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.
Highlights
From the ‡Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand; §Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand; ¶Institute of Zoology, National Taiwan University, Taipei, Taiwan, Republic of China; ʈNational Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, 12120, Thailand; **Shrimp Genetic Improvement Center, Surat Thani 84100, Thailand; ‡‡Department of Anatomy, Faculty of Science, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand; ¶¶Institute of Bioinformatics and Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan, Republic of China
The White spot syndrome virus (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
Construction of WSSV Protein Arrays—WSSV proteins fused with the GAL4 DNA-binding domain (BD) or activating domain (AD) were arranged in a matrix or array format to facilitate the search for pairwise protein interactions
Summary
WSSV and Shrimp—The WSSV Taiwan isolate (WSSV-TW) originally isolated from a batch of WSSV-infected Penaeus monodon collected in Taiwan in 1994 [30, 31] was used as the template for WSSV ORFs amplification. In the second reaction WSSV fragments were amplified to contain flanking regions homologous to the yeast two-hybrid vectors, pGBKT7–1 and pGADT7. At 2 days post injection (dpi) of dsRNA or NaCl, shrimp in each group (24 –34 individuals each) were challenged with crude WSSV stock at a 3-day LD50 dose. PCR reactions were carried out in a 20 l reaction solution containing 500 ng of DNA sample, 0.2 M of each forward and reverse primer, 200 M of dNTP mix, 1.5 mM of MgCl2, 2.5 U of TaqDNA polymerase (Invitrogen), and 1ϫ reaction buffer. Amplification of shrimp -actin using primers listed in Table I and the same reaction components and thermal cycling protocol described for ICP11 above served as an internal control
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