The study of binding between VP28 of WSSV and Rab7 of

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1 The study of binding between VP28 of WSSV and Rab7 of giant tiger prawn Penaeus monodon Yi-Cheng Huang, Hong Sun, Yu-San Han Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan Abstract: White spot syndrome virus (WSSV) was first emerged in south Asia in the early 1990 s. It has a wide range of hosts among crustaceans and causes up to 100% mortality within 7 to 10 days in cultured shrimps. VP28, one of the most abundant envelope proteins of WSSV, has been considered to be the most important viral protein in WSSV infection. In previous study, Rab7 protein of the shrimp host had been identified as one of the putative VP28 binding protein. However, the structure of E.coli expressed VP28 might be different to wild type VP28 due to lacking posttranslational modification. Furthermore, Rab7 is not presented on cell membrane and is not involved in vesicle formation. To reconfirm the interaction between Rab7 and eukaryotic VP28 protein, full length of VP28 was recombined into baculovirus and infected an insect cell line (sf-9). The E.coli expressed Rab7 and anti-vp28 monoclonal antibody, AP-1, were used for compete enzyme-linked immunosorbent assay (ELISA). Results showed that Rab7 did not compete with AP-1 on binding VP28, which indicated that Rab7 may not directly involve in WSSV infection on the plasma membrane, and the role of Rab7 needs further studies.

2 Introduction White spot syndrome virus (WSSV) is one the most devastating pathogens of shrimps. Since first emerged in south Asia in the early 1990 s, it has quickly spread worldwide[1, 2]. WSSV has a wide range of hosts among crustaceans and therefore speed up the viral transmission[3]. The infection of shrimps could reach up to 100% mortality within 3 to 7 days from the onset in cultured fields, which causes huge economic losses to shrimp farming industries[1]. WSSV is a double stranded DNA, rod shaped, and enveloped virus, whose genome has been completely sequenced and classified into the family Nimaviridae and the genus Whispovirus[4]. Currently, there are about 39 structural proteins of WSSV have been identified, of which 22 are envelope proteins constructing the infection-related structure [5, 6]. There is general agreement that envelope proteins play a critical role during early events of virus infection, especially in attachment. VP28, VP26, VP24, and VP19 are 4 major envelope proteins of WSSV. A binding assay in vitro using VP28-EGFP showed that VP28 has ability to bind shrimp cells. Effective neutralization of WSSV using anti-vp28 polyclonal antibody implied that VP28 may have an important role in viral attachment and penetration[7]. A study using the biotin label transfer technique and far-western analysis demonstrated that VP26 could interact with a viral capsid protein VP51, suggests that VP26 may functions as a matrix-like linker protein between the viral envelope and nucleocapsid[8]. A similar method using far-western analysis showed that VP24 interacts with VP28 and probably functions at an early stage of WSSV infection[9].

3 Although considerable progress has been made in virus and host interaction, the study of mechanisms of virus entry events continues to be a challenging problem. Several studies have been made using WSSV envelope protein to investigate the potential binding proteins of host shrimp cells. Using E. coli expressed VP28, Rab7 has been identified as a VP28 binding protein [10]. However, a previous study using proteins purified from WSSV infected tissue revealed that VP28 is threonine phosphorylated. Suggesting that there may be a posttranslational modification during virus maturation, which does not exist in E. coli expression system[6]. Furthermore, Rab7 is thought to be not presented on cell membrane and is not involved in vesicle formation and inner transportation in yeast, vero and HeLa cells [11, 12]. Putting these together, the interaction between Rab7 to VP28 during early stage of WSSV infection remains to be validated. To further study the interaction between Rab7 and wild typed VP28, a baculovirus expression system had been chosen for VP28 preparation. The E.coli expressed Rab7 and anti-vp28 monoclonal antibody, AP-1, were used for compete enzyme-linked immunosorbent assay (ELISA).

4 Materials and Methods 1. Shrimps Shrimps (Penaeus monodon) were collected from a culture field in south Taiwan. The total RNA was extracted using TRI Reagent (SIGMA) according to the manufacturer s protocol. The RNA extracts was reverse transcribed to cdna in a 20 μl final volume containing 2.5 μg of extracted RNA, 200 ng 3 RACE oligo-dt and 10 mm deoxyribonucleotide triphosphate (dntp) mix. The mixture was heated at 70 C for 5 min, and cooled down to 4, then added RT buffer (50 mm Tris-HCL, 75 mm KCL, 3 mm MgCl 2 ) and 200 units M-MuLV Reverse transcriptase (Promega). This mixture was incubated for 5 min at 25 C and 60 min at 42 C. The prepared cdna was incubated at 70 C for 15 min to inactivate the M-MuLV-RT and then stored at 20 C. 2. Expression of shrimp Rab7 protein Aliquots of 1.5 μl of the first standard cdna reaction were amplified in a 25 μl reaction volume containing a final concentration of 1 PCR buffer, 20 mm MgCl 2, 10mM dntp mix, 2 U of Taq DNA polymerase (Bioman) and 10mM of each primers (F:5 -CGA CGA TAg gta ccc ATG GCA TCT CGC AAG AAG AT-3, R1: 5 -TCG AGA ctc gag GTG ATG GTG ATG GTG ATG TTA GCA AGA GCA TGC ATC CT-3, R2: 5 -TCG AGA ctc gag TTA GTG ATG GTG ATG GTG ATG GCA AGA GCA TGC ATC CT-3 ). Primers were designed to amplify full length of P. monodon Rab7 (PmRab7) sequence, which have a 5 KpnI, 3 XhoI restriction sites and a poly-histidine

5 tag located on C-terminal end. The purity and size of the amplified product was checked by 1.2% agarose gel electrophoresis after staining with ethidium bromide. The 636 bp, double-stranded cdna was purified using the Gel-M TM Gel Extraction KIT (VIOGENE) according to manufacturer s instructions. The PCR amplicon was cloned into pet-17b vector (Novagen). The recombinant plasmid was transformed into Escherichia coli strain DH5α. After the insert was sequenced and aligned with PmRab7 gene from PubMed (DQ231062) using ClustalW2 ( the plasmid was extracted and transformed into E.coli strain BL21. Protein production was accomplished by standard methods for bacterial growth, followed by induction with IPTG (isopropyl-β-d-thiogalactopyranoside). The His 6 -PmRab7 (rpmrab7) was further confirmed with standard SDS-PAGE and western-blot using monoclonal anti-polyhistidine antibody (SIGMA) and subject to compete ELISA. 3. Preparation of WSSV VP28 with baculovirus expression system Spodoptera frugiperda (Sf9) insect cells were grown in monolayers at 28 C in Grace s insect medium (Gibco) contained 10% fetal bovine serum (Invitrogen). Recombinant baculovirus containing the VP28 coding sequences were kindly received from Taiwan Leading Biotech Inc. Monolayer Sf9 cultures were infected with the recombinant baculovirus for 60 hours. The infected cells were collected and centrifuge at 2,500 x g for 5 minutes, lysed with PBS contained 2% triton X-100. After incubating for 45 minutes on ice, centrifuge at 40,000 x g for 45 minutes. Collect supernatant which contained VP28 protein and confirmed with standard SDS-PAGE and western-blot using anti-vp28 monoclonal antibody, AP-1(Taiwan Leading Biotech).

6 4. Compete enzyme-linked immunosorbent assay After a coating with VP28 on 96-well plate at room temperature for overnight, AP-1 with 1:3 ratio serially dilutions and Rab7 protein with fit concentration were added simultaneously into a different single well of ELISA plate. In a parallel experiment, AP-1 concentration was fixed but the Rab7 were serial diluted (1:3). After binding for 1 hour, ELISA was performed using Peroxidase AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch) and tetramethyl benzidine (TMB) substract(sigma). The optical density (OD) was measured at 450nm using uninfected sf-9 cells (no VP28) as a negative control.

7 Results 1. Expression of shrimp Rab7 protein The PmRab7 and His-tag fusion PmRab7 genes were cloned into pet-17b vector and expressed in E. coli BL21 strain under induction of 1mM IPTG. After induction, induced and non-induced pet-17b-pmrab7 (containing PmRab7), pet-17b-h6-pmrab7 (containing His6-PmRab7) and pet-17b (containing vector) were analyzed by SDS-PAGE (Fig. 1). Expression protein (approximately 26 kda) corresponding to PmRab7 and His6-PmRab7 proteins were observed in the induced bacteria (Fig. 1 lane 3 to 6). No protein was found at the same position in both induced and non-induced pet-17b-bl21 (Fig. 1 lane 1 and 2). Western blot analysis showed that anti-his antibody reacted with His-tagged PmRab7 protein. This result showed that the PmRab7 gene was highly expressed in pet-17b. Recombinant His-tagged PmRab7 was acquired and this band can react with an anti-his tag antibody (Fig. 2). 2. Preparation of WSSV VP28 with baculovirus expression system The expression of VP28 protein was analysed by SDS-PAGE (Fig. 3A). Although the expression level is relatively low (only a very weak band was observed), the western-blot assay using anti-vp28 monoclonal antibody indicated the correct position of VP28 (Fig. 3B), which were subjected to compete enzyme-linked immunosorbent assay.

8 3. Compete enzyme-linked immunosorbent assay After confirmation of PmRab7 and VP28 expression by SDS-PAGE and western-blot analysis, the interaction between PmRab7 and VP28 was assayed by competitive enzyme-linked immunosorbent assay. When using serial diluted AP-1 to compete with PmRab7. The interference for binding between AP-1 and VP28 was not found (Fig. 4A). On the other hand, PmRab7 of different concentration were also unable to affect the affinity between AP-1 and VP28 (Fig. 4B).

9 Discussion Considering the nature of Rab protein family, it is part of the Ras superfaimly of small GTPases. There are numbers of Rab GTPases are conserved from yeast to humans. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane traffic pathways[13]. Rab7, which regulates transport between early and late endosomes, is located in late endosomes and lysosomes. It has been proposed that it is not needed for the initial maturation of early autophagosomes to late autophagic vacuoles, but that it participated in the final maturation of late autophagic vacuoles, which might demonstrate the absence of Rab7 on the surface of plasma membrane [12-14]. Therefore, Rab7 might not present on host cell surface and serve as VP28 receptor. Although the interaction between Rab7 and E.coli expressed VP28 has been found [10]. The difference between prokaryotic and eukaryotic expression system cannot be omitted. A previous study showed that VP28 purified from WSSV was threonine phosphorylated, indicating the importance of posttranslational modification [3]. There are several cellular proteins have been reported as potential WSSV binding protein. Such as beta-integrin and PmCBP (chitin-binding protein). This indicate that multiple molecules may participate in viral attachment [15, 16]. A recent research showed that four major envelope protein of WSSV could bind to form a complex[17], indicating that ligand-receptor complex may involve several different viral envelope proteins.

10 In this study, the viral envelope protein VP28 has been expressed using Sf-9 cells, which possess the posttranslational modification. The Rab7 was expressed in E.coli BL21 strain, and the interaction between PmRab7 and VP28 was assayed by competitive enzyme-linked immunosorbent assay. The results reported on here do not support the findings of previous research; Whereas Rab7 did not compete with AP-1 on binding VP28. Since AP-1 has been proved to bind VP28 on a critical epitope, which could block WSSV infection significantly, indicating that Rab7 may bind to another epitope of VP28. The successful neutralization of WSSV by anti-rab7 antibody in previous study might due to interference with the transport between cytosolic endosome and lysosomes [10]. The results of the present study suggest Rab7 may not function as VP28 binding protein during early stage of WSSV infection. The findings of this study highlight the significant difference between prokaryotic and eukaryotic expression system, which could result in distinct outcome. Future research should investigate the correlation of WSSV envelope protein complex to cellular binding protein and the role of Rab7 needs further studies.

11 References 1. Chou HY HC, CH W, HC C, CF L: Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan. Diseases of Aquatic Organisms 1995, 23: Nunan LM, Poulos BT, Lightner DV: The detection of White Spot Syndrome Virus (WSSV) and Yellow Head Virus (YHV) in imported commodity shrimp. Aquaculture 1998, 160: CF L, CH H, SE P, CH C, HC H, YL C, CF C, KF L, MS S, CH W, GH K: White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Diseases of Aquatic Organisms 1996, 27: Mayo MA: Virus taxonomy - Houston Arch Virol 2002, 147: Tsai JM, Wang HC, Leu JH, Wang AH, Zhuang Y, Walker PJ, Kou GH, Lo CF: Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion. J Virol 2006, 80: Xie X, Xu L, Yang F: Proteomic analysis of the major envelope and nucleocapsid proteins of white spot syndrome virus. J Virol 2006, 80: Yi G, Wang Z, Qi Y, Yao L, Qian J, Hu L: Vp28 of shrimp white spot syndrome virus is involved in the attachment and penetration into shrimp cells. J Biochem Mol Biol 2004, 37: Wan Q, Xu L, Yang F: VP26 of white spot syndrome virus functions as a linker protein between the envelope and nucleocapsid of virions by binding with VP51. J Virol 2008, 82: Xie X, Yang F: White spot syndrome virus VP24 interacts with VP28 and is involved in virus infection. J Gen Virol 2006, 87: Sritunyalucksana K, Wannapapho W, Lo CF, Flegel TW: PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp. J Virol 2006, 80: Miaczynska M, Zerial M: Mosaic organization of the endocytic pathway. Exp Cell Res 2002, 272: Vonderheit A, Helenius A: Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes. PLoS Biol 2005, 3:e233.

12 13. Stenmark H, Olkkonen VM: The Rab GTPase family. Genome Biol 2001, 2:REVIEWS Jager S, Bucci C, Tanida I, Ueno T, Kominami E, Saftig P, Eskelinen EL: Role for Rab7 in maturation of late autophagic vacuoles. J Cell Sci 2004, 117: Chen LL, Lu LC, Wu WJ, Lo CF, Huang WP: White spot syndrome virus envelope protein VP53A interacts with Penaeus monodon chitin-binding protein (PmCBP). Dis Aquat Organ 2007, 74: Li DF, Zhang MC, Yang HJ, Zhu YB, Xu X: Beta-integrin mediates WSSV infection. Virology 2007, 368: Zhou Q, Xu L, Li H, Qi YP, Yang F: Four Major Envelope Proteins of White Spot Syndrome Virus Bind to Form a Complex. J Virol 2009.

13 Figures M Fig. 1 SDS-PAGE gel of PmRab7 protein expressed in E.coli BL21. Lane1 and lane2 are bacteria containing pet-17b as a negative control, lane3 and lane4 are E.coli containing PmRab7 recombinant plasmid, lane5 and lane6 are E.coli containing His-tag fusion PmRab7 recombinant plasmid, M is protein marker. The arrow indicates the location of Rab7.

14 M Fig. 2 Western blot analysis of anti-his antibody reacted with His-tagged PmRab7. Lane1 and lane2 are bacteria containing pet-17b as a negative control, lane3 and lane4 are E.coli containing PmRab7 recombinant plasmid, lane5 and lane6 are E.coli containing His-tag fusion PmRab7 recombinant plasmid, M is protein marker. The arrow indicates the location of Rab7.

15 A B Fig. 3 WSSV VP28 protein expression in Sf-9. WSSV envelope protein VP28 was expressed in an insect cell line (Sf-9). The expression products were analysed by (A) 1.2% SDS-PAGE analysis and (B) Western-blot analysis. M: protein marker, U: uninfected Sf-9 cells (no VP28), I: infected Sf-9 cells. The arrow indicates the location of VP28.

16 A B Fig.4 Competition between PmRab7 and AP-1 on VP28. (A) rpmrab7 was applied to a ELISA plate incubated with different amount of AP-1. (B) Constant AP-1 was competing with serial diluted rpmrab7. A test with uninfected Sf-9 cell lysate was also performed as negative control. The binding specificity was determined with Peroxidise AffiniPure Goat Anti-Mouse IgG.