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

doi:10.1016/j.fsi.2009.06.018

Fish & Shellfish Immunology

Volume 27, Issue 3, September 2009, Pages 460-465

https://doi.org/10.1016/j.fsi.2009.06.018 Get rights and content

Abstract

White spot syndrome virus (WSSV) can cause the most serious viral disease of shrimp and has a wide host range among crustaceans. Although researches show a lot about its genome and structure, information concerning the mechanism of how WSSV infects' cells is lacking. In this study, some experiments were applied to confirm the biological meaning of the protein–protein interaction between WSSV envelope protein, VP53A, and Penaeus monodon chitin-binding protein (PmCBP). Immunofluorescent study indicated that PmCBP is located on the cell surface of host cells. PmCBP amounts of about 34 kDa can be detected in both P. monodon and Litopenaeus vannamei tissues by Western blotting. In the in vivo neutralization experiment, both rVP53A and rPmCBP that were produced by Esherichia coli can promote resp. a 40% and 20% survival rate of the shrimp which were challenged by WSSV. Furthermore, a yeast-two-hybrid result revealed that PmCBP could interact with at least 11 WSSV envelope proteins. Those findings suggest that PmCBP may be involved in WSSV infection.

Introduction

White spot syndrome (WSS), due to its main clinic sign of “white spots” in the exomesoderm under the carapase, is the most serious viral disease of shrimp that causes huge losses in the shrimp industry around the world. The causative agent of WSS is commonly known as the white spot syndrome virus (WSSV). WSSV is an enveloped, ellipsoid, large, double-stranded DNA virus and it has a wide host range among not only shrimp species but also many other crustaceans [1], [2]. The complete genome sequences of the three WSSV isolates have been determined to encompass approximately 180 putative open reading frames (ORFs) [3], [4], [5]. Recent research on the WSSV structure using proteomic methods has revealed that its structural proteins increased to more than 40 [2]. Because WSSV has a wide host range among crustaceans, developing efficient antiviral strategies or therapeutic methods is of the greatest importance for aquaculture.

It is clear that there are receptors on most host cell membranes that can interact with the virus envelope proteins and facilitate their penetration. Although researches show a lot about WSSV genome and structure, information concerning the mechanism of how it infects cells is lacking. To unravel the mystery, research activities in investigating the protein–protein interactions between shrimp and virus, shrimp itself or virus itself are now taking into consideration [6], [7], [8], [9]. To date, more than one shrimp protein was supposed to mediate WSSV infection [10], [11], [12]. 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. In this study, PmCBP is further characterized and its localization in shrimp cells is also verified. In vivo neutralization experiments showed that both rVP53A and rPmCBP can reduce and delay mortality upon WSSV challenge. It indicated that PmCBP may be a cellular receptor or co-receptor for WSSV or a type of adhesion protein helping WSSV infection.

Section snippets

Recombinant PmCBP over-expression in Esherichia coli and antiserum production

The DNA fragment encoding the partial-length PmCBP was amplified from shrimp cDNA by PCR with the CBP-partial-F/CBP-partial-R primer set (5′-GTTCGAATTCTGCTGGATTTGGGGCAGCA-3′/5′-TGACTCGAGTTAAATCTTTTCCTTTGGAAG-3′; the underlined bases indicate respectively the BamHI and XolI restriction sites that were used for subsequent cloning) and ligated to pGEM^®-T Easy (Promega) plasmid. After confirming the sequence, the resultant plasmid, pGEM-T–PmCBP-C was cleaved with BamHI and XolI, and the amplified

Cellular localization of PmCBP

Since PmCBP was supposed to locate on cell surface, hemocytes were applied to identify the speculation and to confirm if PmCBP colocalize with VP53A in vivo. The hemocyte cells withdrawn from WSSV-infected P. monodon were fixed, treated with mouse anti-PmCBP and rabbit anti-VP53A antibodies, and analyzed by confocal microscopy. As shown in Fig. 1, strong fluorescent signals from both PmCBP (Fig. 1B) and from VP53A (Fig. 1C) were observed in the plasma membrane, and the merged image shows that

Discussion

In the previous study, PmCBP was identified to be able to interact with the WSSV envelope protein VP53A in vitro [13]. However, there was no concrete evidence to identify exactly where PmCBP was located. While analyzing with many bioinformatics programs and/or information published on websites, PmCBP was speculated to be located on the cell surface. As shown in Fig. 1, immunofluorescence assay verified the speculation. Those programs and websites also speculated a transmembrane domain (TMD)

Acknowledgments

This investigation was supported financially by the National Science Council Grant (NSC 96-2311-B-019-003). We thank the Center for Marine Bioscience and Biotechnology, National Taiwan Ocean University, for their partial finance support. We also appreciate Mr. Vince J. Genis, Miss Cleo Fu, and Miss Angela Wang for their positive commentaries on the manuscript.

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¹: These authors contribute equally to this work.

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

Abstract

Introduction

Section snippets

Recombinant PmCBP over-expression in Esherichia coli and antiserum production

Cellular localization of PmCBP

Discussion

Acknowledgments

Virology

Virology

FEBS Lett

J Biotechnol

Virology

Virology

Fish Shellfish Immunol

Aquaculture

Immunity

Virology

J Biol Chem

Cell