Virus Research 179 (2014) 140–146

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Encapsulation and delivery of plasmid DNA by virus-like nanoparticles engineered from Macrobrachium rosenbergii nodavirus Pitchanee Jariyapong a,c , Charoonroj Chotwiwatthanakun d,∗∗ , Monsicha Somrit a , Sarawut Jitrapakdee b , Li Xing e , Holland R. Cheng e , Wattana Weerachatyanukul a,∗ a

Department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Road, Phyathai, Bangkok 10400, Thailand Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Phyathai, Bangkok 10400, Thailand c School of Medicine, Walailak University, Thasala District, Nakhonsrithammarat, Thailand d Nakhonsawan Campus, Mahidol University, Nakhonsawan, Thailand e Department of Molecular and Cell Biology, University of California, Davis, CA 95616, United States b

a r t i c l e

i n f o

Article history: Received 11 June 2013 Received in revised form 29 September 2013 Accepted 24 October 2013 Available online 31 October 2013 Keywords: Macrobrachium rosenbergii nodavirus Virus-like particle Biological container Encapsulation

a b s t r a c t Virus-like particles (VLPs) are potential candidates in developing biological containers for packaging therapeutic or biologically active agents. Here, we expressed Macrobrachium rosenbergii nodavirus (MrNv) capsid protein (encoding amino acids M1-N371 with 6 histidine residuals) in an Escherichia coli BL21(DE3). These easily purified capsid protein self-assembled into VLPs, and disassembly/reassembly could be controlled in a calcium-dependent manner. Physically, MrNv VLPs resisted to digestive enzymes, a property that should be advantageous for protection of active compounds against harsh conditions. We also proved that MrNv VLPs were capable of encapsulating plasmid DNA in the range of 0.035–0.042 mol ratio (DNA/protein) or 2–3 plasmids/VLP (assuming that MrNV VLPs is T = 1, i made up of 60 capsid monomers). These VLPs interacted with cultured insect cells and delivered loaded plasmid DNA into the cells as shown by green fluorescent protein (GFP) reporter. With many advantageous properties including self-encapsulation, MrNv VLPs are good candidates for delivery of therapeutic agents. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Biological containers such as viral capsids, heat shock proteins or ferritin are well-established as potential containers for delivery of therapeutic agents at the nanoscale. Among the best examples are recombinant capsid protein cages called virus like particles (VLPs) that serve as biological weapons against many diseases (Bolhassani et al., 2011). Reconstruction of these protein containers relies either on their self-assembly properties or on molecular modification of their structures to direct their interaction/association with their targets (Uchida et al., 2007). Inherently, VLPs employ a self-assembling property to form functional templates upon their recombinant expression in a relevant expression system. In addition, the feasibility of VLP molecular modifications, both chemically and genetically, fulfill the desired functionality as a biological container (Arora et al., 2012; El Mehdaoui et al., 2000; Hoque et al.,

∗ Corresponding author at: Department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Road, Rachathewi, Bangkok 10400, Thailand. ∗∗ Corresponding author at: Mahidol University, Nakhonsawan Campus, Phayuhakiri, Nakhonsawan 60130, Thailand. Tel.: +66 2 2015883. E-mail addresses: [email protected] (C. Chotwiwatthanakun), [email protected] (W. Weerachatyanukul). 0168-1702/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2013.10.021

1999; Niikura et al., 2002; Renoux et al., 2008). The inherent property of viral capsids is to function in host cell recognition, fusion, and entry makes VLPs ideal for nanoscale delivery of therapeutic materials capable of inducing immunological responses at targeted sites (Ludwig and Wagner, 2007; Uchida et al., 2007). They have also been used for encapsulating DNA in the gene therapy and vaccine development. For example, VLPs of mouse polyoma virus (Krauzewicz et al., 2000), human papillomavirus (Combita et al., 2001) and hepatitis E virus (Takamura et al., 2004) have been used to encapsulate and deliver plasmid DNA into the cells, resulting in expression of reporter genes both in vitro and in vivo. Because the aforementioned VLPs are derivatives of infectious viruses that cause harmful human diseases, their application as biological containers is still questioned and still requires further investigation for safety validation. Macrobrachium rosenbergii nodavirus (MrNv) (Family Nodaviridae) is found together with extra-small virus (XSV) as the cause of a white tail disease (WTD) that leads to a large-scale mortality in cultured giant freshwater prawns (Arcier et al., 1999; Sri Widada et al., 2003). The disease is so named because of hyaline necrosis of muscle fibers in the tail associated with lethargy, anorexia and opaqueness of the abdominal region (Sri Widada et al., 2003). MrNv is a non-enveloped, icosahedral virus of ∼26 nm diameter (Saroja and Bhaskaran, 2011). Similar nodaviruses with variable diameters

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are found in insects and fish (Tang et al., 2002). The genome of MrNv is composed of two linear positive-sense single strand RNAs including RNA1 (3.2 kbp, encoding an RNA-dependent RNA polymerase, RdRp) and RNA2 (1.2 kbp, encoding the single capsid protein of a 43-kDa) (Sijun and Yong, 2009). In fish and insect nodaviruses, the capsid protiens are rich in positively charged amino acid residues (arginine and lysine) at the N-terminus (Tang et al., 2002). This property permits interaction with negatively charged RNA during viral assembly (Schneemann et al., 1993). Expression of recombinant MrNv capsid protein in Escherichia coli and its self-assembly into VLPs has been recently reported (Goh et al., 2011). Taking advantage of the existing information, we thus aimed to develop protocol for constructing VLP from shrimp viral capsid protein (MrNv-VLP) as a nanocontainer which can be used for delivering plasmid DNA into target insect cells. 2. Materials and methods 2.1. Viral RNA isolation and cloning of MrNv capsid protein MrNv-infected prawns were collected from hatcheries located at Chachengsaow province, Thailand. Their tissues were homogenized with Isol-RNA lysis reagent (5-PRIME, Gaithersburg, MD). The total RNA was obtained by phenol/chloroform extraction and the RNA was precipitated with ethanol before re-suspension in nuclease-free water. Contamination of genomic DNA was eliminated by treatment with 1U DNAseI for 1 h at 37 ◦ C and cDNA was synthesized using reverse transcription reaction in 1× first strand buffer (5 mM dithiothreitol, 2 mM dNTPs, 1 ␮g of RNA, 1 ␮g random hexamers (Roche, Germany) and 200 U of reverse transcriptase (Invitrogen, USA)) at 50 ◦ C for 1 h. A full length MrNv capsid gene at the position of M1 –N371 (Genbank accession No. EU 150129) with a sequence of 6 histidines added at the C-terminus (MrNvCAP-6His) was amplified by the following primers: (F) 5 -CCATGGCTAGAGGTAAACAAAATTCTA-3 and (R) 5 CTCGAGCTAATGATGATGATGATGATGATTATTGCCGACGATAGCTCTGATA-3 . The PCR amplification protocol consisted of preheating at 94 ◦ C for 5 min followed by 35 cycles of 94 ◦ C for 30 s, 55 ◦ C for 30 min and 72 ◦ C for 1 min. The PCR products were separated on 1% TBE (89 mM Tris, 89 mM Boric acid, 2.5 mM EDTA; pH 8.0) agarose gels, stained with ethidium bromide and visualized under UV light. The expected DNA band at 2.1 kbp was excised and extracted using a DNA gel extraction kit (Qiagen, Germany) and ligated into pGEM-T Easy vector (Promega, USA). The DNA sequence of the pGEM-MrNvCAP-6His insert was verified by DNA sequencing (Macrogen, Seoul, Korea) before it was cut from the vector with NcoI and XhoI and ligated into a pET16b expression vector (Novagen, Darmstadt, Germany). Transformation of the recombinant vector into competent E. coli BL21 (DE3) was performed by the heat shock method (i.e., 42 ◦ C for 45 s followed by immediate incubation on ice for 5 min). The cells were then inoculated in SOC medium (Invitrogen, Grand Island, NY) on an incubator shaker (37 ◦ C, 250 rpm, 1 h). The mixture was spread on LB agar plates containing ampicillin (50 ␮g/ml) and incubated overnight (37 ◦ C). Colonies of transformed cells were screened by PCR using the following pair of primers: (F) 5 -GAGGTAAACAAAATTCTAATC -3 and (R) 5 -ATTATTGCCGACGATAGCTCTG-3 . Recombinant plasmids were extracted from positive clones, verified by restriction endonuclease digestion and DNA sequencing. 2.2. Expression of recombinant MrNv capsid protein and its purification Protein expression and purification were performed according to previously described methods (Goh et al., 2011). The positive

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clones carrying pET16b-MrNvCap-6his gene transformed into E. coli BL21 (DE3) were inoculated in LB broth containing 50 ␮g/ml of ampicillin and incubated at 37 ◦ C overnight followed by further incubation in the same medium at 25 ◦ C until the absorbance reached 0.6–0.8 at 600 nm (A600 ). Protein expression was induced by adding IPTG (1 mM) to the culture medium followed by further incubation for time-course expression (1–5 h). After induction, the cells were pelleted by centrifugation (5400 × g, 4 ◦ C, 10 min) and the pellet was re-suspended in PBS containing 500 mM NaCl and 2 mM phenylmethylsulfonyl fluoride (PMSF) for sonication at 100 Hz (20 s, 10 cycles). The sonicated samples were pelleted (12,000 × g, 10 min) and the supernatant collected and loaded onto a nickel-based agarose (Ni-NTA) chromatographic column (Qiagen, Germany). After several washes with 20 mM imidazole in 500 mM NaCl, the bound proteins were eluted with elution buffer (250 mM imidazole, 500 mM NaCl, pH 7.4). The eluents was further subjected to discontinuous sucrose gradient centrifugation using a modification of the method of Xing et al. (1999). The fractionated proteins in all interphases were collected for protein concentration assay using NanoDrop 2000 spectrophotometry (Thermo Fisher Scientific, Delaware), and self-assembly of the MrNvCAP-6his protein to form virus-like particle (VLP) was verified by negative staining TEM. 2.3. Protein profiling and Western blotting of expressed protein Proteins obtained from several purification steps using 3 h cultured E. coli including whole cell lysates, flow-through, washing solution, elution solution and centrifugal fractions were resolved by 10% SDS-PAGE gels stained with 1% Coomassie brilliant blue-R250. The resolved proteins of E. coli lysates at 0 and 3 h were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA) for Western immunoblotting. The membranes were submersed in 2% bovine serum albumin (BSA) and 5% skim milk to block non-specific antibody staining (room temperature, 2 h) followed by exposure at 1:2000 dilution to anti-MrNv monoclonal antibody (a kind gift from Prof. Dr. Paisan Sithigorngul, Srinakarinwiroj University, Bangkok, Thailand). This antibody has been shown to give high specificity against MrNV capsid protein (Longyant et al., 2012). Secondary antibody was then added with the horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (1:5000 dilution) and the reactivity of antibody-antigen was detected by an enhanced chemiluminescence method using an ECL kit (Amersham Biosciences, Piscataway, NJ). 2.4. VLP disassembly/re-assembly and plasmid DNA encapsulation We tested the efficiency of two different VLP disassembling conditions as a prerequisite step for the DNA packaging experiment. In brief, 5 ␮g of the purified MrNv-VLPs were incubated with a buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl containing either 1 mM ethylenediaminetetraacetic acid (EDTA) or 1 mM ethyleneglycoltetraacetic acid (EGTA) in the presence of 20 mM dithiothreitol, DTT (1 h, room temperature). The treated VLPs were subjected to ultracentrifugation (200,000 × g, 4 ◦ C, 2 h) and the pellet was resuspended in PBS. They were plated on a carbon coated grid, negatively stained by 2% uranyl acetate and observed under a Tecnai 20 transmission electron microscope operated at 120 kV. For encapsulating plasmid DNA into MrNv-VLP, a Pie1 plasmid DNA was constructed to carry an enhanced green fluorescent protein gene (Pie1-EGFP) (Borirak et al., 2009). Approximately 5 ␮g plasmid DNA was added into the disassembled VLPs in the disassembling buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA and 20 mM DTT). To resume VLP assembly, an increasing concentration of CaCl2 was slowly added to the mixture to reach a final concentration of 5 mM followed by further incubation for 1 h.

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VLPs were pelleted by ultracentrifugation and used for gel electrophoresis and delivery assay using Sf9 cells and for VLP hydrolytic tolerance tests in vitro as described below. In an alternative experiment, plasmid-loaded VLPs were heated at 95 ◦ C for disruption before treatment with 10 IU DNaseI (37 ◦ C, 1 h) to degrade the plasmid DNA released from the disrupted VLPs. 2.5. Gel electrophoresis and calculation of plasmid DNA: MrNv VLP loading ratio For DNA electrophoresis, the plasmid DNA encapsulated into VLPs was first incubated in SDS-PAGE loading dye and heated at 95 ◦ C for 5 min to break up all tightly assembled VLPs. Denatured VLPs with the released plasmid DNA and the serially diluted purified Pie1-EGFP plasmids (0.1–1 ␮g) were electrophoresed in 0.5% agarose gels, stained with ethidium bromide and visualized by a GelDoc Imager (BioRad, Hercules, CA). In case of protein electrophoresis, VLPs loaded with plasmid DNA and the serially diluted MrNv recombinant proteins (0.5–4 ␮g) were denatured as mentioned above. They were then resolved by 10% SDS-PAGE followed by silver staining. To confirm that plasmid DNA was encapsulated into the inner cavity of VLP, 1 U of DNAseI was added into plasmid loaded VLP suspension to allow any exterior DNA digestion (if any) followed by treating and analyzing by electrophoresis. To calculate the amount of loaded DNA and VLPs after an encapsulation experiment, standard curves of plasmid DNA (3.1 kbp band) and MrNv capsid proteins (41.5 kDa band) were generated from intensities of known amounts of the respective DNA and proteins. The band intensities of the loaded plasmid DNA and MrNv proteins were thus quantified densitometrically in the same boxed areas using an ImageJ software (NIH, Bethesda, MA, USA) against the standard curves. Thereafter, the amount of loaded plasmids or MrNv proteins were converted into mole number based on the molecular weight of a single plasmid DNA molecule (1151 kDa) and a molecule of MrNv protein (41.5 kDa). Based on the assumption that a quaternary assembled MrNv VLP may have a triangulation number of T = 1 (which will provide the smallest inner cavity for encapsulating plasmid DNA), 1 VLP could thus theoretically contain 60 copies of capsid proteins with a molecular mass of 2490 kDa. The proportional mole ratio between the loaded DNA per assembled MrNv VLP unit in the given loading condition was then formulated and expressed as moles of plasmid DNA either per single copy of MrNv protein or per entire VLP. 2.6. In vitro hydrolytic digestion of MrNv-VLPs We further tested the resistance of the recombinant MrNvVLPs against strong hydrolytic conditions, a property that would be critical for oral delivery protocols. In vitro digestion was carried out as previously described (Schneemann et al., 1993) with some modifications (Jariyapong et al., 2013). Five microgram of purified MrNv-VLPs were incubated with three types of purified digestive enzymes at 37 ◦ C: 30 mU pancreatic trypsin (50 mM Tris–Cl (pH7.4), 150 mM NaCl and 5 mM CaCl2 ), chymotrypsin (50 mM Tris–Cl (pH 8.0), 150 mM NaCl and 5 mM CaCl2 ) or bovine pepsin (10 mM citrate buffer pH 4.0) in a final volume of 15 ␮l. Control experiments was done with BSA under the same conditions. After 1 h incubation, SDS-PAGE loading dye was added to the mixture to stop the reactions. Integrity of the treated VLPs was then analyzed by comparative intensity and mobility of the MrNvCAP protein bands in silver-stained gels. 2.7. Cell culture and plasmid DNA delivery by MrNv VLPs To test whether MrNv-VLPs were able to deliver plasmid DNA containing EGFP into targeted cells, we selected a Spodoptera

frugiperda insect cell line (Sf9; Invitrogen, Camarillo, CA). The cells were cultured at 27 ◦ C in SF900 II serum-free medium (Gibco, Grand Island, NY) supplemented with 100 U/ml penicillin and 100 U/ml streptomycin. After reaching confluence, the cells were washed with growth medium and sub-cultured in a 6-well plate at a density of 10,000 cells/well and maintained in the medium for 1 day. Thereafter, 600 ng plasmid DNA (determined by A260 ) encapsulated into MrNv-VLP was added to the Sf9 cell layers followed by further incubation in the same medium for 24 and 48 h. Control cells were those incubated with unloaded VLPs or purified plasmids. Internalization of the plasmid loaded VLP into live Sf9 cells was measured by expression of EGFP assessed by examination using an Olympus FV1000 confocal microscope with an argon laser line and a 520 nm emission filter in Kalman’s line-by-line scanning mode. Cultured cells treated with plasmid DNA or MrNv alone served as negative controls. 3. Results 3.1. Expression of MrNv capsid protein in E. coli Using the pET16b-MrNv-Cap6His recombinant plasmid (having an insert for the complete sequence of MrNvCAP containing 6 histidine residues) at the C-terminus for time course expression in E. coli, it was found that recombinant MrNvCAP was expressed at the highest level 3 h post-IPTG induction and remained constant in 4 and 5 h post-induction (Fig. 1A). This expression time was considerably shorter than that previously reported by Goh et al. (2011). By sucrose gradient purification, a single banded protein of 41.5 kDa was obtained (Fig. 1A, arrowhead) and the protein yield was 1.5 mg/L culture medium. This protein was verified as MrNvCAP protein by Western blot using a monoclonal anti-MrNv antibody that revealed a single immunoreactive band at 41.5 kDa (Fig. 1B). 3.2. Self-assembly, disassembly and reassembly of MrNv VLPs Investigation of self-assembly of the recombinant MrNvCAP in physiological buffer by TEM negative staining revealed that most of the MrNvCAP formed mulberry-like particles that were homogenously spherical (∼27 nm in diameter) with a dark central cavity (Fig. 2a). Thus, the recombinant MrNvCAP proteins were capable of self-assembly into VLPs without interference from the six histidine residues tagged onto the C-terminus. As a prerequisite for an encapsulation experiment, a successful disassembly/reassembly method had to be established. For this, two chelating agents (EDTA and EGTA) were tested in the presence of the reducing agent DTT. Surprisingly, the VLP structure was not disrupted by treatment with EDTA + DTT. Instead, the VLPs became 1.8 times larger (∼50 nm) than the native VLPs (∼27 nm) (Fig. 2b). On the other hand, VLPs incubated with EGTA + DTT were completely disrupted and complete VLPs could not be observed by TEM (Fig. 2c). Since EGTA has more affinity for Ca2+ than EDTA (Brinley et al., 1975), we concluded that assembly of MrNv VLPs was highly Ca2+ -dependent and this was confirmed by the use of CaCl2 for VLP reassembly into particles that closely resembled the originally self-assembled VLPs (Fig. 2d). This demonstrated that disassembly and reassembly of MrNv-VLPs could be controlled in vitro without altering capsid morphology. 3.3. Resistance of MrNv VLPs to in vitro hydrolytic digestion Testing the stability of MrNv VLPs under strong hydrolytic conditions to measure their suitability for oral delivery systems revealed that treatment with 30 mU of trypsin, chymotrypsin or pepsin for 1 h changed the band intensity of the 41.5 kDa MrNv

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Fig. 1. Expression and purification of recombinant MrNv capsid protein. The full length of MrNv capsid protein encoding amino acids M1 –N371 and hexahistidine was expressed in E. coli (Rosetta) at several time points. (A) SDS-PAGE of crude whole cell lysate from IPTG-induced bacteria at 0–5 h, flow though (FT) and purified protein by nickel affinity chromatography (fraction B) followed by discontinuous sucrose gradient centrifugation (sucrose), yielding a single band at 41.5 kDa. (B) Western blotting of 0 and 3 h harvested proteins probed with a monoclonal anti-MrNv antibody.

Fig. 2. Electron micrographs of negatively stained MrNv VLPs obtained under different conditions. Panel a: purified VLPs after sucrose gradient centrifugation, Panel b: VLPs treated with EDTA + DTT, Panel c: VLPs treated with EGTA + DTT. Panel d: VLP reassembly in the presence of 5 mM CaCl2 . Bars = 50 nm.

VLP but not its band mobility (Fig. 3). As control, BSA was also treated with proteolytic enzymes and a ladder of small MW protein bands was shown for trypsin digestion. The same result was obtained from the cleavage of BSA with chymotrypsin and pepsin (data not shown). Among the three enzymes tested, chymotrypsin

had the strongest effect on VLPs’ stability with approximately 60% reduction in VLP band intensity (Fig. 3). By surprising contrast, trypsin and pepsin had very little effect on the VLPs in that band intensity was only slightly reduced (95% of the cells in the form of a punctate staining pattern throughout their cytoplasm (Fig. 5e and f). No fluorescent signals were observed in cells incubated with unloaded VLPs or

Although MrNv has been discovered for a considerable period of time (Arcier et al., 1999; Qian et al., 2003), detailed studies on its viral capsid protein were not reported until recently (Goh et al., 2011). In contrast, more extensive data has been available of other nodaviruses in fish and insects (Fisher and Johnson, 1993; Tang et al., 2001, 2002; Wery et al., 1994). The lack of MrNv information is partly due to the lack of continuous crustacean cell lines that can be used for the virus replication and propagation (Sudhakaran et al., 2007). Studied on malabaricus grouper nervous necrosis virus and flock house virus capsid proteins have been carried out by expression in Sf21 insect cells, and they have been shown to spontaneously form icosahedral VLPs with T = 3 quasi-symmetry (Lin et al., 2001; Schneemann et al., 1993). As revealed by negative TEM staining, MrNv recombinant capsid protein expressed in E. coli also engages in self-assembly to form VLPs that exhibit an icosahedral architecture similar to that of the native virion (Goh et al., 2011). Nevertheless, the high resolution structure of the virions or VLPs (e.g., viral capsid angulation and significant functional domains) remains to be elucidated. In this study, we expressed MrNv capsid proteins in a more timeefficient manner than previously reported (Goh et al., 2011) and showed that they retained many inherent properties of the native nodavirus proteins. Two key properties were spontaneous selfassembly into MrNv VLPs similar in morphology to ∼27 nm native MrNv viral particles (Bonami and Sri Widada, 2011), presumedly with the basic amino acids grouped at the N-terminus of the MrNv peptide folded inward to bind with the negatively charged nucleic acid, as suggested for other noda viruses (Lin et al., 2001; Renoux et al., 2008; Tang et al., 2001). We believe that the latter property was a key for our successful encapsulation of the plasmid DNA into the VLP central cavity. The requirement for metal ions and disulfide bridging for VLP stability is variable among non-enveloped viruses. While mouse polyoma virus (Qian et al., 2003), simian virus 40 (Montenarh and Henning, 1983) and hepatitis-E virus (Takamura et al., 2004) require both disulfide bonds and metal ions, human papillomavirus (Sijun and Yong, 2009) needs only disulfide bridging for capsid stability. By contrast, capsid assembly of shrimp infectious hypodermal and hematopoietic necrosis virus (Hou et al., 2009) is independent of both disulfide bonding and metal ions. Here, we provided evidence that MrNv VLP may require both for their assembly and integrity in that EGTA and DTT were required for VLP disassembly and that CaCl2 was required for VLP reassembly (Fig. 2c). Furthermore, the use of EDTA and DTT could not completely disrupted the VLP structure (Fig. 2b), but instead caused expansion in its diameter, firmly indicating the importance of Ca2+ for VLP integrity. Another valuable physical property of MrNv the VLPs was their stability under harsh hydrolytic conditions (Fig. 3). This physical property has long been known for human hepatitis-E virus (HEV) that can tolerate digestive juices and still interact with the host epithelium to initiate infections (Li et al., 2001; Jariyapong et al., 2013), although the mechanism of tolerance is still unknown. In this regard, we further analyzed the linear capsid sequence of MrNv using an Expasy Peptide Cutter software and found that potential sites for hydrolysis by trypsin, chymotrypsin and pepsin exist abundantly throughout the capsid protein sequence. Thus, it is likely that the quaternary structure of the MrNv capsid protein protects these potential cleavage sites from enzyme accessibility. Further work on the atomic structure of the protein is needed to prove this hypothesis.

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Fig. 5. Confocal fluorescence micrographs of Sf9 cells incubated with MrNv VLPs loaded with GFP tagged plasmids. At 0 h post-VLP treatment, no fluorescent signal was observed within Sf9 cells (panel a). Note a moderate fluorescent signal in some cells after treating with plasmid loaded VLPs for 24 h (panel c) and becomes more intense signals in most cells at 48 h (panel). Right panels are the superimposed fluorescent micrographs and their corresponding DIC micrographs. Bars = 125 ␮m.

With many advantageous physical properties including controllable disassembly/reassembly and hydrolytic resistance, MrNv VLPs exert potential for application in encapsulating therapeutic agents such as double stranded RNA, antibodies and gene therapy vectors. In fact, many attempts have already been made to load DNA vectors into non-enveloped viruses with a variable degree of success (Combita et al., 2001; Krauzewicz et al., 2000; Takamura et al., 2004). Moving on from MrNv packaging and delivery of plasmid DNA carrying GFP, experiments are on-going to test whether this system can be used to trace MrNv VLPs in vivo in whole shrimp and to deliver curative substances such as dsRNA. It is also possible that capsid modifications either by genetic engineering to insert active peptide on the VLP surface domain (Uchida et al., 2007) or by chemical conjugation of active ligands such as aptamers (Tong et al., 2009; Stephanopoulos et al., 2010) may pave the way toward delivering MrNv VLPs into specific target cells for curative purpose in mammalian system. Acknowledgements This research was supported by Office of Higher Education Commission, Career Development Grant from Faculty of Science, Mahidol University and National Research Universities Initiative (to Mahidol University). We would also like to thank Prof. Dr.

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Encapsulation and delivery of plasmid DNA by virus-like nanoparticles engineered from Macrobrachium rosenbergii nodavirus.

Virus-like particles (VLPs) are potential candidates in developing biological containers for packaging therapeutic or biologically active agents. Here...
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