PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp
PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp
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DOI：
10.1128/jvi.00349-06
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发表时间：
2006-11-01
影响因子：
5.4
通讯作者：
Flegel, Timothy W.
中科院分区：
文献类型：
--
作者：
Sritunyalucksana, Kallaya;Wannapapho, Wanphen;Flegel, Timothy W.
我们的目的是从对虾中分离和鉴定白斑综合症病毒(WSSV)结合蛋白。用重组WSSV囊膜蛋白(RVP28)包被对虾血细胞膜蛋白印迹后，用抗VP28抗体检测印迹上的反应带。在液-质联用鉴定的三个膜相关分子中，有一个25 kDa的蛋白同时与rVP28和WSSV结合。由于其一级结构与小分子GTP结合蛋白Rab7高度同源，我们将其命名为斑节对虾Rab7(PmRab7)。获得了全长的PmRab7基因，谷胱甘肽S转移酶下拉实验证实了其与rVP28的特异性结合。逆转录聚合酶链式反应检测到PmRab7在多种组织中均有表达，实时荧光定量聚合酶链式反应分析表明该基因在多种组织中均有表达。PmRab7与rVP28或WSSV的结合呈剂量依赖关系，并可被抗Rab7抗体抑制。在体内中和试验中，WSSV+PmRab7(15%)或WSSV+抗Rab7抗体(5%)攻击后的死虾数显著低于WSSV单独攻击后(95%)。与WSSV注射组相比，WSSV+PmRab7组和WSSV+抗Rab7组未见WSSV型组织病理学改变。结论：PmRab7基因与对虾WSSV感染有关。这是第一次鉴定出直接与WSSV主要病毒包膜蛋白结合的虾蛋白。
Our aim was to isolate and characterize white spot syndrome virus (WSSV)-binding proteins from shrimp. After a blot of shrimp hemocyte membrane proteins was overlaid with a recombinant WSSV envelope protein (rVP28), the reactive bands on the blot were detected using anti-VP28 antibody. Among three membrane-associated molecules identified by liquid chromatography-tandem mass spectrometry, there was a 25-kDa protein that bound to both rVP28 and WSSV. Since it had a primary structure with high homology to the small GTP-binding protein Rab7, we named it Penaeus monodon Rab7 (PmRab7). The full-length PmRab7 cDNA was obtained, and results from a glutathione S-transferase pull-down assay confirmed specific binding to rVP28. Reverse transcriptase PCR analysis revealed PmRab7 expression in many tissues, and real-time PCR analysis revealed that expression was constitutive. Binding of PmRab7 to rVP28 or WSSV occurred in a dose-dependent manner and was inhibited by anti-Rab7 antibody. In an in vivo neutralization assay, the number of dead shrimp after challenge with WSSV plus PmRab7 (15%) or WSSV plus anti-Rab7 antibody (5%) was significantly lower than after challenge with WSSV alone (95%). In contrast to the WSSV-injected group, shrimp injected with WSSV plus PmRab7 or WSSV plus anti-Rab7 showed no WSSV-type histopathology. We conclude that PmRab7 is involved in WSSV infection in shrimp. This is the first study to identify a shrimp protein that binds directly to a major viral envelope protein of WSSV.