Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus
- Guan-Hua Lai†1,
- Ming-Kuem Lin†2,
- Yi-Yang Lien3,
- Jai-Hong Cheng4,
- Fang-Chun Sun5,
- Meng-Shiunn Lee6,
- Hsi-Jien Chen7 and
- Meng-Shiou Lee2Email authorView ORCID ID profile
© The Author(s). 2018
Received: 3 August 2017
Accepted: 18 April 2018
Published: 4 May 2018
Chicken anaemia virus (CAV) is commonly found in poultry. VP1 is the sole structural protein of CAV, which is the major component responsible for capsid assembly. The CAV virion consists of the VP1 protein and a viral genome. However, there is currently no information on the protein-nucleic acid interactions between VP1 and DNA molecules.
In this study, the recombinant VP1 protein of CAV was expressed and purified to characterize its DNA binding activity. When VP1 protein was incubated with a DNA molecule, the DNA molecule exhibited retarded migration on an agarose gel. Regardless of whether the sequence of the viral genome was involved in the DNA molecule, DNA retardation was not significantly influenced. This outcome indicated VP1 is a DNA binding protein with no sequence specificity. Various DNA molecules with different conformations, such as circular dsDNA, linear dsDNA, linear ssDNA and circular ssDNA, interacted with VP1 proteins according to the results of a DNA retardation assay. Further quantification of the amount of VP1 protein required for DNA binding, the circular ssDNA demonstrated a high affinity for the VP1 protein. The preferences arranged in the order of affinity for the VP1 protein with DNA are circular ssDNA, linear ssDNA, supercoiled circular dsDNA, open circular DNA and linear dsDNA.
The results of this study demonstrated that the interaction between VP1 and DNA molecules exhibited various binding preferences that were dependent on the structural conformation of DNA. Taken together, the results of this report are the first to demonstrate that VP1 has no sequence-specific DNA binding activity. The particular binding preferences of VP1 might play multiple roles in DNA replication or encapsidation during the viral life cycle.
Chicken anaemia virus (CAV) is a common viral agent in chickens worldwide. CAV belongs to the genus Gyrovirus of the Anelloviridae, which have characteristics of circular single-stranded DNA viruses . This virus frequently results in immunosuppression and anaemia in young chickens due to the destruction of T lymphoid tissue and aplasia of bone marrow, respectively, during virus infection [2–5]. Over 55% of the mortality rate and 80% of the morbidity rate were reported once the chicks were infected with CAV . Therefore, determining how to prevent CAV infection in the poultry industry has becomes an important challenge. The CAV virion lacks an envelope around its capsid coat, and it shows significantly high resistance to environmental stress or chemical agents. Currently, an attenuated live vaccine is available and effective for immunization of chickens for controlling CAV infection. However, young chicks less than 2 weeks-old are susceptible to CAV infection when the live vaccine was used . This consequence has led to the development of a subunit vaccine, including DNA or protein based vaccine, over the past decade.
CAV is a relatively small virus approximately 23 nm in diameter. A total of three open reading frames (ORFs) were involved in the viral genome and have a length of 2.3 kb . These ORFs respectively encode a 51 kDa VP1 protein, a 28 kDa VP2 protein and a 13 kDa VP3 protein. VP2 has dual-specificity phosphatase activity. VP3 is also referred to as apoptin with apoptosis-inducing activity. VP1 is the sole structural protein, which is the major component responsible for capsid assembly [5, 7, 8]. Currently, VP2 and VP3 proteins have been the focus of investigations of virus pathogenicity [9, 10]. In addition to its importance in the viral life cycle, VP3 has also demonstrated apoptosis-inducing activity as well as medical applications for anti-cancer treatments for humans in many previous studies [11–14]. VP1 can interact with VP2 and then significantly elicit the production of virus-neutralizing antibodies in the host in terms of immunogenicity studies . Therefore, VP1 is thought to be a good candidate for an immunogen to develop a subunit vaccine .
In this study, to gain insight into the role of the capsid protein VP1 in the life cycle of CAV, we have investigated the physical interactions of CAV VP1 with the viral DNA. A recombinant E. coli expression system was used to express the recombinant VP1 of CAV following our previous study . The intracellular localization of the CAV VP1 was observed in MDCC-MSB1 cells or CHO-K1 cells using fluorescent green protein in the nucleoplasmic compartment. The DNA-binding activity of VP1 was also systemically examined. To the best of our knowledge, this is the first report to verify the DNA binding activity of the CAV capsid protein, VP1.
Functional prediction of the CAV VP1 protein
Previous studies have shown that only the VP1 protein is located in the CAV virion. Thus, VP1 is also thought to be a DNA-binding protein that is responsible for the encapsidation of a viral genome during virus assembly. Presently, VP3 is the only one of three CAV viral proteins that has exhibited DNA binding activity in previous studies . However, the function of VP1 on nucleic acid binding is still unknown. To gain insight into the role of the capsid protein VP1 in DNA binding, the bioinformatics software DP-Bind (http://lcg.rit.albany.edu/dp-bind/) was applied to analyse the features of DNA binding motifs within the amino acid sequence of the VP1 protein. Computational results of the DNA binding motif from the VP1 protein are shown in Fig. 1. Four potential DNA binding motifs were predicted by the DP-Bind program, and the putative motif position spanned from amino acids residues 3 to 22, 27 to 47, 62 to 67 and 333 to 349. According to these predicted results, VP1 might be having potential activity to bind DNA molecules. However, further investigation is still needed to verify the DNA binding activity of VP1.
Expression and purification of the recombinant CAV viral protein, VP1 and VP3
CAV VP1 is a nuclear protein that binds DNA with no sequence specificity
To elucidate whether the CAV VP1 protein is a DNA-binding protein, purified recombinant VP1 protein was added to circular dsDNA, pCAV, pcDNA3.1 and pGEM-T plasmids and incubated for 1 hour under 37 °C. After incubation, the occurrence of protein-DNA interaction was analysed by DNA movement on agarose gel. As illustrated in Fig. 2b, c and d, the migrations of pCAV, pcDNA3.1 and pGEM-T plasmid on the agarose gel were significantly reduced and shifted towards a pattern with a higher molecular weight. This result is very similar to the reduction in DNA migration that arose from binding VP3 to DNA (Fig. 2b, c and d). In contrast, no reduction in DNA migration occurred when the VP1 protein was absent or when GST protein was loaded with the addition of circular dsDNA plasmid. Moreover, when VP1 protein was pre-treated with 1% sodium dodecyl sulfate (SDS), the denatured VP1 no longer had DNA binding activity (Fig. 2b, c and d).
With respect to pCAV, which is a pcDNA3.1 plasmid carrying the entire CAV genome, the VP1 protein also displayed its DNA binding activity in terms of altered DNA migration pattern, as illustrated in Fig. 2b. In other words, regardless of whether the plasmid DNA used in the protein-DNA binding reaction was from pcDNA3.1 or pCAV, there was no significant effect on the resulting pattern of DNA migration (Fig. 2b, c and d). However, it is worth noting that the DNA migration of the circular dsDNA plasmid in the agarose gel displayed open circular dsDNA (form I, with a higher molecular weight pattern) and supercoiled dsDNA (form II, with a lower molecular weight pattern), simultaneously (Fig. 2b). The VP1 protein was bound to supercoiled dsDNA. which demonstrated that DNA shifting was more obvious than open circular dsDNA.
CAV VP1 binds DNA with a conformational preference
The ratio of unbound free DNA residue was determined by calculating the image intensity of free DNA fragments on an electrophoretic agarose gel after a VP1-DNA binding assay combining increasing amounts of recombinant GST-VP1 protein with certain DNA molecule conformations
Concentrations of GST-VP1 protein (ng)
Open circular dsDNA (Form I)
Supercoiled dsDNA (Form II)
Linear ssDNA (−)
M13 phage DNA
DNA replication in the DNA viruses usually occurs in the nuclei of infected cells. Thus, viral DNA needs to cross the nuclear envelope through protein-mediated nuclear transportation to establish a productive infection after infecting the cells . CAV is a non-enveloped, small DNA virus containing a circular ssDNA genome . VP1, which is a major capsid protein of CAV, interacts with cells during virus infection, and the viral genome should theoretically be carried by VP1 to enter cells. The main question is what protein is conferred a function to direct a viral genome into the nucleus for sequential DNA replication? VP1 is thought to have a functional role to bind and direct the viral genome into the nucleus. However, there is still a lack of direct evidence to support this speculation. In our results from computational prediction, four putative DNA binding motifs were combined with the amino acid sequence of the CAV VP1 protein. Although the exact DNA binding motif was not determined in this study, it did not affect the characterization of VP1 DNA binding activity. After performing a DNA-binding assay to examine DNA migration, the VP1 protein of CAV was confirmed to have DNA-binding activity. Additional experiments are still needed for identifying major DNA binding motifs within VP1 protein. The confirmed DNA-binding activity in VP1 might be useful to further verify the underlying mechanisms of viral DNA replication. In addition, GFP-VP1 has demonstrated nucleo-cytoplasm shuttling activity (Fig. 3). The results imply VP1 is a nuclear protein to binds DNA molecules, such as those in the viral genome and travels into the nucleus during its early life cycle. In fact, we have not only predicted the presence of nuclear localization signals (NLSs) with PSORT II but also predicted nuclear exporting signals (NESs) with NetNES server (http://www.cbs.dtu.dk/services/NetNES/). These putative NLS motifs were within the amino acid sequence of VP1, which spanned from amino acid residues 3 to 19 (NLS1) and 24 to 47 (NLS2). For NESs, the putative motifs within the VP1 protein spanned from amino acid residues 76 to 84 (NES1), 109 to 119 (NES2) and 375 to 387 (NES3) (Fig. 1a). In terms of the observation of fluorescent of GFP-VP1, this result was confirmed according to computational predictions. In the early life cycle of the virus, viral DNA replication is an important stage for the establishment of productive infection . Previous studies have reported circular, negative ssDNA of the CAV genome might be replicated through rolling-circle amplification . During this stage, previous researchers isolated the viral replicative form (RF) DNA, which is an open circular dsDNA obtained from MDCC-MSB1 cells after being infected with the virus for 30 h . In addition to the presence of closed and open circular dsDNA, circular ssDNA with genome-sized and small linear dsDNA of 800 bp were observed in the later stages of DNA replication . In this study, all similar DNA molecules with different conformations, including linear ssDNA, which is derived from circular ssDNA, were used to examine the possible functional roles of the VP1 protein in DNA-binding activities. Additionally, other putative DNA binding motifs, especially for the initiation of rolling-circle amplification (RCR) within the VP1 protein, had also been reported and predicted in a previous study . Three putative motifs were proposed that spanned from amino acid 313 to 320 (FATLTALG), 350 to 358 (GQRWHTLVP), and 399 to 408 (TATYALKEPV). These motifs might be interaction sites, such as the origin (Ori) site of the CAV genome, for interacting with VP1 for regulating DNA replication .
Generally, the DNA binding protein of most DNA viruses showed DNA binding activity with no sequence specificity. In this study, VP1 showed DNA binding characteristics with no sequence specificity similar to other DNA viruses, such as human papillomavirus and polyomavirus [27, 28]. Therefore, some other strategy of VP1 binding to the CAV viral genome should be adopted by the virus. With respect to the binding preferences of the VP1 protein to different DNA molecule conformations, VP1 was found to interact with circular ssDNA and exhibit a higher preference for this conformation, as shown in Table 1. This outcome might truly reflect the conditions of viral encapsidation for coating circular ssDNA of the CAV genome. Actually, circular ssDNA is prone to forming secondary structures with a high probability. This possibility was examined and confirmed with a computational prediction from the Mfold program (http://unafold.rna.albany.edu/?q=mfold/DNA-Folding-Form) (Additional file 1: Figure S1A, B). Similarly, linear ssDNA also has a high probability for forming secondary DNA structures (Additional file 1: Figure S2A, B). Thus, the binding preference of VP1 to linear ssDNA is surpassed only by the preference for circular ssDNA (Table 1). This difference truly meets our expectations. In fact, the sequence of linear ssDNA(−) was complemented with linear ssDNA(+). Then, linear ssDNA(+) was found to have significant VP1-DNA interaction in terms of the results of the DNA migration assay. The binding preference of VP1 to the linear plus-strand of ssDNA(+) is slightly lower than the linear plus-strand of ssDNA(−) (53.1% for 300 μg of VP1) (Additional file 1: Figure S3A, B). Other DNA molecules, such as supercoiled dsDNA, linearized ds DNA and opened dsDNA, displayed lower binding preferences to VP1, which might be a mechanism for VP1 protein to competitively bind various DNA molecules during different stages of the life cycle.
Taken together, this report is the first to show that VP1 has no sequence specificity for its DNA-binding activity and that its particular binding preferences might play multiple roles in DNA replication or encapsidation during a viral life cycle.
In summary, the characterization of DNA binding activity of the VP1 protein was investigated in this study. VP1 was demonstrated to show DNA binding characteristics with no sequence specificity. In addition, the DNA binding activities of VP1 exhibited a differential preference to interact with various DNA molecules with different conformations. This information could be helpful for determining the biological roles of VP1 in the CAV viral life cycle.
Cell cultures, bacterial strains and plasmids
For Chinese Hamster Ovary (CHO-K1) cells, cells were purchased in 2014 from the Bioresource Collection and Research Center (BCRC 6006) in Taiwan. CHO-K1 cells were maintained in Ham’s F12 medium (HyClone, USA) supplemented with 10% FBS (HyClone, USA), 1% P/S (Penicillin/Streptomycin solution) (Gibco, USA). Chicken lymphoblast MDCC-MSB1 cells were purchased from the CLS Cell Lines Service GmbH in Germany in 2015. MDCC-MSB1 cells were grown in RPMI 1640 medium (HyClone, USA) supplemented with 10% FBS (HyClone, USA) and 1% P/S (Penicillin/Streptomycin solution) (Gibco, USA). All cells were cultured in appropriate tissue culture flasks and maintained in a cell culture incubator with 5% CO2 at 37 °C before experiments.
All expression constructs used in this study were maintained in the E. coli strain Top10F’ (Invitrogen, USA). The E. coli strain BL21 (DE3)-pLysS was transformed with protein expression plasmids and followed by IPTG induction to produce recombinant proteins as described in a previous study .
The construction of an expressed plasmid used for detecting subcellular localization was described below. The full-length of CAV VP1 was amplified by PCR using the specific primer sets wt-VP1-f: 5’-CCCGAATTCATGGCAAGACGAGCTCGC-3′, wt-VP1-r: 5’-CGCGTCGACTCAGGGCTGCGTCCCCCAGTA-3′ from the CAV VP1 template that was kindly provided by Dr. Yi-Yang Lien. The PCR product was then cloned into expression vector pEGFP-C2 (#6083–1, Clontech, USA) between EcoR I and Sal I sites to generate a recombinant plasmid named pEGFP-VP1.
Expression and purification of the CAV VP1 and VP3 proteins
To purify the recombinant CAV VP1 and VP3 proteins, the previously created recombinant E. coli strains BL21 (DE3)-pLysS expressing VP1 and VP3 were used to express recombinant proteins . The recombinant E. coli cells were cultured, and the harvested cells were disrupted and prepared following a previously described procedure . Cells were spun down from 50 ml of culture supernatant and resuspended in GST resin binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3). After cell disruption, the resulting cell supernatant was loaded onto a GSTrap FF affinity column (GE healthcare, Piscataway, NJ) for protein purification following the operational conditions described in a previous study . The total protein concentration of recombinant CAV VP1 and VP3 proteins was determined using a Micro BCA kit (Pierce, Rockford, IL) with bovine serum albumin as the reference protein. Purified VP1 and VP3 proteins were dialyzed against DNA-binding buffer (50 mM Tris-HCl, pH 7.5, 120 mM KCl, 1.0 mM EDTA, 0.5 mM DTT, and 30 mg/ml BSA) and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Purified proteins were stored at − 20 °C until required.
Generation of nucleic acids used for the DNA-binding assay
Different DNA species, including circular dsDNA, linear dsDNA and circular ssDNA were used for assessing DNA-binding activity. The circular dsDNA, pcDNA3.1 (#V80020, Invitrogen, USA), pGEM-T easy vector (#A1360, Promega, USA) and pCAV were used for the DNA binding assay. The pCAV plasmid DNA was composed of a full-length Australian CAV strain CAU269/7 (GenBank #AF227982). The linear dsDNA was prepared from a pcDNA3.1 and pGEM-T plasmid by the cutting restriction enzyme EcoRI. Pure M13mp18 single-stranded DNA along with circular ssDNA materials were purchased from New England BioLabs (#N4040S, NEB, USA). All DNA molecules were diluted to 50 ng/ml with DNA-binding buffer and store at − 20 °C until required.
Preparation of linear single-stranded DNA
The linear ssDNA was also used for the DNA binding assay. The preparation of linear ssDNA followed the protocol described in Marimuthu et al.  using the biotin-streptavidin separation method. First, a biotinylated DNA fragment containing the whole CAV genome was amplified by PCR using the EmeraldAmp Max PCR Master kit (Takara, Japan) from pCAV with a designed primer set, including the reverse biotinylated primer Biotin-CAV-r: biotin-labelled-GATTGT GCGGTGAACGAATTAG, and the forward regular primer CAV-f: GAATTCCGAGTGGTTACTATTC. After PCR amplification, the biotinylated PCR product was then immobilized on 40 μl Dynabeads M-280 Streptavidin magnetic beads (Invitrogen, USA) and incubated at 4 °C overnight. After washing the DNA-bonded beads twice with B/W buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl), the washed beads were incubated in 150 μl elution buffer (0.1 M NaOH, 1 mM EDTA, pH 13.0) to perform alkaline denaturation. Under the high alkaline environment, the desired non-biotinylated strand can be separated from the biotinylated strand and suspended in the supernatant. After magnet adsorption, the supernatants were collected, and the linear ssDNA was further purified by a PCR clean-up kit (Geneaid, Taiwan). The linear ssDNA was diluted to 50 ng/ml with DNA-binding buffer and store at − 20 °C until required.
DNA binding assay
Purified GST, GST-VP1 and GST-VP3 proteins were diluted to 500 ng/μl with DNA-binding buffer and 500 ng of proteins were mixed with 100 ng of each DNA variant in a total of 20 μl of DNA-binding buffer. Then, each mixture was incubated for 30 min under 37 °C. The resulting sample was subjected to electrophoresis using a 0.8% agarose gel in a TAE buffer and, then the DNA was stained with ethidium bromide for the analysis of DNA migration.
Cell transfection of CHO-K1 and MDCC-MSB1 cells
CHO-K1 cells were transfected by X-tremeGene HP DNA transfection reagent (Sigma, USA) according the manual’s protocol with a mixture containing 2 μg of plasmid pEGFP-VP1 and 4 μl of transfection reagent in 2.5 ml serum-free Opti-MEM medium (Gibco, USA). After incubating the mixture for 20 min at room temperature, the mixture was added drop by drop into cultured CHO-K1 cells in a 6-well plate. The 24 to 48 h post-transfection, the transfection effect was checked with a confocal fluorescent microscope.
For the transfection of MDCC-MSB1 cells, the 4 × 106 log-phase grown MDCC-MSB1 cells were gently pipetted with 15 μg of plasmid pEGFP-VP1 first in serum-free RPMI 1640 medium and then the mixture was transferred into a 0.4-cm gap electroporation cuvette and the cuvette was harvested on ice for 5 min. The electroporation of MDCC-MSB1 cells was performed with a Gene Pulser II (Bio-Rad, USA) with a Time Constant Protocol set at 34 ms and an operating voltage of 300 V. After electroporation, the transfected cells were then cultured into complete medium in a 6-well plate for 24 to 48 h. Post-transfection, the expression of recombinant EGFP-VP1 proteins were analysed by confocal fluorescence microscopy to make sure the transfection was effective.
Sample preparation for confocal microscopy observation
After transfecting CHO-K1 cells and MDCC-MSB1 cells with plasmid pEGFP-C2 or pEGFP-VP1, the fluorescent images were captured by a confocal fluorescence microscope in terms of the observation of protein fluorescent to verify the EGFP-expressed cells and EGFP-VP1-expressed cells. Transfected cells were collected and fixed with 4% formaldehyde in the dark. After washing the fixed cells twice to remove residual formaldehyde, the cells were stained in 0.1% PBS-T with 1 μg/ml DAPI for 5 min at 37 °C in the dark. Then, the stained cells were mounted with gelvatol medium (Sigma, USA) on a glass slide for confocal observation. Confocal laser scanning microscope (CLSM) images were captured from a Leica TCS SP8 confocal microscope and the images were integrated with LAS X Leica Confocal Software. EGFP fluorescence was observed through excitation at 488 nm and DAPI emitted blue fluorescence upon binding to DNA that was observed through excitation by UV light.
Comparing the DNA conformational preference of VP1 protein through DNA analysis
After dividing the signal intensity at a certain concentration of GST-VP1 proteins by the signal intensity in the absence of proteins (blank), the calculated ratio of unbound DNA residue was obtained to determine the DNA conformational preference for the VP1 protein.
This work of the authors would like to thank the Ministry of Science and Technology, Taiwan (TW) for financially supporting. We also thank the Medical Research Core Facilities Center, office of Research & Development at China Medical University (Taichung, Taiwan, R.O.C.) for supporting experiments and data analysis on confocal microscopic observation.
This research was financially supported through grants from the Ministry of Science and Technology, Taiwan (TW) (grant number: NSC101–2321-B-039-007- and NSC102–2321-B-039-007) to Dr. Meng-Shiou Lee.
Availability of data and materials
All data generated or analysed in this study are included in the article and additional supporting files. All materials are available from the corresponding author, on reasonable request.
GHL and MKL contributed equally to first author in this work. MSL* participated in the study design, performed the experiments and drafted the manuscript. GHL and YYL performed the experiments and participated in the construction of the plasmids. MKL and JHC participated in the experiments on protein-DNA binding assay, and MSL6 participated in the protein purification step. FCS participated in the data analysis, and the manuscript drafting. HJC, GHL and MKL coordinated the study and participated in drafting the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All experiments and participated authors in this study (grant number: NSC101–2321-B-039-007- and NSC102–2321-B-039-007) were approved by the committee on the ethics of genetic engineering, China Medical University. No animals were used in this work.
Consent for publication
All data obtained from this work were approved by all authors for publication.
The authors declare that they have no competing interests. All information generated in this study there is no any commercial conflict of interests.
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