Molecular and structural characterization of fluorescent human parvovirus B19 virus-like particles

BBRC Biochemical and Biophysical Research Communications 331 (2005) 527–535 www.elsevier.com/locate/ybbrc

Molecular and structural characterization of fluorescent human parvovirus B19 virus-like particles Leona Gilbert, Jouni Toivola, Daniel White, Teemu Ihalainen, Wesley Smith, Laura Lindholm, Matti Vuento, Christian Oker-Blom * Department of Biological and Environmental Science, P.O. Box 35, FIN-40014 University of Jyva¨skyla¨, Finland Received 15 March 2005 Available online 8 April 2005

Abstract Although sharing a T = 1 icosahedral symmetry with other members of the Parvoviridae family, it has been suggested that the fivefold channel of the human parvovirus B19 VP2 capsids is closed at its outside end. To investigate the possibility of placing a relatively large protein moiety at this site of B19, fluorescent virus-like particles (fVLPs) of B19 were developed. The enhanced green fluorescent protein (EGFP) was inserted at the N-terminus of the structural protein VP2 and assembly of fVLPs from this fusion protein was obtained. Electron microscopy revealed that these fluorescent protein complexes were very similar in size when compared to wild-type B19 virus. Further, fluorescence correlation spectroscopy showed that an average of nine EGFP domains were associated with these virus-like structures. Atomic force microscopy and immunoprecipitation studies showed that EGFP was displayed on the surface of these fVLPs. Confocal imaging indicated that these chimeric complexes were targeted to late endosomes when expressed in insect cells. The fVLPs were able to efficiently enter cancer cells and traffic to the nucleus via the microtubulus network. Finally, immunoglobulins present in human parvovirus B19 acute and past-immunity serum samples were able to detect antigenic epitopes present in these fVLPs. In summary, we have developed fluorescent virus-like nanoparticles displaying a large heterologous entity that should be of help to elucidate the mechanisms of infection and pathogenesis of human parvovirus B19. In addition, these B19 nanoparticles serve as a model in the development of targetable vehicles designed for delivery of biomolecules.
2005 Elsevier Inc. All rights reserved. Keywords: Human parvovirus B19; Virus-like particles; Green fluorescent protein; Atomic force microscopy; Fluorescence correlation microscopy; Baculovirus; Intracellular trafficking

Human parvovirus B19, a member of the Parvoviridae family, is a small non-enveloped virus containing a single-stranded DNA genome. B19 infects erythroid precursors of bone marrow [1,2] that leads to a wide range of diseases including erythema infectiosum [3], anemias, aplastic or hypoplastic crisis, prolonged bone marrow failure [4], and fetal hydrops and/or fetal death [5,6]. The biphasic clinical course of a B19 infection begins with intranasal viral inoculation accompanied by malaise, itching, myalgia, and pyrexia-like symptoms.

*

Corresponding author. Fax: +358 14 260 2221. E-mail address: christian.oker-blom@jyu.fi (C. Oker-Blom).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.208

Approximately 18 days after infection, the second phase of symptoms consists of rashes, itching or arthralgia. Production of IgM antibodies starts at 12 days post-infection (p.i.) and persists for at least 3 months p.i. [7]. Specific IgG antibodies appear several days after IgM and persist for life protecting against subsequent secondary infections. The structure of recombinant B19 virus-like particles ˚ resolution [8]. The has been determined to be of
3.5-A B19 particle is a non-enveloped, icosahedral virion with a diameter of 18–26 nm [9,10]. The DNA genome of 5600 bases appears in both polarities in equal amounts and codes for three proteins; the non-structural protein (NS1) associated with DNA replication and apoptosis

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[11], the larger capsid protein (VP1) associated with cellular receptor interaction, and the more plentiful (96% [12]) structural protein (VP2) associated with antigen recognition by IgM. This abundant capsid protein has been shown to assemble into virus-like particles (VLPs) without requiring partnership with VP1 [9,13]. Diagnosis of a B19 infection is mainly based on the detection of antibodies raised against the two viral structural proteins, VP1 and VP2, and it has been noted that IgM and IgG have different reactivity against structural and linear B19 epitopes [14–18]. Commercial ELISA kits, as well as, Western blotting assays using recombinant VP1/VP2 and VP2 antigens are available for detection of a B19 infection. Recently fluorescence correlation spectroscopy (FCS) was used to distinguish between antibodies present in acute-phase or past-immunity serum samples [19]. As an alternative to timeconsuming testing and expensive equipment, development of accurate and user-friendly tests for serodiagnostic tests is highly desirable. Chimeric partners of the B19 VPs have been of great interest recently. The unique region of VP1 has been replaced with hen egg white lysozyme and VLP assembly occurred with the co-infection of a helper-virus coding VP2 [20]. Structural proteins of B19 have been constructed, which are truncated at their N-terminal ends or engineered to contain insertions within the native protein backbone to define essential domains needed for self-assembly [21]. In addition, green fluorescent protein (GFP) has been used as a fusion partner for identification of a potential nuclear localization signal (NLS) in B19 [22]. For improvement of a B19 recombinant vaccine, others have created an empty capsid enriched in VP1 [23]. Recently, the enhanced green fluorescent protein (EGFP) was placed at the N-terminus of the canine parvovirus (CPV) VP2 protein without affecting capsid assembly [24]. Here, we have placed EGFP on the surface of B19 VLPs and thoroughly analyzed the characteristics of the corresponding fluorescent virus-like particles (fVLPs) by a variety of techniques including fluorescence correlation spectroscopy (FCS) and atomic force microscopy (AFM). We show that they represent a novel imaging tool for studying the biological properties of B19, as well as, open the doors of using human parvovirus B19 VLPs as a therapeutic agent, and as a reagent for the development of fast and simple fluorescence-based diagnostic assays. Materials and methods Plasmid constructs. The sequence encoding VP2 of B19 was amplified by PCR using the template plasmid p2BAC [25] containing the genes for both capsid proteins VP1 and VP2 as a template. The sense oligonucleotide primer for VP2 was 5 0 -CC ATG AAG CTT CT ATG ACT TCA GTT AAT TC-3 0 (HindIII site and start codon

underlined). The antisense oligonucleotide primer for VP2 was 5 0 -AC GAC GAA TTC TTA CAA TGG GTG CAC ACG-3 0 (EcoRI site and stop codon underlined). The PCR products of VP2 were cloned into plasmid pGEM-T Easy (Promega, Madison, WI). The VP2 coding sequence was isolated from pGEM-T Easy as a HindIII/EcoRI fragment and cloned into the corresponding restriction sites of pEGFP-C1 (Clontech, Palo Alto, CA). The resulting plasmid was named pEGFPVP2-C1. The EGFP-VP2 construct was placed under control of the polyhedrin promoter by the following scheme. The sequence encoding EGFP-VP2 was amplified by PCR using the plasmid pEGFP-VP2-C1 as a template. The sense oligonucleotide primer was 5 0 -G TCC GAA GCG CGC ATG GTG AGC AAG GGC-3 0 (NotI and the start codon underlined). The antisense oligonucleotide primer was 5 0 -C GGC ACA CGT GGG TAA CCG CCG GCG GA-3 0 (PauI site and stop codon underlined). The PCR products were digested with NotI and PauI, and cloned into the corresponding restrictions sites of pFastBacI (Gibco BRL, Grand Island, NY). The resulting plasmid was named pEGFPVP2FastBac and used for generating the recombinant baculovirus. Propagation of recombinant baculoviruses. Spodoptera frugiperda (Sf9, Gibco BRL, Grand Island, NY) insect cells were maintained at +28 C as monolayer cultures in 25 cm2 T25 plastic flasks (Greiner GmbH, Frickenhausen, Germany) in serum free HyQ SFX insect cell culture medium (HyClone, Logan, UT). The recombinant baculovirus, AcEGFP-VP2, was generated using the Bac-to-Bac system (Gibco BRL). In addition to this virus construct, recombinant baculoviruses coding for VP2 alone, the VP1/VP2 genes, and EGFP were used [24– 26]. All viruses were propagated and amplified according to established procedures [27]. Production and purification of VLPs. Amounts of 8 · 107 Sf9 cells in 40 ml culture medium were infected with the recombinant virus AcEGFP-VP2 at a multiplicity of infection (MOI) of 10 plaque forming units (pfus)/ml. Cells were incubated at 28 C for 48 h and samples of 500 ll were taken for SDS–PAGE, immunoblotting, and immunofluorescence microscopy. The purification procedures for the EGFP-VP2, VP2, V1/VP2 VLPs, and soluble EGFP were carried out as previously described [24]. For atomic force microscopy experiments, EGFP-VP2 VLPs were purified as above with the exception of using a 45% CsCl2 gradient as described [28]. SDS–PAGE and immunoblotting. Molecular weight markers (BioRad, Richmond, IL) and samples were boiled in Laemmli sample buffer for 3 min and the proteins were separated on 10% polyacrylamide slab gels, transferred to nitrocellulose membranes, and analyzed by immunoblotting. Proteins were detected with monoclonal mouse anti-VP antibodies (anti-VP) [29,30] or polyclonal rabbit anti-GFP antibodies (anti-GFP, Promega). Detection of the primary antibodies was performed using AP-conjugated goat anti-mouse/rabbit IgG (Promega), and signal was developed with NBT and BCIP (Sigma). Immunofluorescence microscopy. At 48 h p.i., infected insect cells were pelleted by low speed centrifugation (800g, 1 min, RT), and after a series of three washes with PBS (pH 7.4), fixed with 4% paraformaldehyde (PFA–PBS, 20 min, RT). Pelleted cells (10,000g, 1 min, RT) were then permeabilized (1% BSA, 0.1% Triton X-100, and 0.01% sodium azide in PBS) for 20 min at RT. After concentrating the cells (10,000g, 1 min, RT), pellets were incubated with primary antibodies diluted (1/1000) in permeabilization buffer for 45 min at RT. The antiVP was used to identify the recombinant parvoviral proteins. Cells were then rinsed with permeabilization buffer (20 min, RT) and visualized with fluorescently labeled Alexa-633 (violet)-conjugated antimouse secondary antibody (anti-mouse Alexa-633, Molecular Probes, Eugene, OR). After immunofluorescent labeling, cells were washed in permeabilization buffer and immunolabeled with the next set of antibodies. A rabbit polyclonal antibody directed against the cation independent mannose-6-phosphate receptor (Ci-MPR) was used to detect late endosomes [31] at a dilution of 1/50. Alexa-546 (red)-conjugated anti-mouse secondary antibody (anti-mouse Alexa-546, Molecular Probes) was then used to visualize Ci-MPR. Cells were

L. Gilbert et al. / Biochemical and Biophysical Research Communications 331 (2005) 527–535 washed with 50 ll PBS, pelleted (10,000g, 1 min, RT), and finally embedded with 2–7 ll of MOWIOL-DABCO (30 mg/ml; Sigma). Samples were examined by confocal fluorescence microscopy (Carl Zeiss Laser Scanning Microscope, Axiovert 100M, LSM510, Jena, Germany). Electron microscopy. Specimens for electron microscopy were prepared from the samples obtained by sucrose gradient purification. Aliquots of 6 ll of the resuspended samples were applied to metal grids and left to stand for 1–2 min at RT. Excess liquid was blotted away using Whatman 3 MM paper and 6 ll of negative stain (2% potassium phosphotungstate, pH 6) was added to the grids. Excess stain was removed and the grids were left to dry at RT. Samples were examined at 60 kV with a JEOL JEM-1200 EX transmission electron microscope (Jeol, Tokyo, Japan). Fluorescence correlation spectroscopy (FCS). Measurements were carried out using the ConfoCor 2 fluorescence correlation microscope (Carl Zeiss, Jena, Germany). The excitation wavelength of EGFP was 488 nm and the emission photons were collected using a 530–600 nm bandpass filter. The pinhole was adjusted by using Rhodamine 6G dye (Molecular Probes, Eugene, OR) at a count rate of 200 kHz. LabTek II 8-well chambered borosilicate glass plates (Nalge Nunc International, Naperville, IL, USA) were used as carriers and the measuring time was 20-s (40 repeats). The diffusion time and the normalized autocorrelation G (t) were calculated using software provided by the manufacturer (Carl Zeiss). The diffusion coefficients and hydrodynamic radii for the particles were calculated from the measured diffusion times using 10 autocorrelation measurements. The calculations have been described in more detail previously [32,33]. For calculation of the number of EGFP moieties present in the EGFP-VP2 VLPs, samples were diluted (1:200) followed by treatment with 3 mM SDS. Immunoprecipitation of fluorescent fVLPs. To investigate if EGFP moieties were displayed on the outside of EGFP-VP2 VLPs, purified fVLPs were incubated 4 h at 4 C with polyclonal rabbit anti-GFP antibodies (anti-GFP, Promega) and then immunoprecipitated as described below. VP2 VLPs were used as a control. In addition, Sf9 cells infected with AcEGFP-VP2, AcVP2, and AcEGFP were treated with Triton X-100 and clarified as described above. Clarified lysates were then incubated with B19 acute-phase, past-immunity, and negative serum samples [25] for 4 h at 4 C. Protein A–Sepharose beads (Amersham Biosciences, Uppsala, Sweden) were then added and the mixture was incubated for 1 h at 4 C. After a series of washes with TENT buffer, immunocomplexes were pelleted and the fluorescence was monitored in microtiter plates (Victor I, LKG, Turku, Finland). The fluorescence of EGFP was excited at 488 nm and detected at 543 nm. Feeding and immunostaining of Hep G2 cells. Approximately 1 · 106 Hep G2 cells/ml were maintained in 10 ml DMEM (Gibco BRL, Grand Island, NY) supplemented with 10% FCS (DMEM–FCS) and grown overnight on coverslips (diameter 13 mm) at 37 C. Next day, the growth medium was replaced with a mixture of 40 ll DMEM–FCS and 40 ll (1.31 mg/ml) of purified EGFP-VP2 VLPs or soluble EGFP. After binding for 1 h on ice, cells were washed with PBS and 5 ml DMEM was added to the coverslips. Cells were incubated at 37 C for 4 or 6 h, fixed with 4% paraformaldehyde in PBS (PFA/PBS) for 15 min at RT, and left in 1% PFA/PBS at 4 C until prepared for confocal microscopy. Fed cells were rinsed twice with PBS (pH 7.4) prior to fixation in 50 ll of 4% PFA–PBS (20 min, RT) and permeabilized (1% BSA, 0.1% Triton X-100, and 0.01% sodium azide in PBS) for 20 min at RT. The permeabilized cells were exposed to anti-VP2 and visualized by anti-mouse Alexa-633 (described above). Alternatively cells were exposed to mouse monoclonal anti-a-tubulin antibodies (anti-a-tubulin, Amersham Biosciences, Uppsala, Sweden) followed by visualization with anti-mouse Alexa-546 (see above). Cells were incubated with antibodies for 1 h at RT, washed twice with 50 ll PBS, and embedded as previously described. Atomic force microscopy. Amounts of 10–20 ll cesium chloride gradient purified EGFP-VP2 fVLPs (1.31 lg/ml in PBS) were incubated on freshly cleaved mica (SPI Supplies, West Chester, PA) in a

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moist chamber for 40 min at RT. Samples were carefully dried using Whatman 3 MM paper continued by washing with PBS. Washing was concluded by placing 40 ll PBS on the mica followed by drying with Whatman 3 MM paper. To complete drying, samples were incubated in a desiccator for 1–3 h. Prior to atomic force microscopy (AFM) imaging samples were quickly washed using distilled water and dried with a He2 stream. Dried samples were imaged with a Dimension 3100 Atomic Force Microscope (Veeco Instruments, Santa Barbara, CA) operating in tapping mode. RTESP tips (Veeco Instruments) were used with a resonant frequency of approximately 300 kHz, and a scanning speed of 1–5 Hz. NANOSCOPE 6.11 was utilized to perform height analyses of all AFM data collected to confirm that the images obtained corresponded to EGFP-VP2 VLPs and were used to calculate the diameter of the fVLPs from the measured AFM data. The size of the fVLPs was calculated using the surface area from the imaged VLPs. The surface area was estimated from half of the oblate and its bottom area that was attached to the mica. The oblateÔs equatorial radius was the radius of the imaged VLP and the polar radius the height of the VLP. The final size of the fVLP was calculated based on the surface area of the oblate as follows [34]: S ¼ 3pa2 þ p

c2 1 þ e ; ln 1e e

ð1Þ

where a and c are the radius and the height of the imaged VLP, respectively. In the equation, e is the ellipticity of the imaged VLP as defined below: rffiffiffiffiffiffiffiffiffiffiffiffiffi c2 e  1  2: ð2Þ a Molecular modeling. A molecular model of the EGFP-VP2 capsid structure was constructed from PDB file 1S58. A VP2 15mer was assembled corresponding to a face of the capsid around one fivefold axis. In one of the five VP2 monomers around each of the fivefold axes the EGFP domains were modeled against PDB file 1S6Z, with the linker region arbitrarily modeled as an a-helix extending from the internal N-terminus of VP2, out through the pore in the fivefold axis cannon structure, similar to the CPV structure PDB entry 4DPV, and connected to the EGFP domain. Models were built and molecular graphics were rendered using BODIL and PYMOL.

Results Assembly of EGFP-VP2 into fluorescent VLPs The main goal of this study was to elucidate whether a molecular fusion of EGFP and B19 VP2 (Fig. 1A) would be able to fold correctly, assemble into fluorescent viruslike particles (fVLPs), and be able to externally display the fluorescent fusion partner on the fVLPs. The identity and molecular characteristics of the corresponding VP2 fusion construct were compared to those of non-fused VP2 (Fig. 1A), VP1/VP2 (Fig. 1B), and soluble EGFP (Fig. 1D). Sf9 cells were infected with recombinant baculoviruses harboring the corresponding genetic constructs, and the resulting protein products were studied by immunoblotting (Figs. 2A and B) and electron microscopy (Figs. 2C–E). Monoclonal antibodies raised against B19 (anti-VP) were able to identify proteins with apparent molecular weights of 58 kDa (Fig. 2A, lanes 1 and 2), 83 kDa (Fig. 2A, lane 2), and 84 kDa (Fig. 2A, lane 3) corresponding to VP2, VP1, and EGFP-VP2,

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rus infected cells were lysed and the cytoplasmic components were separated by sucrose gradient centrifugation. Fluorescent bands containing fVLPs were negatively stained and analyzed by electron microscopy (Figs. 2C–E). As shown in Fig. 2E, the EGFP-VP2 fusion protein assembled into VLPs with diameters of approximately 22 nm. The fVLPs had a similar appearance compared to those assembled from VP2 (Fig. 2C) and VP1/VP2 (Fig. 2D), see below. B19 fVLPs accumulate in the late endosomal compartment of Sf9 cells

Fig. 1. Cartoon of the recombinant baculovirus constructs. (A) AcVP2, (B) AcVP1/VP2, (C) AcEGFP-VP2, and (D) AcEGFP.

respectively. Accordingly, the polyclonal anti-GFP antibody identified the EGFP-VP2 fusion protein, as well as, soluble EGFP with apparent molecular weights of 84 kDa (Fig. 2B, lane 3) and 26 kDa (Fig. 2B, lane 4), respectively. Some breakdown of the EGFP-VP2 fusion protein could, however, be observed when the antiGFP antibody was used (Fig. 2B, lane 3). To investigate whether the EGFP-VP2 fusion protein was able to assemble into fVLPs, recombinant baculovi-

The characteristics of fVLPs in cells were further studied by immunofluorescence microscopy (Fig. 3). Sf9 cells infected with the recombinant baculoviruses AcVP2 (Fig. 3A), AcVP1/VP2 (Fig. 3B), AcEGFP (Fig. 3C), or AcEGFP-VP2 (Figs. 3D–J) were collected at 48 h p.i. and then immunostained with appropriate primary (anti-VP and Ci-MPR) and secondary antibodies (anti-mouse Alexa-633 and anti-mouse Alexa-546). Confocal imaging of Sf9 cells infected with the recombinant baculoviruses AcVP2 (Fig. 3A) and AcVP1/VP2 (Fig. 3B) showed that the viral proteins were expressed at high levels. Soluble EGFP was also expressed at sufficient levels to be viewed directly (Fig. 3C). Mid-section confocal imaging of cells expressing EGFP-VP2 (Figs. 3D–F) shows that the VP2 proteins (Fig. 3D) were

Fig. 2. Characterization of the fluorescent human parvovirus B19 virus-like particles (fVLPs). Immunoblot analysis of the recombinant proteins produced from insect cells (A,B). Identification of the recombinant proteins from cell lysates was carried out using anti-VP2 or anti-GFP antibodies as shown. The recombinant protein products (arrows: VP2 = 58 kDa, VP1 = 83 kDa, EGFP-VP2 = 83.7 kDa, and EGFP = 26.0 kDa) and molecular weight markers (kDa) are indicated. Electron micrographs of negatively stained VLPs comparing VP2, VP1/VP2, and EGFP-VP2 (C–E, respectively).

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Fig. 3. Confocal imaging of Sf9 cells infected with the recombinant baculoviruses (A) AcVP2, (B) AcVP1/VP2, (C) AcEGFP, (D–F) AcEGFP-VP2, and (G–J) AcEGFP-VP2. Cells were fixed with paraformaldehyde 48 h post-infection (p.i.), immunostained where applicable, and viewed with a confocal laser scanning microscope. (A–F) Images are single confocal optical mid-sections of approximately 0.7 lm thickness. (A,B) Monoclonal mouse anti-VP (anti-VP) antibody and Alexa-633 (violet)-conjugated anti-mouse secondary antibody (anti-mouse Alexa-633) was used to visualize VP epitopes. (C) Direct viewing of EGFP. (D) VP epitopes were detected as above in EGFP-VP2 expressing cells. (E) EGFP fluorescence in EGFPVP2 expressing cells. (F) Merged image of (D,E). Cells expressing EGFP-VP2 (G–J) were first stained for VP epitopes as above. Further staining was carried out with a rabbit polyclonal antibody directed against the cation-independent mannose-6-phosphate receptor (Ci-MPR, I) and visualized by Alexa-546 (red)-conjugated secondary antibody (anti-mouse Alexa-546). (J) Merge. (G–J) are projection images of three mid-sections of 0.7 lm thickness.

3I) colocalized with the VP2 staining and EGFP (Fig. 3J), showing that these large vacuolar structures are a part of the late endosomal compartment.

expressed and that there was colocalization with its fusion partner, EGFP (Fig. 3E), seen in the merged image as white (Fig. 3F). Further, the expressed EGFP-VP2 proteins appeared in globular cellular structures (Figs. 3D–F). When investigating these structures, additional Sf9 cells were infected with AcEGFP-VP2 (Figs. 3G–J) for 48 h and immunostained with anti-VP antibodies that were visualized with anti-mouse Alexa-633, violet. Double labeling continued with Ci-MPR, a late endosome marker, which was visualized with anti-mouse Alexa-546, red. This projection image (Figs. 3G–J) of middle sections of the cells showed similar staining as in Figs. 3D–F. The VP2 proteins (Fig. 3G) and EGFP (Fig. 3H) again accumulated in these large vacuolar structures. The late endosomal marker staining (Fig.

Determination of the average number of fluorescent EGFP domains displayed on the fVLPs Changes in particle brightness, number of the fluorescent molecules in the observation volume, and diffusion time of the EGFP-VP2 VLPs were observed by FCS in the presence and absence of 3 mM SDS. When fVLPs were analyzed in the absence of SDS, high counts per molecule (CPM) were observed (Table 1). In contrast, when the VLPs were treated with 3 mM SDS (concentration close to the critical micelle concen-

Table 1 Size and the number of fVLPs in the observation volume (0.2 fl) analyzed by fluorescence correlation spectroscopy (FCS) and size by atomic force microscopy (AFM) SDS (mM) 0 3

CR (kHz) 1.2 ± 0.6 1.9 ± 0.7

CPM (kHz) 21 ± 21.1 5.1 ± 2.7

D (cm2 s1) 7

1.76 · 10 5.01 · 107

FCS Rh (nm)

N

AFM diameter (nm)

14 ± 2 5±0

1 9±5

28.4 ± 6.4 —

Count rate (CR), counts per molecule (CPM), diffusion coefficients (D), hydrodynamic radii (FCS Rh), the relative number (N) of fVLP before or after treatment with SDS, and AFM diameter.

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tration, 6–8 mM for SDS) [35], fluorescent units were released simultaneously raising the total count rate (CR) and decreasing the CPM (from 21 to 5 kHz) (Table 1). Further treatment with 5 mM SDS did not affect the particle number or the diffusion time (data not shown). In the presence of detergents, the hydrodynamic radius of the fluorescent units decreased threefold (14–5 nm) and the number of these units (N) increased more than 10-fold (Table 1). EGFP-VP2 VLPs have EGFP displayed on the outside of the VLP The fluorescence intensity of the immunoprecipitated complexes with anti-GFP antibodies was observed with a fluorescence monitor. Fluorescent readings of 10,429 and 100 units were recorded for EGFP-VP2 and VP2 VLPs, respectively. Immunoreactivity of human sera with EGFP-VP2 protein complexes The reactivity of anti-B19 parvovirus antibodies present in human serum samples (acute-phase, past-immunity, and negative) against human parvovirus B19 with the EGFP-VP2 complexes is shown in Fig. 4. Lysates of AcEGFP-VP2 (black), AcVP2 (gray), and AcEGFP (stripes) infected Sf9 cells were mixed with sera and immunocomplexes were precipitated using protein A– Sepharose beads. The acute-phase serum immunoprecipitated the EGFP-VP2 complexes. Similarly, the past-immunity serum precipitated the EGFP-VP2 complexes, but at a lower level. The negative sera showed

negligible reactivity with the fluorescent complexes. Lysates containing non-fluorescent VP2 or soluble EGFP were used for each type of serum which also showed very low background. In summary, there is a higher binding capacity of acute-phase sera antibodies to EGFP-VP2 complexes compared to past-immunity sera and negative serum samples. Together, these results also support that the VP2 fusion partner is folded correctly since the antigenic epitopes are recognized by the corresponding B19 positive serum samples. Entry and trafficking of B19 fVLPs in Hep G2 cells Feeding experiments of Hep G2 cells with purified fVLPs were carried out to study entry and trafficking in this B19 non-permissive cell line (Fig. 5). Four or six hours post-feeding, cells were immunostained with the anti-VP antibody that was visualized with antimouse Alexa-633 (violet) secondary antibody or fVLPs were visualized directly with EGFP. The fVLPs at 6 h post-feeding were clearly able to enter the Hep G2 cell line (Fig. 5A) as seen by colocalization of the VP2 protein with its EGFP fusion partner inside the cell. Hep G2 cells fed 4 h post-feeding with EGFP-VP2 VLPs were also stained with an anti-a-tubulin antibody (Fig. 5B), which was visualized using an anti-mouse Alexa-546 secondary antibody, red. Colocalization of the fVLPs with these intracellular structures is seen in the merged image, yellow. The fVLPs were able to bind and enter a non-permissive cell line and traffic in close connection with the microtubulus network. In contrast, Hep G2 cells fed with soluble EGFP (Fig. 5C) showed no binding of this protein and consequently no green fluorescence was observed inside the cells. Characterization of the B19 fVLPs by atomic force microscopy and molecular modeling

Fig. 4. Fluorescence measurement of immunoprecipitated particles with human sera against B19 infections. Cell lysates of AcEGFP-VP (black), AcVP2 (gray), and AcEGFP (stripes) infections were incubated with acute-phase, past-immunity, and negative human sera for B19 infection.

The molecular model of the EGFP-VP2 fVLP structure is schematically illustrated in Figs. 6B and C. It is in complete agreement with the immunoprecipitation data where anti-GFP antibodies were used in that the EGFP was located on the outside of the capsid (see above). Further, the capsid sizes measured by FCS and AFM (approximately 28 and 28.4, respectively; Table 1) were similar to the modeled capsid size, indicating that the fVLPs have a native 60mer icosahedral capsid structure. Several EGFP domains were seen by AFM (Fig. 6A) in positions analogous to their positions in the molecular model (Figs. 6B and C). It is clear from the FCS data (Table 1) that there is heterogeneity in the number of EGFP domains on each fVLP, such that not every fivefold axis might have an EGFP domain protruding from it, which is the case in the AFM image (Fig. 6A).

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Fig. 5. Confocal imaging of Hep G2 cells fed with purified EGFP-VP2 VLPs (A,B) and EGFP (C). At 4 or 6 h post-feeding cells were fixed with paraformaldehyde, immunostained, and viewed with a confocal laser-scanning microscope. (A) Cells were immunostained with anti-VP antibody (anti-VP2) and visualized by anti-mouse Alexa-633, violet at 6 h post-feeding. EGFP was viewed directly. Colocalization of the VLPs with its fusion partner EGFP is seen in the merged picture, yellow. Fed cells at 4 h post-feeding with EGFP-VP2 VLPs were viewed directly after staining with antia-tubulin antibody (B) and visualized by anti-mouse Alexa-546, red. Merged image of colocalization of the VLPs with the intracellular structures, yellow. (C) Negative control, EGFP fed cells were immunostained for a-tubulin as above. Note the absence of yellow in the merged image.

Fig. 6. Visualization of fluorescent B19 VLPs. (A) Characterization of fVLPs by atomic force microscopy (AFM). CsCl2 purified EGFP-VP2 VLPs were applied to freshly cleaved mica. (A) Phase contrast AFM image. Bar = 50 nm. (B) Molecular model of a 15mer unit from the fVLP made with BODIL and PYMOL. (C) Cartoon representation of the fVLP with the 15mer unit outlined in yellow.

Discussion The recombinant protein constructs depicted in Figs. 1A–D were expressed abundantly in baculovirus infected insect cells (Fig. 2). The recombinant B19 proteins VP1 and VP2 as well as soluble EGFP migrated with expected molecular weights, i.e., 83, 58, and 26 kDa, respectively [2,12]. In addition, the EGFP-VP2 fusion protein migrated with an apparent molecular weight of 84 kDa as predicted. Here, however, some breakdown

products could be observed when the protein was probed using antibodies directed against GFP [24]. Confocal imaging of Sf9 cells infected with AcEGFP-VP2 showed abundant staining when anti-VP was used (Fig. 3). Localization of the viral proteins in VP2, VP1/VP2, and EGFP-VP2 expressing Sf9 cells was similar as compared to that previously seen with recombinant canine parvoviral proteins [24,36]. Localization of overexpressed recombinant proteins in the late endosomes has been seen before [28,37,38] and may explain

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targeting to this cellular compartment also in the case of EGFP-VP2. Assembly of purified VLPs of VP2, VP1/VP2, and EGFP-VP2 was verified by electron microscopy and showed similar properties (Figs. 2C–D) as previously described for other parvoviruses [9,13,19,39–41]. To show that the EGFP moieties were incorporated into the B19 VLPs, an immunoprecipitation experiment was conducted with a polyclonal anti-GFP antibody and fVLPs purified by sucrose gradient purification. This experiment showed that these fVLPs could be pulled down with an antibody directed against GFP and thereby also suggest that the EGFP moiety is displayed on the surface of these VLPs. With this information, FCS was used to determine if the EGFP-VP2 complexes were indeed assembled into fVLPs. The size of the radius of these fVLPs (14 nm; Table 1) corresponded to the size (18–26 nm) of observed wild-type virus or B19 parvovirus-like particles [9,19,39]. In addition, the numbers of fluorescent units in the observed volume for EGFPVP2 was 1 compared to those of the denatured EGFPVP2 fusion proteins treated with SDS. It is obvious that more than one fluorescent fusion is incorporated into the fVLPs, as they were brighter compared to the count rate (CR) of soluble EGFP alone [data not shown, see 24]. The fVLPs treated with 3 mM SDS showed that single fluorescent molecules were released. A clear rise in particle number concluded an average of nine fluorescent chimeric proteins that were incorporated in one fVLP (Table 1). Atomic force microscopy (AFM) confirmed the findings obtained from the FCS (Fig. 6 and Table 1) in that the EGFP molecules are associated with the VP2 VLPs and that they are exposed on the surface of the corresponding particles. The AFM studies gave a diameter of about 37 nm for the EGFP-VP2 VLPs. The effect of the tip increasing the actual sample radius was approximated to 10 nm [42]. Taking this into account, and using the oblate of several particles, a diameter of 28.4 nm for EGFP-VP2 VLPs was obtained (Table 1). While the molecular model in Figs. 6B and C is largely schematic, the experimental data support the model in that the EGFP is outside the capsid, and is likely to be connected to the internal N-terminus of VP2 via the linker, which protrudes through the pore at the fivefold axis. To further study the molecular characteristics of these fVLPs, immunoprecipitation experiments were conducted using acute-phase, past-immunity, and negative human serum samples. The IgM antibodies showed an increased binding to the fVLPs as compared to IgG binding. This finding is in agreement with the fact that early antibody response consists of IgM directed against VP2 and that the IgG molecule would bind to VP2 as the primary target [14]. VP1 specific antigenic epitopes are not present in the fVLPs described here. These results are thus in support of the assumption that this fluo-

rescent recombinant virus-like particle is structurally intact. The ability of these fVLPs to enter cells and follow the traditional endocytotic route of parvoviruses [28,37] was tested (Fig. 5). CPV has recently been shown to bind microtubulus structures [24,38]. Human cancer cells (Hep G2) were therefore fed with purified fVLPs and studied by confocal microscopy. The EGFP-VP2 fusion construct was able to enter these cells and vesicular trafficking to the perinuclear region was accomplished as seen with fVLPs of CPV [24]. In addition, the EGFP fusion described here did not affect endocytosis of the fVLPs under these experimental conditions (Fig. 5). The actual entity that was translocated along this path warrants additional studies. Another matter is that the EGFP partner did not alter the critical surface conformation responsible for entry into the cell and that in a form of soluble EGFP, it was not able to enter the cell. Together, this analysis shows that physical size of such fluorescent B19 VLPs can be determined by FCS as well as by AFM, and that these entities display proper antigenic epitopes that could be utilized in fast diagnostics. In addition, these fVLPs could be used in experiments that characterize the early phases of a B19 infection and thus also be powerful in studying mechanisms related to, e.g., altered tropism and gene therapy.

Acknowledgments The Ci-MPR antibody was provided by Varpu Marjoma¨ki (University of Jyva¨skyla¨, Jyva¨skyla¨, Finland). Warm thanks are given to Prof. Markus Ahlskog and Prof. Klaus Hedman for their discussions. This study was funded in part by the Academy of Finland (Contract # 10216) and the K. Albin Johansson Foundation.

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