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.
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