White spot syndrome virus (WSSV), a unique member within the virus family Nimaviridae, is the most notorious aquatic virus infecting shrimp and other crustaceans and has caused enormous economic losses in the shrimp farming industry worldwide. Therefore, a comprehensive understanding of WSSV morphogenesis, structural proteins, and replication is essential for developing prevention measures of this serious parasite. The viral genome is approximately 300kb and contains more than 180 open reading frames (ORF). However, most of proteins encoded by these ORF have not been characterized. Due to the importance of WSSV structural proteins in the composition of the virion structure, infection process and interaction with host cells, knowledge of structural proteins is essential to understanding WSSV entry and infection as well as for exploring effective prevention measures. This review article summarizes mainly current investigations on WSSV structural proteins including the relative quantities, localization, function and protein-protein interactions. Traditional proteomic studies of 1D or 2D gel electrophoresis separations and mass spectrometry (MS) followed by database searches have identified a total of 39 structural proteins. Shotgun proteomics and iTRAQ were initiated to identify more structural proteins. To date, it is estimated that WSSV is assembled by at least 59 structural proteins, among them 35 are defined as the envelope fraction (including tegument proteins) and 9 as nucleocapsid proteins. Furthermore, the interaction within several major structural proteins has also been investigated. This identitification and characterization of WSSV protein components should help in the understanding of the viral assembly process and elucidate the roles of several major structural proteins.
Citation: Yan-wei TAN, Zheng-li SHI. Proteomic Analyses of the Shrimp White Spot Syndrome Virus[J]. VIROLOGICA SINICA, 2008, 23 (3): 157-166 https://doi.org/10.1007/s12250-008-2924-0
Received: 30 November, 2007; Accepted: 28 December 2007
Data Availability: All relevant data are within the paper and its Supporting Information files.
Corresponding author: Zheng-li SHI: Tel/Fax: +86-27-87197240, E-mail: email@example.com.
BIOLOGICAL AND MOLECULAR CHARACTERS OF WSSV
In recent year, the annual production of shrimp in the farming industry has declined due to mass mortalities in shrimp ponds predominantly caused by viruses (33). So far, more than 20 viruses have been reported, among them, the white spot syndrome virus (WSSV) is the most devastating since it can cause up to 100% cumulative mortality within 3-7 days and leads to enormous losses to the shrimp farming industry (28). WSSV infects cultured and wild penaeid shrimp as well as most species of marine and fresh water crustaceans including crayfishes, crabs and lobsters (3, 6, 10, 15, 30). Due to its broad host range, WSSV is not only a major threat to shrimp farming, but also to the worldwide marine ecology (13).
Histopathological studies on WSSV infected shrimp show that the prime tissue targets are mainly those of ectodermal and mesodermal origin (31, 35, 52, 57). The initial infection starts in the stomach, gills, cuticular epidermis and the connective tissue of the hepatopancreas. At later stages, the lymphoid organ, antennal gland, muscle tissue, hematopoietic tissue, heart, hindgut and parts of the midgut also become infected. The nervous system and the compound eyes are only infected at the very late stages. The stomach, gills, cuticular epidermis, lymphoid organ, hematopoietic tissue and antennal gland are all heavily infected with WSSV at late stages of infection and become necrotic (4, 31).
The virion of WSSV is a large, ovoid particle of about 275 nm in length and 120 nm in width, with a tail-like appendage at one end (12). So far, neither the function nor the composition of this appendage is known. The virion consists of a rod-shaped nucleocapsid with a tight-fitting capsid layer, surrounded by a loose-fitting trilaminar envelope, which consists mainly of the WSSV encoded proteins VP28 and VP19 (12, 36, 45, 48). VP28 is most likely located on the surface of the virus particle and plays a key role in WSSV infection (50). The nucleocapsid is formed by stacks of rings (about 14 in total), which are in turn formed by regular spaced globular subunits of about 8nm in diameter, arranged in two parallel rows (12, 36). The nucleocapsid contains the viral genome and consists mainly of the WSSV encoded proteins VP664, VP51C, VP60B and VP15 (42, 46, 55, 66).
The virion of WSSV contain a circular, supercoiled, double-stranded (ds) DNA genome, estimated to be ~300 kilobase pairs (kb). Three full-length genomes (307 287, 305 107 and 292 967bp in size, respectively) of WSSV isolates originating from Taiwan (WSSV-TW, GenBank accession NO. AF440570) (52), China (WSSV-CN, GenBank accession NO. AF332093) (61) and Thailand (WSSV-TH, GenBank accession NO. AF 369029) (49) have been sequenced. Between the isolates of WSSV, a few restriction fragment length polymorphisms (RFLPs) were reported, indicating the presence of some genomic variation (32, 36, 53). WSSV-TH was the first to be completely sequenced (49). Computer-assisted analysis identified 184 putative ORFs encoding proteins ranging in size from 50 to 6 077 aa in WSSV-TH genome. However, only 12 of the 184 WSSV ORFs (6%) could be assigned a putative function involved in DNA replication, nucleotide metabolism and protein modification (49).
The WSSV genome is further characterized by the presence of nine direct repeat regions with different sizes, designated homologous region (11) 1 to 9. These hrs are dispersed throughout the WSSV genome and consist of three to eight identical repeat units of 250bp or parts thereof. The hrs are largely located in intergenic regions, although several short ORFs are annotated within the WSSV hrs (49). Similar but (slightly) smaller repeat regions have been identified in ascoviruses and baculoviruses (1, 9). It was demonstrated that the hrs function as enhancers of transcription and origins of DNA replication (14, 20, 38) in Baculoviruses. However, a recent study on homologous region of Autographa californica multiple nucleopolyhedrovirus indicated that no single homologous repeat region is essential for DNA replication. Thus, the functional significance of multiple origin regions is still unclear (2).
WSSV was supposed to be within the family Baculoviride. But phylogenetic analysis based on several enzyme genes including the ribonucleotide reductase large and small subunits, the protein kinases, the endonuclease, the chimeric thynmidine-hymidylate kinase and the DNA polymerase demonstrated that the WSSV was distantly related to other known viral families (8, 29, 43, 44, 47, 49, 51, 54). Based on these phylogenetic analyses and its unique morphology, WSSV had been accommodated in a new virus family, Nimaviridae, in the 8th report of International Committee on Taxonomy for Viruses (ICTV). This virus family consists of a single genus (Whispovirus) and the WSSV as its sole species so far (34, 51).
Identification of structural proteins by proteomic methods
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.
Localization in virion
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.
Function and interaction
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.
Envelope proteins involved during infection
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.
WSSV possess the largest genome of all animal viruses. Like other large DNA viruses such as herpesvirus, baculovirus and poxvirus, the component and structure of WSSV are much more complicated than expected. High throughput proteomics techniques proved to be very useful in studying the large DNA virus, particularly in the lack of permissive cell lines. Recently, a stable isotopic labeling, cleavable isotopecoded affinity tags (cICATs) was coupled with 2D-LC-MS/MS to quantitatively identified 33 WSSV and host proteins involved in virus infection. The advantage of this method is to identify the low quantity proteins and compare the proteins expression level during different infection stage, thus allow us to understand the whole infection process in which how the virus and host protein are involved.
However, the understanding of WSSV structure and the function of its structural proteins is still in the future. The application of classical methods such as western-blot, immunogold labeling and sera neutralization test are still needed to investigate in more detail of the function of virus genes. The other high throughput screening methods such as the two hybrid yeast systems will be used in the future to study the interaction between virus proteins, and between them and host proteins.
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