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

Abstract

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...

Characterization of a prophenoloxidase from hemocytes of the shrimp Litopenaeus vannamei that is down-regulated by white spot syndrome virus

“…1). Comparison of proPO-a and proPO-b sequences indicated that both had a microsatellite DNA site at 3 0 -UTR, but the microsatellite DNA of proPO-b contained a compound imperfect SSR, ((CT) 38 (CA) 8 (AA)(CA) 3 (TA)(CA) 14 ), which differed from the perfect SSR ((CT) 20 ) from proPO-a. The cDNA sequences of proPO-a and proPO-b prior to the microsatellite DNA site were almost identical (99% identity), but differed after the microsatellite DNA site (Fig.…”

Penaeus monodon chitin-binding protein (PmCBP) is involved in white spot syndrome virus (WSSV) infection

“…In the previous study, authors performed a yeast-two-hybrid screening of a Penaeus monodon cDNA library, using VP53A, an envelope protein of WSSV, as bait. One of the molecules that specifically interacted with VP53A was the P. monodon chitin-binding protein (PmCBP) [13]. Although PmCBP was confirmed with the capability of binding to VP53A and chitin, its biological function remains unknown.…”

A gC1qR Prevents White Spot Syndrome Virus Replication in the Freshwater CrayfishPacifastacus leniusculus

“…Some of these appear to play a critical role in anti-WSSV activity since they are associated with an ability of infected crustaceans to survive viral infection (5,17,20,30). Moreover, some proteins of shrimp, such as a chitin-binding protein and ␤-integrin, have been proposed as cellular receptors for WSSV (4,14,30). However, the mechanism by which they function as receptors is not known.…”