WSSV PROTEINS PROJECT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 白斑综合征病毒新的非结构蛋白VP9的鉴定：其结构揭示了具有特定金属结合位点的铁氧还蛋白折叠 摘要 对虾白斑综合症病毒(WSSV)是对虾养殖业的主要病原。由WSV230开放阅读框编码的WSSV全长蛋白VP9，通过免疫印迹、质谱学和免疫电子显微镜等方法首次在斑节对虾感染组织、鳃和胃中鉴定为一种新的非结构蛋白。实时荧光定量RT-PCR结果表明，VP9的转录始于WSSV感染的早期和晚期，是WSSV的主要基因。全长VP9的结构由2.2°分辨率的X射线确定。VP9是第一个被报道的WSSV蛋白结构。VP9的晶体结构显示具有二价金属离子结合部位的铁氧还蛋白折叠。硫酸镉被发现是结晶所必需的。Cd~(2+)离子结合在均二聚体的单体界面之间。不同的二价金属离子与VP9进行了滴定，并用核磁共振波谱分析了它们之间的相互作用。滴定数据表明，VP9同时与锌和镉结合。VP9采用与乳头瘤病毒E2蛋白DNA结合区相似的折叠。根据我们目前的研究，我们推测VP9可能参与了WSSV的转录调控，在乳头状瘤病毒感染宿主细胞过程中，VP9的功能类似于E2蛋白。 白斑综合征病毒两种主要囊膜蛋白VP26和VP28的晶体结构揭示了它们之间的进化关系 摘要 对虾白斑综合征病毒(WSSV)是一种能感染对虾的强毒病原体。它具有杆状形态，一端有尾状附属物。包膜由四种主要蛋白质组成。包膜结构蛋白在病毒感染中起着至关重要的作用，被认为是第一个与宿主相互作用的分子。本文报道了WSSV两种主要囊膜蛋白VP26和VP28在2.2和2.0分辨率下的定位和晶体结构。仅这两种蛋白就约占囊膜蛋白的60%，它们的结构代表了WSSV的前两种结构囊膜蛋白。VP26、VP28和其他病毒蛋白的结构比较揭示了WSSV囊膜蛋白与其他病毒结构蛋白之间的进化关系。这两种蛋白质都采用带有突出的N-末端区域的X-桶结构。我们用免疫电子显微镜研究了VP26和VP28的定位。这项研究表明VP26和VP28位于病毒的外表面，并在WSSV包膜中观察到蘑菇状结构，这是对VP26的首次令人信服的观察。有趣的是，VP26和VP28晶体结构的三聚体与蘑菇状结构匹配得很好。根据我们的研究结合文献，我们推测VP26和VP28的N端跨膜区可能锚定在病毒被膜上，使核心跨膜桶突出到被膜外，并可能与宿主受体相互作用或与宿主细胞膜融合，从而有效地转移病毒感染。此外，将三聚体宿主相互作用模式扩展到其他结构相似的病毒蛋白是很有诱惑力的。我们的发现有可能进一步扩展到针对WSSV的药物和疫苗开发。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Identification of a Novel Nonstructural Protein VP9 from White Spot Syndrome Virus: its Structure Reveals a Ferredoxin Fold with Specific Metal Binding Sites Abstract White Spot Syndrome Virus (WSSV) is a major pathogen in shrimp aquaculture. VP9, a full length protein of WSSV, encoded by ORF WSV230, was identified for the first time, in the infected Penaeus monodon shrimp tissues, gill and stomach as a novel, nonstructural protein by western blot, mass spectrometry and immuno-electron microscopy. Real-time RT-PCR demonstrated that the transcription of VP9 started from the early to the late stage of WSSV infection as a major mRNA species. The structure of full length VP9 was determined by X-ray at 2.2¿¿¿ resolution. VP9 is the first structure to be reported for WSSV proteins. The crystal structure of VP9 revealed a ferredoxin fold with divalent metal ion binding sites. Cadmium sulphate was found to be essential for crystallization. The Cd2+ ions were bound between the monomer interfaces of the homodimer. Various divalent metal ions have been titrated against VP9 and their interactions were analyzed using NMR spectroscopy. The titration data indicated that VP9 binds with both Zn2+ and Cd2+. VP9 adopts a similar fold as the DNA binding domain of papillomavirus E2 protein. Based on our present investigations, we hypothesize that VP9 might be involved in the transcriptional regulation of WSSV, a function similar to E2 protein during papillomavirus infection of the host cells. Crystal Structure of Two Major Envelope Proteins VP26 and VP28 from White Spot Syndrome Virus (WSSV) Shed Light on their Evolutionary Relationship Abstract White spot syndrome virus (WSSV) is a virulent pathogen known to infect penaeid shrimp. It has bacilliform morphology with a tail-like appendage at one end. The envelope consists of four major proteins. Envelope structural proteins play a crucial role in viral infection and are believed to be the first molecules to interact with the host. Here we report the localization and crystal structure of two major envelope proteins VP26 and VP28 from WSSV at 2.2 and 2.0¿¿¿ resolutions respectively. These two proteins alone account for approximately 60% of the envelope and their structures represent the first two structural envelope proteins of WSSV. Structural comparisons among VP26, VP28 and other viral proteins reveal an evolutionary relationship between WSSV envelope proteins and structural proteins from other viruses. Both proteins adopt ¿¿¿x-barrel architecture with a protruding N-terminal region. We have investigated the localization of VP26 and VP28 using immuno-electron microscopy. This study suggests that VP26 and VP28 are located on the outer surface of the virus and are observed as a mushroom-like structure in the WSSV envelope and this is the first convincing observation for VP26. Interestingly, the trimer of the VP26 and VP28 crystal structures matches well with the mushroom-like structures. Based on our studies combined with the literature, we speculate that the predicted N-terminal transmembrane region of VP26 and VP28 may anchor on the viral envelope membrane making the core ¿¿¿x-barrel to protrude outside the envelope and possibly to interact with the host receptor or to fuse with the host cell membrane for effective transfer of the viral infection. Furthermore, it is tempting to extend the trimer host interaction mode to other structural viral proteins of similar structures. Our finding has the potential to extend further towards drug and vaccine development against WSSV.