With the completion of the WSSV genomic sequence, attention has been focused on the functional analysis of the encoded proteins, particularly the structural proteins. Since these proteins are the first molecules to interact with host and, therefore, play critical roles in cell targeting as well as in triggering the host defenses. Even though several major structural proteins, such as VP35, VP28, VP26, VP24, VP19 and VP15 (7, 16, 47, 48), have been successfully identified by SDS-PAGE coupled with Western blotting and/or protein N-terminal sequencing, but it is not always easy to identify every structural protein. The WSSV exhibits a large number of protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which is indicative the complexity of the virus particle.
In recent years, proteomic methods have been developed to provide powerful methods in large-scale analysis of proteins. The application of mass spectrometry followed by database searches of sequenced genomes has been proven to be a fast and sensitive for the comprehensive understanding of gene products (37). Proteins from purified virions are firstly separated by gradient SDS-PAGE and then the visible bands are excised from the gel followed by trypsin digestion and mass spectrometry to get the resulting peptide sequence data. A previous study were able to identify 18 structural proteins from WSSV by using one dimensional (1D) SDS-PAGE and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) or nano-electrospray ionization quadrupole time of flight (ESI Q-TOF) mass spectrometers (18). Later, 33 WSSV structural proteins were resolved by 2D SDS-PAGE using the online LC-ESI Q-TOF mass spectrometer and this has increased structural proteins identified to 39 by these two proteomic studies (41). Due to the low abundance of some structural proteins, the gel-based proteomic studies are not always efficient and accurate. In a recent study, in order to achieve a better understanding of the structural proteome of WSSV, shotgun proteomics using offline coupling of LC system with MALDI TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. The resulting data from shotgun proteomics has identified 45 viral proteins, 13 of which are reported for the first time, the remains are identified in the previous studies (27). Therefore, the overall numbers of viral structural proteins that have been identified are 59.
Up to now, the entry pathway or assembly of WSSV has not been defined due to the lack of a permissive cell culture. Therefore, a comprehensive determination of the localization of structural proteins in the virion is important to elucidate viral assembly, infection and virion morphogenesis. The conventional methods such as immunogold electron microscopy (IEM) and western blot analysis have been used to localize 14 viral proteins, including VP28, VP26, VP31, VP51C, VP36B, VP68, VP41A, VP12B, VP180, VP124, VP39, VP110 and VP24 as envelope proteins (17, 18, 23, 24, 26, 60, 63-65, 67, 68) while VP466 as nucleocapsid protein (21). Tsai et al initiated a more detailed study on WSSV structural proteins, in which they presumed there is an intermediate layer called "tegument" between envelope and nucleocapsid proteins. In their study, Triton X-100 was used in combination with various concentrations of NaCl and led to indentify 7 envelope proteins, 5 tegument proteins and 6 nucleocapsid proteins (42). Recently, a more complementary and systematic method, iTRAQ, has localized 12 novel envelope proteins and 2 novel nucleocapsid proteins (27). Further, a novel envelope protein WSV010 has been identified by shotgun proteomics approach using offline coupling LC system with MALDI-TOF/TOF MS/MS (5). In total, through different proteomic methods, 35 proteins were currently identified as envelope proteins (including tegument proteins) and 9 as nucleocapsid proteins (Table 1). The elucidation of localization of WSSV structural proteins could facilitate the investigation of the molecular mechanisms of virus assembly and virus entry.
Table 1. The localization of structural proteins in WSSV so far characterized
The nucleocapsid contains the viral genome and consists mainly of the WSSV encoded proteins VP664 and VP15 (21, 45, 46, 48). VP664, a remarkable large protein of around 664 kDa, was thought to be the major core protein responsible for the striated appearance of the nucleocapsids (21) and is quite evenly distributed at intervals on the outer surface of the nucleocapsid (42). Moreover, VP664 molecules evidently extend from the nucleocapsid to the outside surface of the tegument, which may increase the flexibility of the nucleocapsid and allows it to assume its olive-like shape in the mature virion (42). VP15, a highly basic protein with no hydrophobic regions, is a histone-like, double-stranded DNA-binding protein that tends to binds double-stranded DNA with a clear preference to supercoiled DNA, suggesting that VP15 is involved in packing the viral genome within the nucleocapsid (56).
Envelope proteins play vital roles in initiating a virus infection, including binding to receptors or penetrating into host cells by membrane fusion. VP28 is the most abundant envelope protein located on the surface of the virus particle and is supposed to play a key role in WSSV binding to shrimp cells as an attachment protein facilitating virus enter the cytoplasm (47, 62). It has been reported VP28 can bind to shrimp cells in low-pH environment and interact with host cells through PmRab7 (39). In combination with recent data, the results presented here indicated that VP28 must play a crucial role in systemic WSSV infection in shrimp. VP26 was reported to be localized in the tegument (42) and was supposed to associate loosely with both the envelope and the nucleocapsid and might function as a matrix-like linker protein between the envelope and the nucleocapsid. However, through recent immunogold labeling experiment, the "status" of VP26 was finally characterized as an envelope protein since all the gold particles localize on the outer surface of the envelope, not on the nucleocapsid or within the space between them (40). VP24 is likewise a tegument protein but we do not yet have western blotting results.
The crystalline structures of VP28 and VP26 have been determined and both of them adopt β-barrel architecture with a protruding N-terminal region. The predicted N-terminal transmembrane region of VP26 and VP28 may anchor as trimers on the viral envelope membrane, making the core β-barrel protrude outside the envelope to interact with the host receptor or to fuse with the host cell membrane for the effective transfer of the viral infection (42). Proteinprotein interactions are essential for virion morphogenesis. Using far-western and coimmunoprecipitation experiment, Xie et al. reported that VP28 interact with both VP26 and VP24 by forming a complex (59). Recently, WSV010 has been identified as a novel envelope protein and has interaction with a major viral structural protein VP24 (5). Thus, VP24 maybe also act as a linker protein for VP28, VP26 and VP24 to form a complex, which plays an important role in viral morphogenesis and infection.
To sum up, the interaction within envelope proteins of WSSV play important role in the infection process and virion morphogenesis. Tsai et al. proposed a model showing the morphogenesis of WSSV in vitro through electronic micrograph results (42). Firstly, the empty nucleocapsid forms and various envelope proteins, probably including VP28, VP26, VP24, VP31, VP36B and WSV010 assemble around it. Next, the fibrillar component (a complex of DNA and VP15 and perhaps other proteins) is folded or packed inside the nucleocapsid and the virion becomes fatter. Finally, the open end of the nucleocapsid is closed, and the virion matures. Further exploration of the biochemical interactions of WSSV structural proteins might help to elucidate the exact molecular mechanisms of virion morphogenesis.
Sera neutralization experiment is commonly used to identify the envelope proteins involved in virus infection in vivo and in vitro. Through this method, several research groups have identified 7 envelope proteins which could delay WSSV infection significantly: VP28, VP31, VP36A, VP36B (VP281), VP466, VP68 and VP76 (19, 24, 25, 50, 58). Furthermore, their results demonstrated that the whole WSSV infection is initiated by multiple envelope proteins rather than one alone. These proteins could be the candidates for screening receptors in shrimp cell surface and are useful to discover the infection mechanism.