Virus Research 200 (2015) 24–29
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Identification of the interaction domains of white spot syndrome virus envelope proteins VP28 and VP24 Zaipeng Li a,b,1 , Weiyu Chen a,1 , Limei Xu a , Fang Li a,∗ , Feng Yang a,∗ a State Key Laboratory Breeding Base of Marine Genetic Resources, Key Laboratory of Marine Genetic Resources of State Oceanic Administration & Fujian, Third Institute of Oceanography, Xiamen 361005, PR China b School of Life Science, Xiamen University, Xiamen 361005, PR China
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Article history: Received 2 December 2014 Received in revised form 18 January 2015 Accepted 19 January 2015 Available online 28 January 2015 Keywords: WSSV VP28 VP24 Interaction domains
a b s t r a c t VP28 and VP24 are two major envelope proteins of white spot syndrome virus (WSSV). The direct interaction between VP28 and VP24 has been described in previous studies. In this study, we confirmed this interaction and mapped the interaction domains of VP28 and VP24 by constructing a series of deletion mutants. By co-immunoprecipitation, two VP28-binding domains of VP24 were located at amino acid residues 46–61 and 148–160, while VP24-binding domain of VP28 was located at amino acid residues 31–45. These binding domains were further corroborated by peptide blocking assay, in which synthetic peptides spanning the binding domains were able to inhibit VP28–VP24 interaction, whereas same-size control peptides from non-binging regions did not. © 2015 Elsevier B.V. All rights reserved.
1. Introduction White spot syndrome virus (WSSV) is a major viral pathogen of cultured shrimp with high infectivity and mortality (Chou et al., 1995; Lo et al., 1996; Takahashi et al., 1994; Wongteerasupaya et al., 1995). Ultrastructural studies have shown that WSSV is an enveloped and rod-shaped virus of 70–150 by 250–380 nm (Wang et al., 1995) and contains a double-strand circular DNA genome of about 300 kb encoding approximately 180 predicted ORFs (van Hulten et al., 2001; Yang et al., 2001). Currently, WSSV has been classified as a single species within the genus Whispovirus, family Nimaviridae (Mayo, 2002). So far, more than 40 structural proteins of WSSV have been identified by proteomic methods (Li et al., 2007; Tsai et al., 2004, 2006; Xie et al., 2006). Among them, about 30 proteins locate in viral envelope (Li et al., 2007; Tsai et al., 2004, 2006; Xie et al., 2006), including four major (high abundance) envelope proteins VP28, VP26, VP24 and VP19. VP28, encoded by ORF wsv421, has been implicated to involve in cell attachment during infection (Sritunyalucksana et al., 2006; Yi et al., 2004). VP24, encoded by ORF wsv002, was reported to interact with VP28 and involved in virus infection (Xie and Yang, 2006).
Increasing evidences indicate that intricate interactions exist between WSSV envelope proteins. For example, the four major structural proteins can form a multi-protein complex by the direct interactions between VP28–VP26, VP28–VP24, VP28–VP19, VP26–VP24 and VP24–VP19 (Chang et al., 2010; Xie et al., 2006; Zhou et al., 2009). Furthermore, VP24 also displayed specific interactions with some low-abundance envelope proteins like WSV010, VP38/VP38A, VP51A/VP52A and VP33/VP36B (Chang et al., 2010; Chen et al., 2007; Jie et al., 2008; Lin et al., 2010). According to experimental results of two-dimensional blue native/SDS PAGE, VP24 and VP26 might function as hub proteins to recruit low-abundance WSSV structural proteins to complete virion morphogenesis (Li et al., 2011). Based on the current published data, we believe that VP24 and VP28 are two very important viral envelope proteins of WSSV, and the interaction between them is involved in viral infection, assembly and morphogenesis. In this study, we constructed a series of deletion mutations in VP24 and VP28, and determined the binding regions required for their interaction by co-immunoprecipitation (co-IP). The synthetic peptides spanning the binding regions could effectively disrupt the interaction between VP24 and VP28 and may serve as potential anti-WSSV agents. 2. Materials and methods 2.1. Construction of expression plasmids
∗ Corresponding authors. Tel.: +86 0592 2195015; fax: +86 0592 2085376. E-mail addresses: lifang [email protected] (F. Li), [email protected] (F. Yang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.virusres.2015.01.017 0168-1702/© 2015 Elsevier B.V. All rights reserved.
In our previous study, VP24 lacking the first N-terminal 25 amino acids (aa) has been demonstrated to interact with VP28
Z. Li et al. / Virus Research 200 (2015) 24–29
(Xie and Yang, 2006). In this study, a series of deleted VP24 or VP28 mutants were constructed by PCR amplification with WSSV genomic DNA (Yang et al., 1997). VP24 mutants were cloned into pET-V5 plasmid (generated by replacing the His-tag in pET-28a (Novagen) with a V5-tag). VP28 mutants were cloned into plasmid pET-His to produce N-terminal His6 -tagged fusion proteins. Primer sequences used in PCR were listed in Supplementary Tables 1 and 2.
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Table 1 Synthetic peptides used in the studies. Name
No. of residues
Amino acid sequences
P-VP2446–61 P-VP24148–160 P-VP24193–208 P-VP2831–44 P-VP28181–194
16 13 16 14 14
SEIINLTINGVARGNH GREFSANKFVLYF SMRFSPGNDLFKVGEK NTVTKTIETHTDNI IAATAGGNLFDMYV
2.4. Western blotting analysis 2.2. Expression of recombinant proteins The plasmids for recombinant expression were either transformed alone or co-transformed into Escherichia coli BL21 (DE3). The transformant was grown in 5 ml LB medium with ampicillin and/or kanamycin at 37 ◦ C until OD600 reached 0.6–0.8. Expression of recombinant proteins was induced by 0.1 mM IPTG for 24 h at 18 ◦ C. Cells were harvested by centrifugation and lysed on ice by sonication in 4 ml binding buffer (50 mM Tris–HCl/pH 8.0, 150 mM NaCl, 0.5% triton X-100) supplemented with a protease inhibitor cocktail tablet (Roche). The lysate was centrifuged at 40,000 × g for 20 min and the supernatant was collected for further analysis.
The proteins were separated by 14% SDS-PAGE and transferred to a PVDF membrane (Immobilon-P, Millipore). The membrane was blocked by incubation in BløkTM -CH reagent (Millipore) for 1 h at room temperature (RT), followed by addition of primary antibody (monoclonal anti-His or anti-V5 antibody, Sigma) and incubation for 1 h at RT. Next, the membrane was washed three times with TBST (50 mM Tris–HCl/pH 8.0, 150 mM NaCl, 0.1% Tween 20) and incubated with alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (Pierce) diluted in BløkTM -CH reagent. After three washes with TBST, the AP signal was detected using NBT/BCIP substrate (Roche). 2.5. Peptide blocking assay
2.3. Co-immunoprecipitation Briefly, 100 l of lysate supernatant containing His- and V5tagged proteins was incubated with 10 l of anti-V5-agarose beads (Sigma) for 0.5 h at room temperature with gentle rotation. Beads were then washed five times with washing buffer (50 mM Tris–HCl/pH 8.0, 300 mM NaCl, 0.5% triton X-100, 0.01% SDS). The immunoprecipitated complex was dissociated from the beads by boiling in SDS-PAGE sample buffer for 10 min.
The peptides used for blocking experiments (Table 1) were synthesized at >90% purity by Shanghai Science Peptide Biological Technology Co., Ltd. Each peptide was dissolved in binding buffer to 1, 3 and 5 mg/ml, equivalent to 0.6, 1.8 and 3.0 mM, for blocking experiments. V5-VP2426–208 and His-VP2831–204 were expressed separately in E. coli BL21 and the bacterial extracts were prepared by sonication. For blocking VP24 binding to VP28, 100 l of the lysate supernatant containing His-VP2831–204 was preincubated with 300 l of
Fig. 1. Schematic representation of VP24 mutants (black boxes) (left panel) and the summary of the interactions of these mutants with VP28 (right panel), which summarizes the results of co-IP in Fig. 2. The mutants are marked with the residue’s numbers. White boxes indicate V5-tag and angled lines indicate deleted regions. “+” and “−” indicate interaction and lack of interaction with VP28, respectively.
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the corresponding peptide at RT for 1 h by gently shaking, and then was mixed with 100 l of the lysate supernatant containing V5VP2426–208 and 10 l of anti-His agarose beads. After incubation at RT for 30 min by gently shaking, the beads were washed five times with washing buffer to remove the unbound V5-VP2426–208 , and the bound V5-VP2426–208 was detected by Western blotting with anti-V5 antibody. For blocking VP28 binding to VP24, 100 l of the lysate supernatant containing V5-VP2426–208 was preincubated with 300 l of the corresponding peptide for 1 h at RT by gently shaking, and then was mixed with 100 l of the lysate supernatant containing HisVP2831–204 and 10 l of anti-V5 agarose beads. After incubation for 30 min at RT by gently shaking, the beads were washed five times with washing buffer to remove the unbound His-VP2831–204 , and the bound His-VP2831–204 was detected by Western blotting with anti-His antibody. 3. Results 3.1. There are two VP28-binding domains in VP24 To delineate the VP28-interacting domain(s) in VP24, we constructed a series of deleted forms of V5-VP24 (Fig. 1, left panel). pET-V5-VP2426–208 and pET-V5 served as the positive and negative controls, respectively, because the interaction of VP2426–208 with VP28 has been previously demonstrated (Xie and Yang, 2006). Firstly, V5-tagged VP2426–208 , VP2426–172 , VP2426–135 , VP2426–98 , VP2462–208 , VP2499–208 or VP24136–208 (Fig. 1A, left panel) were co-expressed with His-VP2831–204 in E. coli. Cell lysates were immunoprecipitated with anti-V5 agarose beads and the immunoprecipitated complexes were analyzed by Western blotting with anti-V5 antibody and anti-His antibody, respectively. As shown in Fig. 2A, the truncated forms of VP24 were expressed with the expected molecular weight (upper panel), and all of the mutants showed interaction with VP28 (lower panel). The results of the binding assays are summarized in Fig. 1A (right panel), suggesting that there are at least two VP28-binding sites at the N- and C-terminal regions of VP24. Next, we made five additional deleted mutants of VP24, VP2446–147 , VP2462–147 , VP2487–147 , VP2487–172 and VP2487–188 (Fig. 1B, left panel) and they were co-expressed with HisVP2831–204 . Co-IP experiments revealed that VP28 interacted with VP2446–147 , VP2487–172 and VP2487–188 , but not VP2487–147 and VP2462–147 (Fig. 2B). The binding data are summarized in Fig. 1B (right panel), which suggest that two regions encompassing aa 46–61 and 148–172 of VP24 are involved in the interaction between VP24 and VP28. To define the VP28-binding sites on VP24 more precisely, three deleted mutants, VP2446–61 , VP24148–160 and VP24148–172 , were generated (Fig. 1C, left panel). As shown in Fig. 2C, after deletion of 16 residues at the N-terminal region, VP2446–61 lost its VP28-binding ability. Likewise, the deletions of 13 residues from the C-terminal region abolished VP28-binding capability of VP24148–160 . The binding results are presented in Fig. 1C (right panel). Based on the above data, we conclude that there are two VP28-binding domains in VP24, spanning aa 46–61 and 148–160. 3.2. N-terminal region of VP28 is involved in the interaction with VP24 To determine VP24-binding site on VP28, N-terminal and Cterminal truncation mutants of VP28 fused with His-tag were generated (Fig. 3, left panel) and co-expressed with V5-tagged VP2426–208 . Western blotting assays showed that all His-tagged VP28 mutants were expressed and detected with anti-His antibody
Fig. 2. Co-IP of VP24 mutants and VP28. Serial constructs of VP24 mutants fused with V5-tag were co-transformed with His-tagged VP28. Cells were harvested and lysed. The lysates were immunoprecipitated with anti-V5-agarose beads, and the resulting proteins were eluted with SDS-PAGE sample buffer and separated by SDSPAGE (14%). The expression of VP24 mutants was detected by Western blotting (WB) with anti-V5 antibody and co-precipitated VP28 were detected by Western blotting with anti-His antibody (1:3000). pET-V5-VP2426–208 was used as the positive control while empty vector pET-V5 (−) as the negative one. (A) Identification of interactions between VP28 with VP2426–172 , VP2426–135 , VP2426–98 , VP2462–208 , VP2499–208 and VP24136–208 . (B) Identification of interactions between VP28 with VP2446–147 , VP2462–147 , VP2487–147 , VP2487–188 and VP2487–172 . (C) Identification of interactions between VP28 with VP2446–61 , VP24148–172 and VP24148–160 .
(Fig. 4, upper panel). VP2831–204 , VP2831–169 and VP2831–134 could be co-precipitated with VP2426–208 , while VP2845–204 , VP2866–204 and VP28101–204 could not (Fig. 4, lower panel), suggesting that the region spanning aa 31–44 in VP28 is critical for binding of VP24. 3.3. Peptides P-VP2446–61 and P-VP24148–160 inhibit the binding of VP24 to VP28 In order to estimate whether both the VP28-binding domains in VP24 are functional, synthesized peptides (Table 1), P-VP2446–61 and P-VP24148–160 (spanning two VP28-binding sites of VP24, aa 46–61 and 148–160, respectively) were used for peptide blocking experiments, while P-VP24193–208 , from the non-binding region (corresponding to aa 193–208 of VP24) was used as control peptide. As shown in Fig. 5, P-VP2446–61 and P-VP24148–160 were able to inhibit the binding of VP24 to VP28 in a dose-dependent manner, whereas control peptide P-VP24193–208 did not inhibit
Z. Li et al. / Virus Research 200 (2015) 24–29
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Fig. 3. Schematic representation of VP28 mutants (black boxes) (left panel) and the summary of the interactions of these mutants with VP24 (right panel), which summarizes the results of co-IP in Fig. 4. The mutants are marked with the residue’s numbers and white boxes are for His-tag. “+” and “−” indicate interaction and lack of interaction with VP24, respectively.
VP24–VP28 interaction. The results demonstrate that P-VP2446–61 or P-VP24148–160 can effectively block VP24–VP28 interaction in vitro, which further indicate that VP2446–61 and VP24148–160 regions are crucial for VP24 interaction with VP28. 3.4. Peptides P-VP2831–44 spanning VP24-binding domain inhibits the binding of VP28 to VP24 To demonstrate that the N-terminal region of VP28 is indeed responsible for VP24 binding, we tested the ability of P-VP2831–44 peptide corresponding to aa 31–44 of VP28 to block VP24/VP28 complex formation by peptide blocking assays. A 14-mer control peptide P-VP28181–194 from the non-interacting region was used as a control. As shown in Fig. 6, the interaction between VP24 and VP28 was efficiently inhibited by P-VP2831–44 peptide in a dose-dependent manner. In contrast, P-VP28181–194 peptide failed to exhibit an effect on the interaction. The results suggest that
Fig. 5. Peptides P-VP2446–61 and P-VP24148–160 block VP24 binding to VP28 in vitro. VP28 and VP24 were expressed separately in E. coli BL21 and 100 l bacterial lysate containing His-VP2831–204 was preincubated with 300 l P-VP2446–61 (A) or P-VP24148–160 (B) of indicated concentrations (1, 3 and 5 mg/ml) for 1 h. Then 100 l of lysate containing V5-VP2426–208 and 10 l of anti-His agarose beads were added to the mixture. After incubation for 0.5 h by gently shaking, the beads were washed five times, and the immunoprecipitated complex was analyzed by Western blotting (WB) with anti-V5 antibody. P-VP24193–208 was used as control peptide.
Fig. 4. Co-IP of VP24 with VP28 mutants. Serial constructs of VP28 with His-tag (VP2831–204 , VP2831–169 , VP2831–134 , VP2845–204 , VP2866–204 and VP28101–204 ) were co-transformed with V5-tagged VP24. Cells were harvested and lysed. The lysates were directly subjected to SDS-PAGE (14%) and Western blotting (WB) with antiHis antibody (1:3000) for checking the expression of VP28 mutants (upper panel), or were immunoprecipitated with anti-V5-agarose beads for detecting VP24 (middle panel) and co-precipitated VP28 (lower panel) by Western blotting with anti-V5 antibody and anti-His antibody (1:3000).
Fig. 6. Peptide P-VP2831–44 blocks VP28 binding to VP24 in vitro. VP28 and VP24 were expressed separately in E. coli BL21 and 100 l bacterial lysate containing V5-VP2426–208 was preincubated with 300 l P-VP2831–44 or P-VP28181–194 (control peptide) of indicated concentrations (1, 3 and 5 mg/ml) for 1 h. Then 100 l of lysate containing His-VP2831–204 and 10 l of anti-V5 agarose beads were added to the mixture. After incubation for 0.5 h by gently shaking, the beads were washed five times, and the immunoprecipitated complex was analyzed by Western blotting (WB) with anti-His antibody.
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P-VP2831–44 can associate with both aa 46–61 region and aa 148–160 region of VP24 to block VP28 binding to VP24.
4. Discussion A direct interaction between VP28 and VP24 has been demonstrated direct interaction by far-Western blotting, co-IP and yeast two-hybrid experiments (Chang et al., 2010; Xie et al., 2006; Xie and Yang, 2006; Zhou et al., 2009). However, their specific binding regions remain unclear. In the present study, we performed an extensive mutational analysis to identify the interaction domains of VP28 and VP24. Since a strong hydrophobic region is present at the N-terminus of VP24 or VP28 (van Hulten et al., 2000a, 2000b), the hydrophobic region of both proteins was removed to achieve soluble expression in E. coli BL21, viz. VP2426–208 (Xie and Yang, 2006) and VP2831–204 (Tang et al., 2007). A total of 15 truncated and deletion constructs of VP24 were generated (Fig. 1), and all mutants were soluble after expression and could therefore be easily used for co-IP analysis (Fig. 2). We found that three C-terminal deleted mutants VP2426–172 , VP2426–135 , VP2426–98 and three Nterminal deleted mutants VP2462–208 , VP2499–208 , VP24136–208 can all interact with VP28 (Fig. 2A), which suggest that there are at least two distinct VP28-binding sites within VP24. This is consistent with the findings of Chang et al. (2010), who reported that both the N-terminal of VP24 (VP241–105 ) and C-terminals of VP24 (VP2499–208 ) interacted with VP28 N-terminal (VP281–133 ). Next, mutants VP2446–147 , VP2487–172 and VP2487–188 but not VP2462–147 and VP2487–147 interacted with VP28 (Fig. 2B), indicating that the region aa 46–61 or aa 148–171 of VP24 is required for the interaction with VP28. Final, two VP28-binding sites, aa 46–61 in the N-terminus and aa 148–160 in the C-terminus of VP24, were confirmed by using three deletion mutants VP2446–61 , VP24148–172 and VP24148–160 (Fig. 2C). We next identified the VP24-binding site on VP28. The results showed that N-terminal deleted VP2845–204 , VP2866–204 and VP28101–204 could not be immunoprecipitated with VP24 (Fig. 4), suggesting that the interacting domain of VP28 with VP24 is located between aa 31–44 in the N-terminus. The above findings were further supported by peptide blocking experiments. The peptides with sequence identical to the VP28- or VP24-binding regions, P-VP2446–61 , P-VP24148–160 and PVP2831–44 , are able to block the interaction of VP28 with VP24 and this effect is sequence-specific, because peptides of equal length from non-binding regions, P-VP28181–194 and P-VP24193–208 fail to inhibit the interaction (Figs. 5 and 6). Hence, we can draw the conclusion that the VP24-binding domain of VP28 (residues 31–44) is able to interact with two distinct VP28-binding domains in VP24, residues 46–61 and residues 148–160, respectively. The crystal structure of VP28 exhibits a single -barrel, and an ␣-helix protrusion extended approximately 20 A˚ from the -barrel architecture. This protruding ␣-helix is composed of the N-terminal 15 residues (Thr 32 to Asn 47) of VP28 (Tang et al., 2007). Interestingly, in the current study, we found that the VP24-binding domain (N-terminal residues 31–44) in VP28 is consistent with the ␣-helix protrusion, indicating that the ␣-helix protrusion plays a role in VP24 binding. Since complex interactions exist between VP28, VP26 and VP24, including self-interaction of VP28 and VP24 (Chang et al., 2010; Li et al., 2011; Zhou et al., 2009), and furthermore the information of the crystal structure of VP24 has not been reported yet, the actual binding state of two VP28-binding domains and one VP24-binding domain is difficult to determine in vivo. However, we speculate that due to the effect of the steric hindrance, it is more likely that two VP28-binding domains of VP24 interact with VP24binding domain from two different VP28, although the possibility
of two VP28-binding domains of VP24 simultaneously binding to one VP24-binding domain of VP28 cannot be eliminated. In summary, we first defined three interacting domains in VP24 and VP28, and we then used this information to construct short peptides capable to prevent VP24–VP28 interaction. These results will shed light on the molecular mechanism of WSSV assembly and provide valuable information on the search for potential anti-WSSV agents. Acknowledgments This work was supported by Natural Science Foundation of China (No. 31272698), National Basic Research Program of China (973 Program, No. 2012CB114401) and Special Fund for Agroscientific Research in the Public Interest (No. 201103034). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2015.01.017. References Chang, Y.S., Liu, W.J., Lee, C.C., Chou, T.L., Lee, Y.T., Wu, T.S., Huang, J.Y., Huang, W.T., Lee, T.L., Kou, G.H., Wang, A.H., Lo, C.F., 2010. A 3D model of the membrane protein complex formed by the white spot syndrome virus structural proteins. PLoS ONE 5, e10718. Chen, J., Li, Z., Hew, C.-L., 2007. Characterization of a novel envelope protein WSV010 of shrimp white spot syndrome virus and its interaction with a major viral structural protein VP24. Virology 364, 208–213. Chou, H.Y., Huang, C.Y., Wang, C.H., Chiang, H.C., Lo, C.F., 1995. Pathogencity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan. Dis. Aquat. Organ. 23, 165–173. Jie, Z., Xu, L., Yang, F., 2008. The C-terminal region of envelope protein VP38 from white spot syndrome virus is indispensable for interaction with VP24. Arch. Virol. 153, 2103–2106. Li, Z., Lin, Q., Chen, J., Wu, J.L., Lim, T.K., Loh, S.S., Tang, X., Hew, C.L., 2007. Shotgun identification of the structural proteome of shrimp white spot syndrome virus and iTRAQ differentiation of envelope and nucleocapsid subproteomes. Mol. Cell. Proteomics 6, 1609–1620. Li, Z., Xu, L., Li, F., Zhou, Q., Yang, F., 2011. Analysis of white spot syndrome virus envelope protein complexome by two-dimensional blue native/SDS PAGE combined with mass spectrometry. Arch. Virol. 156, 1125–1135. Lin, Y., Xu, L., Yang, F., 2010. Tetramerization of white spot syndrome virus envelope protein VP33 and its interaction with VP24. Arch. Virol. 155, 833–838. Lo, C.F., Ho, C.H., Peng, S.E., Chen, C.H., Hsu, H.C., Chiu, Y.L., Chang, C.F., Liu, K.F., Su, M.S., Wang, C.H., Kou, G.H., 1996. White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Dis. Aquat. Organ. 27, 215–225. Mayo, M.A., 2002. A summary of taxonomic changes recently approved by ICTV. Arch. Virol. 147, 1655–1663. Sritunyalucksana, K., Wannapapho, W., Lo, C.F., Flegel, T.W., 2006. PmRab7 is a VP28binding protein involved in white spot syndrome virus infection in shrimp. J. Virol. 80, 10734–10742. Takahashi, Y., Itami, T., Kondo, M., Maeda, M., Fujii, R., Tomonaga, S., Supamattaya, K., Boonyaratpalin, S., 1994. Electron microscopic evidence of bacilliform virus infection in kuruma shrimp (Penaeus japonicus). Fish Pathol. 29, 121-121. Tang, X., Wu, J., Sivaraman, J., Hew, C.L., 2007. Crystal structures of major envelope proteins VP26 and VP28 from white spot syndrome virus shed light on their evolutionary relationship. J. Virol. 81, 6709–6717. Tsai, J.M., Wang, H.C., Leu, J.H., Hsiao, H.H., Wang, A.H., Kou, G.H., Lo, C.F., 2004. Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus. J. Virol. 78, 11360–11370. Tsai, J.M., Wang, H.C., Leu, J.H., Wang, A.H., Zhuang, Y., Walker, P.J., Kou, G.H., Lo, C.F., 2006. Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion. J. Virol. 80, 3021–3029. van Hulten, M.C., Goldbach, R.W., Vlak, J.M., 2000a. Three functionally diverged major structural proteins of white spot syndrome virus evolved by gene duplication. J. Gen. Virol. 81, 2525–2529. van Hulten, M.C., Westenberg, M., Goodall, S.D., Vlak, J.M., 2000b. Identification of two major virion protein genes of white spot syndrome virus of shrimp. Virology 266, 227–236. van Hulten, M.C., Witteveldt, J., Peters, S., Kloosterboer, N., Tarchini, R., Fiers, M., Sandbrink, H., Lankhorst, R.K., Vlak, J.M., 2001. The white spot syndrome virus DNA genome sequence. Virology 286, 7–22. Wang, C.H., Lo, C.F., Leu, J.H., Chou, C.M., Yeh, P.Y., Chou, H.Y., Tung, M.C., Chang, C.F., Su, M.S., Kou, G.H., 1995. Purification and genomic analysis of baculovirus
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Identification of the interaction domains of white spot syndrome virus envelope proteins VP28 and VP24 Zaipeng Li a,b,1 , Weiyu Chen a,1 , Limei Xu a , Fang Li a,∗ , Feng Yang a,∗ a State Key Laboratory Breeding Base of Marine Genetic Resources, Key Laboratory of Marine Genetic Resources of State Oceanic Administration & Fujian, Third Institute of Oceanography, Xiamen 361005, PR China b School of Life Science, Xiamen University, Xiamen 361005, PR China
a r t i c l e
i n f o
Article history: Received 2 December 2014 Received in revised form 18 January 2015 Accepted 19 January 2015 Available online 28 January 2015 Keywords: WSSV VP28 VP24 Interaction domains
a b s t r a c t VP28 and VP24 are two major envelope proteins of white spot syndrome virus (WSSV). The direct interaction between VP28 and VP24 has been described in previous studies. In this study, we confirmed this interaction and mapped the interaction domains of VP28 and VP24 by constructing a series of deletion mutants. By co-immunoprecipitation, two VP28-binding domains of VP24 were located at amino acid residues 46–61 and 148–160, while VP24-binding domain of VP28 was located at amino acid residues 31–45. These binding domains were further corroborated by peptide blocking assay, in which synthetic peptides spanning the binding domains were able to inhibit VP28–VP24 interaction, whereas same-size control peptides from non-binging regions did not. © 2015 Elsevier B.V. All rights reserved.
1. Introduction White spot syndrome virus (WSSV) is a major viral pathogen of cultured shrimp with high infectivity and mortality (Chou et al., 1995; Lo et al., 1996; Takahashi et al., 1994; Wongteerasupaya et al., 1995). Ultrastructural studies have shown that WSSV is an enveloped and rod-shaped virus of 70–150 by 250–380 nm (Wang et al., 1995) and contains a double-strand circular DNA genome of about 300 kb encoding approximately 180 predicted ORFs (van Hulten et al., 2001; Yang et al., 2001). Currently, WSSV has been classified as a single species within the genus Whispovirus, family Nimaviridae (Mayo, 2002). So far, more than 40 structural proteins of WSSV have been identified by proteomic methods (Li et al., 2007; Tsai et al., 2004, 2006; Xie et al., 2006). Among them, about 30 proteins locate in viral envelope (Li et al., 2007; Tsai et al., 2004, 2006; Xie et al., 2006), including four major (high abundance) envelope proteins VP28, VP26, VP24 and VP19. VP28, encoded by ORF wsv421, has been implicated to involve in cell attachment during infection (Sritunyalucksana et al., 2006; Yi et al., 2004). VP24, encoded by ORF wsv002, was reported to interact with VP28 and involved in virus infection (Xie and Yang, 2006).
Increasing evidences indicate that intricate interactions exist between WSSV envelope proteins. For example, the four major structural proteins can form a multi-protein complex by the direct interactions between VP28–VP26, VP28–VP24, VP28–VP19, VP26–VP24 and VP24–VP19 (Chang et al., 2010; Xie et al., 2006; Zhou et al., 2009). Furthermore, VP24 also displayed specific interactions with some low-abundance envelope proteins like WSV010, VP38/VP38A, VP51A/VP52A and VP33/VP36B (Chang et al., 2010; Chen et al., 2007; Jie et al., 2008; Lin et al., 2010). According to experimental results of two-dimensional blue native/SDS PAGE, VP24 and VP26 might function as hub proteins to recruit low-abundance WSSV structural proteins to complete virion morphogenesis (Li et al., 2011). Based on the current published data, we believe that VP24 and VP28 are two very important viral envelope proteins of WSSV, and the interaction between them is involved in viral infection, assembly and morphogenesis. In this study, we constructed a series of deletion mutations in VP24 and VP28, and determined the binding regions required for their interaction by co-immunoprecipitation (co-IP). The synthetic peptides spanning the binding regions could effectively disrupt the interaction between VP24 and VP28 and may serve as potential anti-WSSV agents. 2. Materials and methods 2.1. Construction of expression plasmids
∗ Corresponding authors. Tel.: +86 0592 2195015; fax: +86 0592 2085376. E-mail addresses: lifang [email protected] (F. Li), [email protected] (F. Yang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.virusres.2015.01.017 0168-1702/© 2015 Elsevier B.V. All rights reserved.
In our previous study, VP24 lacking the first N-terminal 25 amino acids (aa) has been demonstrated to interact with VP28
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(Xie and Yang, 2006). In this study, a series of deleted VP24 or VP28 mutants were constructed by PCR amplification with WSSV genomic DNA (Yang et al., 1997). VP24 mutants were cloned into pET-V5 plasmid (generated by replacing the His-tag in pET-28a (Novagen) with a V5-tag). VP28 mutants were cloned into plasmid pET-His to produce N-terminal His6 -tagged fusion proteins. Primer sequences used in PCR were listed in Supplementary Tables 1 and 2.
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Table 1 Synthetic peptides used in the studies. Name
No. of residues
Amino acid sequences
P-VP2446–61 P-VP24148–160 P-VP24193–208 P-VP2831–44 P-VP28181–194
16 13 16 14 14
SEIINLTINGVARGNH GREFSANKFVLYF SMRFSPGNDLFKVGEK NTVTKTIETHTDNI IAATAGGNLFDMYV
2.4. Western blotting analysis 2.2. Expression of recombinant proteins The plasmids for recombinant expression were either transformed alone or co-transformed into Escherichia coli BL21 (DE3). The transformant was grown in 5 ml LB medium with ampicillin and/or kanamycin at 37 ◦ C until OD600 reached 0.6–0.8. Expression of recombinant proteins was induced by 0.1 mM IPTG for 24 h at 18 ◦ C. Cells were harvested by centrifugation and lysed on ice by sonication in 4 ml binding buffer (50 mM Tris–HCl/pH 8.0, 150 mM NaCl, 0.5% triton X-100) supplemented with a protease inhibitor cocktail tablet (Roche). The lysate was centrifuged at 40,000 × g for 20 min and the supernatant was collected for further analysis.
The proteins were separated by 14% SDS-PAGE and transferred to a PVDF membrane (Immobilon-P, Millipore). The membrane was blocked by incubation in BløkTM -CH reagent (Millipore) for 1 h at room temperature (RT), followed by addition of primary antibody (monoclonal anti-His or anti-V5 antibody, Sigma) and incubation for 1 h at RT. Next, the membrane was washed three times with TBST (50 mM Tris–HCl/pH 8.0, 150 mM NaCl, 0.1% Tween 20) and incubated with alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (Pierce) diluted in BløkTM -CH reagent. After three washes with TBST, the AP signal was detected using NBT/BCIP substrate (Roche). 2.5. Peptide blocking assay
2.3. Co-immunoprecipitation Briefly, 100 l of lysate supernatant containing His- and V5tagged proteins was incubated with 10 l of anti-V5-agarose beads (Sigma) for 0.5 h at room temperature with gentle rotation. Beads were then washed five times with washing buffer (50 mM Tris–HCl/pH 8.0, 300 mM NaCl, 0.5% triton X-100, 0.01% SDS). The immunoprecipitated complex was dissociated from the beads by boiling in SDS-PAGE sample buffer for 10 min.
The peptides used for blocking experiments (Table 1) were synthesized at >90% purity by Shanghai Science Peptide Biological Technology Co., Ltd. Each peptide was dissolved in binding buffer to 1, 3 and 5 mg/ml, equivalent to 0.6, 1.8 and 3.0 mM, for blocking experiments. V5-VP2426–208 and His-VP2831–204 were expressed separately in E. coli BL21 and the bacterial extracts were prepared by sonication. For blocking VP24 binding to VP28, 100 l of the lysate supernatant containing His-VP2831–204 was preincubated with 300 l of
Fig. 1. Schematic representation of VP24 mutants (black boxes) (left panel) and the summary of the interactions of these mutants with VP28 (right panel), which summarizes the results of co-IP in Fig. 2. The mutants are marked with the residue’s numbers. White boxes indicate V5-tag and angled lines indicate deleted regions. “+” and “−” indicate interaction and lack of interaction with VP28, respectively.
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the corresponding peptide at RT for 1 h by gently shaking, and then was mixed with 100 l of the lysate supernatant containing V5VP2426–208 and 10 l of anti-His agarose beads. After incubation at RT for 30 min by gently shaking, the beads were washed five times with washing buffer to remove the unbound V5-VP2426–208 , and the bound V5-VP2426–208 was detected by Western blotting with anti-V5 antibody. For blocking VP28 binding to VP24, 100 l of the lysate supernatant containing V5-VP2426–208 was preincubated with 300 l of the corresponding peptide for 1 h at RT by gently shaking, and then was mixed with 100 l of the lysate supernatant containing HisVP2831–204 and 10 l of anti-V5 agarose beads. After incubation for 30 min at RT by gently shaking, the beads were washed five times with washing buffer to remove the unbound His-VP2831–204 , and the bound His-VP2831–204 was detected by Western blotting with anti-His antibody. 3. Results 3.1. There are two VP28-binding domains in VP24 To delineate the VP28-interacting domain(s) in VP24, we constructed a series of deleted forms of V5-VP24 (Fig. 1, left panel). pET-V5-VP2426–208 and pET-V5 served as the positive and negative controls, respectively, because the interaction of VP2426–208 with VP28 has been previously demonstrated (Xie and Yang, 2006). Firstly, V5-tagged VP2426–208 , VP2426–172 , VP2426–135 , VP2426–98 , VP2462–208 , VP2499–208 or VP24136–208 (Fig. 1A, left panel) were co-expressed with His-VP2831–204 in E. coli. Cell lysates were immunoprecipitated with anti-V5 agarose beads and the immunoprecipitated complexes were analyzed by Western blotting with anti-V5 antibody and anti-His antibody, respectively. As shown in Fig. 2A, the truncated forms of VP24 were expressed with the expected molecular weight (upper panel), and all of the mutants showed interaction with VP28 (lower panel). The results of the binding assays are summarized in Fig. 1A (right panel), suggesting that there are at least two VP28-binding sites at the N- and C-terminal regions of VP24. Next, we made five additional deleted mutants of VP24, VP2446–147 , VP2462–147 , VP2487–147 , VP2487–172 and VP2487–188 (Fig. 1B, left panel) and they were co-expressed with HisVP2831–204 . Co-IP experiments revealed that VP28 interacted with VP2446–147 , VP2487–172 and VP2487–188 , but not VP2487–147 and VP2462–147 (Fig. 2B). The binding data are summarized in Fig. 1B (right panel), which suggest that two regions encompassing aa 46–61 and 148–172 of VP24 are involved in the interaction between VP24 and VP28. To define the VP28-binding sites on VP24 more precisely, three deleted mutants, VP2446–61 , VP24148–160 and VP24148–172 , were generated (Fig. 1C, left panel). As shown in Fig. 2C, after deletion of 16 residues at the N-terminal region, VP2446–61 lost its VP28-binding ability. Likewise, the deletions of 13 residues from the C-terminal region abolished VP28-binding capability of VP24148–160 . The binding results are presented in Fig. 1C (right panel). Based on the above data, we conclude that there are two VP28-binding domains in VP24, spanning aa 46–61 and 148–160. 3.2. N-terminal region of VP28 is involved in the interaction with VP24 To determine VP24-binding site on VP28, N-terminal and Cterminal truncation mutants of VP28 fused with His-tag were generated (Fig. 3, left panel) and co-expressed with V5-tagged VP2426–208 . Western blotting assays showed that all His-tagged VP28 mutants were expressed and detected with anti-His antibody
Fig. 2. Co-IP of VP24 mutants and VP28. Serial constructs of VP24 mutants fused with V5-tag were co-transformed with His-tagged VP28. Cells were harvested and lysed. The lysates were immunoprecipitated with anti-V5-agarose beads, and the resulting proteins were eluted with SDS-PAGE sample buffer and separated by SDSPAGE (14%). The expression of VP24 mutants was detected by Western blotting (WB) with anti-V5 antibody and co-precipitated VP28 were detected by Western blotting with anti-His antibody (1:3000). pET-V5-VP2426–208 was used as the positive control while empty vector pET-V5 (−) as the negative one. (A) Identification of interactions between VP28 with VP2426–172 , VP2426–135 , VP2426–98 , VP2462–208 , VP2499–208 and VP24136–208 . (B) Identification of interactions between VP28 with VP2446–147 , VP2462–147 , VP2487–147 , VP2487–188 and VP2487–172 . (C) Identification of interactions between VP28 with VP2446–61 , VP24148–172 and VP24148–160 .
(Fig. 4, upper panel). VP2831–204 , VP2831–169 and VP2831–134 could be co-precipitated with VP2426–208 , while VP2845–204 , VP2866–204 and VP28101–204 could not (Fig. 4, lower panel), suggesting that the region spanning aa 31–44 in VP28 is critical for binding of VP24. 3.3. Peptides P-VP2446–61 and P-VP24148–160 inhibit the binding of VP24 to VP28 In order to estimate whether both the VP28-binding domains in VP24 are functional, synthesized peptides (Table 1), P-VP2446–61 and P-VP24148–160 (spanning two VP28-binding sites of VP24, aa 46–61 and 148–160, respectively) were used for peptide blocking experiments, while P-VP24193–208 , from the non-binding region (corresponding to aa 193–208 of VP24) was used as control peptide. As shown in Fig. 5, P-VP2446–61 and P-VP24148–160 were able to inhibit the binding of VP24 to VP28 in a dose-dependent manner, whereas control peptide P-VP24193–208 did not inhibit
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Fig. 3. Schematic representation of VP28 mutants (black boxes) (left panel) and the summary of the interactions of these mutants with VP24 (right panel), which summarizes the results of co-IP in Fig. 4. The mutants are marked with the residue’s numbers and white boxes are for His-tag. “+” and “−” indicate interaction and lack of interaction with VP24, respectively.
VP24–VP28 interaction. The results demonstrate that P-VP2446–61 or P-VP24148–160 can effectively block VP24–VP28 interaction in vitro, which further indicate that VP2446–61 and VP24148–160 regions are crucial for VP24 interaction with VP28. 3.4. Peptides P-VP2831–44 spanning VP24-binding domain inhibits the binding of VP28 to VP24 To demonstrate that the N-terminal region of VP28 is indeed responsible for VP24 binding, we tested the ability of P-VP2831–44 peptide corresponding to aa 31–44 of VP28 to block VP24/VP28 complex formation by peptide blocking assays. A 14-mer control peptide P-VP28181–194 from the non-interacting region was used as a control. As shown in Fig. 6, the interaction between VP24 and VP28 was efficiently inhibited by P-VP2831–44 peptide in a dose-dependent manner. In contrast, P-VP28181–194 peptide failed to exhibit an effect on the interaction. The results suggest that
Fig. 5. Peptides P-VP2446–61 and P-VP24148–160 block VP24 binding to VP28 in vitro. VP28 and VP24 were expressed separately in E. coli BL21 and 100 l bacterial lysate containing His-VP2831–204 was preincubated with 300 l P-VP2446–61 (A) or P-VP24148–160 (B) of indicated concentrations (1, 3 and 5 mg/ml) for 1 h. Then 100 l of lysate containing V5-VP2426–208 and 10 l of anti-His agarose beads were added to the mixture. After incubation for 0.5 h by gently shaking, the beads were washed five times, and the immunoprecipitated complex was analyzed by Western blotting (WB) with anti-V5 antibody. P-VP24193–208 was used as control peptide.
Fig. 4. Co-IP of VP24 with VP28 mutants. Serial constructs of VP28 with His-tag (VP2831–204 , VP2831–169 , VP2831–134 , VP2845–204 , VP2866–204 and VP28101–204 ) were co-transformed with V5-tagged VP24. Cells were harvested and lysed. The lysates were directly subjected to SDS-PAGE (14%) and Western blotting (WB) with antiHis antibody (1:3000) for checking the expression of VP28 mutants (upper panel), or were immunoprecipitated with anti-V5-agarose beads for detecting VP24 (middle panel) and co-precipitated VP28 (lower panel) by Western blotting with anti-V5 antibody and anti-His antibody (1:3000).
Fig. 6. Peptide P-VP2831–44 blocks VP28 binding to VP24 in vitro. VP28 and VP24 were expressed separately in E. coli BL21 and 100 l bacterial lysate containing V5-VP2426–208 was preincubated with 300 l P-VP2831–44 or P-VP28181–194 (control peptide) of indicated concentrations (1, 3 and 5 mg/ml) for 1 h. Then 100 l of lysate containing His-VP2831–204 and 10 l of anti-V5 agarose beads were added to the mixture. After incubation for 0.5 h by gently shaking, the beads were washed five times, and the immunoprecipitated complex was analyzed by Western blotting (WB) with anti-His antibody.
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P-VP2831–44 can associate with both aa 46–61 region and aa 148–160 region of VP24 to block VP28 binding to VP24.
4. Discussion A direct interaction between VP28 and VP24 has been demonstrated direct interaction by far-Western blotting, co-IP and yeast two-hybrid experiments (Chang et al., 2010; Xie et al., 2006; Xie and Yang, 2006; Zhou et al., 2009). However, their specific binding regions remain unclear. In the present study, we performed an extensive mutational analysis to identify the interaction domains of VP28 and VP24. Since a strong hydrophobic region is present at the N-terminus of VP24 or VP28 (van Hulten et al., 2000a, 2000b), the hydrophobic region of both proteins was removed to achieve soluble expression in E. coli BL21, viz. VP2426–208 (Xie and Yang, 2006) and VP2831–204 (Tang et al., 2007). A total of 15 truncated and deletion constructs of VP24 were generated (Fig. 1), and all mutants were soluble after expression and could therefore be easily used for co-IP analysis (Fig. 2). We found that three C-terminal deleted mutants VP2426–172 , VP2426–135 , VP2426–98 and three Nterminal deleted mutants VP2462–208 , VP2499–208 , VP24136–208 can all interact with VP28 (Fig. 2A), which suggest that there are at least two distinct VP28-binding sites within VP24. This is consistent with the findings of Chang et al. (2010), who reported that both the N-terminal of VP24 (VP241–105 ) and C-terminals of VP24 (VP2499–208 ) interacted with VP28 N-terminal (VP281–133 ). Next, mutants VP2446–147 , VP2487–172 and VP2487–188 but not VP2462–147 and VP2487–147 interacted with VP28 (Fig. 2B), indicating that the region aa 46–61 or aa 148–171 of VP24 is required for the interaction with VP28. Final, two VP28-binding sites, aa 46–61 in the N-terminus and aa 148–160 in the C-terminus of VP24, were confirmed by using three deletion mutants VP2446–61 , VP24148–172 and VP24148–160 (Fig. 2C). We next identified the VP24-binding site on VP28. The results showed that N-terminal deleted VP2845–204 , VP2866–204 and VP28101–204 could not be immunoprecipitated with VP24 (Fig. 4), suggesting that the interacting domain of VP28 with VP24 is located between aa 31–44 in the N-terminus. The above findings were further supported by peptide blocking experiments. The peptides with sequence identical to the VP28- or VP24-binding regions, P-VP2446–61 , P-VP24148–160 and PVP2831–44 , are able to block the interaction of VP28 with VP24 and this effect is sequence-specific, because peptides of equal length from non-binding regions, P-VP28181–194 and P-VP24193–208 fail to inhibit the interaction (Figs. 5 and 6). Hence, we can draw the conclusion that the VP24-binding domain of VP28 (residues 31–44) is able to interact with two distinct VP28-binding domains in VP24, residues 46–61 and residues 148–160, respectively. The crystal structure of VP28 exhibits a single -barrel, and an ␣-helix protrusion extended approximately 20 A˚ from the -barrel architecture. This protruding ␣-helix is composed of the N-terminal 15 residues (Thr 32 to Asn 47) of VP28 (Tang et al., 2007). Interestingly, in the current study, we found that the VP24-binding domain (N-terminal residues 31–44) in VP28 is consistent with the ␣-helix protrusion, indicating that the ␣-helix protrusion plays a role in VP24 binding. Since complex interactions exist between VP28, VP26 and VP24, including self-interaction of VP28 and VP24 (Chang et al., 2010; Li et al., 2011; Zhou et al., 2009), and furthermore the information of the crystal structure of VP24 has not been reported yet, the actual binding state of two VP28-binding domains and one VP24-binding domain is difficult to determine in vivo. However, we speculate that due to the effect of the steric hindrance, it is more likely that two VP28-binding domains of VP24 interact with VP24binding domain from two different VP28, although the possibility
of two VP28-binding domains of VP24 simultaneously binding to one VP24-binding domain of VP28 cannot be eliminated. In summary, we first defined three interacting domains in VP24 and VP28, and we then used this information to construct short peptides capable to prevent VP24–VP28 interaction. These results will shed light on the molecular mechanism of WSSV assembly and provide valuable information on the search for potential anti-WSSV agents. Acknowledgments This work was supported by Natural Science Foundation of China (No. 31272698), National Basic Research Program of China (973 Program, No. 2012CB114401) and Special Fund for Agroscientific Research in the Public Interest (No. 201103034). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2015.01.017. References Chang, Y.S., Liu, W.J., Lee, C.C., Chou, T.L., Lee, Y.T., Wu, T.S., Huang, J.Y., Huang, W.T., Lee, T.L., Kou, G.H., Wang, A.H., Lo, C.F., 2010. A 3D model of the membrane protein complex formed by the white spot syndrome virus structural proteins. PLoS ONE 5, e10718. Chen, J., Li, Z., Hew, C.-L., 2007. Characterization of a novel envelope protein WSV010 of shrimp white spot syndrome virus and its interaction with a major viral structural protein VP24. Virology 364, 208–213. Chou, H.Y., Huang, C.Y., Wang, C.H., Chiang, H.C., Lo, C.F., 1995. Pathogencity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan. Dis. Aquat. Organ. 23, 165–173. Jie, Z., Xu, L., Yang, F., 2008. The C-terminal region of envelope protein VP38 from white spot syndrome virus is indispensable for interaction with VP24. Arch. Virol. 153, 2103–2106. Li, Z., Lin, Q., Chen, J., Wu, J.L., Lim, T.K., Loh, S.S., Tang, X., Hew, C.L., 2007. Shotgun identification of the structural proteome of shrimp white spot syndrome virus and iTRAQ differentiation of envelope and nucleocapsid subproteomes. Mol. Cell. Proteomics 6, 1609–1620. Li, Z., Xu, L., Li, F., Zhou, Q., Yang, F., 2011. Analysis of white spot syndrome virus envelope protein complexome by two-dimensional blue native/SDS PAGE combined with mass spectrometry. Arch. Virol. 156, 1125–1135. Lin, Y., Xu, L., Yang, F., 2010. Tetramerization of white spot syndrome virus envelope protein VP33 and its interaction with VP24. Arch. Virol. 155, 833–838. Lo, C.F., Ho, C.H., Peng, S.E., Chen, C.H., Hsu, H.C., Chiu, Y.L., Chang, C.F., Liu, K.F., Su, M.S., Wang, C.H., Kou, G.H., 1996. White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Dis. Aquat. Organ. 27, 215–225. Mayo, M.A., 2002. A summary of taxonomic changes recently approved by ICTV. Arch. Virol. 147, 1655–1663. Sritunyalucksana, K., Wannapapho, W., Lo, C.F., Flegel, T.W., 2006. PmRab7 is a VP28binding protein involved in white spot syndrome virus infection in shrimp. J. Virol. 80, 10734–10742. Takahashi, Y., Itami, T., Kondo, M., Maeda, M., Fujii, R., Tomonaga, S., Supamattaya, K., Boonyaratpalin, S., 1994. Electron microscopic evidence of bacilliform virus infection in kuruma shrimp (Penaeus japonicus). Fish Pathol. 29, 121-121. Tang, X., Wu, J., Sivaraman, J., Hew, C.L., 2007. Crystal structures of major envelope proteins VP26 and VP28 from white spot syndrome virus shed light on their evolutionary relationship. J. Virol. 81, 6709–6717. Tsai, J.M., Wang, H.C., Leu, J.H., Hsiao, H.H., Wang, A.H., Kou, G.H., Lo, C.F., 2004. Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus. J. Virol. 78, 11360–11370. Tsai, J.M., Wang, H.C., Leu, J.H., Wang, A.H., Zhuang, Y., Walker, P.J., Kou, G.H., Lo, C.F., 2006. Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion. J. Virol. 80, 3021–3029. van Hulten, M.C., Goldbach, R.W., Vlak, J.M., 2000a. Three functionally diverged major structural proteins of white spot syndrome virus evolved by gene duplication. J. Gen. Virol. 81, 2525–2529. van Hulten, M.C., Westenberg, M., Goodall, S.D., Vlak, J.M., 2000b. Identification of two major virion protein genes of white spot syndrome virus of shrimp. Virology 266, 227–236. van Hulten, M.C., Witteveldt, J., Peters, S., Kloosterboer, N., Tarchini, R., Fiers, M., Sandbrink, H., Lankhorst, R.K., Vlak, J.M., 2001. The white spot syndrome virus DNA genome sequence. Virology 286, 7–22. Wang, C.H., Lo, C.F., Leu, J.H., Chou, C.M., Yeh, P.Y., Chou, H.Y., Tung, M.C., Chang, C.F., Su, M.S., Kou, G.H., 1995. Purification and genomic analysis of baculovirus
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