Adsorption characteristics of an enteric virus-binding protein to norovirus, rotavirus and poliovirus
- Takahiro Imai†1, 2,
- Daisuke Sano2Email author,
- Takayuki Miura†2,
- Satoshi Okabe†2,
- Keishi Wada†1,
- Yoshifumi Masago†1 and
- Tatsuo Omura†1
© Imai et al; licensee BioMed Central Ltd. 2011
Received: 27 July 2011
Accepted: 16 December 2011
Published: 16 December 2011
Water contamination with human enteric viruses has posed human health risks all over the world. Reasonable and facile methodologies for recovering and quantifying infectious enteric viruses in environmental samples are needed to address the issues of waterborne viral infectious diseases. In this study, a bacterial protein that has a binding capability with several enteric viruses is discovered, and its binding characteristics were investigated for utilizing it as a viral adsorbent in virus recovery and detection technologies.
A gene of an enteric virus-binding protein (EVBP), derived from a monomer of a bacterial chaperon protein GroEL, was successfully acquired from a genomic DNA library of activated sludge microorganisms with nested PCR. Equilibrium dissociation constants between EVBP and norovirus-like particles (NoVLPs) of genotypes GI.7 and GII.4, estimated with quartz crystal microbalance method, were 240 and 210 nM, respectively. These values of equilibrium dissociation constant imply that the binding affinity between EVBP and NoVLPs is 1 to 3-log weaker than that in general antigen-antibody interactions, but about 2-log stronger than that in weak specific interactions of proteins with cations and organic polymers. The adsorptions of EVBP to norovirus, group A rotavirus and poliovirus type 1 were found to be significant in enzyme-linked immunosorbent assay. Meanwhile, the binding of native GroEL tetradecamer to viral particles was weaker than that of EVBP, presumably because of a steric hindrance. The small molecule of EVBP could have an advantage in the access to the surface of viral particles with rugged structure.
EVBP that has a broad binding spectrum to enteric viruses was newly discovered. The broad binding characteristic of EVBP would allow us to utilize it as a novel adsorbent for detecting diverse enteric viruses in clinical and environmental samples.
Water contamination with human enteric viruses is a growing concern, because the dissemination of human enteric viruses in reservoir, river water and seawater could pose human health risks for infectious viral diseases via drinking water [1, 2], recreation water , irrigation water , marine cultured products [5, 6], and so on. The quantity of infectious enteric viruses in water, which has to be accurately determined for assessing risks of waterborne viral diseases, is often very difficult to analyze, because of the low concentration of enteric viruses in water environments , the inability of molecular detection methods to distinguish infectious and non-infectious virions [8, 9], and the presence of inhibitory substances for the processes of genome extraction and amplification . Reasonable and facile methodologies for recovering and quantifying infectious enteric viruses in environmental samples are needed to address the issues of waterborne viral infectious diseases.
One rationalized approach to collect infectious virions from environmental samples would be the employment of viral antigen recovery methods by using virus-specific antibodies . The integrity of viral capsid structure, including antigenic sites, is prerequisite to maintain infectivity, and viral particles bound to specific antibodies might be structurally intact. However, diverse enteric viruses in Caliciviridae, Picornaviridae and Reoviridae have been recognized as waterborne agents , and antibody may be too specific to capture all possible strains of a target virus . The preparation of an array of antibodies for each enteric virus of interest would be enormously costly and unrealistic. The discovery of alternative viral adsorbents, which have a broad binding spectrum to enteric viruses and ensure the infectivity of recovered virions, could be very valuable for accurately investigating health risks posed by infectious enteric viruses in water environments.
Virus-binding proteins, which were discovered from a bacterial culture derived from activated sludge , could be alternative viral adsorbents, because these bacterial proteins can stably interact with human viral particles . Recently, norovirus-binding proteins (NoVBPs) were acquired with an affinity to a norovirus (NoV) capsid peptide . It was expected that these NoVBPs, which are derived from homologues of a chaperonin protein GroEL, had a binding capability to several NoV genotypes, presumably because of the critical role of hydrophobic effect . This feature of a broad binding spectrum has raised the expectation that the acquired NoVBPs might be utilized as adsorbents for the recovery and detection of multiple genotypes of NoV in environmental samples. Furthermore, the main contribution of hydrophobic effect might allow us to utilize these proteins as adsorbents for other enteric viruses, if non-specific interactions of these proteins with impurities in environmental samples are well excluded.
In this study, a gene of NoVBP was acquired from an extracted genomic DNA of activated sludge microorganisms, and the adsorption characteristics of the gene product (a candidate of enteric virus-binding protein: EVBP) to particles of enteric viruses were investigated. Values of the equilibrium dissociation constant were estimated with quarts crystal microbalance (QCM) method. Furthermore, the interaction between the EVBP candidate and enteric virus particles, including NoV genotypes GI.7, GII.3, GII.4, GII.6, group A rotavirus (RV-A) and poliovirus type 1 (Sabin) (PV1), was analyzed by enzyme-linked immunosorbent assay (ELISA). Based on these binding assays, the applicability of the gene product as EVBP was discussed.
Production and purification of the EVBP candidate
Equilibrium dissociation constant estimated with QCM
Binding assay of the EVBP candidate with enteric virus particles by ELISA
In order to confirm the binding capability of the EVBP candidate to enteric viruses, ELISA using particles of PV1, RV-A, NoV GI.7, GII3, GII.4, and GII.6 was conducted. The binding assay of a native GroEL, a tetradecamer molecule (Sigma, Japan), was also performed in order to investigate the influence of the complex formation on virus-binding ability. The EVBP candidate and GroEL complex were biotinylated prior to the binding assays, and the binding of biotinylated proteins to immobilized viral particles was detected in ELISA with avidin carrying horseradish peroxidase.
The present study investigated the applicability of EVBP as a viral adsorbent for diverse enteric viruses in families of Picornaviridae, Caliciviridae and Reoviridae, which are dominant etiological agents of gastroenteritis cases all over the world . A broad binding spectrum to human enteric viruses with a sufficient binding affinity is an indispensable binding characteristic of EVBP for utilizing it as a virus adsorbent. However, too low binding specificity is not preferred, because it would cause the non-specific adsorption of impurities co-recovered with viral particles from environmental samples, which must hamper the interaction between EVBP and viral particles.
Although the utilization of EVBP in the detection of enteric viruses from environmental samples was emphasized in this study, there are also possible applications of EVBP in virus detection from clinical samples, especially in the first screening for diagnostics of gastroenteritis. It would be possible to utilize EVBP as a ligand in chromatography and protein microarray techniques . The display on bacterial cell surface  and the immobilization on carriers  could also facilitate the application of this protein as a viral adsorbent in clinical settings. The key aspect in the utilization of EVBP is to curb the non-specific interactions with impurities in clinical and environmental samples, because the hydrophobic effect is likely to be the main factor in the binding between viral particles and EVBP. Appropriate blocking reagents would be required when EVBP is utilized as a detection probe for enteric viruses. BSA was employed as a blocking reagent in the ELISA, and it was found that the binding of EVBP to BSA is negligible.
The binding spectrum of EVBP indicated in this study could be broader than that of histo-blood group antigens (HBGAs) specific to human norovirus , because other enteric viruses such as enterovirus and rotavirus are not using HBGAs as primary receptors on susceptible cells. The employment of porcine gastric mucin including HBGAs  could be able to capture other enteric viruses because of the presence of adhesive glycoproteins in mucin, although the non-specific adsorption of impurities also has to be overcome in the usage of mammalian gastric mucin as a viral adsorbent. Since pH, ionic strength and ion composition in surrounding water are ruling the adsorption of viral particles , it is important to take the measure of natural substances with the propensity to adhere to EVBP in various conditions in the further study. These efforts would offer new techniques using this novel adsorbent for detecting diverse enteric viruses from clinical and environmental samples.
EVBP that has a broad binding spectrum to enteric viruses was newly discovered. EVBP is derived from a monomer of a bacterial chaperon protein GroEL, and this small hydrophobic protein molecule of EVBP could have an advantage in the access to the surface of viral particles with rugged structure. The broad binding characteristic of EVBP would allow us to utilize it as a novel adsorbent for detecting diverse enteric viruses in clinical and environmental samples.
Test virus and cells
Buffalo green monkey kidney (BGMK) cells and fetal rhesus monkey kidney cells (MA-104) were cultured in Eagle's minimal essential medium (MEM) with Earle's salts containing 10% (vol/vol) fetal bovine serum, 0.075% NaHCO3, 2 mM L-glutamine, 10 mM nonessential amino acids, 100 mg/ml penicillin, and 100 U/ml streptomycin. Cells were grown to a confluent monolayer at 37°C with 5% CO2 in a humidifying incubator. PV1 and RV-A was propagated in BGMK and MA-104 cells for 3 to 5 days at 37°C, respectively. PV1 was provided by Dr. Kazuyoshi Yano, Tokyo Metropolitan Institute of Public Health, Japan. RV-A was kindly supplied by Prof. Osamu Nakagomi, Graduate School of Biomedical Sciences, Nagasaki University, Japan. Virus stock was prepared by the freeze-thaw method followed by polyethylene glycol precipitation  and kept in 200 mL aliquots at 20°C until use. NoVLPs of genotypes GI.7, GII.3, GII.4 and GII.6 were prepared as described elsewhere .
Acquisition of a gene coding a possible enteric virus-binding protein
Primers used in this study
Sequence (5' - 3')
Construction of the cloning vector for EVBP candidate
A clone of the target gene was amplified by PCR with primers EVBP-FW and EVBP-RV (Table 1). EVBP-FW contains 5'-CACC-3' region, which is used for PCR Directional TOPO cloning (Invitrogen, CA, USA). PCR master was used for the DNA amplification. The PCR profile was run with 1 cycle of 95°C for 5 min, 35 cycles of 95°C for 0.5 min, 50°C for 0.5 min, and 72°C for 1 min, and 1 cycle of 72°C for 10 min. The PCR product size is 1634. PCR product was cloned with pENTR/SD/D-TOPO (Invitrogen Corp., Carlsbad, CA, USA). The cloned vector, pENTR/KW/EVBP, was used for the transformation of E. coli cells (One Shot TOP10 Chemically Competent Cell, Invitrogen Corp., Carlsbad, CA, USA). The target gene in pENTR/KW/EVBP was subcloned into pDEST17 (Invitrogen Corp., Carlsbad, CA, USA) with LR reaction. The pDEST17 carrying the target gene (pDEST17/TI/EVBP) was used for the transformation of E. coli DH5 alpha and purified as described above.
Production of EVBP candidate with E. coli
Purified pDEST17/TI/EVBP was used for the transformation of E. coli BL21-AI (Invitrogen Corp., Carlsbad, CA, USA). A preculture containing the expression plasmid was grown overnight at 37°C in LB medium with ampicillin (50 mg/ml) and used to inoculate (1/50) LB medium. The culture was grown for 4 hrs at 37°C, and protein expression was induced by the addition of 20% L-arabinose to a final concentration of 0.5%. After overnight-growth at 37°C, cells were immediately collected by centrifugation at 9,000 xg for 10 min at 4°C. The extraction of soluble proteins was performed by BugBuster (Novagen, Darmstadt, Germany). Histidine-tagged EVBP candidate was purified with His Trap FF crude (GE Healthcare, UK). Purified EVBP candidate was desalted by dialysis against 20 mM NH4HCO3 (pH: 8.0) at 4°C for at least 12 hr, and concentrated with a vacuum and centrifugal dehydrator (CVE-100, EYELA, TOKYO RIKAKIKAI CO. Ltd., Tokyo, Japan). EVBP candidate in the pellet was suspended in 100 μl of double-autoclaved milliQ water, and processed for SDS-PAGE and silver staining as described previously .
Estimation of equilibrium dissociation constant
QCM method was employed to estimate the equilibrium dissociation constant between the EVBP candidate and NoVLPs. Firstly, 50 μL of NoVLP suspension of GI.7 or GII.4 in PBS at the concentration of 1010 particles/mL were inoculated on the surface of a sensor chip and left for 1 hr. NoV GII.4 is employed in the QCM test because this genotype is the most important etiological agent over the world . NoV GI.7 is also detected in epidemiological studies relatively frequently among NoV GI strains . The sensor chip was washed with MilliQ water and installed in QCM machine (Single-Q0500, BioLab). Then, 1 mg/ml of the EVBP candidate suspended in PBS (pH: 6.5) was inoculated on the surface of a sensor chip, and the frequency of chip was recorded. The amount of the EVBP suspension inoculated on the sensor chip was changed from 1 to 8 μl. The equilibrium dissociation constant in Langmuir binding isotherm equation was estimated with the associated analytical software Q-UP.
Binding assay of EVBP candidate with ELISA
A 50 μl portion of virus (109 copies/well) or NoVLP (109 particles/well) suspension was added to each well of a microtiter and left for 1 hr to coat the well. Triplicate wells were used for each sample. Then, wells were washed twice with PBS and blocked with 5% BSA in PBS. After incubation at room temperature for 2 hrs, the wells were washed twice with PBS, and the biotinylated EVBP candidate in 50 μl of PBS containing 5% BSA was applied to the wells. Plates were incubated at room temperature for 1 hr and washed twice with PBS and then horseradish peroxidase (HRP)-modified streptavidin (Wako, Osaka, Japan) in 50 μl of PBS containing 5% BSA was inoculated to each well. After incubation at room temperature for 1 hr, wells were washed four times with PBS, and HRP-modified streptavidin bound was measured by coloring with o-phenylenediamine (P-7288, SIGMA CHEMICAL CO., ST. Louis, MO., USA) and H2O2 in citrate-phosphate buffer for 30 min. The coloring reaction was stopped with 2 M H2SO4. The absorbance at 492 nm (A492) was determined with a plate reader (Multiskan MS, Labsystems, Finland).
Acknowledgements and Funding
This work was funded by Grant-in-Aid for Scientific Research (S: 19106009 and Young Scientist: 20-5366) from the Japan Society for the Promotion of Science (JSPS), and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency (JST).
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