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Journal of Virology, July 2005, p. 8764-8772, Vol. 79, No. 14
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.14.8764-8772.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

RING-H2 Protein WSSV249 from White Spot Syndrome Virus Sequesters a Shrimp Ubiquitin-Conjugating Enzyme, PvUbc, for Viral Pathogenesis

Zhilong Wang, Hui Kheng Chua, Ayu A. R. A. Gusti, Fang He, Beau Fenner, Ivanus Manopo, Hai Wang, and Jimmy Kwang*

Animal Health Biotechnology Group, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore

Received 13 January 2005/ Accepted 30 March 2005


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ABSTRACT
 
Modification of proteins by ubiquitin is essential for numerous cellular processes. The RING-H2 finger motif has been implicated in ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Four proteins, WSSV199, WSSV222, WSSV249, and WSSV403, from white spot syndrome virus (WSSV) contain the RING-H2 motif. Here we report that WSSV249 physically interacts with a shrimp ubiquitin-conjugating enzyme, PvUbc, and mediates ubiquitination through its RING-H2 motif in the presence of E1 and PvUbc. Mutations of the putative zinc coordination residues in the RING-H2 domain of WSSV249, however, ablate ubiquitination efficiency. In addition, the RING-H2 domain of WSSV249 is capable of ubiquitination with UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, and UbcH10, respectively, exhibiting a low degree of E2 specificity. Significantly, the expression of WSSV249 and PvUbc increased during infection, as revealed by real-time PCR. Furthermore, in situ hybridization showed that WSSV249 and PvUbc display similar expression patterns in infected shrimps, and immunofluorescence and immunohistochemistry assays showed an increase of PvUbc in infected shrimp cells. These results suggest that the RING-H2 protein WSSV249 from WSSV may function as an E3 ligase via sequestration of PvUbc for viral pathogenesis in shrimp.


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INTRODUCTION
 
White spot syndrome virus (WSSV) is currently the most serious viral pathogen of aquacultured shrimp worldwide. It causes up to 100% mortality within 3 to 10 days of infection, resulting in major economic losses to the shrimp farming industry (36). WSSV is also able to infect a wide range of aquatic crustaceans, including crabs, lobsters, and freshwater crayfish (19, 38). A better understanding of WSSV in recent years has led to its reclassification under the new monotypic family Nimaviridae (genus Whispovirus) (53), and various diagnostic methods have been developed for its detection (16, 23, 37). Proteomics has also been used for the study of WSSV structural proteins (51), and characterization of WSSV envelope proteins has led to the development of an oral vaccine against the virus in shrimp (57). Although the use of molecular techniques has become an indispensable tool for the study of WSSV, more in-depth research on the interaction of the virus with its host is needed for effective control over the disease.

Viruses have evolved the ability to utilize the host protease machinery to direct cellular protein degradation for their survival and replication (49). Ubiquitination plays key roles in viral infection and facilitates activities required for various aspects of the virus life cycle, from entry (21) through replication (17, 41) and enhanced cell survival (49, 56) to viral release (24, 60). Recently, several plant homeodomain-containing viral proteins have been identified as E3 ubiquitin ligases which promote immune evasion by downregulating proteins that govern immune recognition (14).

Ubiquitin-dependent proteolysis regulates protein abundance and serves as a central regulatory function in many biological processes such as cell cycle regulation, signal transduction, transcriptional regulation, DNA repair, inflammatory response, and antigen presentation in eukaryotic cells. In the ubiquitin-dependent proteolytic pathway, ubiquitin is linked to substrates through a well-organized process involving the sequential action of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Polyubiquitinated proteins are then targeted to the 26S proteosome for degradation (13, 15, 25, 44).

A previous study (20) has revealed the involvement of the RING finger domain in specific ubiquitination events by acting as the E3 ubiquitin protein ligase. In general, a RING finger protein possesses the motif Cys-X2-Cys-X(9-39)-Cys-X(1-3)-His-X(2-3)-Cys-X2-Cys-X(4-48)-Cys-X2-Cys, where X can be any amino acid. It is characterized by a highly conserved spacing that binds two zinc ions in a cross-brace structure for conformational stability. The RING finger family has two subclasses: RING-HC and RING-H2 (where His replaces Cys4), which are important motifs with established functions in RNA processing, cell cycle control, apoptosis, peroxisomal biogenesis, and viral replication (3, 4, 18, 45, 50).

WSSV contains a large double-stranded DNA genome of 290 to 305 kbp, and three isolates have been sequenced (10, 11, 53, 59). Four WSSV proteins, WSSV199, WSSV222, WSSV249, and WSSV403, are predicted to encode the RING-H2 motif, but their function has yet to be determined. Our previous studies revealed that WSSV249 was detected in WSSV-infected shrimp RNA but not in specific-pathogen-free shrimp RNA (33), implying the pathogenesis of WSSV249 in virus infection. Here we report that WSSV249 mediates ubiquitination through its RING-H2 motif in the presence of E1 and a shrimp ubiquitin-conjugating enzyme, PvUbc. The RING-H2 domain of WSSV249 exhibits a low degree of E2 specificity and is capable of ubiquitination with UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, and UbcH10, respectively. Furthermore, the expression of WSSV249 and PvUbc increased in a time course study, and WSSV249 and PvUbc displayed similar expression patterns in infected shrimps. These results suggest that the RING-H2 protein WSSV249 from WSSV may function as an E3 ligase by sequestering PvUbc for viral pathogenesis in shrimp.


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MATERIALS AND METHODS
 
Construction of shrimp cDNA library. Total RNAs were extracted from normal WSSV-free shrimps (Penaeus vannamei) (33). First-strand cDNAs were reverse transcribed from 0.3 µg of total shrimp RNAs, using Powerscript reverse transcriptase with SMART Oligo II and CDS primers (Clontech). Nonnormalized double-stranded cDNAs were amplified from the newly synthesized first-strand cDNAs using SMART PCR primer (Clontech). Normalization of first-strand cDNAs and SMART-amplified cDNAs were performed according to the protocols described by Zhulidov et al. (63). The amplified normalized cDNAs were purified using the QIAquick PCR purification kit (QIAGEN), ligated into SrfI-modified pGADT7 vector (Clontech), and subsequently transformed into Escherichia coli (XL1-Blue) cells by electroporation. The shrimp cDNA library was screened for inserts by PCR with primers T7 (5'-TAATACGACTCACTATAGGGC-3') and AD3 (5'-AGATGGTGCACGATGCACAG-3') (Clontech) on 40 randomly selected colonies. The library was then arrayed in 30 plates to obtain 1:500,000 independent colonies, which were eluted by LB ampicillin medium and stored at –70°C.

Construction of plasmids. To express fusion proteins with glutathione S-transferase (GST), plasmid pGEX-4T-3 (Amersham Biosciences) was utilized. BamHI-SmaI fragments of WSSV249 RING finger domain and PvUbc obtained by PCR amplification using primers 249RF-5B (5'-CGGGATCCGACCATGGCTTAAACCCATC-3') and 249RF-3S (5'-GCGGGCCCTCAGTTGACCCTCACTCTGTTTCC-3'), as well as E2-5B (5'-CGGGATCCATGGCGGCTACAAGGCGGCTAC-3') and E2-3S (5'-TCCCCCGGGTTAGTCTGCAGGCCTCTTTTC-3'), were introduced in frame at the C terminus of pGEX-4T-3, resulting in plasmids pGEX-249 and pGEX-PvUbc, respectively. Point mutations of the conserved cysteine and histidine residues within the RING finger domain of WSSV249 were generated by PCR. The amino acid changes are as follows: Cys313->Ser, Cys 333->Ser, His335->Tyr, and Cys341->Ser, resulting in plasmids pGEX-C313S, pGEX-C333S, pGEX-H335Y, and pGEX-C341S, respectively. All the constructs were validated in frame by sequencing using an ABI PRISM dye terminator cycle sequencing kit.

Protein expression and antibody preparation. Recombinant GST-RING and GST-PvUbc were expressed in Escherichia coli BL21(DE3) and purified from bacterial extracts using B-PER GST spin purification kit (Pierce) according to the manufacturer's instructions. Polyclonal antibodies against PvUbc were raised in guinea pig by immunization of GST-PvUbc with adjunct montanide ISA (Seppic) as described by Catty and Raykundalia (8).

In vitro ubiquitination assay. Ubiquitination was performed as previously described by Imai et al. (27) with slight modifications. The assay mixture of 20 µl contained 40 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2 mM ATP, 2 mM dithiothreitol, 6.3 µg of ubiquitin, 0.25 µg of E1, 0.5 µg of E2, and 2 µg of E3 (RING finger protein, WSSV249-RING-H2). E1 and human E2s (UbcH1, UbcH2, UbcH3, UbcH5a, GST-UbcH5b, UbcH5c, UbcH6, UbcH9, and UbcH10) were purchased from Boston Biochem, Inc.. The reaction mixtures were incubated at 30°C for 3 h.

Western blot analysis. Protein samples were separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel, transferred onto a nitrocellulose membrane, and probed with mouse anti-Ub (Santa Cruz Biotechnology) and anti-GST (Pierce). The monoclonal antibodies anti-Ub and anti-GST were diluted 1:1,000 (vol/vol) and the immunoreactive proteins detected using an Amersham Biosciences ECL system.

Yeast two-hybrid screen. The Matchmaker GAL4 Two-Hybrid System 3 from Clontech was utilized for yeast two-hybrid screening. The AD fusion library was constructed with healthy shrimp cDNA and cloned into the unique sites of EcoRI and BamHI of the pGADT7 vector. The bait gene, encoding the RING finger domain of WSSV249, was PCR amplified from the WSSV genome, using primers 249RF-5 (5'-AGAGAATTCGACCATGGCTTAAACCCATC-3') and 249RF-3 (5'-GTGCTGCAGGTTGACCCTCACTCTGTTTCC-3'), and cloned using the EcoRI and PstI sites of the GAL4 DNA-binding fusion vector, pGBKT7. Bait and fish plasmids were used to cotransform the yeast strain AH109, and the transformants were selected on His– Leu– Trp– medium. Positive colonies were further selected on Ade– His– Leu– Trp– {alpha}-5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside medium. Plasmids harboring library cDNAs were recovered from the blue colonies, and their nucleotide sequences were determined by sequencing.

In situ hybridization. In situ hybridization was performed on paraffin-embedded tissue sections essentially as described by Jowett (31). Briefly, both WSSV-free shrimps and WSSV-infected shrimps were fixed in 4% (wt/vol) paraformaldehyde-phosphate-buffered saline (PBS), dehydrated, and embedded in paraffin. Shrimp tissue sections 6 µm thick were made and attached to 3-aminopropyltriethoxy-silane-coated slides. Digoxigenin (DIG)-labeled antisense or sense riboprobes of WSSV249 and PvUbc were synthesized from PCR fragments of WSSV249 and PvUbc flanked with T7 promoter sequence by in vitro transcription using T7 RNA polymerase (Stratagene) and 10x DIG labeling mix (Roche). Signal was detected by using 1:5,000-diluted anti-DIG-alkaline phospatase (at 4°C overnight), Fab fragment (Roche), and substrates 5-bromo-4-chloro-3-indolyl phosphate and Nitro Blue Tetrazolium. Images were taken by using an Olympus microscope (IX71).

Time course RT-PCR. RNA samples were prepared from noninfected as well as WSSV-infected shrimps at 3 h, 6 h, 12 h, 24 h, and 48 h postinfection. Total RNAs were extracted from the frozen shrimp heads (500 mg) using TRIzol-LS reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. For reverse transcription (RT)-PCR, total RNA samples were first treated with 200 U of RNase-free DNase I at 37°C for 30 min to remove any contaminating genomic DNA, extracted with phenol-chloroform, and precipitated by ethanol. About 1 µg of the treated total RNAs were used in the synthesis of first-strand cDNAs, using oligo(dT) primer (Roche Diagnostics, USA) and 2 µl of the reaction was used in RT-PCR. Primers 249-5 (5'-GACCATGGCTTAAACCCATC-3') and 249-3 (5'-GTTGACCCTCACTCTGTTTCC-3') were used to amplify WSSV249, and primers E2-5 (5'-CAGGGATATACAAGTGGATGA-3') and E2-3 (5'-TTAGTCTGCAGGCCTCTTTTCT-3') were used to amplify PvUbc. The cycling parameters for RT-PCR are as follows: denaturation at 94°C for 3 min; amplification for 30 cycles at 94°C for 30 s, 55°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 10 min. The internal control, ß-actin, was amplified under the same PCR conditions using the ß-actin forward (5'-GCGTATCCTTCGTAGATGGG-3') and reverse (5'-CCCAGAGCAAGAGAGGTATC-3') primer pair.

Real-time PCR. SYBR Green I real-time PCR was performed as previously described by Khadijah et al. (33) and Komurian-Pradel et al. (34). The LightCycler fast start DNA masterplus SYBR Green I kit was purchased from Roche. Primers 249-F (5'-TCCCTATCTACGGGTCAG-3') and 249-R (5'-GTTTAGTTGAGCGGGC-3') and PvUbc-F (5'-ACAAGTGGATGAGAGCA-3') and PvUbc-R (5'-ATGATGGGGAGACATACC-3') were used to amplify a 183-bp WSSV249 fragment and a 195-bp PvUbc fragment, respectively.

Primary cell cultures of lymphoid organ and immunofluorescence assay (IFA). The development of primary cell cultures of lymphoid organ tissue from P. vannamei followed the description of Wang et al. (54). Excised lymphoid organs were separated and incubated with double-strength Leibovitz's L15 (Sigma) supplemented with 20% fetal bovine serum in a 96-well plate at 28°C. The unattached tissue was removed 24 h after seeding and the confluent cells inoculated with WSSV in fresh medium. At 24 h postinfection, the cell cultures were fixed with 100% ethanol, incubated with anti-PvUbc serum (1:50 [vol/vol] dilution in PBS) and fluorescein isothiocyanate-conjugated anti-guinea-pig immunoglobulin G (1:50 [vol/vol] dilution in PBS; DAKO) sequentially, and examined using an Olympus IX71 fluorescence microscope.

Immunohistochemistry assay. Immunohistochemistry assays followed the description of Bradley-Dunlop et al. (7). The primary antibody anti-PvUbc was diluted 1:200 (vol/vol) (at 4°C overnight), and the secondary antibody (goat anti-guinea-pig; DAKO) was diluted 1:1,000 (vol/vol). Preimmune rabbit serum served as the negative control. Signals were detected with 3,3'-diaminobenzidine tetrahydrochloride (Pierce), counterstained with hematoxylin (Sigma-Aldrich), mounted with Aqua PolyMount (Polysciences), and examined under an Olympus IX71 microscope.


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RESULTS
 
WSSV249 contains a RING-H2 domain. Characterization studies were performed to investigate the structure and functions of WSSV249. In order to determine the involvement of WSSV249 in WSSV infection of Penaeus vannamei, a genome search of WSSV249 showed that it encodes a 783-amino-acid protein with a RING-H2 domain and a repetitive region. Further analysis of the RING-H2 domain showed that the 47 amino acids consist of a conserved cysteine-rich region known as C3H2C3 [CX2CX(9-39)CX(1-3)HX(2-3)HX2CX(4-48)CX2C] (Fig. 1A). Three EF-hand calcium binding domains, each consisting of 12 amino acids, were also identified as domains I, II, and V, respectively (Fig. 1A).



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  		FIG. 1. Structural motifs of WSSV249 and time course study of WSSV249 and PvUbc expression. (A) Structural motifs of WSSV249. Domains I (amino acids 144 to 156), II (amino acids 207 to 219), and V (amino acids 692 to 704), respectively, represent EF-hand calcium binding domains. Domain IV (amino acids 454 to 639) represents a repeat region. Domain III (amino acids 310 to 357) represents the position of the RING finger and is indicated with the locations of the first and last zinc-coordinating cysteine residues. In the lower row, the numbers refer to the positions of eight zinc-coordinating residues, and the mutated zinc-coordinating residues for functional analysis are indicated with an asterisk. (B) Time course study of WSSV249 and PvUbc by RT-PCR. The transcription of WSSV249 was detected as early as 3 h postinfection, while low expression of PvUbc was detected in noninfected samples (mock). ß-Actin acted as the housekeeping control gene. (-), negative control; M, mock (noninfected sample); 3, 6, 12, 24, and 48, hours after WSSV infection. (C) Time course study of WSSV249 and PvUbc expression by real-time PCR. Expression is expressed as number of copies/reaction as determined by comparison with plasmid controls. Error bars represent the standard error of the mean taken from three independent experiments.

Transcription of WSSV249 was increased in a time course study. A time course study was performed to compare and quantify WSSV249 expression from infected and noninfected shrimps. RT-PCR analysis showed that WSSV249 can be detected as early as 3 h postinfection (Fig. 1B). The amount of WSSV249 transcript increased from 6 to 48 h postinfection compared to that for the positive control, ß-actin, which showed constant amplification throughout the whole time course study (Fig. 1B). Moreover, the increased amount of WSSV249 mRNA was demonstrated by real-time PCR. From 12 to 48 h postinfection, the expression of WSSV249 increased approximately 36-fold (Fig. 1C), which is consistent with our RT-PCR findings (Fig. 1B). These results suggested that the gene encoding the RING-H2 protein WSSV249 could be related to viral pathogenesis.

WSSV249-RING-H2 interacts with PvUbc in vitro. In order to identify possible shrimp proteins interacting with the RING finger domain of WSSV249, a truncated WSSV249 including the RING-H2 motif (WSSV249-RING-H2) was used as a bait protein for screening the shrimp cDNA library in a yeast two-hybrid assay. Positive transformants from the His– Leu– Trp– plates were replated on a high-stringency plate (Ade– His– Leu– Trp– {alpha}-5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) for further selection. Sequence analysis revealed a U3 small nucleolar ribonucleoprotein component (snoRNP), a transcription factor (TAFII p250), and a novel shrimp ubiquitin-conjugating enzyme (Fig. 2). This shrimp ubiquitin-conjugating enzyme showed high homology to ubiquitin-conjugating enzymes from other species and was named Penaeus vannamei Ubc (PvUbc). The PvUbc gene is 465 bp long, with a coding region of 154 amino acids (Fig. 3A). Sequence alignment showed a high level of conservation of PvUbc with its orthologs from other animals, having >75% amino acid homology with ubiquitin-conjugating enzyme of Drosophila melanogaster, Danio rerio, Homo sapiens, and Mus musculus (Fig. 3B). The physical interaction between WSSV249-RING-H2 and PvUbc suggests that WSSV249 could be functional in the ubiquitin pathway.



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  		FIG. 2. WSSV249-RING-H2 interacted with shrimp proteins PvUbc, TAFII p250, and snoRNP in a yeast two-hybrid assay. Specific interaction between the RING-H2 domain of WSSV249 and PvUbc, TAFII p250, and snoRNP in yeast AH109 cells were detected on a plate lacking tryptophan, leucine, and histidine. Plasmids pGBKT7-53 plus pGADT7-T were indicated as the positive control, while pGBKT7-Lam plus pGADT7-T were used as the negative control. Plasmids pGBKT7 plus pGADT7-cDNA and pGBKT7-RING plus pGADT7 were also employed as controls.



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  		FIG. 3. Sequence analysis of PvUbc. (A) Complete nucleotide and amino acid sequence of PvUbc. The coding sequence is represented in capital letters. (B) Alignment of PvUbc and its orthologs. Identical amino acids are indicated with an asterisk. Included are DmUbc (Drosophila melanogaster; AAS93730, DrUbc (Danio rerio; NP_001002072), HsUbc (Homo sapiens; NP_003338), and MmUbc (Mus musculus; NP_033482). The shrimp E2 PvUbc is 76.0% identical to DmUbc, 82.5% identical to DrUbc, and 81.2% identical to HsUbc and MmUbc. The alignment was performed by the Clustal method in MegAlign.

WSSV249-RING-H2 mediates ubiquitination. To determine whether WSSV249-RING-H2 functions as an E3 ligase, GST fusion proteins for WSSV249-RING-H2 and PvUbc were constructed. Recombinant proteins GST-249 (WSSV249-RING-H2) and GST-PvUbc were expressed and purified (Fig. 4A). The ubiquitination activity of WSSV249-RING-H2 was tested with E1 and PvUbc. Western blot analysis using a ubiquitin antibody, anti-Ub, showed polyubiquitination bands together with E1 and PvUbc. No polyubiquitination smear occurred in control assays lacking either E1, PvUbc, or WSV249-RING-H2 (Fig. 4B, lanes 3, 4, and 1). Similarly, polyubiquitination bands were detected only in the reaction with E1, PvUbc, and WSSV-RING-H2 (Fig. 4C, lane 2) using a GST antibody (anti-GST), and no polyubiquitination band was detected in other reactions (Fig. 4C, lanes 1, 3, and 4). These results suggest that the function of WSSV249-RING-H2 as an E3 ligase is dependent on the presence of both E1 and PvUbc.



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  		FIG. 4. Ubiquitination assay with WSSV249-RING-H2 and PvUbc. (A) Expression and purification of GST-RING and GST-PvUBc fusion proteins. The GST protein was loaded as a control. The numbers indicate the size of the marker protein. (B) RING-H2 domain of WSSV249 mediating ubiquitination is dependent on PvUbc. Ubiquitination assays were carried out to evaluate the E3 ligase activity of the WSSV249 RING-H2 domain in the presence or absence of ubiquitin-activating enzyme (E1), PvUbc (E2), or GST. The samples were resolved on an SDS polyacrylamide gel and detected using antiubiquitin antibody in Western blots. (C) RING-H2 domain of WSSV249 mediating ubiquitination is dependent on PvUbc when detected with anti-GST antibody in Western blots. (D) An intact RING-H2 domain of WSSV249 is necessary for E3 ligase activity. The wild-type (WT) (lane 1) and mutated versions (C313S, C333S, H335Y, and C341S) of WSSV-249-RING-H2 (lanes 2 to 5) were evaluated for mediation of their own ubiquitination in a similar ubiquitination assay.

To demonstrate the importance of the WSSV249 RING finger domain in ubiquitination, WSSV249-RING-H2 point mutants were generated by substituting Cys313, Cys333, and Cys341 with serine and His335 with tyrosine, respectively. Our results demonstrate that modification of the RING-H2 motif disrupts the E3 ligase activity of this protein, as shown in lanes 2 to 5 of Fig. 4D. The distinct polyubiquitination smear is observed only in lane 1 for the wild-type strain of WSSV249-RING-H2.

In order to investigate whether WSSV249-RING-H2 mediates ubiquitination through dependence on other ubiquitin-conjugating enzymes, the ubiquitination activity of WSSV249-RING-H2 was assessed using E1 and a range of ubiquitin-conjugating enzyme E2s (UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH9, and UbcH10). Surprisingly, WSSV249-RING-H2 was able to reconstitute ubiquitination activity in cooperation with seven different ubiquitin-conjugating enzymes: UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, and UbcH10 (Fig. 5). Specific polyubiquitination bands were also detected. Strong ubiquitination activity was observed in the reactions with UbcH1, UbcH5b, and UbcH6 as detected by the multiple bands, as opposed to the weaker activity displayed with UbcH10, with only a few ubiquitination bands (Fig. 5, lanes 2, 6, 16, and 18). These results not only suggest that WSSV249-RING-H2 may function as an E3 ligase but also indicate that it exhibits a low degree of E2 specificity.



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  		FIG. 5. WSSV-RING-H2 exhibits a low degree of E2 specificity. GST-fused WSSV249 RING-H2 was evaluated for ubiquitination by incubation with ATP, ubiquitin, E1, and either one of the E2 enzymes (UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH9, and UbcH10), followed by resolving it on a 10% SDS gel and immunoblotting with anti-Ub antibody. For UbcH1 (lane 2), UbcH2 (lane 4), UbcH5a (lane 10), UbcH5b (lane 6), UbcH6 (lane 16), and UbcH10 (lane 18), polyubiquitination bands were detected.

Detection of an increased expression of PvUbc in a time course study. WSSV249 expression increased in infected shrimps (Fig. 1B and C), and so we investigated whether the expression of PvUbc was also related to WSSV infection. RT-PCR analysis showed that PvUbc expression increased from 3 to 48 h postinfection, compared to the positive control, ß-actin (Fig. 1B). Weak PvUbc expression was also detected in uninfected samples (mock) (Fig. 1B). These results were further demonstrated by real-time PCR. From 0 to 48 h postinfection, the expression of PvUbc increased approximately 2.2-fold (Fig. 1C). These data suggested that PvUbc is induced by WSSV infection.

PvUbc and WSSV249 display a similar expression pattern in infected shrimps. To further investigate the tissue distribution of WSSV249 and PvUbc, in situ hybridization was performed using DIG-labeled antisense RNA probes. Results indicated that both WSSV249 and PvUbc showed strong expression in the subcuticular epithelium cells (Fig. 6E and G) and stomach lining cells of infected shrimps (Fig. 6M and O). High expression of WSSV249 and PvUbc was also detected in hemocytes lodged in the connective tissues of infected shrimps (Fig. 6U and W). However, no expression of either WSSV249 or PvUbc was detected in noninfected shrimps (Fig. 6A, C, I, K, Q, and S). In addition, DIG-labeled sense RNA probes, WSSV249-sense and PvUbc-sense, were employed as controls, and both WSSV249-sense and PvUbc-sense did not display any positive signal in noninfected or infected shrimps (Fig. 6B, F, D, H, J, N, L, P, R, V, T, and X). The expression of WSSV249 and PvUbc in hemocytes is of significance, since hemocytes have been implicated as part of the shrimp immune defense against WSSV infection. Our results suggest that WSSV infection initiates a similar expression pattern between WSSV249 and PvUbc and further support the hypothesis that WSSV249 could sequester PvUbc during viral pathogenesis.



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  		FIG. 6. Detection of WSSV249 and PvUbc mRNAs in the experimentally infected shrimps by in situ hybridization. The infected Penaeus vannamei was sampled 72 h postinfection. In the subcuticular epithelium of the exoskeleton, strong expression of WSSV249 (E) and PvUbc (G) was detected only in infected shrimps, as indicated by arrows. Expression was also detected in the stomach lining cells of the infected shrimps for both WSSV249 (M) and PvUbc (O). In hemocytes present in the connective tissues, strong expression of WSSV249 (U) and PvUbc (W) was detected. No expression for WSSV249 and PvUbc was observed in noninfected shrimps (A and C from the subcuticular epithelium, I and K from the stomach lining cells, Q and S from hemocytes). DIG-labeled sense RNA probes WSSV249-sense and PvUbc-sense were employed as negative controls; neither displayed any positive signal in noninfected or infected shrimps (B, F, D, and H from the subcuticular epithelium, J, N, L, and P from the stomach lining cells, R, V, T, and X from hemocytes). Scale bar = 30 µm.

Increased expression of PvUbc in infected shrimp cells. Based on the increased expression of PvUbc in infected shrimps (Fig. 1B and C), we continued to investigate whether expression of the protein PvUbc was increased in infected shrimps. Primary cell cultures of lymphoid organs from Penaeus vannamei were used for the indirect fluorescent-antibody assay. When primary cell cultures were inoculated with WSSV, stronger signals were detected among cells using anti-PvUbc (Fig. 7B and D) than among uninfected control cells (Fig. 7A and C). Furthermore, an immunohistochemistry assay was performed to test the expression of PvUbc. The results showed that the organ cells, such as stomach epithelial cells from infected shrimps, displayed high expression of PvUbc (Fig. 8D), while PvUbc expression was not detected in cells from uninfected shrimps (Fig. 8C). These data suggested that WSSV infection could induce PvUbc expression.



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  		FIG. 7. Detection of PvUbc in primary cell cultures by IFA. Primary cell cultures of lymphoid organ tissue were inoculated with WSSV (D) or medium (C) (as the control) and detected by anti-PvUbc by fluorescence microscopy (magnification, x200). Primary cell cultures in (A) and (B) under light were used as controls. The arrows indicated the positive signals for PvUbc.



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  		FIG. 8. Increased expression of PvUbc in infected shrimp cells by immunohistochemistry assay. Sample slides from noninfected shrimps and infected shrimps were detected with preimmune rabbit serum as a negative control (A, from noninfected shrimps; B, from infected shrimps) or the anti-PvUbc (C, from noninfected shrimps; D, from infected shrimps). Arrows indicate the positive reaction of PvUbc in the stomach epithelial cells. Scale bar = 20 µm.


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DISCUSSION
 
Viruses employ many fascinating strategies to infiltrate the host line of defense. One of the more interesting and relevant mechanisms is through manipulation of the host's own ubiquitination pathway, where the host proteins are redirected for degradation in the 26S proteosome. This specific process often involves an E3 ubiquitin ligase that is directly encoded by either the virus or the host genome (2). Since ubiquitin-mediated proteolysis of cellular proteins is an important process in many basic cellular processes, perturbations in this system often lead to pathogenesis of many diseases (12). In this study, we have identified and characterized an E3 ligase, RING-H2 protein WSSV249, and analyzed its interaction with a novel ubiquitin-conjugating enzyme, PvUbc from Penaeus vannamei, via the ubiquitination pathway. This interaction could play a key role in the pathogenesis of the WSSV in evading the host defense machinery.

As demonstrated in the yeast two-hybrid assay, WSSV249 exhibited multiple interactions with its host proteins, such as the ubiquitin-conjugating enzyme PvUbc, a transcription factor (TAFII p250), and a U3 snoRNP. TAFII p250 has been described as a key transcription factor involved in mediating monoubiquitination of H1 in vitro and also displays ubiquitin-activating activity (35, 43), while snoRNP has been suggested to be involved in RNA processing and modification (61). This interesting attribute of WSSV249 suggests that it could be involved in utilizing the shrimp's biochemical machinery during WSSV replication and infection, which will be investigated in future studies.

Previous studies have suggested that RING E3 ligases exhibit specific preferences for E2 enzymes in proteolytic degradation (30, 42). Contrary to these findings, WSSV249 seems to display low E2 specificity in the ubiquitination assay. Other than PvUbc, WSSV249 is capable of ubiquitination with UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, and UbcH10. Of particular interest are the interactions between WSSV249 and UbcH1 (E2-25K/Hip-2), a crucial factor in regulating Aß neurotoxicity, and UbcH10, a cancer-related E2 ubiquitin-conjugating enzyme. UbcH1 may play a role in the pathogenesis of Alzheimer's disease (47), while UbcH10 is highly expressed in various human primary tumors and has the ability to promote cell growth and malignant transformation (40). Both E2 enzymes play key roles in human-related diseases, which is of significance to our WSSV research, since previous studies have shown that WSSV249 was detected in WSSV-infected shrimp but not in healthy shrimp (33). This further suggests that the E3 ligase WSSV249 could play a role in pathogenesis of WSSV infection.

E3s are the central determinants of specificity in ubiquitination, which is in turn dependent on specific E2-E3 and E3-substrate interactions (13, 15, 25, 44). The specificity of E2-E3 interaction is determined by only a few amino acids, and it is possible that E2 enzymes do interact with a variety of E3 ligases (42, 62). As observed in our ubiquitination assays, the low specificity for different classes of E2 enzymes is a unique characteristic of WSSV249. Although there are other RING finger proteins that showed a shared preference for different E2s (6, 27, 46, 49), none possesses the same unique and diverse affinity WSSV249 has for the different families of E2 enzymes. The low specificity for E2 enzymes by WSSV249 could also be one of the factors contributing to the broad host range observed with WSSV infections, though further experimental evidence is needed to prove this hypothesis.

Similar to other in situ hybridization studies on WSSV-infected shrimps (9, 16, 52), expression of WSSV249 and PvUbc was detected in the epithelial lining of the stomach, subcuticular epidermis of the cephalothorax, and in aggregating hemocytes in the connective tissues. In a review by Lo and Kou (37), the epidermis and stomach are likely targets for WSSV infection, and the expression of viral genes could be detected in shrimp with low-level infection. Crustaceans lack a true adaptive immune system and instead rely on an innate immune response, in which hemocytes are one of the cellular effectors employed by crustaceans in a defense against pathogenic invasion (1, 22). As suggested by van de Braak et al. (52), WSSV infection could have induced the aggregation of hemocytes to the sites of infection and may themselves be targets for WSSV infection (58). Our previous electron microscopy study also demonstrated that WSSV infects two types of shrimp hemocytes (55). Thus, this would likely explain the expression of WSSV249 and PvUbc in the hemocytes of the connective tissues. Another interesting observation is the induced expression of both WSSV249 and PvUbc in the same tissues of infected shrimps. Of the many functional properties reported for E2 enzymes, one of them is as a stress response factor (29). During infection, the increasing degree of stress experienced by the host shrimp, as well as the presence of foreign proteins, may trigger the production of PvUbc for degradation of shrimp host proteins via the ubiquitination pathway, which ultimately leads to host cell pathogenesis.

The study of shrimp viruses has been hampered by the lack of a reliable and convenient in vitro cell culture system (48). A previous report (26) claimed development of a cell line, "PMO," from shrimp lymphoid organ. However, increasing doubts on the true origin of the "PMO" cell line prompted us to carry out a sequence analysis using "PMO" DNA as a template and universal primers 5'-CGCCTGTTTAACAAAAACAT-3' and 5'-CCGGTCTGAACTCAGATCATGT-3' for shrimp mitochondrial 16S rRNA (5) in a PCR analysis. Sequence analysis of the PCR product showed that the "PMO" cell line 16S rRNA shares 100% homology with Anguilla japonica mitochondrial 16S rRNA (data not shown), which led us to conclude that "PMO" is actually derived from Anguilla japonica, a species of eel. We therefore employed the use of primary cell cultures from shrimps for WSSV studies. Several investigators have reported on successful development of WSSV-infected primary cultures from lymphoid organs from Penaeus monodon (32, 54), the blue shrimp, Litopenaeus stylirostris (48), and the kuruma shrimp, Marsupenaeus japonicus (28). Primary shrimp cell cultures were also developed from ovaries of black tiger shrimp, P. monodon (32), and the use of primary ovarian cultures of the kuruma shrimp, Marsupenaeus japonicus, was reported to facilitate characterization of WSSV infection (39). In this study, we developed primary cell cultures from the lymphoid organs of P. vannamei and used them for indirect fluorescent-antibody studies. The establishment of a continuous shrimp cell line thus remains a challenge for shrimp virus research.

In this study, we have demonstrated a physical interaction between the RING-H2 domain of WSSV249 and PvUbc and characterized the WSSV249-RING-H2 function as an E3 ubiquitin ligase. Both WSSV249 and PvUbc expression increased in a time course study, suggesting that WSSV249 and PvUbc could be related to virus pathogenesis. In addition, WSSV249 and PvUbc displayed a similar pattern with strong expression in infected shrimps, implying that WSSV249 and PvUbc are associated with WSSV infection. Moreover, our data showed that WSSV249-RING-H2 exhibited a low degree of E2 specificity. This low degree of E2 specificity from WSSV249-RING-H2 could be of significance for WSSV pathogenesis. Furthermore, a specific interaction of the PRRSV nucleocapsid protein with the host cell protein fibrillarin (one component of snoRNPs) was demonstrated in the nucleolus, implying a potential linkage of viral strategies for the modulation of host cell functions, possibly through rRNA precursor processing and ribosome biogenesis (61), and TFIIH, a transcription factor, was identified as a target for the Rift Valley hemorrhagic fever virus (35). Whether WSSV employs a similar mechanism for targeting snoRNP or TAFII p250, like PRRSV or Rift Valley hemorrhagic fever virus, needs to be investigated in future studies.


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ACKNOWLEDGMENTS
 
We express our gratitude to Lin Sheng Wong (Genomex Technologies Pte. Ltd.) for his technical assistance with the construction of the shrimp cDNA library. We also thank Yinglin Ang, Poh Nee Er, and He Qigai for their help in experiments.

This work was supported by Temasek Life Sciences Laboratory.


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FOOTNOTES
 
* Corresponding author. Mailing address: Animal Health Biotechnology Group, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore. Phone: 65-68727473. Fax: 65-68727007. E-mail: kwang{at}tll.org.sg. Back


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Journal of Virology, July 2005, p. 8764-8772, Vol. 79, No. 14
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.14.8764-8772.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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