RING-H2 Protein WSSV249 from White Spot Syndrome Virus Sequesters a Shrimp Ubiquitin-Conjugating Enzyme, PvUbc, for Viral Pathogenesis

Wang, Zhilong; Chua, H.; Gusti, Ayu A. R. A.; He, Fang; Fenner, Beau J.; Manopo, Ivanus; Wang, Hai; Kwang, Jimmy

doi:10.1128/jvi.79.14.8764-8772.2005

Cited by 56 publications

(51 citation statements)

References 63 publications

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“…Recent research on WSSV structure using proteomic methods has revealed that its structural proteins have increased in number to 39 (Tsai et al 2004). Now, research activities have begun to focus on the protein-protein interactions between the shrimp and virus, between shrimp proteins, and between virus proteins (Lu & Kwang 2004, Tonganunt et al 2005, Wang et al 2005, Xie & Yang 2005. According to previous studies by Tsai et al (2004Tsai et al ( , 2006, VP53A encoded by WSSV gene wssv067 is an envelope protein.…”

Section: Introductionmentioning

confidence: 99%

White spot syndrome virus envelope protein VP53A interacts with Penaeus monodon chitin-binding protein (PmCBP)

Chen¹,

Lü²,

Wu³

et al. 2007

Dis. Aquat. Org.

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White spot syndrome virus (WSSV) is the causative agent of a severe disease of cultivated shrimp. Using purified WSSV virions, VP53A encoded by open reading frame wssv067 was identified as a structural protein by SDS-PAGE and proteomics. Immunoelectron microscopy with a gold-labeled secondary antibody revealed that VP53A was distributed on the viral envelope. In order to further explore the link between WSSV067 and host proteins, we performed a yeast 2-hybrid screening of a Penaeus monodon cDNA library, using WSSV067C as bait. One of the molecules that specifically interacted with WSSV067C was the P. monodon chitin-binding protein (PmCBP). An in vitro binding assay showed that c-myc-WSSV067C was capable of co-precipitating HA-PmCBP-C. Furthermore, PmCBP was expressed in almost all organs but appeared to be up-regulated at the late stage of WSSV infection.KEY WORDS: Penaeus monodon · White spot syndrome virus · VP53A · Envelope protein · Chitin-binding protein Resale or republication not permitted without written consent of the publisherDis Aquat Org 74: [171][172][173][174][175][176][177][178] 2007 2000, Liu et al. 2001). This virus source has been maintained in our laboratory since 1994 (GenBank Accession No. AF440570). It was previously known simply as the WSSV Taiwan isolate (Lo et al. 1999), but to distinguish it from other WSSV Taiwan isolates, it will henceforth be referred to as the WSSV Taiwan-1 strain (WSSV T-1 strain). WSSV T-1 strain was used as the basis for all of the WSSV genome sequence research conducted in the present study.Antibody preparation. The DNA sequence encoding 200 deduced amino acids from the WSSV067 C-terminus was amplified from WSSV-infected shrimp by PCR with the 067CFBamHI/067CRSalI primer set (5'-GGGGATCCGATAACGGTTGAAGGAGTC-3'/5'-GG AAGCTTTTACATCAACACTGTAACTGC-3'; the underlined bases indicate respectively the BamHI and HindIII restriction sites that were used for subsequent cloning) and ligated to pGEM-T Easy (Promega). After confirming the sequence, the resulting plasmid pGEM-T-VP53AC was cleaved with BamH1 and Sal1, and the amplified fragment was then cloned to pET28b(+) (Novagen) at BamHI and HindIII sites. The resulting pET-VP53AC clone was transformed into BL21 Codon Plus Escherichia coli cells (Stratagene). For protein expression and purification, cells were grown overnight at 37°C in Luria-Bertani (LB) media supplemented with 50 µg ml -1 kanamycin and 34 µg ml -1 chloramphenicol. The cells were inoculated into new media at a ratio of 1:300, and grown at 37°C for 1.5 to 2 h. Expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubation was continued for another 1.5 to 3 h. The induced bacteria were spun down at 4°C, suspended in ice-cold phosphate-buffered saline (PBS) containing 10% glycerol and a Protease Inhibitor Cocktail tablet (Roche Molecular BioChemicals), and sonicated for 30 s on ice. The insoluble debris was collected by centrifugation, suspended with PBS containing 1.5% sodium lauryl sarcosine, and solubiliz...

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Section: Introductionmentioning

confidence: 99%

White spot syndrome virus envelope protein VP53A interacts with Penaeus monodon chitin-binding protein (PmCBP)

Chen¹,

Lü²,

Wu³

et al. 2007

Dis. Aquat. Org.

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“…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).…”

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confidence: 99%

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

Lin

Lim

et al. 2007

J Virol

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Shrimp subcuticular epithelial cells are the initial and major targets of white spot syndrome virus (WSSV) infection. Proteomic studies of WSSV-infected subcuticular epithelium of Penaeus monodon were performed through two approaches, namely, subcellular fractionation coupled with shotgun proteomics to identify viral and host proteins and a quantitative time course proteomic analysis using cleavable isotope-coded affinity tags (cICATs) to identify differentially expressed cellular proteins. Peptides were analyzed by offline coupling of two-dimensional liquid chromatography with matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. We identified 27, 20, and 4 WSSV proteins from cytosolic, nuclear, and membrane fractions, respectively. Twentyeight unique WSSV proteins with high confidence (total ion confidence interval percentage [CI%], >95%) were observed, 11 of which are reported here for the first time, and 3 of these novel proteins were shown to be viral nonstructural proteins by Western blotting analysis. A first shrimp protein data set containing 1,999 peptides (ion score, >20) and 429 proteins (total ion score CI%, >95%) was constructed via shotgun proteomics. We also identified 10 down-regulated proteins and 2 up-regulated proteins from the shrimp epithelial lysate via cICAT analysis. This is the first comprehensive study of WSSV-infected epithelia by proteomics. The 11 novel viral proteins represent the latest addition to our knowledge of the WSSV proteome. Three proteomic data sets consisting of WSSV proteins, epithelial cellular proteins, and differentially expressed cellular proteins generated in the course of WSSV infection provide a new resource for further study of WSSV-shrimp interactions.White spot syndrome virus (WSSV) has been a catastrophic pathogen of cultured peneid shrimps since its first appearance in the early 1990s (32). The initial and major target of this virus is shrimp epithelia, including subcuticular, stomach, and gill epithelia. WSSV-infected epithelial cells show hypertrophied nuclei containing massive amounts of viruses (26). Genomic studies revealed that the virus consists of a double-stranded DNA of about 300 kbp with more than 180 predicted open reading frames (ORFs) (9,43,54). So far, the majority of proteins encoded by the predicted ORFs have not been detected, and functions of many of these presumptive proteins remain elusive. Information on virus-host interactions is therefore very limited.Proteomics has been demonstrated to be an important platform technology and has contributed to our understanding of virus-host interaction (4, 37). Shotgun two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) is a promising approach for high-throughput identification of proteins (21, 48). Cleavable isotope-coded affinity tags (cICATs) coupled with 2D-LC-MS/MS enable the quantitative pairwise comparison of protein expression levels in uninfected and infected cells (7,15). Previous proteomic studies on WSSV had identified more than 40 ...

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“…The E3 ligase activity of WSSV277 is involved in regulation of antiapoptosis by ubiquitin-mediated degradation of tumor suppressor-like (TSL) protein (12), and it is required for efficient WSSV replication (14). WSSV304 serves as a ubiquitin ligase E3 to sequester a shrimp ubiquitin-conjugating enzyme, Penaeus vannamei Ubc (PvUbc), for viral pathogenesis (31). WSSV462, which has E3 ligase function, is a regulator for the latency state of WSSV (13).…”

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confidence: 99%

“…AAL89172.1), and WSSV462 (GenBank accession no. AAL89330.1), contain the RING-H2 motifs and may function as the E3 ligases (31). The E3 ligase activity of WSSV277 is involved in regulation of antiapoptosis by ubiquitin-mediated degradation of tumor suppressor-like (TSL) protein (12), and it is required for efficient WSSV replication (14).…”

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Enzyme E2 from Chinese White Shrimp Inhibits Replication of White Spot Syndrome Virus and Ubiquitinates Its RING Domain Proteins

Chen

Wang

Zhao

et al. 2011

J Virol

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Recent studies have shown that the ubiquitin (Ub) proteasome pathway (UPP) is closely related to immune defense. We have identified a ubiquitin-conjugating enzyme, E2, from the Chinese white shrimp, Fenneropenaeus chinensis (FcUbc). Injection of recombinant FcUbc protein (rFcUbc) reduced the mortality of shrimp infected with white spot syndrome virus (WSSV) and inhibited replication of WSSV. rFcUbc, but not a mutant FcUbc (mFcUbc), bound to WSSV RING domains (WRDs) from four potential E3 ligase proteins of WSSV in vitro. Importantly, rFcUbc could ubiquitinate the RING domains (named WRD2 and WRD3) of WSSV277 and WSSV304 proteins in vitro and the two proteins in WSSV-infected Drosophila melanogaster Schneider 2 (S2) cells. Furthermore, overexpression of FcUbc increased ubiquitination of WSSV277 and WSSV304 during WSSV infection. In summary, our study demonstrates that FcUbc from Chinese white shrimp inhibited WSSV replication and could ubiquitinate WSSV RING domain-containing proteins. This is the first report about antiviral function of Ubc E2 in shrimp.

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RING-H2 Protein WSSV249 from White Spot Syndrome Virus Sequesters a Shrimp Ubiquitin-Conjugating Enzyme, PvUbc, for Viral Pathogenesis

Cited by 56 publications

References 63 publications

White spot syndrome virus envelope protein VP53A interacts with Penaeus monodon chitin-binding protein (PmCBP)

White spot syndrome virus envelope protein VP53A interacts with Penaeus monodon chitin-binding protein (PmCBP)

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

Enzyme E2 from Chinese White Shrimp Inhibits Replication of White Spot Syndrome Virus and Ubiquitinates Its RING Domain Proteins

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