The Packaging of Different Cargo into Enveloped Viral Nanoparticles

Aug 9, 2012 - †Department of Biology, ‡Department of Chemistry, and §Indiana University Proteomics Facility, Indiana University, Bloomington, Ind...
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The Packaging of Different Cargo into Enveloped Viral Nanoparticles Fan Cheng,† Irina B. Tsvetkova,‡ Y-Lan Khuong,† Alan W. Moore,† Randy J. Arnold,§ Nancy L. Goicochea,‡,∥ Bogdan Dragnea,*,‡,⊥ and Suchetana Mukhopadhyay*,† †

Department of Biology, ‡Department of Chemistry, and §Indiana University Proteomics Facility, Indiana University, Bloomington, Indiana 47405, United States ⊥ Horia Hulubei Foundation, Magurele 077125, Romania S Supporting Information *

ABSTRACT: Viral nanoparticles used for biomedical applications must be able to discriminate between tumor or virus-infected host cells and healthy host cells. In addition, viral nanoparticles must have the flexibility to incorporate a wide range of cargo, from inorganic metals to mRNAs to small molecules. Alphaviruses are a family of enveloped viruses for which some species are intrinsically capable of systemic tumor targeting. Alphavirus virus-like particles, or viral nanoparticles, can be generated from in vitro self-assembled core-like particles using nonviral nucleic acid. In this work, we expand on the types of cargo that can be incorporated into alphavirus core-like particles and the molecular requirements for packaging this cargo. We demonstrate that different core-like particle templates can be further enveloped to form viral nanoparticles that are capable of cell entry. We propose that alphaviruses can be selectively modified to create viral nanoparticles for biomedical applications and basic research. KEYWORDS: alphavirus, core-like particle, viral nanoparticle, in vitro assembly, cargo

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INTRODUCTION There is a growing need to develop transport vesicles, including viral nanoparticles (VNPs), to deliver organic molecules and contrasting imaging agents to appropriate targets.1 VNPs are defined here as viral particles that carry nongenomic cargo and can bind and enter a new host cell to deliver the cargo. VNPs are frequently derived from plant viruses or bacteriophages. The advantage of using these viruses as VNPs stems from their ability to self-assemble in vitro and package various types of cargo.2 To apply VNPs in the clinical setting, a balance between the ability to package diverse material (or “cargo”) within the particle and the ability to target VNPs to specific cells is critical. Although some plant viruses, like Cowpea mosaic virus, can be internalized into animal tissues and be used to visualize tumor vasculature, target areas of inflammation, and monitor angiogenesis over time in mice,3−6 the majority of plant-derived VNPs are nonenveloped and require surface modifications to allow for targeted delivery and reduced immune response in vivo.5−8 The second group of VNPs used for therapeutics is composed of the oncolytic animal viruses.9−11 The advantage of these viruses is their ability to discriminate human tumor cells from healthy cells, and several genetically modified viruses of this group are already in clinical trials.12,13 Disadvantages of using oncolytic viruses for drug delivery or imaging include difficulty loading chemical or inorganic cargo into these particles and the potential for introducing a modified virus genome into the host.7 Thus, there is a need to develop VNP © 2012 American Chemical Society

platforms that combine the benefits of the diverse cargoencapsidation found in plant viruses with the cellular selectivity found in oncolytic animal viruses. We designed a novel viral platform that uses the alphavirus system and incorporates the advantages of both of the above VNP systems. Alphaviruses are small, icosahedral, enveloped viruses that contain a positive-strand RNA genome that is surrounded by the capsid protein (CP) to form the nucleocapsid core. A host-derived lipid membrane embedded with viral glycoprotein spikes is external to the core.14,15 We see four major advantages to using alphaviruses as VNP vectors. First, Sindbis virus, an alphavirus, is capable of systemic tumor targeting without further surface modification. The Sindbisderived viral vectors circulate in the bloodstream and induce efficient tumor suppression and eradication, as demonstrated in tissue culture and mouse models.16−20 Thus, little or no receptor modification is necessary to achieve cell-specificity. Second, alphavirus CPs can self-assemble into nucleocapsid cores in vitro, which allows them to incorporate nonviral genomic cargo such as small pieces of single-stranded DNA, Special Issue: Viral Nanoparticles in Drug Delivery and Imaging Received: Revised: Accepted: Published: 51

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RNA, or gold nanoparticles.21−24 Self-assembled particles are named core-like particles (CLPs) because viral genomes are not incorporated within the particle, thus eliminating the concern for viral mutations leading to disease and viral gene integration into the host. Third, previous research has demonstrated that CLPs introduced into cells expressing viral glycoproteins acquire an envelope. These particles, called VNPs, resemble the proteinaceous composition and structure of intact viruses but are devoid of a viral genome. Furthermore, VNPs can target new cells, undergo fusion, disassemble, and release their cargo within the cytoplasm of the recipient cell.21,25 Fourth, structural studies of alphavirus particles14,24,26−30 and atomic structures of viral spike proteins31,32 allow for a structure-based engineering platform to modify surface spikes for additional targeting specificity or incorporation of surface probes. Alphavirus CLP assembly is hypothesized to be driven through electrostatic interactions between the CP and the cargo, similar to other systems.2 The alphavirus CP consists of two domains. The N-terminal domain is composed of residues 1−100. This region is rich in proline and glycine and contains ∼30 basic residues, suggesting the N-terminus interacts with viral RNA.33−36 Atomic structures of the CP alone have the Nterminus disordered,37,38 and in the cryo electron microscopy structure of the virus, the N-terminus is positioned inward, making contacts with the viral RNA.14,37,39 The C-terminal domain (residues 101−270) is a chymotrypsin-like domain that comprises a majority of the ordered nucleocapsid core seen in the alphavirus structure.14,24 There are minimal CP−CP contacts in the C-terminal domain14,40 suggesting electrostatic interactions between the basic charged N-terminal domain of CP and the acidic residues of the viral RNA drive nucleocapsid core formation. Previous work has shown the CPs from the alphaviruses Sindbis virus, Western Equine Encephalitis virus, and Ross River virus (RRV) self-assemble into CLPs in the presence of single-stranded nucleic acid.23,24 Additional work has shown that RRV CLPs will form with size-selected gold nanoparticles that are surface modified with polyethylene glycol and DNA oligomers.22 In this work, we assembled RRV CLPs using a range of cargo types and with labeled CP. We determined that electrostatic interactions between the cargo and CP are necessary for CLP assembly and found that there is an optimum ratio of negative charge from the cargo and the positive charge from the CP at which CLP assembly occurs. Finally, using our established approach for enveloping CLPs with viral enveloped proteins,21 we demonstrated that VNPs can be formed using different CLP templates. Thus, we engineered a viral nanoparticle with the potential to maintain the cellular targeting specificity of an oncolytic virus while carrying nongenomic cargo.

CA) or LiCl/ethanol precipitation, and were analyzed by gel electrophoresis to verify size and determine concentration. Alexa Fluor 488 (AF488)-labeled and unlabeled 48mer DNA oligomer (5′-CCGTTAATGCATGTCGAGATATAAAGCATAAGGGACATGCATTAACGG-3′) were purchased from IDT DNA (Integrated DNA Technologies, Coralville, IA). Heparin and fluorescein isothiocyanate (FITC)-labeled heparin were purchased from Sigma-Aldrich (St. Louis, MO) and Polysciences, Inc. (Warrington, PA), respectively. In Vitro Assembly of CLPs. RRV CP was cloned into a pET29b vector (Novagen, EMD Chemical Inc., Gibbstown, NJ) and expressed in Rosetta 2 cells (Novagen, EMD Chemical Inc.). RRV CP purification and CLP assembly was performed as described previously.23,24 Briefly, equal volumes of 1.2 mg/ mL cargo and 2.0 mg/mL RRV CP in HNE (20 mM HEPES, 150 mM NaCl, and 0.1 mM EDTA) were mixed and incubated for 10 min at room temperature. CLP formation was confirmed by agarose gel shift assay, sucrose density centrifugation, and transmission electron microscopy (TEM).23,24 CLPs remained intact, as determined by TEM, for up to 30 days when stored at either 4 °C or −20 °C. TEM. CLP samples (5 μL) were applied to 400-mesh carbon-coated Formvar copper grids and stained with 1% uranyl acetate. The grids were examined on a JEOL 1010 TEM (Tokyo, Japan) at 80 kV. Images were recorded using a Gatan UltraScan 4000 charge-coupled device (CCD) camera (Pleasanton, CA). The CLP TEM diameters were determined using at least two grids, prepared independently, with over 300 particles imaged per grid. Quantification of Cargo in the Assembled CLP. To determine the amount of cargo encapsidated in RRV CLPs, AF488-labeled 48mer DNA, 32P-labeled mRNA, and fluorescent heparin were used. Large-scale CLPs were made by mixing 100 μL of 1.2 mg/mL 32P-labeled firefly luciferase mRNA, 32Plabeled Sindbis-replicon-GFP mRNA,21 or fluorescent heparin with 100 μL of 2.0 mg/mL RRV CP in HNE. CLPs were then pelleted through a 20% sucrose cushion. Pellets were resuspended in 200 μL of HNE buffer. Protein concentrations were determined by the Bradford assay. Heparin concentration was determined by measuring the fluorescence using a BIOTEK synergy 2 multimode microplate reader (BIO-TEK, Winooski, VT). RNA concentrations were determined by measuring the radioactivity of 32P in a Packard 1600 TR liquid scintillation analyzer (Canberra Packard, Zellik, Belgium). Differential Scanning Fluorimetry (DSF). A real-time polymerase chain reaction (PCR) device, Mx3005p (Stratagene, LA Jolla, CA), was used to monitor the stability of CLPs by the increase in the fluorescence of the SYPRO Orange dye (Invitrogen, Carlsbad, CA).43−45 CLPs were prepared at room temperature and diluted in HNE buffer to provide a final protein concentration of 0.2 mg/mL or 0.4 mg/mL. Twenty microliter CLP samples were loaded into 96-well PCR microplates (Eppendorf, Hauppauge, NY) in the real-time PCR device. For all the experiments, the final concentration of SYPRO Orange dye was 10× (the absolute concentration is not disclosed by the manufacturer). MicroAmp optical adherent film was used to cover the microplate in the real-time PCR device. CLP samples were heated from 25 to 95 °C, in increments of 0.3 °C per 30 s. The fluorescence intensity was measured every 30 s. The negative first derivatives of fluorescence intensities (−RT where RT is the first derivative) were plotted as a function of temperature by using the

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EXPERIMENTAL SECTION CLP Cargo Reagents. Capped and polyadenylated luciferase mRNA, Sindbis replicon-Green fluorescent protein (GFP), and RRV mRNA were generated by in vitro transcription using Sp6 polymerase and standard reaction components.24,41,42 32P-labeled RNA transcripts were synthesized in the presence of 32P-CTP in reactions. Fluorophorelabeled RNA was generated by in vitro transcription using Alexa Fluor 546 (AF546)-14-uridine-5′-triphosphate (UTP) (Invitrogen Life Technologies, Grand Island, NY), at a labeled:unlabeled UTP ratio of 1:100. All RNA transcripts were purified using either an RNeasy Mini Kit (Qiagen, Valencia, 52

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Figure 1. Alphavirus CLPs containing nonviral genome cargo. CLPs made with (a) heparin, (b) AF488-labeled 48mer DNA oligo, (c) FITC-labeled heparin, and (d) 20 nm PEG-coated Au-nanoparticles. Fluorescence images of (e) AF488-labeled 48mer DNA oligo CLPs and (f) FITC-labeled heparin on coverslips showing individual particles and incorporation of fluorescent cargo. Scale bar for a−d.

internally developed software package. We performed at least 3 measurements on four biological samples. Fluorophore Modification of CP. Purified RRV CP was mixed with a slight excess of tris(2-carboxyethyl)phosphine in phosphate buffered saline (PBS) at 1.1:1 molar ratio and incubated at room temperature for 30−60 min. Labeling was performed by adding 2 mg/mL AF488 maleimide (Invitrogen, Grand Island, NY) in a 12:1 AF488:protein molar ratio. The mixture was covered in foil and left overnight at 4 °C in the dark. Excess dye was removed and the sample was washed three times with PBS using a 0.5 mL 10K cutoff Amicon Ultra-2 Ultracel 10 membrane (Amicon-Millipore, Billerica, MA). Concentrations of labeled protein and fluorescent dye were measured using a Nanodrop 1000 (Thermo Scientific, Wilmington, DE). Protein concentration was measured by absorbance at 280 nm with an extinction coefficient of 39,420 M−1 cm−1, and background was corrected for any signal due to fluorophore showing absorbance at 280 nm. The amount of fluorophore in samples was determined by absorbance at 495 nm using an extinction coefficient of 71,000 M−1 cm−1. VNP Formation. The assembly of alphavirus VNPs using CLPs has been described previously.21 A 35 mm dish containing 2 × 106 baby hamster kidney (BHK) cells was transfected with pRRV-GP, a plasmid that expresses the RRV glycoproteins. Fifteen hours later, 2.5 μg of purified CLPs (approximately 2.2 × 1011 particles) was transfected into the pRRV-GP-transfected cells using 10 μL of Lipofectamine 2000. After 1 h, cells were washed three times with PBS to remove residual CLPs and Lipofectamine reagent and 0.5 mL of minimum essential medium + 10% fetal bovine serum was added to the cells. After an additional 1 h, the medium containing VNPs was collected and concentrated by ultracentrifugation through a 27% sucrose cushion for TEM analyses. Purified VNPs remained intact, as determined by TEM, up to 1 week when stored at 4 °C. Confocal Fluorescence Microscopy. Imaging of fluorescently labeled CLPs and VNPs was done with a Revolution XD fluorescence microscopy system (Andor Technology, Inc., South Windsor, CT) equipped with an inverted Nikon Ti

microscope and a Yokogawa confocal scanning unit with Nipkow disk. Samples were excited through a high-numerical aperture 60× oil-immersed objective (CFI APO TIRF, NA 1.49, Nikon, Melville, NY) with a 488 nm laser (25 mW) and 561 nm laser (50 mW). The resulting fluorescence was collected back through the objective, passed through a quadwavelength dichroic mirror and an emission filter to eliminate residual laser light, and recorded by a EM CCD camera iXon DU-897-BV (Andor Technology, Inc.). Images were processed and analyzed using Andor iQ and ImageJ (National Institutes of Health, Bethesda, MD) software. Cloning and Expression of Sindbis Virus Glycoproteins Containing the Venus Fluorescent Protein. The Venus fluorescent protein46 was cloned into a pCAGGS vector expressing the glycoprotein spike proteins (E3-E2-6K-E1) from Sindbis virus strain TE12 to produce SINV GP-Venus. Venus fluorescent protein is a derivative of green fluorescent protein. The Venus gene (with linkers at both the 5′- and 3′-ends) was inserted between the E3 and E2 genes of Sindbis. The 5′-linker was GGCGCGCCAGGATCAGCA, encoding the amino acid sequence Gly-Ala-Pro-Gly-Ser-Ala, and the 3′-linker was GCCGGCCCAGGAAGCGGA, encoding the amino acid sequence Ala-Gly-Pro-Gly-Ser-Gly. Transfection of the SINV GP-Venus plasmid was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Eight microliters of Lipofectamine 2000 was used to transfect approximately 2.3 × 106 BHK cells (35 mm dish) with 1 μg of plasmid DNA, per the manufacturer’s instructions. Sixteen hours post-transfection, cells were imaged using an Olympus1×71 fluorescence microscope (Olympus, Center Valley, PA).

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RESULTS CLP Formation Using Labeled Cargo and Labeled CP. Alphavirus CLP assembly is driven by electrostatic interactions between the negatively charged RNA and the basic N-terminus of the CP. In this work, we extend our study of alphavirus CLP assembly by testing a variety of negatively charged cargo, including non-nucleic acid materials and fluorescent-labeled cargo, and fluorescent-labeled capsid protein. 53

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We could assemble CLPs containing heparin, AF488-labeled 48mer DNA, in vitro transcribed RNA of different lengths that incorporated AF546-14-UTP, and FITC-labeled heparin (Figure 1, Table 1). Electron micrographs showed that the Table 1. Size of CLPs and VNPs Made with Different Cargo av diam (nm) ± SD (n)a cargo

CLP

cytoplasmic cores RNAb AF546-RNAc 48mer DNA oligonucleotide AF488-48mer DNA oligonucleotide heparin FITC-heparin Au-nanoparticles (20 nm diameter) AF488-CP glutamic acid poly-L-lysine

41 ± 2 (100) 40 ± 2 (100) 39 ± 3 (18) 41 ± 3 (100) 41 ± 2 (40) 40 ± 2 (100) 40 ± 2 (42) 39 ± 3 (100) 42 ± 3 (19) no CLPs no CLPs

VNP 68 ± ND 62 ± 69 ± 79 ± ND 72 ± ND 73 ± ND ND

4 (20)

Figure 2. Alphavirus CLPs made from labeled CP. Left, TEM image of CLPs made from AF488 labeled CP. Right, fluorescence image of the CLPs on a coverslip.

3 (5) 5 (19) 12 (13)

well as DNA and RNA, and the number of negative charges per cargo molecule varies over a ∼200-fold range. We found (Table 2) that the number of cargo molecules encapsidated in a

6 (23) 5 (20)

Table 2. Ratio of Cargo to CP in CLPsa

a

Diameter given in nanometers. SD, standard deviation; ND, not determined. n represents number of particles counted. bRNA is in vitro transcribed RNA, either 2000, 7800, or 11700 bases in length. cSee text for method of incorporation and specific fluorophore being used.

cargo cytoplasmic cores, Viral RNA ssDNA ssRNA

CLPs were of approximately the same size (39−41 nm) and spherical shape compared to wild-type cores and other in vitro assembled CLPs (Figure 1, Table 1). No CLP formation was detected with glutamic acid or poly-L-lysine under any condition tested. When CLPs containing AF488-labeled 48mer DNA and FITC-labeled heparin were viewed under the confocal fluorescence microscope, bright, equal intensity, diffraction-limited spots representing the CLPs were clearly seen (Figure 1). We next determined if CLPs could be formed using fluorescent-labeled RRV CP by targeting the four cysteine residues in the C-terminus of RRV CP (amino acid residues 115, 122, 137, and 173) to label with AF488. We hypothesized that minor modifications at the C-terminus of CP would not interfere with in vitro CLP assembly because there are minimal contacts between the C-terminal domains of CP molecules. Purified CP was incubated with AF488 maleimide under reducing conditions. After excess dye was removed from the CP, we determined from absorbance measurements that there were on average two AF488 fluorophore molecules per molecule of CP. Mass spectroscopy analysis showed that the CP was modified with AF488, but identification of the location and efficiency of the cysteine residues modified was inconclusive (Supporting Information). AF488-CP was incubated with unlabeled 48mer DNA oligomers to form CLPs. TEM images showed that labeled CPs assembled in vitro to form CLP and the assembled particles showed no difference in structure and size compared to wild-type cores (Figure 2, Table 1). Furthermore, with confocal microscopy, single CLP particles were clearly visualized (Figure 2). Requirement of Charge Neutralization for CLP Assembly. Structural studies show that CLPs containing nucleic acid cargo are similar in size, morphology, and symmetry to authentic nucleocapsid cores.24 To test the possibility that assembly entails a constant ratio of negative to positive charges, we quantitated the amounts of different anionic cargo incorporated into CLPs. Cargo molecules included heparin and DNA-derivatized Au-nanoparticle as

20 nm Aunanoparticle heparin

length of nucleotide

no. of cargo molecules per CLP

net negative charge from cargo per CLP

negative charge per capsid protein

11,703

1

11,703

49

48 2,384 8,781 ND

∼200 3−4 1 1

9,600 7,200−9,500 8,781 10,000

40 30−40 37 42

NA

150−180

10,800

45

a

We assumed there are 240 CP per CLP. Each CP has 30 basic amino acids in the N-terminus of the CP. The cargo is predicted to interact with these basic residues. These values represent the average of two experiments. ND, not determined; NA, not applicable.

CLP was inversely related to the number of negative charges on a cargo molecule. Assembled CLPs always contained 7,200− 11,700 negative charges; or between 30 and 50 charges per CP molecule. Thus the total quantity of negative changes packaged in a CLP is roughly conserved, despite the diversity of cargo tested here. Stability of CLPs. From the results shown in Table 2, we observed no difference between the CLPs containing different cargo. In a complementary approach, we performed DSF on CLPs containing different cargo. In DSF, when CLPs begin to denature or the CP unfolds as a result of increasing temperature, SYPRO Orange dye can bind to the newly exposed hydrophobic regions of CP and produce a fluorescent signal. The fluorescence intensity as a function of temperature is monitored. To avoid potential interference with the SYPRO Orange signal, we did not use fluorophore-labeled cargo or CPs for these studies. Like cytoplasmic cores isolated from virus-infected cells47 and in previous studies using CLPs with 48mer DNA,24 CLPs with pure single-stranded DNA or RNA showed dissociation temperatures of approximately 52 °C (Figure 3). In contrast, CLPs containing heparin and Au-nanoparticles showed a slightly higher dissociation temperature, approximately 54 °C (Figure 3). The thermal denaturation of the CLPs containing Au-nanoparticles is broader than that of the other CLP samples, possible explanations including heterogeneity in the particles or 54

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Figure 3. Disassembly of CLPs containing different cargo at two different temperatures. Nucleic acid containing CLPs disassembles at slightly lower temperatures compared to CLPs containing heparin and Au-nanoparticles. When the salt concentration is increased from 150 mM to 300 mM, CLPs disassemble at a lower temperature consistent with electrostatic interactions between CP and cargo driving assembly. Data is plotted as the negative slope of the thermal denaturation curve as a function of temperature.

containing RNA,21 could enter a new host cell. As proof of principle for nongenomic cargo VNPs, we infected BHK cells with VNPs that contained Au-nanoparticles; the Au-nanoparticles were tagged with fluorescein, while cell membranes were stained with FM4-64. Figure 5 shows four images of the same field of view, taken 2.5 s apart. It is evident that the cargo (green particle, white arrow) is within the cell and is moving during the imaging. Its velocity is consistent with previous studies on alphavirus-infected cells.48 Expression of Fluorescent Protein Fused to the Glycoprotein Spikes. To maximize the utility of the alphavirus VNP system, we expressed a fluorescent protein as a fusion to the spike proteins, expressed from the DNA vector, which allowed tracking of VNP spike components both in vitro and in vivo. We introduced the gene for the fluorescent protein Venus between the E3 and E2 genes of Sindbis to express E3Venus-E2-6K-E1. Cells transfected with the vector can be seen expressing Venus fluorescent protein (Figure 6). After translation and processing of the viral glycoprotein spikes, Venus remains covalently bound to the E2 protein, as determined by Western blot (data not shown). Spike proteins can be tracked individually or in conjunction with labeled cargo and/or CP.

an increased multiplicity in disassembly or denaturation pathways. To further confirm that CLP assembly is a result of nonspecific CP−cargo electrostatic interactions, we tested the stability of CLPs at a higher salt concentration (300 mM NaCl). The results showed a lower dissociation temperature in buffer containing 300 mM NaCl compared to the same CLPs made in 150 mM NaCl (Figure 3). We observed a shift in the dissociation temperature from 52 to 49 °C for DNA and RNAcontaining CLPs and a shift from 54 to 51 °C for heparincontaining CLPs. These results support our hypothesis that nonspecific electrostatic interactions are the predominant driving force in alphavirus capsid assembly. VNP Formation from Various CLPs. Alphaviruses require their glycoprotein surface spikes for cell attachment, entry, and subsequent delivery of cargo. Thus, for CLPs to become VNPs for biomedical applications, it is necessary to have a strategy of coating CLPs with viral glycoproteins. We used a previously described21 method to make VNPs from CLPs encapsidating different cargo and from CLPs with fluorescent-labeled CP (Figure 4). The VNPs obtained with this method were

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DISCUSSION

The results from this work have made several new contributions to the field of VNPs. First, although the formation of alphavirus VNPs has been demonstrated in the past,21,25 we extended the range of cargo that can be encapsidated within alphavirus CLPs and subsequent VNPs. We were able to incorporate small fluorescent-labeled oligonucleotides, large pieces of RNA and mRNAs potentially coding for small peptides, small molecules like heparin, and Aunanoparticles (Figure 1, Table 1). The rationale for using nonnucleic acid materials was to determine if small molecules used for therapeutics could be encapsidated. The rationale for using labeled cargo and CP was to allow monitoring and tracking of VNPs in vivo to determine their targeting specificity, lifetime, and circulation within a host. Our results support the hypothesis that electrostatic interactions are driving alphavirus CLP formation. Using different sized cargo (Table 2), the number of cargo molecules incorporated into CLPs varied with different cargo. The smaller

Figure 4. VNPs can be formed from different CLP templates. VNPs made from CLPs containing (a) FITC-labeled heparin, (b) AF488labeled 48mer DNA, and (c) AF488-labeled CP. Scale bar is 50 nm.

spherical particles with an average diameter of 70 nm (Table 1). Western blot analysis showed the ratio of CP to viral glycoprotein was similar among the different VNP samples and wild-type particles (data not shown), suggesting the CLPs are completely enveloped by the viral glycoprotein spikes and lipid membrane. Entry of Au-VNPs into Cells. We demonstrated above that we can form VNPs containing cargo other than nucleic acid. It was of interest to determine where these VNPs, like those 55

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Figure 5. VNP containing Au-nanoparticle enters BHK cells. The four panels represent 2.5 s time intervals; movement of the VNP was monitored from 0 to 7.5 s. The Au-nanoparticle, labeled with FITC, is shown in green, and the host cell is stained with FM4-64 and is outlined in red. The Aunanoparticle was used to make RRV CP CLPs and covered with viral glycoproteins to form VNPs. Over time, the particle (indicated with the white triangle) is seen moving within a cross-section of a BHK cell.

assembly. Recent work using mutated Brome mosaic virus CP determined that, although there might be an optimal ratio of cargo to CP charge interactions, other factors like CP−CP interactions may influence the size and amount of cargo packaged in a virus particle.54 Our results with alphavirus CLPs support the hypothesis that both electrostatic interactions between cargo and CP and additional CP−CP interactions direct CLP self-assembly. If electrostatic interactions were the only parameter directing CLP self-assembly, varying the amount of cargo or the overall charge would yield CLPs of various diameters. Because this does not occur (Table 1), we postulated there must be additional CP−CP interactions dictating the conserved 40 nm size restriction for particle self-assembly. Though individual CP−CP interactions may be weak, the presence of 120 concerted CP−CP interactions in a CLP (240 copies of CP per particle) likely increases their contribution to particle stability. The utility of the alphavirus system as a VNP platform is evident when CLPs containing different cargo could be enveloped into VNPs (Figure 4, Table 1), and the potential for encapsidating labeled CP (Figures 2 and 4) and viral glycoproteins (Figure 6) to track particles. Furthermore, the VNPs can enter a recipient host cell (Figure 5) without further modification to the virus particle. The encapsidation of heparin and Au-nanoparticles into VNPs suggests that antiviral or antitumor therapies could also be packaged into VNPs, although a helper molecule may be needed to provide the anionic charge required to form the initial CLP. Inorganic cargos that would be useful candidates for this system include ultrasmall superparamagnetic iron oxide nanoparticles, which have been used to image lymph nodes containing micrometastases in patients with prostate cancer,55 and Aunanoparticles, which can act as highly sensitive contrast agents for single-proton emission computed tomography imaging.56 Other studies combined magnetic resonance imaging with biological targeting57 using gadolinium or iron oxide based nanoparticles with a fluorescent dye. These multifunctional “nanoclinic” nanoparticles consist of a thin silica shell encapsulating magnetic iron oxide and fluorescent dyes for enhanced magnetic resonance and optical imaging.57 As described before, the limiting step in the production of VNPs is the efficiency in introducing the CLPs into viral glycoprotein-expressing cells.21 Several methods have been used by our group21 and others,25 yet overall yields remained low. As a means of characterizing the efficiency of the VNP production process, we transfected isolated cytoplasmic nucleocapsid cores and measured the activity of the VNPs produced. We transfected 1 × 108 cytoplasmic nucleocapsid cores, as determined by Bradford assay, to measure total protein concentration. At the end of the VNP assembly procedure we obtained 1 × 103 infectious VNPs. Using 100 as the ratio of total particles to infectious particles,58 we calculated

Figure 6. Expression of SINV spike-Venus fusion proteins in BHK cells. BHK cells were transfected with a DNA vector expressing E3Venus-E2-6K-E1 for Venus-fused viral glycoprotein spikes. Left, brightfield image, and right, fluorescence image. Images were taken at 16 h posttransfection, at 4× magnification.

the cargo molecule, the more molecules of cargo were packaged into the CLP. However, the total negative charge from the cargo per CLP was consistently between 8,000 and 11,000 and the negative charge per CP (240 copies of CP per CLP) ranged from 30 to 49. If we assume that only the 30 basic residues at the N-terminus of each CP are interacting with cargo, we obtain a ratio of 1.1−1.6 negative charges from the cargo for each basic charge from the CP, which is close to the 1.6 ratio between the genomic charge and the net charge on the capsid peptide arms for many other viruses conjectured by Belyi and Muthukumar.49 Our DSF results (Figure 3) showed CLP stability is increased with lower ionic strength buffer, consistent with the electrostatic interactions between cargo and CP driving assembly. Together, these results demonstrate that there is a window of cargo to CP electrostatic interactions for CLP formation. In previous literature, a linear correlation between ssRNA viral genome length and the number of charged residues on the CP has been observed with a ratio estimated to be between 1.61 and 2.49,50 Examples include Flock house virus, which has a charge ratio of 1.4, and Brome mosaic virus, which ranges from 1.56 to 1.79 depending on the RNA fragment being encapsidated. Consistent with the electrostatic interaction driving assembly, previous work also showed that deletions of the basic residues in the CP results in reduced packaging of viral RNA.51,52 Although L-Glu carries a negative charge at neutral pH, it did not serve as substrate for successful CLP assembly. In previous studies using 12mer and 6mer oligonucleotides, CLPs were also not formed. We hypothesized that these smaller oligomers do not meet the requirement of minimum substrate length for the formation of CP dimer.23,53 Together, these results are consistent with a size requirement for the polyanion interacting with the basic residues of the CP. The positive charge of poly-Llysine does not favor interaction with CP, even though its elongated structure is long enough for potential CP dimer formation. While there might be an optimal ratio of cargo to CP charge interactions, additional factors may contribute to CLP 56

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that 1 × 105 total VNPs were released. Although this quantity is measurable and allows for subsequent assays, it is only a 0.1% yield from starting material. Use of VNPs in diagnostic imaging is well established in terms of using nonenveloped viruses like plant viruses and bacteriophages. However, a majority of these VNPs must be chemically or genetically modified to allow the VNPs to achieve specificity during cell targeting. The targeting specificity of alphaviruses has been demonstrated previously,16−19 and we determined the parameters needed to incorporate different cargo into our alphavirus VNP system. Thus, the potential use of alphavirus VNPs for diagnostic, therapeutic, and other biomedical applications in medicine is very promising.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1 and Table S1 showing mass spectrometry data identifying that RRV CP has been labeled with AF488. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*S.M.: Department of Biology, Indiana University, 212 S. Hawthorne Drive, Bloomington, IN 47405; phone, 812-8563686; fax, 812-856-5710; e-mail, [email protected]. B.D.: Department of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington, IN 47405; phone, 812-856-3686; fax, 812-855-8300; e-mail, [email protected]. Present Address ∥

University of South Florida Morsani College of Medicine, Nanomedicine Research Center and Division of Translational Medicine, Department of Internal Medicine and Department of Molecular Medicine, Tampa, FL 33612. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank members of the Indiana University Virology group for stimulating discussions on this research and Alan Rein for constructive comments on the manuscript. B.D. acknowledges support from the National Institutes of Health (Grant GM081029) and the National Science Foundation (Grant CHE 1014947).

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ABBREVIATIONS USED VNP, viral nanoparticle; CLP, core-like particle; CP, capsid protein REFERENCES

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