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 open reading frame wsv230, was identified for the first time in the infected Penaeus monodon shrimp tissues, gill, and stomach as a novel, nonstructural protein by Western blotting, mass spectrometry, and immunoelectron microscopy. Real-time reverse transcription-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 both X-ray and nuclear magnetic resonance (NMR) techniques. It 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 sulfate was found to be essential for crystallization. The Cd 2؉ 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 Zn 2؉ and Cd 2؉ . VP9 adopts a similar fold as the DNA binding domain of the 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 that of the E2 protein during papillomavirus infection of the host cells.Shrimp aquaculture has become an important industry worldwide during the last few decades. Intensive cultivation and massive worldwide trade of the shrimp and other aquaculture industries have led to the emergence and spread of numerous viral pathogens. Out of these viral pathogens, shrimp white spot syndrome virus (WSSV) is the most devastating to crustacean species. To date, there is no effective treatment available for WSSV infection.WSSV belongs to a new virus family, Nimaviridae, under a new genus, Whispovirus, which shares low sequence homology with other DNA viruses (16,24). WSSV is an enveloped virus with a 305-kb double-stranded circular DNA genome. Its genome was completely sequenced, with 180 predicted open reading frames (ORFs) (27). Most of these WSSV genes bear poor sequence homology with other known proteins, and thus the function of these genes cannot be predicted. However, a large amount of work has been carried out on the identification and characterization of WSSV structural proteins, including envelope and other capsid proteins. To date, a total of 39 structural proteins are known from WSSV (12, 23). A structure-based functional study is a promising approach to elucidate the function of such WSSV proteins. Besides the envelope and capsid proteins, nonstructural proteins are required for replication of the viral genome, production of the virus particle, and inhibition of the host cell functions. These proteins are therefore potential candidates for drug design and the development of vaccines.Here we report, for the first time, the identification of a novel, nonstructural WSSV protein, VP9. We have demon...

White Spot Syndrome Virus Proteins and Differentially Expressed Host Proteins Identified in Shrimp Epithelium by Shotgun Proteomics and Cleavable Isotope-Coded Affinity Tag

“…Full-length wsv051, -076, -294, and -477 genes were cloned into pET-32a (ϩ) vector (Novagen Inc.) and expressed in Escherichia coli BL21, and protein expression was confirmed by both DNA sequencing and MALDI-TOF-MS. Purified fusion proteins were subjected to SDS-PAGE analysis prior to immunizing rabbits to generate polyclonal antibodies. Polyclonal antibodies against WSSV VP9, VP26, and VP28 were generated previously (28,40). Anti-VP664 was kindly provided by C.-F.…”

“…The following antibodies were purchased: anti-actin (A2066; Sigma), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (FL-335; Santa Cruz Biotechnology), and anti-histone 2A (H-124; Santa Cruz Biotechnology). Western blotting was carried out as described elsewhere (28).…”

“…However, our knowledge of viral nonstructural proteins and the host cellular response during WSSV infection remains poor. To date, only a few nonstructural proteins, encoded by highly conserved gene sequences, such as DNA polymerase (9), ribonucleotide reductase (27), and others (14,16,18,27,28,47), have been confirmed by traditional gene cloning and immunoassays. Differential expression of host proteins was mainly investigated at the mRNA level using cDNA microarrays and expressed sequence tags (11,12,17,36,39,44).…”

Shotgun Identification of the Structural Proteome of Shrimp White Spot Syndrome Virus and iTRAQ Differentiation of Envelope and Nucleocapsid Subproteomes

“…The reliability of our findings was verified by ORF-specific RT-PCR for those newly identified proteins. Surprisingly wsv230, which was defined as nonstructural protein VP9 (wsv230) in a previous study (35), was detected in the virion by both shotgun and iTRAQ approaches. The postulation that wsv230 is a non-structural protein was based on the Western blot and IEM results.…”

Detection of White Spot Syndrome Virus in Brazil using Negative Staining, Immunoelectron Microscopy and Immunocytochemistry Techniques

“…In other experiments, WSSV particles were enhanced by this technique (Wu & Yang;Liu et al, 2006;Tsai et al, 2006;Chang et al, 2008).…”