interactions were obtained from 143 baits. An independent coimmunoprecipitation assay (co-IP) was performed to validate the selected protein interaction pairs identified from the yeast two-hybrid approach. The program Cytoscape was employed to create a WSSV protein–protein interaction (PPI) network. The topology of the WSSV PPI network was based on the Barabási-Albert model and consisted of a scale-free network that resembled other established viral protein interaction networks. Using the RNA interference approach, knocking down either of two candidate hub proteins gave shrimp more protection against WSSV than knocking down a nonhub gene. The WSSV protein interaction map established in this study provides novel guidance for further studies on shrimp viral pathogenesis, host-viral protein interaction and potential targets for therapeutic and preventative antiviral strategies in shrimp aquaculture.White spot syndrome virus (WSSV)1 is the causative agent of white spot disease (WSD) and is one of the most serious viral pathogens that threaten the shrimp culture industry worldwide. Because WSD causes rapid and high mortality up to 100% within 3–10 days after viral infection (1), it causes dramatic economic losses on farms. WSSV is a large enveloped, ovoid to bacilliform, double-stranded DNA (dsDNA) virus with a genome of ∼300 kb (See reviews in (2, 3)). The WSSV genome has been completely characterized for isolates from Thailand (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AF369029","term_id":"58866698","term_text":"AF369029"}}AF369029), China (accession number {"type":"entrez-nucleotide","attrs":{"text":"AF332093","term_id":"721172032","term_text":"AF332093"}}AF332093) and Taiwan (accession number {"type":"entrez-nucleotide","attrs":{"text":"AF440570","term_id":"19481591","term_text":"AF440570"}}AF440570). To expand its basic genetic information, various genomic and proteomic approaches have been applied to gain more insight into the molecular mechanisms of WSSV pathogenesis (See reviews in (2, 3)). However, the roles of most of the WSSV proteins still remain to be elucidated. This is due to the fact that many of its putative open reading frames (ORFs) lack homology to known proteins in the database. Protein–protein interaction studies can provide a valuable framework for understanding the roles of protein functions. Interaction studies of WSSV proteins have particularly focused on viral structural proteins (4–15). However, so far there has been no report on a protein–protein interaction (PPI) network for WSSV or any other crustacean virus. By contrast, several PPI networks for cellular organisms such as Saccharomyces cerevisiae (16, 17), Helicobacter pylori (18), Drosophila melanogaster (19), Caenarhabitis elegans (20), Plasmodium falciparum (21), and Homo sapiens (22, 23) and pathogens such as bacteriophage T7 (24), vaccinia virus (25), hepatitis C virus (26), and herpesviruses (27–29) have already been established. Therefore, the present study aimed to obtain a more fundamental understanding of WSSV protein interactions. A comprehensive yeast two-hybrid assay was employed to generate viral fusion proteins with DNA binding (BD) and activation (AD) domains in an array format that effectively allowed searching every possible binary interaction in WSSV. The interaction results from the yeast two-hybrid assays were subsequently validated by coimmunoprecipitation (co-IP). Topological properties of the WSSV PPI network were assessed and compared with previously published viral networks. Candidate viral hub proteins with high numbers of interacting partners were identified in this study and their significance was investigated using an RNA interference approach. |
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