Binding of shrimp cellular proteins to Taura syndrome viral capsid proteins VP1, VP2 and VP3

Virus Research 122 (2006) 69–77

Binding of shrimp cellular proteins to Taura syndrome viral capsid proteins VP1, VP2 and VP3 Saengchan Senapin a,b,∗ , Amornrat Phongdara c a

b

National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand c Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkla 90112, Thailand Received 20 January 2006; received in revised form 23 June 2006; accepted 28 June 2006 Available online 4 August 2006

Abstract Viruses are a major cause of production losses in the world shrimp-farming industry. Despite this, little is known about viral–host interactions in shrimp due in part to the lack of continuous shrimp cell lines. Here, the yeast two-hybrid assay system was employed to study interactions between three Taura syndrome viral capsid proteins (VP1–VP3) and proteins from a cDNA library of the black tiger shrimp Penaeus monodon. VP1 interacted with ␤-actin, elongation factor 1␣ (EF1␣), lysozyme (Lys) and laminin receptor/ribosomal protein p40 (Lamr/p40) containing a putative palindromic laminin binding region LMWWML. VP2 interacted with ␤-actin and EF1␣, while VP3 bound to the same proteins as VP1 except for Lamr/p40. In vitro pull-down assays confirmed these interactions. The most interesting interaction was specific binding between VP1 and Lamr/p40 since Lamr/p40 has been identified as the mammalian cell receptor for several arthropod-borne viruses (arboviruses). A search of mosquito vector and Drosophila sequences at available databases revealed the presence of putative Lamr/p40 proteins with high homology to the Lamr/p40 from shrimp. © 2006 Elsevier B.V. All rights reserved. Keywords: TSV; Capsid; Yeast two-hybrid; Laminin receptor; Shrimp

1. Introduction Taura syndrome virus (TSV) is a major marine shrimp pathogen that has caused serious economic losses in the shrimp farming industry (Lightner, 1996). It first caused disease outbreaks in Ecuador in 1992 but its viral etiology was not discovered for several years (Hasson et al., 1995). It has since become widely distributed throughout the Americas (Hasson et al., 1999a) and Asia (Tu et al., 1999; Yu and Song, 2000). Infection in the Pacific white shrimp Penaeus vannamei (also called Litopenaeus vannamei) occurs in three phases (Hasson et al., 1995, 1999b,c). Gross signs of acute TSV infection in P. vannamei include soft shell and the appearance of red body, tail and/or appendages due to chromatophore expansion. Animals in the recovery phase have black lesions in the cuticle and those in the chronic phase look grossly normal. TSV has non-enveloped,

∗

Corresponding author. Tel.: +662 2015889; fax: +662 3547344. E-mail address: [email protected] (S. Senapin).

0168-1702/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2006.06.013

icosahedral virions 31–32 nm in diameter (Bonami et al., 1997) and a genome of single-stranded positive-sense RNA comprised of 10,205 nucleotides containing two open reading frames, designated ORF1 and ORF2 (Bonami et al., 1997; Mari et al., 2002). It has been classified in the family Dicistroviridae near the genus Cripavirus (cricket paralysis virus) (Mayo, 2002, 2005; Fauquet et al., 2004). ORF1 encodes a putative nonstructural protein of 234 kDa and ORF2 codes for three major capsid proteins of 55, 40, and 24 kDa called VP1, VP2, and VP3, respectively. Viral infection involves a number of steps. Initial processes crucial for the viral life cycle include attachment of the virion to host cell surface receptor(s) followed by cell entry and genome delivery (Flint et al., 2003). The virus releases its genome into the cell either directly through the plasma membrane after cell attachment or by fusion of the viral membrane with the endosome membrane after endocytosis (White, 1990; Sodeik, 2000; Smith and Helenius, 2004). However, all of these and other steps in viral–host interaction for shrimp viral pathogens are poorly defined, due in part to the lack of continuous shrimp cell lines. The present study aimed to identify shrimp proteins that interact

70

S. Senapin, A. Phongdara / Virus Research 122 (2006) 69–77

2. Methods

plate, 0.2 ␮M of each primer, 1 ␮l of SuperScript One-Step RT/Platinum Taq Mix (Invitrogen), and 1× reaction buffer. The RT reaction was carried out at 50 ◦ C for 30 min. The mixture was then denatured at 94 ◦ C for 2 min, and amplification was performed using a cycling scheme of 94 ◦ C for 15 s, 50–60 ◦ C for 30 s, and 72 ◦ C for 1 min/kb, for 40 cycles. The cDNA products were purified followed by insertion in frame with the Gal4-binding domain (BD) of pGBKT7, creating pBD-VP1, pBD-VP2, and pBD-VP3, respectively. The constructs were subsequently sequenced to confirm gene sequences and correct reading frames.

2.1. Strains and general techniques

2.4. Yeast two-hybrid assay

The strain of Saccharomyces cerevisiae used in this study was AH109 (MATa, trp1–901, leu2–3, 112, ura3–52, his3–200, gal4Δ, gal80Δ, LYS2::GAL1UAS -GAL1TATA -HIS3, MEL1, GAL2UAS GAL2TATA -ADE2, URA3::MEL1UAS -MEL1TATA -lacZ) from Clontech. Yeast cells were cultured at 30 ◦ C either in a complete YPD medium (1% yeast extract, 1% peptone, 2% glucose) or in a synthetic defined (SD) medium supplemented with required essential nutrients. Plates contained 2% Bacto agar. Transformation of yeast cells was performed by the lithium acetate/dimethyl sulfoxide method (Gietz et al., 1995). Escherichia coli XL1-Blue and BL21 pLysS (DE3) were used for general cloning and protein expression, respectively. DNA manipulation was performed according to established protocols (Sambrook and Russell, 2001; Burke et al., 2000).

Yeast two-hybrid screens were carried out based on the Clontech Matchmaker GAL4 Two-Hybrid System. S. cerevisiae AH109 was co-transformed with pBD-VP1, pBD-VP2 or pBDVP3 and a Gal4(AD)-fused P. monodon hemocyte cDNA library previously established (Tonganunt et al., 2005). Cells were then plated on SD medium lacking tryptophan and leucine and scored for growth at 30 ◦ C. Interactions between BD and AD constructs were indicated by both growth on media lacking adenine and histidine and by a blue color change due to X-␣gal (Apollo Scientific, UK) contained in the medium. Rescued library plasmids from positive clones were simultaneously cotransformed into AH109 with pBD-VP1 and the specificity of the interactions was confirmed by growth on SD/-Leu/-Trp/Ade/-His/X-␣-gal plates. The empty vector pGADT7 was used as a negative control and interaction between murine p53 bait fusion and SV40 prey fusion (BD Biosciences) served as a positive control. Library plasmids obtained were sequenced by Macrogen Co. Ltd. (South Korea). cDNA sequences were compared to known DNA and protein sequences using the BLAST network service (Altschul et al., 1990). Potential interactions between pBD-VP2 and pBD-VP3 and identified library plasmids were tested in a yeast two-hybrid system as described above.

with TSV components. It was expected that a better understanding would be achieved as to how shrimp respond to viruses at the molecular level. Since capsid proteins are the structural molecules of TSV and other non-enveloped viruses, they are the logical candidates for initial recognition and/or interaction with host cells. We therefore use TSV capsid proteins VP1 to VP3 as bait to screen for interacting proteins from a black tiger shrimp (P. monodon) hemocyte cDNA library using the yeast two-hybrid assay system.

2.2. RNA preparation Naturally TSV-infected white shrimp (P. vannamei) were provided by the Shrimp Biotechnology Business Unit, Thailand. Hemolymph of animals was collected in an equal volume of anticoagulant AC1 solution (Soderhall and Smith, 1983) and the hemocytes were obtained by centrifugation at 800 × g for 15 min. Total RNA was then prepared using Trizol reagent (Invitrogen). RNA concentration and quality were measured by spectrophotometric analysis at 260 and 280 nm. Diagnosis using an IQ2000 TSV detection kit (Farming IntelliGene Biotechnology Corporation, Taiwan) verified that the RNA sample was TSV positive. 2.3. Construction of TSV capsid bait plasmids TSV VP1, VP2, and VP3 capsid genes were amplified using RT-PCR. Primers were designed based on a complete sequence of the TSV genome (GenBank accession no. NC 003005). These were VP1F (5 -GCATG CCA TGG GAT CAA AAG ATA GGG ATAT-3 ) and VP1R (5 -GAAAA CTG CAG ATG TGT GGA TGG ATA TATA-3 ), VP2F (5 -GCATG CCA TGG GAG CTA ACC CAG TTT GAAAT-3 ) and VP2R (5 -GAAA CTG CAG AAA TCC GAA CAT TGA AGCT-3 ), and VP3F (5 -GCATG CCA TGG GAG CTG GTC TGG ACT ACTC-3 ) and VP3R (5 -GAAA CTG CAG AGC CAA TTC GGC AGG TCCA3 ), respectively, where underlines represent restriction sites. RT-PCR reactions of 50 ␮l consisted of 100 ng of RNA tem-

2.5. Protein expression in yeast AH109 cells transformed with bait fusion constructs were grown in SD/-Trp at 30 ◦ C overnight, subcultured in the corresponding fresh medium and allowed to grow at 30 ◦ C until a cell density of OD 600 nm of 0.8 was reached. Yeast cells were harvested by centrifugation at 3000 × g for 10 min at 4 ◦ C. Yeast crude cell extracts were prepared as described previously (Kushnirov, 2000). The yeast cell extract was separated by SDS-PAGE and transferred to a nitrocellulose membrane (Hybond ECL, Amersham Biosciences). After blocking with Tris-buffered saline (TBS) containing 5% nonfat milk at room temperature for 1 h, the membrane was washed with TBS, 0.1% Tween-20 (TBST) and incubated overnight at 4 ◦ C with Anti-cMyc antibody (BD Biosciences) appropriately diluted in TBST containing 5% nonfat milk. The blot was then washed with TBST and incubated with AP-conjugated anti-mouse IgG (Zymed) at a 1:5000 dilution. Capsid fusion proteins were detected using NBT/BCIP substrate.

S. Senapin, A. Phongdara / Virus Research 122 (2006) 69–77

71

2.6. Expression and purification of fusion proteins in E. coli GST-fusion proteins (Lys, Lamr/p40, and VP1) and Hisfusion proteins (VP1, VP2, VP3, and Lamr/p40) were expressed in E. coli XL1-Blue and BL21 pLysS (DE3), respectively. Crude bacterial extracts were prepared from IPTG-induced and non-induced cells. Equal amounts of protein were separated on a 12% SDS-PAGE followed by blotting onto nitrocellulose membranes. Detections of GST-fusion proteins and His-fusion proteins were performed using antiGST antibody (Amersham Biosciences) and anti-His antibody (Zymed), respectively. Appropriate anti-mouse and anti-rabbit horseradish peroxidase-coupled secondary antibodies were purchased from Zymed. The detection procedure was performed according to the supplier’s protocol. Subsequently, 100 ml culture scale was prepared for the purification of GST- and His-fusion proteins using glutathione sepharose 4B (Amersham Biosciences) and Ni-NTA (Qiagen) beads, respectively. 2.7. In vitro pull-down assay Purified His-TSV capsid fusion proteins were incubated with 250 ␮l of Ni-NTA (Qiagen) for 1 h with rocking at 4 ◦ C. The beads were washed 10 times with wash buffer before addition of purified GST and GST-tag fusion proteins. Beads were incubated 1 h at room temperature with rocking before the washings. Protein complexes were eluted with buffer containing 250 mM imidazol. A reciprocal pull-down assay was performed between GST-VP1 and His-Lamr/p40 using glutathione sepharose 4B (Amersham Biosciences). His-Lamr/p40 was incubated with GST or GST-VP1 that had been immobilized on glutathione-sepharose 4B. Following 1 h incubation at 4 ◦ C, samples were washed and eluted with reduced glutathione. Bound proteins were analyzed by SDS-PAGE and Western blotting. Signals were detected by chemiluminescent or colorimetric methods. 3. Results 3.1. Identification of VP1-interacting proteins The pBD-VP1 construct was tested for fusion protein expression in yeast. The c-Myc tagged VP1 product of expected size was detected using an antibody against the c-Myc epitope (data not shown). Using pBD-VP1 as bait for screening the P. monodon hemocyte cDNA library, four cDNA clones that produced proteins able to bind to VP1 were found. After DNA sequence analysis and BLAST searching (Altschul et al., 1990), three of the sequences matched previously reported shrimp proteins including ␤-actin, elongation factor 1␣ (EF1␣), and lysozyme (Lys) (GenBank accession numbers AF300705, AY117542 and AF539466, respectively). The fourth clone had strong similarity to laminin receptor and/or ribosomal protein p40 (Lamr/p40) of various organisms. These interactions were confirmed by cotransformation into yeast and analysis in the two-hybrid system (Fig. 1).

Fig. 1. Interaction of TSV capsid proteins with P. monodon proteins. VP1, VP2, and VP3 capsid proteins were constructed in frame with the BD domain and shrimp components with the AD domain. Yeast AH109 cells were cotransformed with BD and AD constructs and grown on SD/-Leu/-Trp plate at 30 ◦ C for 3 days. Growth of three independent transformants on SD/-Leu/-Trp and SD/Leu/-Trp/-Ade/-His/X-␣-gal plates was compared. Interaction (+) was demonstrated as formation of blue yeast colonies on the latter plate, while no interaction (−) was indicated by no growth. Yeast containing murine p53 bait fusion and SV40 prey fusion (BD Biosciences) served as the positive control whereas yeast harboring pBD-VP1 and empty pGADT7 plasmid served as the negative control.

3.2. Interaction tests of yeast two-hybrid isolated cDNAs with VP2 and VP3 The pBD-VP2 and pBD-VP3 constructs harboring two other TSV capsid proteins were shown to be expressed in yeast cells by Western analysis (data not shown). Pair wise interaction tests, as described for pBD-VP1 above, showed that VP2 interacted with ␤-actin and EF1␣, while VP3 bound to ␤-actin, EF1␣, and Lys (Fig. 1). These results indicated that binding to Lamr/p40 occurred with VP1 only. 3.3. In vitro pull-down assay confirmed the yeast two-hybrid results The yeast two-hybrid results indicated that ␤-actin and EF1␣ bound to all three TSV capsid proteins while interactions with Lys and Lamr/p40 were more specific. Therefore, confirmation tests using in vitro pull-down assays were conducted between Lys and Lamr/p40 and VP1 and VP3. Incubation of purified GST-Lamr/p40 and GST-Lys individually with purified Histagged VP1 (His-VP1) or His-VP3 proteins bound to Ni-NTA beads followed by elution and Western blotting revealed that His-VP1 bound to both GST-Lamr/p40 and GST-Lys whereas His-VP3 binds to GST-Lys only (Fig. 2A). This confirmed the results from the yeast two-hybrid assay. Binding was not medi-

72

S. Senapin, A. Phongdara / Virus Research 122 (2006) 69–77

3.5. Sequence analysis of Lamr/p40

Fig. 2. Western blotting of in vitro pull-down assays. (A) Purified GST, GSTLamr/p40, and GST-Lys were incubated with His-VP1 immobilized on Ni-NTA beads (lanes 1, 2, and 3, respectively). Binding tests of GST, GST-Lamr/p40, and GST-Lys with His-VP3 were performed in the same way (lanes 4, 5, and 6, respectively). Bound proteins were run on a 12% SDS-PAGE and analyzed by Western blotting using anti-His and anti-GST antibodies. (B) Reciprocal pulldown assay was performed between VP1 and Lamr/p40. His-Lamr/p40 was incubated with GST-VP1 or GST that had been immobilized on glutathionesepharose beads. The pull-down products were subjected to Western blotting analysis using anti-His antibody. “Input” represents input His-Lamr/p40 protein in the binding tests.

ated via the GST-tag since no GST protein was found together with either His-VP1 or His-VP3. The reciprocal pull-down assays between VP1 and Lamr/p40 (i.e., by switching the vectors that had formerly used for cloning of both genes) revealed the expected band of His-Lamr/p40 in the pull-down product of GST-VP1 but not in that of GST by Western blotting with antiHis antibody (Fig. 2B). This confirmed binding between the two proteins. 3.4. Sequence analysis of Lys Sequence comparisons against P. monodon Lys (GenBank accession no. AF539466) and other available sequences in databases suggested that 24 amino acid residues were missing at the N-terminus of the deduced protein sequence of the Lys clone identified from the yeast two-hybrid assay. In addition, there were three nucleotide differences, one at position 356 that changed lysine 119 to methionine and one at position 105 and one at 240 that did not change the deduced sequence (Fig. 3A). Although sequence error could be introduced during cDNA library construction, it is also possible that the sequence variation was due to genetic polymorphism. The complete Lys ORF (except for the first methionine) was cloned into pGBKT7 plasmid for an additional protein interaction test and found to bind to VP1 (data not shown).

The Lamr/p40 clone sequence obtained in this study was compared to an EST clone identified under a project entitled “Genome research for increasing culture efficiency of the black tiger shrimp (Penaeus monodon) phase I: largescale cDNA sequencing and development of genetic markers” (Phongdara et al., unpublished). It was found to lack the first 19 amino acid residues. The nucleotide sequence of P. monodon Lamr/p40 has previously been submitted into the GenBank under accession number DT044263. The complete Lamr/p40 ORF (except for the first methionine) was also found to bind to VP1 in a yeast two-hybrid assay (data not shown). The P. monodon Lamr/p40 gene encodes a protein of 318 amino acids (Fig. 4) with a predicted molecular weight of 33.8 kDa. The P. monodon Lamr/p40 sequence is an alaninerich protein harboring 65 alanine residues (20.4%). A potential helical transmembrane domain of Lamr/p40 was predicted by the PHDsec program (Rost and Sander, 1993) and located at the alanine-rich C-terminal region (Fig. 3B). Comparison of the deduced protein sequence with the GenBank database using a BLAST search (Altschul et al., 1990) yielded over 400 hits to the deduced amino acid sequences of various organisms. However, we chose only sequences from organisms where it has been demonstrated that the laminin receptors facilitate the entry of pathogenic agents. These organisms are humans (Protopopova et al., 1997), Chinese hamster (Cricetulus griseus) (Wang et al., 1992) and the mosquito Aedes albopictus (Ludwig et al., 1996). Unfortunately, the Lamr/p40 sequence of A. albopictus has not been released, we therefore used instead the putative sequences from two genera of mosquitoes that are listed: A. aegypti (TIGR accession no. TC56656) and Anopheles gambiae (TIGR accession no. TC91618). In addition, since shrimp belong to phylum arthropoda, potential complete Lamr/p40 sequences from other arthropods including the fruit fly (Drosophila melanogaster), bee (Apis mellifera), silkworm (Bombyx mori) and midge (Culicoides sonorensis) (GenBank accession numbers AAN09049, XP 393965, AAV34856, and AAV84247, respectively), were also included in the comparison. An alignment performed using ClustalW program (Thompson et al., 1994) revealed 60–74% sequence identity of shrimp Lamr/p40 with these proteins. Similarity was less pronounced at the C-terminal region (Fig. 4). It has been reported that peptide G (sequence I P C N N K G A H S V G L M W W M L A R), corresponding to positions 161–180 of human Lamr/p40, exhibits laminin-binding activity and that the palindromic sequence L M W W M L is the active site (Castronovo et al., 1991). Our identified Lamr/p40 contains the sequence I P C N N R S P H S I G L M W W M L A R and thus shares 16 out of 20 amino acids with peptide G including the palindromic sequence. The palindromic sequences of B. mori, A. mellifera, D. melanogaster, A. aegypti, and C. sonorensis differ by only one amino acid (i.e., Leu instead of Met) for the second M in the palindrome. However, the two consecutive Trp residues, that often mediate protein–protein interaction (Macias et al., 1997), are still present.

S. Senapin, A. Phongdara / Virus Research 122 (2006) 69–77

73

Fig. 3. The nucleotide and deduced amino acid sequences of full-length P. monodon Lys and Lamr/p40. (A) Sequence comparison of Lys obtained from the GenBank database (accession no. AF539466) and this study is shown. Nucleotide and amino acid differences are indicated by appropriate letters, while identical nucleotides are represented by dots. (B) The in frame stop codon in the 5 -untranslated region of P. monodon Lamr/p40 is underlined. A potential helical transmembrane domain is shaded.

74

S. Senapin, A. Phongdara / Virus Research 122 (2006) 69–77

Fig. 4. Comparison of deduced amino acid sequences of potential laminin receptor proteins. Protein sources were B. mori (GenBank accession no. AAV34856), A. mellifera (GenBank accession no. XP 393965), D. melanogaster (GenBank accession no. AAN09049), A. gambiae (TIGR accession no. TC91618), A. aegypti (TIGR accession no. TC56656), C. sonorensis (GenBank accession no. AAV84247), P. monodon (GenBank accession no. DT044263), H. sapiens (GenBank accession no. AAB22299), and C. griseus (GenBank accession no. CAA80434). Sequences were aligned using ClustalW program (Thompson et al., 1994). Identical amino acids are marked with asterisks, similar amino acids indicated by dots, and gaps introduced to optimize the alignment by dashes. The shaded region indicates a potential palindromic sequence reported to confer laminin-binding activity.

S. Senapin, A. Phongdara / Virus Research 122 (2006) 69–77

4. Discussion Generally, viral attachment proteins including envelope and outer layer capsid proteins are responsible for recognition and interaction with specific host cell receptors prior viral entry. It has been shown that antibodies against white spot syndrome virus (WSSV) VP28 envelope protein were able to prevent WSSV infection in the black tiger shrimp P. monodon (van Hulten et al., 2001; Yi et al., 2004). More recently, additional envelope proteins have been shown to be involved in WSSV infection (Huang et al., 2005; Wu et al., 2005). Despite these discoveries, the mechanism of viral entry into shrimp cells is unclear. Additionally, little is known of protein–protein interactions between shrimp and their pathogens. This study has successfully identified shrimp proteins that interact with viral capsid proteins using a yeast two-hybrid screen. To date, there are only two other reports on the use of this method in shrimp research (Lu and Kwang, 2004; Tonganunt et al., 2005). We found that ␤-actin and EF1␣ bound to all three TSV capsid proteins. These two proteins have previously been reported to interact with viral proteins. ␤-Actin has been reported to bind to WSSV nucleocapsid protein VP26 (Xie and Yang, 2005). Several other studies have indicated that the cytoskeleton component actin can facilitate intracellular transport of viruses or viral genomes (Li et al., 1998; Lyengar et al., 1998; Vanderpasschen et al., 1998; Liu et al., 1999). Translation factor EF1␣ has been shown to interact with the viral matrix (MA) and nucleocapsid domains of human immunodeficiency virus type 1 Gag polyprotein (Cimarelli and Luban, 1999). This interaction between EF1␣ and MA inhibits translation of viral genomic RNA and may thus make it available for packaging into nascent virions. Further study is required to explore possible roles of shrimp EF1␣ interaction with TSV. We found that Lys bound to VP1 and VP3, and this was confirmed by in vitro pull-down assays. Lys is well known for its ability to hydrolyze bacterial cell walls and this has been demonstrated for kuruma shrimp Lys against several Vibrio species that are major bacterial pathogens of shrimp (Hikima et al., 2003). To date, there have been no reports of Lys activity against shrimp viruses. However, Lee-Huang et al. (1999) have described Lys inhibition of HIV. The possible consequences of Lys binding to TSV capsid proteins VP1 and VP3 but not VP2 should be further investigated. Lamr/p40 bound to TSV capsid protein VP1 only. Lamr/p40 was first isolated from mammalian cells as a 67-kDa laminin receptor (Lesot et al., 1983; Malinoff and Wicha, 1983; Rao et al., 1983). So far however, a full-length gene encoding a 37-kDa laminin receptor is the only protein that has been isolated. It has been suggested that the 37-kDa polypeptide serves as a precursor of the 67-kDa protein (Castronovo et al., 1991) and that transition to the mature protein involves dimerization and acylation of the precursor protein (Buto et al., 1997). The binding of basement membrane laminin proteins to specific laminin receptors has been implicated in a wide range of biological processes, including cell adhesion, morphogenesis, migration, and differentiation (Castronovo, 1993). In addition, Lamr/p40 has been implicated as a receptor for cellular prion protein (Rieger et al.,

75

1997; Gauczynski et al., 2001). The Lamr/p40 laminin-binding site which has been reported to comprise the six amino acid palindrome L M W W M L (Castronovo et al., 1991; Magnifico et al., 1996; Ardini et al., 1998; Hundt et al., 2001; Jaseja et al., 2005) is also found in the P. monodon Lamr/p40 sequence. Lamr/p40 has also been found in the 40S ribosomal subunit where it reportedly functions in protein synthesis (Auth and Brawerman, 1992; Rosenthal and Wordeman, 1995). Since the Lamr/p40 sequences are present in both prokaryotes and eukaryotes, it has been hypothesized that they are ribosomeassociated proteins with conserved N-terminal regions that function in translation and with divergent C-terminal sequences that acquired additional roles as receptors during evolution (Ardini et al., 1998). If this is a case, the alanine-rich C-terminal part of shrimp Lamr/p40 is likely to mediate receptor functions. Viruses reported to utilize Lamr/p40 as a receptor include Sindbis virus (Wang et al., 1992), Venezuelan equine encephalitis virus (Ludwig et al., 1996), flavivirus tick-borne encephalitis virus (Protopopova et al., 1997) and dengue virus (Thepparit and Smith, 2004; Tio et al., 2005). Interestingly, all of these viruses are arthropod-borne viruses or arboviruses. The host cells reported to have laminin receptors facilitating viral entry were from humans, hamsters and a mosquito vector. Our search of GenBank and other databases revealed the existence of potential Lamr/p40 sequences in every arthropod whose sequence is available. This information and our results with TSV lead us to suggest that Lamr/p40 type receptors may serve as common viral receptors in both mammalian and arthropods. In any case, Lamr/p40 is a prime candidate for further study as a shrimp cellular receptor for TSV. Acknowledgements This work was supported by grant BT-B-02-SG-B7-4805 from National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand. The authors would like to thank Prof. T.W. Flegel, Centex Shrimp, for advice in preparing the manuscript and S. Suklour, Prince of Songkla University, for help with pull-down assays. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Ardini, E., Pesole, G., Tagliabue, E., Magnico, A., Castronovo, V., Sobel, M.E., Colnaghi, M.I., Menard, S., 1998. The 67-kDa laminin receptor originated from a ribosomal protein that acquired a dual function during evolution. Mol. Biol. Evol. 15, 1017–1025. Auth, D., Brawerman, G., 1992. A 33-kDa polypeptide with homology to the laminin receptor: component of translation machinery. Proc. Natl. Acad. Sci. U.S.A. 89, 4368–4372. Bonami, J.-R., Hasson, K.W., Mari, J., Poulos, B.T., Lightner, D.V., 1997. Taura syndrome of marine penaeid shrimp: characterization of the viral agent. J. Gen. Virol. 78, 313–319. Burke, D., Dawson, D., Stearns, T., 2000. Methods In Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, 2000 ed. Cold Spring Harbor Laboratory Press, NY.

76

S. Senapin, A. Phongdara / Virus Research 122 (2006) 69–77

Buto, S., Tagliabue, E., Ardini, E., Magnifico, A., Ghirelli, C., van den Brule, F., Castronovo, V., Colnaghi, M.I., Sobel, M.E., Menard, S., 1997. Formation of the 67-kDa laminin receptor by acylation of the precursor. J. Cell. Biochem. 69, 244–251. Castronovo, V., 1993. Laminin receptors and laminin-binding proteins during tumor invasion and metastasis. Invasion Metastasis 13, 1–30. Castronovo, V., Taraboletti, G., Sobel, M.E., 1991. Functional domains of the 67-kDa laminin receptor precursor. J. Biol. Chem. 266, 20440– 20446. Cimarelli, A., Luban, J., 1999. Translation elongation factor 1-alpha interacts specifically with the human immunodeficiency virus type 1 Gag polyprotein. J. Virol. 73, 5388–5401. Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A., 2004. Virus Taxonomy, VIIIth Report of the ICTV. Elsevier/Academic Press, London. Flint, S.J., Enquist, L.W., Racaniello, V.R., Skalka, A.M., 2003. Principles of Virology: Molecular Biology, Pathogenesis and Control of Animal Viruses, second ed. American Society Microbiology, Washington. Gauczynski, S., Peyrin, J.M., Haik, S., Leucht, C., Hundt, C., Rieger, R., Krasemann, S., Deslys, J.P., Dormont, D., Lasmezas, C.I., Weiss, S., 2001. The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein. EMBO J. 20, 5863–5875. Gietz, R.D., Schiestl, R.H., Willems, A.R., Woods, R.A., 1995. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355–360. Hasson, K.W., Lightner, D.V., Mari, J., Bonami, J.R., Poulos, B.T., Mohney, L.L., Redman, R.M., Brock, J.A., 1999a. The geographic distribution of Taura Syndrome Virus (TSV) in the Americas: determination by histopathology and in situ hybridization using TSV-specific cDNA probes. Aquaculture 171, 13–26. Hasson, K.W., Lightner, D.V., Mohney, L.L., Redman, R.M., Poulos, B.T., White, B.M., 1999b. Taura syndrome virus (TSV) lesion development and the disease cycle in the Pacific white shrimp Penaeus vannamei. Dis. Aquat. Org. 36, 81–93. Hasson, K.W., Lightner, D.V., Mohney, L.L., Redman, R.M., White, B.M., 1999c. Role of lymphoid organ spheroids in chronic Taura syndrome virus (TSV) infections in Penaeus vannamei. Dis. Aquat. Org. 38, 93– 105. Hasson, K.W., Lightner, D.V., Poulos, B.T., Redman, R.M., White, B.L., Brock, J.A., Bonami, J.R., 1995. Taura Syndrome in Penaeus vannamei: demonstration of a viral etiology. Dis. Aquat. Org. 23, 115–126. Hikima, S., Hikima, J., Rojtinnakorn, J., Hirono, I., Aoki, T., 2003. Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species. Gene 316, 187–195. Huang, R., Xie, Y.L., Zhang, J.H., Shi, Z.L., 2005. A novel envelope protein involved in White spot syndrome virus infection. J. Gen. Virol. 86, 1357–1361. Hundt, C., Peyrin, J.M., Haik, S., Gauczynski, S., Leucht, C., Rieger, R., Riley, M.L., Deslys, J.P., Dormont, D., Lasmezas, C.I., Weiss, S., 2001. Identification of interaction domains of the prion protein with its 37-kDa/67-kDa laminin receptor. EMBO J. 20, 5876–5886. Jaseja, M., Mergen, L., Gillette, K., Forbes, K., Sehgal, I., Copie, V., 2005. Structure-function studies of the functional and binding epitope of the human 37 kDa laminin receptor precursor protein. J. Pept. Res. 66, 9–18. Kushnirov, V.V., 2000. Rapid and reliable protein extraction from yeast. Yeast 16, 857–860. Lee-Huang, S., Huang, P.L., Sun, Y.T., Huang, P.L., Kung, H.F., Blithe, D.L., Chen, H.C., 1999. Lysozyme and RNases as anti-HIV components in betacore preparations of human chorionic gonadotropin. Proc. Natl. Acad. Sci. U.S.A. 96, 2678–2681. Lesot, H., Kuhl, U., von der Mark, K., 1983. Isolation of a laminin-binding protein from muscle cell membrane. EMBO J. 2, 861–865. Li, E., Stupack, D., Bokoch, G.M., Nemerow, G.R., 1998. Adenovirus endocytosis requires actin cytoskeleton reorganization mediated by Rho family GTPases. J. Virol. 72, 8806–8812. Lightner, D.V., 1996. Epizootiology, distribution and the impact on international trade of two penaeid shrimp viruses in the Americas. Rev. Sci. Tech. 15, 579–601.

Liu, B.D., Dai, R.K., Tian, C.J., Dawson, L., Gorelick, R., Liu, X.F., 1999. Interaction of the human immunodeficiency virus type 1 nucleocapsid with actin. J. Virol. 73, 2901–2908. Lu, L.Q., Kwang, J., 2004. Identification of a novel shrimp protein phosphatase and its association with latencey-related ORF427 of white spot syndrome virus. FEBS Lett. 577, 141–146. Ludwig, G.V., Kondig, J.P., Smith, J.F., 1996. A putative receptor for Venezuelan equine encephalitis virus from mosquito cells. J. Virol. 70, 5592–5599. Lyengar, S., Hildreth, J.E., Schwartz, D.H., 1998. Actin-dependent receptor colocalization required for human immunodeficiency virus entry into host cells. J. Virol. 72, 5251–5255. Macias, M.J., Hyvonen, M., Baraldi, E., Schultz, J., Sudol, M., Saraste, M., Oschkinat, H., 1997. Structure of of the WW domain of a kinaseassociated protein complexed with a proline-rich peptide. Nature 382, 646– 649. Magnifico, A., Tagliabue, E., Buto, S., Ardini, E., Castronovo, V., Colnaghi, M.I., Menard, S., 1996. Peptide G, containing the binding site of the 67-kDa laminin receptor, increases and stabilizes laminin binding to cancer cells. J. Biol. Chem. 271, 31179–31184. Malinoff, H.L., Wicha, M.S., 1983. Isolation of a cell surface receptor protein for laminin from murine fibrosarcoma cells. J. Cell Biol. 96, 1475– 1479. Mari, J., Poulos, B.T., Lightner, D.V., Bonami, J.R., 2002. Shrimp Taura syndrome virus: genomic characterization and similarity with members of the genus Cricket paralysis-like viruses. J. Gen. Virol. 83, 915–926. Mayo, M.A., 2002. A summary of taxonomic changes recently approved by ICTV. Arch. Virol. 147, 1655–1656. Mayo, M.A., 2004. Changes to virus taxonomy. Arch. Virol. 150, 189–198. Protopopova, E.V., Konavalova, S.N., Loktev, V.B., 1997. Isolation of a cellular receptor for tick-borne encephalitis virus using anti-idiotypic antibodies. Vopr. Virusol. 42, 264–268. Rao, N.C., Barsky, S.H., Terranova, V.P., Liotta, L.A., 1983. Isolation of a tumor cell laminin receptor. Biochem. Biophys. Res. Commun. 111, 804– 808. Rieger, R., Edenhofer, F., Lasmezas, C.I., Weiss, S., 1997. The human 37-kDa laminin receptor precursor interacts with the prion protein in eukaryotic cells. Nat. Med. 3, 1383–1388. Rosenthal, E.T., Wordeman, L., 1995. A protein similar to the 67 kDa laminin binding protein and p40 is probably a component of the translational machinery in Urechis caupo oocytes and embryos. J. Cell Sci. 108, 245–256. Rost, B., Sander, C., 1993. Prediction of protein secondary structure at better than 70% accuracy. J. Mol. Biol. 232, 584–599. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, third ed. Cold Spring Harbor, NY. Smith, A.E., Helenius, A., 2004. How viruses enter animal cells. Science 304, 237–242. Sodeik, B., 2000. Mechanisms of viral transport in the cytoplasm. Trends Microbiol. 8, 465–472. Soderhall, K., Smith, V.J., 1983. Separation of the haemocyte populations of Carcinus maenas and other marine decapods, and prophenoloxidase distribution. Dev. Comp. Immunol. 7, 229–239. Thepparit, C., Smith, D.R., 2004. Serotype-specific entry of dengue virus into liver cells: Identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor. J. Virol. 78, 12647–12656. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Tio, P.H., Jong, W.W., Cardosa, M.J., 2005. Two dimensional VOPBA reveals laminin receptor (LAMR1) interaction with dengue virus serotypes 1, 2 and 3. Virol. J. 2 (Epub ahead of print). Tonganunt, M., Phongdara, A., Chotigeat, W., Fujise, K., 2005. Identification and characterization of syntenin binding protein in the black tiger shrimp Penaeus monodon. J. Biotechnol. 120, 135–145. Tu, C., Huang, H.T., Chuang, S.H., Hsu, J.P., Kuo, S.T., Li, N.J., Hsu, T.L., Li, M.C., Lin, S.Y., 1999. Taura syndrome in Pacific white shrimp Penaeus vannamei cultured in Taiwan. Dis. Aquat. Org. 38, 159–161.

S. Senapin, A. Phongdara / Virus Research 122 (2006) 69–77 van Hulten, M.C.W., Witteveldt, J., Snippe, M., Vlak, J.M., 2001. White spot syndrome virus envelope protein VP28 is involved in the systemic infection of shrimp. Virology 285, 228–233. Vanderpasschen, A., Hollinshead, M., Smith, G.L., 1998. Intracellular and extracellular vaccinia virions enter cells by different mechanisms. J. Virol. 79, 877–887. Wang, K.S., Kuhn, R.J., Strauss, E.G., Ou, S., Strauss, J.H., 1992. High-affinity laminin receptor is a receptor for Sindbis virus in mammalian cells. J. Virol. 66, 4992–5001. White, J.M., 1990. Viral and cellular membrane fusion proteins. Annu. Rev. Physiol. 52, 675–697.

77

Wu, W.L., Wang, L., Zhang, X.B., 2005. Identification of white spot syndrome virus (WSSV) envelope proteins involved in shrimp infection. Virology 332, 578–583. Xie, X.X., Yang, F., 2005. Interaction of white spot syndrome virus VP26 protein with actin. Virology 336, 93–99. Yi, G.H., Wang, Z.M., Qi, Y.P., Yao, L.G., Qian, J., Hu, L.B., 2004. Vp28 of shrimp white spot syndrome virus is involved in the attachment and penetration into shrimp cells. J. Biochem. Mol. Biol. 37, 726– 734. Yu, C.I., Song, Y.L., 2000. Outbreaks of Taura syndrome in Pacific white shrimp Penaeus vannamei cultured in Taiwan. Fish. Pathol. 35, 21–24.