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Selection of a specific peptide from a nona-peptide library for in vitro inhibition of grass carp hemorrhage virus replication
Virus Research 67 (2000) 119 – 125 www.elsevier.com/locate/virusres
Selection of a specific peptide from a nona-peptide library for in vitro inhibition of grass carp hemorrhage virus replication Bing Wang a,b, Li-Hua Ke b, Hong Jiang b, Chuan-Zhao Li a, Po Tien a,* b
a Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China
Received 24 November 1999; received in revised form 2 February 2000; accepted 3 February 2000
Abstract Grass carp hemorrhage virus (GCHV), a member of Reoviridae, causes severe hemorrhagic disease of grass carp (Ctenopharyngodon idellus) in China. Icosahedral virions of GCHV were used as to assay the effect of specific peptides for the inhibition of GCHV infectivity. A random nona-peptide library displayed on phage fUSE5 was constructed, and the expressed peptides were fused onto the amino terminus of the minor coat protein III. By biopanning, the fused peptides were bound to the biotinylated GCHV. Phages containing specific peptides bound to GCHV were eluted and amplified in Escherichia coli K91. Three rounds of affinity selection enriched the pool of inhibiting peptides. Sixteen clones which inhibited the replication of GCHV in a grass carp kidney cell line were selected. The TCID50 of GCHV was decreased over 10 000 ×. Six clones having the strongest inhibitory effect shared the same DNA sequence, with a deduced amino acid sequence of NH2-Leu-Trp-Val-Gly-Gly-Gly-Arg-Asn-Ala-COOH. A synthesized nona-peptide of identical sequence exhibited similar inhibitory activity towards GCHV replication in vitro. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Grass carp hemorrhage virus; Peptide library; Inhibition of replication; Kidney cell line
1. Introduction Grass carp hemorrhage virus (GCHV) isolated in Hunan Province, China, causes severe hemorrhagic disease of grass carp (Ctenopharyngodon
A part of Dr Wang’s Thesis directed by Professors Tien and Ke. * Corresponding author. Tel.: + 86-10-62554398; fax: + 8610-62554247. E-mail address: [email protected] (P. Tien)
idellus). GCHV has been identified as a member of Reoviridae (Ke et al., 1990). Virions of Reoviridae have icosahedral particles with diameters approximately 60–80 nm, one or two outer protein coats and an inner protein coat. The particle with the outer coat(s) removed is termed the core. Transcriptase activity is associated with the core. The genome of Reoviridae contains 10– 12 segments of linear dsRNA, molecular weights (MWs)= 0.2–3.0× 106, total MW= 12–20×106,
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B. Wang et al. / Virus Research 67 (2000) 119–125
about 14–22% by weight of the virus particle. Each RNA segment has one ORF encoding a protein requiring no further processing. Reoviridae contain 6–10 proteins in the virus particle; including transcriptase and messenger RNA capping enzymes (Tyler and Fields, 1990). GCHV has icosahedral particles measuring approximately 71 nm in diameter. It has a double capsid composed of surface and core proteins. Serological tests indicate that GCHV is antigenically unrelated to reovirus and rotavirus (Ke et al., 1991). The GCHV genome contains 11 segments of ds RNA (Huang et al., 1992). Parmley and Smith (1988) introduced the use of phage display epitope libraries. These libraries are mixtures of filamentous phage clones, each of which displays one peptide sequence on the virion surface (Scott and Smith, 1990). Potential applications of peptide libraries include investigation of specific antibodies and discovery of mimetic drug candidate (Houghten and Peptide, 1993; Scott and Craig, 1994). Here we report the experimental results of the construction of a random nona-peptide library displayed on phage fUSE5 and the selection of specific peptides which inhibit GCHV in vitro replication.
2. Material and methods
tion at 8000 rpm for 30 min. The virus was precipitated from the supernatant by centrifugation at 16 000 rpm for 2 h. The pellet was suspended in 1 × SSC (sodium chloride–citrate sodium), followed by CsCl gradient (30, 40, 50, and 60% w/v) centrifugation (Beckman model L8-80) at 135 000× g at 4°C for 4 h. The virus band was recovered and dialysed in TM buffer (100 mM Tris–HCl, pH 8.0, 10 mM MgCl2) at 4°C for 24h. GCHV stained by 2% phosphotungstic acid (pH 6.9) was observed by electron microscopy (JEM-100). For biotinylation (Oldenburg et al., 1992) 19.6 ml of a virus suspension (0.7 mg/ml) containing 13.72 mg virions was brought to pH 8.5 in a siliconized 1.5 ml microtube by mixing 4.4 ml 1 M NaHCO3 (pH 8.5), followed by addition of 2 ml 2 mM sodium acetate (pH 6.0) containing 0.5 mg/ml of NHS–LC–Biotin. The coupling solution was allowed to stand for 2 h at room temperature, quenched by the addition of 500 ml 1 M ethanolamine (pH 9.0) and incubated for an additional 2 h. Then 20 ml of dialyzed bovine serum albumin (BSA) (50 mg/ml) as a carrier protein and 1 ml Tris buffered saline (TBS) buffer were added to the reaction mixture, followed by concentration and washing once with TBS and TBS–azide, respectively, in a 30 kDa Centricon ultrafilter. The final volume was 160 ml. The concentration of biotinylated GCHV was 85.75 mg /ml.
2.1. Virus and kidney cell line 2.3. Construction of the nona-peptide library GCHV was isolated from grass carp in Hunan Province, China. The virus was cultured in a grass carp kidney cell line (Zuo et al., 1984, 1986) using Eagle MEM medium containing 10% bovine serum and 300 mg/ml L-glutamine, pH adjusted to 7.0 with 7.5% NaHCO3. The culture was incubated at room temperature (Fang et al., 1989; Fang and Ke, 1990).
2.2. Purification and biotinylation of GCHV 6irions The virus was harvested from the cell culture 3 days after inoculation with GCHV by centrifugation (Beckman model J2-21) at 3500 rpm for 30 min. The supernatant was cleared by centrifuga-
Construction of the nona-peptide library was carried out according to Scott and Smith (1990) and Pasqualini and Ruoslahti (1996). The fUSE5 derived from fd-tet could be propagated like other tetracycline-resistance plasmids without the function of pIII. The plasmid was constructed in such a way that only clones bearing frame-restoring inserts could form infectious particles in this library. A large number of random 27-mers were synthesized and digested by BglI before they were inserted near the amino terminus of the minor coat-protein gene (gIII) of fUSE5. A phage particle could only express one of the random 27-mer sequences on its surface as a fusion protein of pIII. Thus, a nona-peptide library composed of
B. Wang et al. / Virus Research 67 (2000) 119–125
approximately 1014 or 329 different nona-peptide epitopes was generated. Vector DNA was prepared by suspension of the pellet of Escherichia coli K802 containing crude fUSE5 in 40 ml buffered glucose. The RF was prepared by alkaline lysis, followed by extraction twice with phenol and once with chloroform. The extracted DNA was precipitated with ethanol, and purified by CsCl – ethidium bromide density gradient centrifugation. The purified DNA was cleaved with SfiI before used. For the synthesis of random nucleic acid and generation of insert fragment, a single-stranded DNA composed of 79 oligonucleotides flanked by the BglI recognition site was synthesized using standard chemosynthesis, resulting in a collection of 27 oligonucleotides encoding all possible nonapeptides. After the double-stranded DNA was synthesized by PCR, it was cleaved by BglI before use for ligation. The ligation mixture containing 5 mg/ml of linearized vector DNA, 10 mg/ml of doublestranded degenerate insert and 10 U/ml T4 DNA ligase in ligation buffer, was incubated at 20°C for 40 h. The ligated product was determined and analysed by 0.8% agarose gel electrophoresis. The ligation product was precipitated by ethanol, resuspended in water and then used in ten separate transfections via electroporations into E. coli MC1061. The ‘shocked’ cells were immediately suspended in SOC medium containing 0.2 mg/ml tetracycline (Tc), gently shaken at 37°C for 60 min, and then plated onto LB agar plates with 20 mg/ml Tc. The positive culture was transferred to 1 l LB medium with 40 mg/ml Tc and grown at 37°C for 24–48 h to amplify the library.
2.4. Selection of specific peptide by binding to biotinylated GCHV Phage clones carrying peptides with the desired specificity were selected by biopanning in which the peptide–phage particles were bound to biotinylated GCHV virions (Daniels and Lane, 1994). The fusion phage particles that did not bind were washed away. The bound peptide– phage were eluted and amplified in E. coli K91 and this process was repeated three times. To
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remove weakly or nonspecifically bound peptide– phage particles, the concentration of biotinylated virus was gradually decreased from 8.6 mg/ml at the first panning to 0.86 and 0.43 mg/ml at the second and third panning, respectively. This biopanning process has been described previously by Sanna et al. (1995).
2.5. Detection and determination of affinity between peptides and 6irus by ELISA To produce and purify the desired phages containing peptides next to the pIII protein, colonies were isolated and inoculated into 3 ml LB medium containing 40 mg/ml Tc and 50 mg/ml Kan, and incubated in a shaker at 37°C for 24 h. Each culture was transferred into a 1.5 ml microfuge tube and centrifuged at 4000 rpm for 1 min to pellet the cells. One ml of the culture supernatant was pipetted into a new microfuge tube containing 150 ml PEG–NaCl solution (Smith and Scott, 1993). After mixing, the phages were allowed to precipitate overnight at 4°C. The virus pellet was collected by centrifugation at 13 000 rpm for 15 min and the pellet was resuspended in 1 ml TBS. The virus mixture was centrifuged for 1 min at 13 000 rpm to remove any cell debris and the supernatant was pipetted into a microfuge tube containing 150 ml PEG– NaCl solution. The phage were precipitated again, collected, and dissolved in 110 ml 0.15 M NaCl. The solution was again centrifuged for 1 min as above and the supernatant was transferred to another microfuge tube containing 12.2 ml 1 M acetic acid. The mixture was left on ice for 10 min before centrifugation at 12 000 rpm for 10 min. The final virion pellet was dissolved in 400 ml TBS buffer and stored at 4°C. The phage titer was 5×1011 particles/ml as determined by the methods described by Cwirla et al. (1990). Partially purified phage suspensions containing 5×1010 virions in 90 ml were placed in each well of a microtiter plate well. Blotto solution (10 ml) (Blotto solution (10×), 0.159% Na2CO3 (w/v), 0.293% NaHCO3 (w/v), 0.02% NaN3 (w/v)) was added and the plate was gently agitated at 4°C overnight. The microtiter plate was washed with TBS–Tween and refilled with 350 ml Blotto
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buffer. It was incubated at 37°C for 2 h. The wells were washed, filled with 100 ml biotinylated GCHV (5 mg/ml) and left at 4°C overnight. The wells were washed as above, filled with 100 ml of avidin–peroxidase (1:1000), and incubated at 37°C for 2 h. The wells were thoroughly washed ten times with TBS – Tween, then filled with 100 ml freshly prepared ABTS solution (38.6% (0.22 mg/ ml 2, 2-azinobis), 61.4% (0.2 M Na2HPO4 + 0.1 M citric acid) and 0.1% (30% w/v H2O2)), and left to react for 1 h at room temperature in the dark. The positive reaction had a blue color, and the ELISA value was determined and calculated by subtracting the value of OD490 from OD410 (Smith and Scott, 1993).
2.6. Detection of anti6iral acti6ity by TCID50 The determination of virus TCID50 (50% tissue culture infective dose) was conducted by the end point method. The GCHV was diluted up to 1015-fold with MEM (pH 7.0) without serum. The mixture of 50 ml of different test samples and 6 ml phage preparation containing 4.5×1010 particles was incubated at 28°C for 30 min. After removing MEM culture fluid from the wells, the mixtures were pipetted into a microtiter plate containing grass carp kidney cells and incubated at room temperature for 30 min. The solution in the wells was poured out, and the wells were washed three times with MEM (pH 7.0) without serum. Approximately 200 ml MEM culture medium containing 2% serum was added to each well and it was incubated at 28°C for 72 h. The cytopathic effect of different groups was observed and determined by light microscopy.
2.7. DNA sequencing A sequence template was prepared from the positive phage clone (200 ml) by precipitation twice with PEG – NaCl as above. The partially purified phage was extracted once with an equal volume of phenol, phenol – chloroform, and chloroform, successively. The final 150 ml of the aqueous phase was transferred to a microfuge tube containing 250 ml TE buffer, 40 ml 3 M sodium acetate and 1 ml cold ethanol. After incu-
bation for 2 h at − 80°C, ssDNA of the phage was precipitated. The DNA pellet was washed with 70% ethanol, dried briefly in vacuum and stored at − 20°C. An 18-base primer complementary to the wildtype gene III sequence which is 33 bases away from the cloning site towards the 3%-terminus in the fUSE5 was used for sequencing. The sequencing reaction using [a-32P]dUTP was performed according to the protocol in the T7 sequencing kit (Pharmacia Biotech.)
2.8. Synthesis of peptide A synthetic nona-peptide designed according to the DNA sequence of positive clones was synthesized, purified by HPLC, and then used for the determination and comparison of the inhibitory activity with positive clones.
3. Results
3.1. Replication of the purified GCHV in kidney cell line and its cytopathic effect Electron microscopy indicated that the purified GCHV particles were spherical, 71 nm in diameter, with discernible subunits. These purified GCHV were used to infect grass carp kidney cells. GCHV replicated well in grass carp kidney cells, producing primarily plaques approximately 0.2 mm in diameter in a monolayer culture. A unique cytopathic effect observed by light microscopy is shown in Fig. 1b. At the optimum temperature for incubation (28°C), its logarithmic growth phase occurred 12–48 h after infection. The virus titer and TCID50 reached 1.78× 10 − 11 PFU/ml and 1.0 × 10 − 10 in a normal atmosphere or 5% CO2, respectively. The presence of virus particles in the culture fluid of the infected cells was further confirmed by ELISA and electron microscopy.
3.2. Selection of specific clones binding to GCHV Biotinylated GCHV particles were used to bind the random peptides displayed on the surface of fUSE5 in the process of selection. The binding
B. Wang et al. / Virus Research 67 (2000) 119–125
reaction was monitored by ELISA. Through three rounds of biopanning, the percentage yield of specific phage was increased from 0.22% to 2.5%. From 300 clones of a random nona-peptide library, sixteen specific phage clones were selected and produced. Each of these clones showed stable and high binding activity to GCHV (Table 1).
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peptide or the synthetic peptide as applied to the cells infected with GCHV was diluted to 10 − 8, the cytopathic plaques were not formed (Fig. 1c). However, plaques were observed in cells infected with GCHV plus wild type phages (Fig. 1d) or with GCHV alone (Fig. 1b).
3.4. DNA sequencing 3.3. Inhibition of GCHV replication Of the positive clones that decreased GCHV TCID50 significantly, six clones showed the strongest inhibition effect on GCHV replication and they reduced GCHV TCID50 more than 10 000-fold compared to controls. The synthetic nona-peptide had a similar inhibition effect on the replication of GCHV (Table 1). Fig. 1 illustrates the inhibition of GCHV replication in cell culture by positive phage clones. When the specific phage
DNA sequencing of the six positive clones, which exhibited the strongest inhibition on GCHV replication, had the identical DNA sequence of 5%-CTGTGGGTTGGGGGTGGG CGGAATGCT-3%. According to this DNA sequence a peptide sequence was deduced as follows: NH2Leu-Trp-Val-Gly-Gly-Gly-Arg-Asn-Ala. It was interesting that it contains three hydrophobic amino acids, Leu, Trp, and Val at its amino terminus.
Fig. 1. The inhibition of GCHV replication in grass carp kidney cells by specific peptide (magnification: × 200). (a) Normal untreated cells, (b) cells + GCHV, (c) cells + GCHV + phage with specific peptide, and (d) Cells +GCHV + wild type phage.
B. Wang et al. / Virus Research 67 (2000) 119–125
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Table 1 Inhibition by selected nona-peptides of replication of grass carp hemorrhage virus in grass carp kidney cells Number of selected nona-peptide clones
ELISA (OD410–OD495) after three round selection
TCID50 of GCHV
53a 55 56a 57 58 59 61a 65 193 194a 195a 198 199 201 202a 203
0.550 0.410 0.690 0.450 0.530 0.470 0.670 0.380 0.660 0.510 0.520 0.530 0.450 0.350 0.670 0.650
10−6.0 10−6.2 10−6.0 10−7.9 10−7.0 10−7.9 10−6.0 10−7.0 10−6.7 10−5.6 10−5.6 10−7.2 10−6.2 10−7.0 10−5.7 10−6.4
Wild−type phage control Synthetic nona-peptideb (100 mg/ml) GCHV control
0.00
10−9.6
0.00 s
10−6.0
0.00
10−10
a Clones with the strongest inhibitory effect on virus replication which have been sequenced. b With sequence of Leu-Trp-Val-Gly-Gly-Gly-Arg-Asn-Ala.
4. Discussion Recent efforts to control the disease caused by GCHV in China have been unsuccessful. Crude vaccines and cell vaccines for GCHV failed to control this epidemic and GCHV replication. It poses additional and potential danger for spreading this disease. Thus, more effective and safer agents to combat GCHV-induced diseases are necessary. The random peptide library is a new technology utilized in the study of molecular recognition. It can provide a unique strategy for the design of pharmaceuticals and recombinant vaccines, and for the screening of anti-viral peptides. Dyson and Murray (1995), using HBcAg as a receptor, selected a specific peptide from a fUSE hexa-peptide
library which can bind to HBV virions. Thompson et al. (1996) obtained a high affinity human monoclonal antibody against an HIV epitope using the phage display. In this study we used intact GCHV as a receptor to screen for a peptide ligand, and obtained a specific peptide with high affinity to GCHV. The peptide screened by the intact virus may block the active site related to replication on the virus surface. The antiviral peptide displayed on the phage did not induce an immunological response of the acceptor. We hypothesize that this peptide might bind to the attachment protein of the virion and inhibit its entry into the host cells, resulting in virus neutralization. This specific peptide might also change the structure of the virus or viral proteins which interferes with the infection process. Thus, the GCHV-specific peptide selected from the peptide library in this study may be potentially applicable in virus control and at the same time establish the foundation for the design of other antiviral peptides or other low molecular weight pharmaceuticals.
Acknowledgements We are grateful to Professor Xiaofeng Wang for her assistance in the preparation of the manuscript and to Professor Ruiwen Zhang for his critical reading and useful suggestions. Our appreciation also goes to Weihua Zhuang for typing the manuscript, figures and tables, and Dong Hong for his technical assistance with the photographs.
References Cwirla, S.E., Peter, E.A., Barrett, R.W., Dower, W.J., 1990. Peptides on phage: a vast library of peptides for identifying ligands. Proc. Natl. Acad. Sci. USA 87, 6378 – 6382. Daniels, D.A., Lane, D.P., 1994. The characterisation of p53 binding phage isolated from phage peptide display libraries. J. Mol. Biol. 243, 639 – 652. Dyson, M.R., Murray, K., 1995. Selection of peptide inhibitors of interactions involved in complex protein assemblies: association of the core and surface antigens of hepatitis B virus. Proc. Natl. Acad. Sci. USA 92, 2194 – 2198.
B. Wang et al. / Virus Research 67 (2000) 119–125 Fang, Q., Ke, L.H., 1990. A plaque assay under normal atmosphere. Virol. Sin. 5 (3), 337–340. Fang, Q., Ke, L.H., Cai, Y.Q., 1989. Growth characteristics and high titer culture of grass carp hemorrhage virus (GCHV)-873 in vitro. Virol. Sin. 4 (3), 315–319. Huang, J., Ke, L.H., Cai, Y.Q., 1992. Study on the reaction cores of grass carp hemorrhage virus (GCHV). Acta Biochem. Biophys. Sin. 24 (2), 132–139. Houghten, R.A., Peptide, L., 1993. Criteria and trends. Trends Genet. 9, 235 – 239. Ke, L.H., Fang, Q., Cai, Y.Q., 1990. Characteristics of a novel isolate of grass carp hemorrhage virus. Acta Hydrobiol. Sin. 14 (2), 153 –159. Ke, L.H., Fang, Q., Yu, L.F., 1991. Serological identification of grass carp hemorrhage virus. Virol. Sin. 6 (3), 252– 255. Oldenburg, K.R., Loganathan, D., Goldstein, I.J., Schultz, P.G., Gallop, M.A., 1992. Peptide ligands for a sugarbinding protein isolated from a random peptide library. Proc. Natl. Acad. Sci. USA 89, 5393–5397. Parmley, S.F., Smith, G.P., 1988. Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73, 305 – 318. Pasqualini, R., Ruoslahti, E., 1996. Organ targeting in-vivo using phage display phage libraries. Nature 380, 364 – 366. Sanna, P.P., Williamson, R.A., Logu, A.D., Bloom, F.E., Burton, D.R., 1995. Directed selection of recombinant
.
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human monoclonal antibodies to herpes virus glycoproteins from phage display libraries. Proc. Natl. Acad. Sci. USA 92, 6439 – 6443. Scott, J.K., Craig, L., 1994. Random peptide libraries. Curr. Opin. Biotechnol. 5, 40 – 48. Scott, J.K., Smith, G.P., 1990. Searching for peptide ligands with an epitope library. Science 249, 386 – 390. Smith, G.P., Scott, J.K., 1993. Libraries of peptides and proteins displayed on filamentous phage. Methods Enzymol. 15, 228 – 257. Thompson, J., Pope, T., Tung, J.S., 1996. Affinity maturation of a high-affinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity. J. Mol. Biol. 256, 77 – 88. Tyler, K.L., Fields, B.N., 1990. Reoviridae: a brief introduction. In: Fields, B.N., Knight, J.C. (Eds.), Virology, 2nd edn. Raven Press, New York, pp. 1271 – 1328. Zuo, W.G., Qian, H.X., Xu, Y.F., 1984. A research note on the establishment of CIK cell line derived from kidney of grass carp (Ctenopharyngodon idellus). Freshw. Fish. 2, 38 – 39. Zuo, W.G., Qian, H.X., Xu, Y.F., 1986. A cell line derived from the kidney of grass carp (Ctenopharygodon idellus). J. Fish. China 10 (1), 11 – 17.
Selection of a specific peptide from a nona-peptide library for in vitro inhibition of grass carp hemorrhage virus replication Bing Wang a,b, Li-Hua Ke b, Hong Jiang b, Chuan-Zhao Li a, Po Tien a,* b
a Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China
Received 24 November 1999; received in revised form 2 February 2000; accepted 3 February 2000
Abstract Grass carp hemorrhage virus (GCHV), a member of Reoviridae, causes severe hemorrhagic disease of grass carp (Ctenopharyngodon idellus) in China. Icosahedral virions of GCHV were used as to assay the effect of specific peptides for the inhibition of GCHV infectivity. A random nona-peptide library displayed on phage fUSE5 was constructed, and the expressed peptides were fused onto the amino terminus of the minor coat protein III. By biopanning, the fused peptides were bound to the biotinylated GCHV. Phages containing specific peptides bound to GCHV were eluted and amplified in Escherichia coli K91. Three rounds of affinity selection enriched the pool of inhibiting peptides. Sixteen clones which inhibited the replication of GCHV in a grass carp kidney cell line were selected. The TCID50 of GCHV was decreased over 10 000 ×. Six clones having the strongest inhibitory effect shared the same DNA sequence, with a deduced amino acid sequence of NH2-Leu-Trp-Val-Gly-Gly-Gly-Arg-Asn-Ala-COOH. A synthesized nona-peptide of identical sequence exhibited similar inhibitory activity towards GCHV replication in vitro. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Grass carp hemorrhage virus; Peptide library; Inhibition of replication; Kidney cell line
1. Introduction Grass carp hemorrhage virus (GCHV) isolated in Hunan Province, China, causes severe hemorrhagic disease of grass carp (Ctenopharyngodon
A part of Dr Wang’s Thesis directed by Professors Tien and Ke. * Corresponding author. Tel.: + 86-10-62554398; fax: + 8610-62554247. E-mail address: [email protected] (P. Tien)
idellus). GCHV has been identified as a member of Reoviridae (Ke et al., 1990). Virions of Reoviridae have icosahedral particles with diameters approximately 60–80 nm, one or two outer protein coats and an inner protein coat. The particle with the outer coat(s) removed is termed the core. Transcriptase activity is associated with the core. The genome of Reoviridae contains 10– 12 segments of linear dsRNA, molecular weights (MWs)= 0.2–3.0× 106, total MW= 12–20×106,
0168-1702/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 0 0 ) 0 0 1 3 2 - 5
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B. Wang et al. / Virus Research 67 (2000) 119–125
about 14–22% by weight of the virus particle. Each RNA segment has one ORF encoding a protein requiring no further processing. Reoviridae contain 6–10 proteins in the virus particle; including transcriptase and messenger RNA capping enzymes (Tyler and Fields, 1990). GCHV has icosahedral particles measuring approximately 71 nm in diameter. It has a double capsid composed of surface and core proteins. Serological tests indicate that GCHV is antigenically unrelated to reovirus and rotavirus (Ke et al., 1991). The GCHV genome contains 11 segments of ds RNA (Huang et al., 1992). Parmley and Smith (1988) introduced the use of phage display epitope libraries. These libraries are mixtures of filamentous phage clones, each of which displays one peptide sequence on the virion surface (Scott and Smith, 1990). Potential applications of peptide libraries include investigation of specific antibodies and discovery of mimetic drug candidate (Houghten and Peptide, 1993; Scott and Craig, 1994). Here we report the experimental results of the construction of a random nona-peptide library displayed on phage fUSE5 and the selection of specific peptides which inhibit GCHV in vitro replication.
2. Material and methods
tion at 8000 rpm for 30 min. The virus was precipitated from the supernatant by centrifugation at 16 000 rpm for 2 h. The pellet was suspended in 1 × SSC (sodium chloride–citrate sodium), followed by CsCl gradient (30, 40, 50, and 60% w/v) centrifugation (Beckman model L8-80) at 135 000× g at 4°C for 4 h. The virus band was recovered and dialysed in TM buffer (100 mM Tris–HCl, pH 8.0, 10 mM MgCl2) at 4°C for 24h. GCHV stained by 2% phosphotungstic acid (pH 6.9) was observed by electron microscopy (JEM-100). For biotinylation (Oldenburg et al., 1992) 19.6 ml of a virus suspension (0.7 mg/ml) containing 13.72 mg virions was brought to pH 8.5 in a siliconized 1.5 ml microtube by mixing 4.4 ml 1 M NaHCO3 (pH 8.5), followed by addition of 2 ml 2 mM sodium acetate (pH 6.0) containing 0.5 mg/ml of NHS–LC–Biotin. The coupling solution was allowed to stand for 2 h at room temperature, quenched by the addition of 500 ml 1 M ethanolamine (pH 9.0) and incubated for an additional 2 h. Then 20 ml of dialyzed bovine serum albumin (BSA) (50 mg/ml) as a carrier protein and 1 ml Tris buffered saline (TBS) buffer were added to the reaction mixture, followed by concentration and washing once with TBS and TBS–azide, respectively, in a 30 kDa Centricon ultrafilter. The final volume was 160 ml. The concentration of biotinylated GCHV was 85.75 mg /ml.
2.1. Virus and kidney cell line 2.3. Construction of the nona-peptide library GCHV was isolated from grass carp in Hunan Province, China. The virus was cultured in a grass carp kidney cell line (Zuo et al., 1984, 1986) using Eagle MEM medium containing 10% bovine serum and 300 mg/ml L-glutamine, pH adjusted to 7.0 with 7.5% NaHCO3. The culture was incubated at room temperature (Fang et al., 1989; Fang and Ke, 1990).
2.2. Purification and biotinylation of GCHV 6irions The virus was harvested from the cell culture 3 days after inoculation with GCHV by centrifugation (Beckman model J2-21) at 3500 rpm for 30 min. The supernatant was cleared by centrifuga-
Construction of the nona-peptide library was carried out according to Scott and Smith (1990) and Pasqualini and Ruoslahti (1996). The fUSE5 derived from fd-tet could be propagated like other tetracycline-resistance plasmids without the function of pIII. The plasmid was constructed in such a way that only clones bearing frame-restoring inserts could form infectious particles in this library. A large number of random 27-mers were synthesized and digested by BglI before they were inserted near the amino terminus of the minor coat-protein gene (gIII) of fUSE5. A phage particle could only express one of the random 27-mer sequences on its surface as a fusion protein of pIII. Thus, a nona-peptide library composed of
B. Wang et al. / Virus Research 67 (2000) 119–125
approximately 1014 or 329 different nona-peptide epitopes was generated. Vector DNA was prepared by suspension of the pellet of Escherichia coli K802 containing crude fUSE5 in 40 ml buffered glucose. The RF was prepared by alkaline lysis, followed by extraction twice with phenol and once with chloroform. The extracted DNA was precipitated with ethanol, and purified by CsCl – ethidium bromide density gradient centrifugation. The purified DNA was cleaved with SfiI before used. For the synthesis of random nucleic acid and generation of insert fragment, a single-stranded DNA composed of 79 oligonucleotides flanked by the BglI recognition site was synthesized using standard chemosynthesis, resulting in a collection of 27 oligonucleotides encoding all possible nonapeptides. After the double-stranded DNA was synthesized by PCR, it was cleaved by BglI before use for ligation. The ligation mixture containing 5 mg/ml of linearized vector DNA, 10 mg/ml of doublestranded degenerate insert and 10 U/ml T4 DNA ligase in ligation buffer, was incubated at 20°C for 40 h. The ligated product was determined and analysed by 0.8% agarose gel electrophoresis. The ligation product was precipitated by ethanol, resuspended in water and then used in ten separate transfections via electroporations into E. coli MC1061. The ‘shocked’ cells were immediately suspended in SOC medium containing 0.2 mg/ml tetracycline (Tc), gently shaken at 37°C for 60 min, and then plated onto LB agar plates with 20 mg/ml Tc. The positive culture was transferred to 1 l LB medium with 40 mg/ml Tc and grown at 37°C for 24–48 h to amplify the library.
2.4. Selection of specific peptide by binding to biotinylated GCHV Phage clones carrying peptides with the desired specificity were selected by biopanning in which the peptide–phage particles were bound to biotinylated GCHV virions (Daniels and Lane, 1994). The fusion phage particles that did not bind were washed away. The bound peptide– phage were eluted and amplified in E. coli K91 and this process was repeated three times. To
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remove weakly or nonspecifically bound peptide– phage particles, the concentration of biotinylated virus was gradually decreased from 8.6 mg/ml at the first panning to 0.86 and 0.43 mg/ml at the second and third panning, respectively. This biopanning process has been described previously by Sanna et al. (1995).
2.5. Detection and determination of affinity between peptides and 6irus by ELISA To produce and purify the desired phages containing peptides next to the pIII protein, colonies were isolated and inoculated into 3 ml LB medium containing 40 mg/ml Tc and 50 mg/ml Kan, and incubated in a shaker at 37°C for 24 h. Each culture was transferred into a 1.5 ml microfuge tube and centrifuged at 4000 rpm for 1 min to pellet the cells. One ml of the culture supernatant was pipetted into a new microfuge tube containing 150 ml PEG–NaCl solution (Smith and Scott, 1993). After mixing, the phages were allowed to precipitate overnight at 4°C. The virus pellet was collected by centrifugation at 13 000 rpm for 15 min and the pellet was resuspended in 1 ml TBS. The virus mixture was centrifuged for 1 min at 13 000 rpm to remove any cell debris and the supernatant was pipetted into a microfuge tube containing 150 ml PEG– NaCl solution. The phage were precipitated again, collected, and dissolved in 110 ml 0.15 M NaCl. The solution was again centrifuged for 1 min as above and the supernatant was transferred to another microfuge tube containing 12.2 ml 1 M acetic acid. The mixture was left on ice for 10 min before centrifugation at 12 000 rpm for 10 min. The final virion pellet was dissolved in 400 ml TBS buffer and stored at 4°C. The phage titer was 5×1011 particles/ml as determined by the methods described by Cwirla et al. (1990). Partially purified phage suspensions containing 5×1010 virions in 90 ml were placed in each well of a microtiter plate well. Blotto solution (10 ml) (Blotto solution (10×), 0.159% Na2CO3 (w/v), 0.293% NaHCO3 (w/v), 0.02% NaN3 (w/v)) was added and the plate was gently agitated at 4°C overnight. The microtiter plate was washed with TBS–Tween and refilled with 350 ml Blotto
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buffer. It was incubated at 37°C for 2 h. The wells were washed, filled with 100 ml biotinylated GCHV (5 mg/ml) and left at 4°C overnight. The wells were washed as above, filled with 100 ml of avidin–peroxidase (1:1000), and incubated at 37°C for 2 h. The wells were thoroughly washed ten times with TBS – Tween, then filled with 100 ml freshly prepared ABTS solution (38.6% (0.22 mg/ ml 2, 2-azinobis), 61.4% (0.2 M Na2HPO4 + 0.1 M citric acid) and 0.1% (30% w/v H2O2)), and left to react for 1 h at room temperature in the dark. The positive reaction had a blue color, and the ELISA value was determined and calculated by subtracting the value of OD490 from OD410 (Smith and Scott, 1993).
2.6. Detection of anti6iral acti6ity by TCID50 The determination of virus TCID50 (50% tissue culture infective dose) was conducted by the end point method. The GCHV was diluted up to 1015-fold with MEM (pH 7.0) without serum. The mixture of 50 ml of different test samples and 6 ml phage preparation containing 4.5×1010 particles was incubated at 28°C for 30 min. After removing MEM culture fluid from the wells, the mixtures were pipetted into a microtiter plate containing grass carp kidney cells and incubated at room temperature for 30 min. The solution in the wells was poured out, and the wells were washed three times with MEM (pH 7.0) without serum. Approximately 200 ml MEM culture medium containing 2% serum was added to each well and it was incubated at 28°C for 72 h. The cytopathic effect of different groups was observed and determined by light microscopy.
2.7. DNA sequencing A sequence template was prepared from the positive phage clone (200 ml) by precipitation twice with PEG – NaCl as above. The partially purified phage was extracted once with an equal volume of phenol, phenol – chloroform, and chloroform, successively. The final 150 ml of the aqueous phase was transferred to a microfuge tube containing 250 ml TE buffer, 40 ml 3 M sodium acetate and 1 ml cold ethanol. After incu-
bation for 2 h at − 80°C, ssDNA of the phage was precipitated. The DNA pellet was washed with 70% ethanol, dried briefly in vacuum and stored at − 20°C. An 18-base primer complementary to the wildtype gene III sequence which is 33 bases away from the cloning site towards the 3%-terminus in the fUSE5 was used for sequencing. The sequencing reaction using [a-32P]dUTP was performed according to the protocol in the T7 sequencing kit (Pharmacia Biotech.)
2.8. Synthesis of peptide A synthetic nona-peptide designed according to the DNA sequence of positive clones was synthesized, purified by HPLC, and then used for the determination and comparison of the inhibitory activity with positive clones.
3. Results
3.1. Replication of the purified GCHV in kidney cell line and its cytopathic effect Electron microscopy indicated that the purified GCHV particles were spherical, 71 nm in diameter, with discernible subunits. These purified GCHV were used to infect grass carp kidney cells. GCHV replicated well in grass carp kidney cells, producing primarily plaques approximately 0.2 mm in diameter in a monolayer culture. A unique cytopathic effect observed by light microscopy is shown in Fig. 1b. At the optimum temperature for incubation (28°C), its logarithmic growth phase occurred 12–48 h after infection. The virus titer and TCID50 reached 1.78× 10 − 11 PFU/ml and 1.0 × 10 − 10 in a normal atmosphere or 5% CO2, respectively. The presence of virus particles in the culture fluid of the infected cells was further confirmed by ELISA and electron microscopy.
3.2. Selection of specific clones binding to GCHV Biotinylated GCHV particles were used to bind the random peptides displayed on the surface of fUSE5 in the process of selection. The binding
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reaction was monitored by ELISA. Through three rounds of biopanning, the percentage yield of specific phage was increased from 0.22% to 2.5%. From 300 clones of a random nona-peptide library, sixteen specific phage clones were selected and produced. Each of these clones showed stable and high binding activity to GCHV (Table 1).
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peptide or the synthetic peptide as applied to the cells infected with GCHV was diluted to 10 − 8, the cytopathic plaques were not formed (Fig. 1c). However, plaques were observed in cells infected with GCHV plus wild type phages (Fig. 1d) or with GCHV alone (Fig. 1b).
3.4. DNA sequencing 3.3. Inhibition of GCHV replication Of the positive clones that decreased GCHV TCID50 significantly, six clones showed the strongest inhibition effect on GCHV replication and they reduced GCHV TCID50 more than 10 000-fold compared to controls. The synthetic nona-peptide had a similar inhibition effect on the replication of GCHV (Table 1). Fig. 1 illustrates the inhibition of GCHV replication in cell culture by positive phage clones. When the specific phage
DNA sequencing of the six positive clones, which exhibited the strongest inhibition on GCHV replication, had the identical DNA sequence of 5%-CTGTGGGTTGGGGGTGGG CGGAATGCT-3%. According to this DNA sequence a peptide sequence was deduced as follows: NH2Leu-Trp-Val-Gly-Gly-Gly-Arg-Asn-Ala. It was interesting that it contains three hydrophobic amino acids, Leu, Trp, and Val at its amino terminus.
Fig. 1. The inhibition of GCHV replication in grass carp kidney cells by specific peptide (magnification: × 200). (a) Normal untreated cells, (b) cells + GCHV, (c) cells + GCHV + phage with specific peptide, and (d) Cells +GCHV + wild type phage.
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Table 1 Inhibition by selected nona-peptides of replication of grass carp hemorrhage virus in grass carp kidney cells Number of selected nona-peptide clones
ELISA (OD410–OD495) after three round selection
TCID50 of GCHV
53a 55 56a 57 58 59 61a 65 193 194a 195a 198 199 201 202a 203
0.550 0.410 0.690 0.450 0.530 0.470 0.670 0.380 0.660 0.510 0.520 0.530 0.450 0.350 0.670 0.650
10−6.0 10−6.2 10−6.0 10−7.9 10−7.0 10−7.9 10−6.0 10−7.0 10−6.7 10−5.6 10−5.6 10−7.2 10−6.2 10−7.0 10−5.7 10−6.4
Wild−type phage control Synthetic nona-peptideb (100 mg/ml) GCHV control
0.00
10−9.6
0.00 s
10−6.0
0.00
10−10
a Clones with the strongest inhibitory effect on virus replication which have been sequenced. b With sequence of Leu-Trp-Val-Gly-Gly-Gly-Arg-Asn-Ala.
4. Discussion Recent efforts to control the disease caused by GCHV in China have been unsuccessful. Crude vaccines and cell vaccines for GCHV failed to control this epidemic and GCHV replication. It poses additional and potential danger for spreading this disease. Thus, more effective and safer agents to combat GCHV-induced diseases are necessary. The random peptide library is a new technology utilized in the study of molecular recognition. It can provide a unique strategy for the design of pharmaceuticals and recombinant vaccines, and for the screening of anti-viral peptides. Dyson and Murray (1995), using HBcAg as a receptor, selected a specific peptide from a fUSE hexa-peptide
library which can bind to HBV virions. Thompson et al. (1996) obtained a high affinity human monoclonal antibody against an HIV epitope using the phage display. In this study we used intact GCHV as a receptor to screen for a peptide ligand, and obtained a specific peptide with high affinity to GCHV. The peptide screened by the intact virus may block the active site related to replication on the virus surface. The antiviral peptide displayed on the phage did not induce an immunological response of the acceptor. We hypothesize that this peptide might bind to the attachment protein of the virion and inhibit its entry into the host cells, resulting in virus neutralization. This specific peptide might also change the structure of the virus or viral proteins which interferes with the infection process. Thus, the GCHV-specific peptide selected from the peptide library in this study may be potentially applicable in virus control and at the same time establish the foundation for the design of other antiviral peptides or other low molecular weight pharmaceuticals.
Acknowledgements We are grateful to Professor Xiaofeng Wang for her assistance in the preparation of the manuscript and to Professor Ruiwen Zhang for his critical reading and useful suggestions. Our appreciation also goes to Weihua Zhuang for typing the manuscript, figures and tables, and Dong Hong for his technical assistance with the photographs.
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