Department of Virology, College of Life Science, Wuhan University, Wuhan, Hubei 430072, PR China
Correspondence
Yipeng Qi
qiyipeng{at}whu.edu.cn
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ABSTRACT |
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INTRODUCTION |
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In 2000, two major structural protein genes of WSSV (VP28 and VP26) were identified (van Hulten et al., 2000a), followed by genes for ribonucleotide reductase, endonuclease, protein kinase and other structural proteins (van Hulten et al., 2000b
, 2002
; Witteveldt et al., 2001
; Liu et al., 2001
; Chen et al., 2002
; Zhang et al., 2002
). In 2001, the entire 305 kb genome sequence was reported (van Hulten et al., 2001
; Yang et al., 2001
). Analysis indicated that it contained 181 open reading frames (ORFs), some of which were similar to known viral genes or eukaryotic genes, but most of which encoded putative proteins without homology to any known protein (Yang et al., 2001
). Because of the lack of similarity to any existing virus family, WSSV was allocated to a new family, the Nimaviridae, and to the genus Whispovirus (http://www.ncbi.nlm.nih.gov/ICTV/). Although research results have provided much useful information about the molecular basis of virus replication and infection, a poor understanding of the virus infection mechanism still makes control difficult. Some research groups have reported work on development of drugs to prevent the disease (Chanratchakool & Chalor Limsuwan, 1998
; Itami et al., 1998
), but until now no effective drug has been found that can prevent or inhibit WSSV infection.
Phage display is an in vivo selection technique by which a library of billions of peptides (Cwirla et al., 1990; Devlin et al., 1990
; Scott & Smith, 1990
) or proteins (McCafferty et al., 1990
) can be produced by the fusion of random nucleic acid sequences to the N terminus of one of the capsid protein genes (pVIII or pIII) of a filamentous bacteriophage. Thus, the genetic material encoding each variant is integrated into the genome of an individual phage, which expresses and displays the modified capsid proteins on the surface of the phage particles. The most significant advantage of this technique is that it provides a natural link between phenotype and genotype, allowing specific screening based on binding affinity to a given target molecule by an in vivo selection process called panning. During the panning procedure, phage that displays a relevant protein is retained by virtue of its binding to the target, while non-adherent phage is washed away. Bound phage can be recovered from the surface, used to reinfect bacteria, reproduced for further enrichment and eventually analysed for binding. This concept was successfully applied to small peptides in 1990 (Cwirla et al., 1990
; Devlin et al., 1990
; Scott & Smith, 1990
). With the rapid development of the technique over the past decade, phage display has been used successfully in numerous applications, including antibody engineering (Hayden et al., 1997
; Chames & Baty, 2000
), peptide and protein drug discovery and manufacture (Kay et al., 1998
), vaccine development (Lesinski & Westerink, 2001
; Klemm & Schembril, 2000
) and identification of ligands (Ladner & Ley, 2001
; Ehrlich & Bailon, 2001
). Several pharmaceutical companies have used the phage-display system to develop protein drugs and there are currently 59 of these, largely peptides and monoclonal antibodies, which have been referred to as biotech drugs (Drews, 2000
).
Human and mammalian diseases have been controlled by antibodies or proteins/peptides with various degrees of success (Guarino et al., 1995; Juliano et al., 2001
). Control of WSSV currently focuses on exclusion of the virus from culture ponds but, once introduced, spread is often rapid and uncontrollable and no effective treatments have been found for infected shrimp. In the absence of relevant research reports, we reasoned that it might be possible to prevent WSSV infection if the active sites of key proteins could be blocked by binding to phage-displayed peptides. Thus, we selected peptides from a random peptide library by virtue of their binding to WSSV and tested them for neutralization of WSSV infection.
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METHODS |
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Primary shrimp cell culture.
Lymphoid organs were obtained from fresh adult P. monodon (100 g) and washed three times with cold, buffered antibiotic solution (1 M PBS containing 1000 IU penicillin ml-1, 1000 µg streptomycin ml-1, 250 µg gentamicin ml-1). After a final wash and further incubation for 10 min in cold, buffered antibiotic solution, the lymphoid organs were minced into fragments that were as small as possible. These fragments were filtered and transferred to wells in a 24-well plate, and 1 ml of the culture medium containing 2x L-15 supplemented with 15 % foetal calf serum (FCS), 1 % glucose, 5 g NaCl l-1, 1 IU penicillin ml-1, 1 µg streptomycin ml-1 and 5 µg gentamicin ml-1 was added to each well. The plate was sealed and incubated at 28 °C until 7080 % confluent monolayers were formed. These cells were then ready for virus infection.
Construction of a random decapeptide library.
An 82-base library oligonucleotide with the following sequence was synthesized by Genecore: 5'-GCTGGCCCAGCCGGCC(NNK)10CGCGCGGCCGCCCACGTAATACACGTGGGCGGCCGC-3'. This oligonucleotide contains the sequence NNK, consisting of two random nucleotides followed by either G or T, and an SfiI and NotI cleavage site and a self-complementary 3' terminus, allowing formation of a self-priming hairpin structure (Maniatis et al., 1976). The complementary strand was synthesized by extension of the 3' end with T4 DNA polymerase. After digestion with SfiI and NotI restriction enzymes, a degenerate sequence flanked by SfiI and NotI sites compatible with those of the pCANTAB5E vector (Amersham Pharmacia) was generated, and the desired fragment was recovered following separation on a 2 % low melting point agarose gel. Meanwhile, the vector was digested, recovered and dephosphorylated. The purified fragments were ligated together using standard methods. The ligation mixture was transformed into E. coli TG1 cells by electroporation using a Gene Pulser electroporation apparatus (Bio-Rad) at 2·5 kV, 200 ohms, 25 µF in 0·4 cm pre-chilled cuvettes.
The transformed cells were recovered at 37 °C by shaking at 250 r.p.m. and plating on to 10 large SOBAG plates for incubation at 30 °C for 16 h. A 100 µl sample was removed and various dilutions were plated on small SOBAG plates to determine the transformation efficiency. The spread colonies were scraped off the plates together with 15 ml 2x YT medium and stored at -70 °C until use.
Rescue of the phage peptide library.
A 2 ml aliquot of the stored library cells was added to 250 ml 2x YT-AG medium and incubated at 37 °C until the OD600 was 0·5, at which time 4x1011 p.f.u. M13K07 helper phage was added, followed by incubation for 30 min at 37 °C with shaking. The infected cells were harvested by centrifugation at 3300 g for 10 min and the pellet was resuspended gently in 50 ml 2x YT medium containing 100 µg ampicillin ml-1 and 25 µg kanamycin ml-1. This was followed by incubation overnight at 30 °C with shaking. The overnight culture was centrifuged at 10 800 g for 10 min and the supernatant mixed thoroughly with 1/5 vol. of PEG/NaCl (20 % PEG 6000, 2·5 M NaCl) followed by storage on ice for 1 h. After the phage had precipitated completely, the supernatant was centrifuged twice at 10 800 g for 10 min; then the pellet was resuspended in 5 ml PBS and the recombinant peptide library was stored at 4 °C until further use.
Panning.
Immunotubes (Maxisorb; Nunc) were coated overnight at 4 °C with WSSV (0·5 mg ml-1) in PBS, pH 7·0, and then washed once with water and blocked with PBS containing 5 % skimmed milk powder at 37 °C for 1 h. The blocking solution was removed and the library (1012 p.f.u.) was added in 4 ml PBS and incubated at 37 °C for 2 h, followed by washing 20 times with PBS containing 0·5 % Tween 20 and 20 times with PBS to remove non-specifically bound phage. Bound phage was eluted with 0·2 M glycine/HCl buffer, pH 2·2. The eluate was neutralized with 0·5 ml 1 M Tris/HCl, pH 9·1. The eluted phage was used to infect E. coli cells for amplification. Amplified phage was rescued using the same procedure as above and subjected to the next round of panning. After three rounds of panning, the phages were characterized by ELISA and positive clones were sequenced using an ABI 377 DNA sequencer with a BigDye Terminator cycle sequencing kit. DNA sequencing was carried out by Genecore.
ELISA.
Phage clones amplified by panning were tested by ELISA for their ability to bind specifically to WSSV. After coating with 0·1 µg WSSV µl-1 at 4 °C for 4 h, the IPTG-induced phages were added to the microtitre plate wells. Phage was allowed to bind to WSSV for 2 h at 4 °C. The ELISA was performed according to a standard protocol (Sambrook et al., 1989) using horseradish peroxidase (HRP)-conjugated mouse anti-E-tag (1 : 10 000 in BSA; Amersham Pharmacia) and TMB substrate [2 mg TMB ml-1 (Sigma) in 1 M sodium acetate, 4 % H2O2). HRP activity was estimated by measuring A405 using a universal microplate reader (EXL-800; Bio-tek). This measurement was repeated twice, using the vector phage as a negative control.
Affinity constant determination.
The affinity (Kaff) constant of peptides for WSSV was determined according to the method of Beatty et al. (1987) using a solid-phase non-competitive enzyme immunoassay.
Virus infection and its inhibition.
The different peptides were extracted from the cells, filtered through a 0·45 µm filter membrane and the concentration determined. Meanwhile, about 200 µl WSSV (106 p.f.u. ml-1) was filtered and adjusted to 5 ml with PBS, and then serially diluted in 2 ml PBS. Various concentrations of the different peptides were mixed with an equal volume of serially diluted WSSV for 1 h at 37 °C before addition to confluent monolayers of primary shrimp cells in a 24-well tissue culture plate (500 µl per well). After adsorption of virus for 1 h at room temperature, the wells were washed twice with L-15 culture medium to remove unattached virus. They were then overlaid with a mixture of agarose and L-15 medium supplemented with 2 % FCS in a volume of 500 µl. After the agarose had cooled and hardened, the plate was sealed and incubated at 28 °C. After 48 h, plaques were counted by microscopy (Olympus).
Animal test.
Healthy freshwater crayfish were purchased from a market and cultured in our laboratory in groups of 15 individuals per aquarium with an individual filter and oxygenator. After culture for 2 days, they were injected intramuscularly as follows: three experimental groups were separately injected with 50 µl of the various peptide extracts and 10 µl virus, one positive control group was injected with 10 µl virus only and one negative control group was injected with 200 µl 0·9 % NaCl. The crayfish were subsequently cultured for a period of 20 days and the mortality was monitored twice a day. Two dead individuals from each group were examined in detail, with midgut and hepatopancreas prepared for pathological sections (Lightner, 1996).
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RESULTS |
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Selection by biopanning of peptides specific for WSSV
To select the population of peptides specifically bound to WSSV, phagemid particles were rescued from the library by infection with helper phage M13K07 and selected for binding to the WSSV surface. Three rounds of rescue, selection and infection were performed. In a parallel experiment, WSSV was treated with 0·1 % SDS and then used for panning. The panning process was monitored by titrating the phage eluted from the WSSV-coated tube at each stage as colony-forming units (c.f.u.). A gradual increase in enriched phage from 6x104 to 1·2x106 c.f.u. was monitored after each round of panning for the antigen, as shown in Table 1. The number of phage eluted after the third round of panning did not increase further, indicating that saturation point had been reached.
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DISCUSSION |
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It is universally acknowledged that the effectiveness of peptides in the drug discovery process is due to their apparent ability to home in on active or biologically relevant sites on target proteins. Short peptides from combinatorial libraries can act as surrogate ligands of proteins that interact with other proteins if they possess the required critical residues. This is because the number of residues critical for binding may be rather small, even though the two interacting proteins can be fairly large (Geysen et al., 1985; Ruoslahti & Pierschbacher, 1986
; Wells, 1996
). The coat proteins of a virus may interact with other structural proteins resulting in changes to virus morphology that may affect cell surface receptors. This is relevant to initiation of virus infection and simultaneously provides many excellent targets for antiviral drugs. For example, it has been found that phage-displayed peptides selected from a 6-mer library were capable of blocking the interaction of the core antigen protein and viral envelope protein of hepatitis B virus. This, in turn, altered virus morphology and blocked infection (Dyson & Murray, 1995
). The same phage library was also screened with adenovirus type 2 (Ad2) penton capsomer and fibre domains (Hong & Boulanger, 1995
).
Our results have demonstrated that peptide 2E6 from a decapeptide library exhibited the highest specificity and efficiency of inhibiting infection by WSSV of the phage-displayed peptides analysed. The sequence data revealed that the motif VAVNNSY might play an important role in binding to the active sites of the key proteins of WSSV. In the same way, peptide 1D6, which had a similar motif to peptide 2E6, had a higher specificity and inhibition efficiency to some degree. However, peptide 1D10, which had a completely different sequence from the other peptides, had the highest affinity but lowest efficiency of inhibiting infection of WSSV. There are two aspects to a possible explanation of our results. First, peptide 1D10 could bind to antigen as a result of its three-dimensional configuration, rather than just its primary structure. Secondly, the peptide could bind with proteins that are non-essential for infection. Thus, it could bind to WSSV but be unable to block infection.
Our in vitro cell culture system for evaluation of infection inhibition efficiency is simple and easy to carry out. It is useful in providing not only quantitative information but also qualitative information on the type of CPE. Thus, it may find application in the testing of other potential antiviral drugs. Plaque assays have always been used for virus purification and qualitative analysis of infection. However, when viruses are serially diluted, a plaque represents infection by a single virus, thus the number and area of plaques reflect the number and virulence of live virus. Thus, a plaque assay can be used quantitatively to reflect the effect of a peptide indirectly by counting the number of plaques. In our study, an in vivo test was performed by substituting the freshwater crayfish for P. monodon shrimps, as the crayfish is a good model for virus infection and is more easily fed and kept alive in the laboratory.
It is known that shrimp do not have an antibody-based immune system like mammals for the prevention of virus infection. Thus, vaccination for disease prevention is not a practical approach. It has been proposed that a vaccination-like process may result in active viral accommodation, but this would also not prevent virus infection (Flegel & Pasharawipas, 1998). In contrast, peptides such as those described here might be used to target the core protein and destroy virus activity or simply to block certain ligands from interacting with their receptors. In this way, it might be possible to protect shrimp from WSSV infection using an appropriate peptide. Taken together, our results show that peptide 2E6 is a potential candidate for such an antiviral peptide drug, since it has a high specificity to WSSV and can effectively block WSSV infection.
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ACKNOWLEDGEMENTS |
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Received 22 November 2002;
accepted 31 March 2003.
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