Murine pneumotropic virus VP1 virus-like particles (VLPs) bind to several cell types independent of sialic acid residues and do not serologically cross react with murine polyomavirus VP1 VLPs

K. Tegerstedt1, K. Andreasson1, A. Vlastos1, K. O. Hedlund2, T. Dalianis1 and T. Ramqvist1

1 Department of Oncology-Pathology, Karolinska Institute, Cancer Center Karolinska R8 : 01, Karolinska Hospital, SE-171 76 Stockholm, Sweden
2 Swedish Institute for Infectious Disease Control and Microbiology and Tumor Biology Center, Karolinska Institute, SE-171 82 Solna, Sweden

Correspondence
Torbjörn Ramqvist
torbjorn.ramqvist{at}cck.ki.se


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The ability of murine pneumotropic virus (MPtV) major capsid protein VP1 to form virus-like particles (VLPs) was examined. MPtV-VLPs obtained were used to estimate the potential of MPtV to attach to different cells and to assess some characteristics of the MPtV cell receptor. Furthermore, to evaluate if MPtV-VLPs could potentially complement murine polyomavirus (MPyV) VP1 VLPs (MPyV-VLPs) as vectors for prime–boost gene therapy, the capability of MPtV-VLPs to serologically cross react with MPyV-VLPs and to transduce DNA into cells was examined. MPtV VP1 obtained in a recombinant baculovirus system formed MPtV-VLPs readily. MPtV-VLPs were shown by FACS analysis to bind to different cells, independent of MHC class I antigen expression. In addition, MPtV-VLPs did not cause haemagglutination of red blood cells and MPtV-VLP binding to cells was neuraminidase resistant but mostly trypsin and papain sensitive, indicating that the MPtV receptor lacks sialic acid components. When tested by ELISA and in vivo neutralization assays, MPtV-VLPs did not serologically cross react with MPyV-VLPs, suggesting that MPtV-VLPs and MPyV-VLPs could potentially be interchanged as carriers of DNA in repeated gene therapy. Finally, MPtV-VLPs were shown to transduce foreign DNA in vitro and in vivo. In conclusion, the data suggest that MPtV-VLPs, and possibly also MPtV, bind to several different cell types, that binding is neuraminidase resistant and that MPtV-VLPs should potentially be able to complement MPyV-VLPs for prime–boost gene transfer in vivo.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Murine pneumotropic virus (MPtV), previously called Kilham polyomavirus, the second murine polyomavirus to be identified, was isolated in 1953 (Kilham & Murphy, 1953). While MPtV produces a fatal interstitial pneumonia in newborn mice, in older animals it induces a non-apparent persistent infection (Greenlee, 1981). During primary infection, MPtV replicates in the vascular endothelial cells of the lung, liver and spleen, whereas later during persistence the virus is found mainly in renal tubules (Greenlee et al., 1994).

The nucleotide and amino acid sequence of MPtV with an amino acid identity of 44 % to simian virus type 40 (SV40) and 36 % to mouse polyomavirus (MPyV) suggests that the viruses are related (Mayer & Dorries, 1991). Furthermore, similar to SV40, it has two non-structural proteins, large T antigen and small T antigen, and lacks the middle T antigen of MPyV, while similar to both SV40 and MPyV, it has three structural proteins, VP1 to VP3.

Based on studies in vivo, the cellular tropism of MPtV has been claimed to be limited to vascular endothelial cells and renal tubuli, whereas MPyV infects a wide range of cell types (Dawe et al., 1987). However, this conclusion must be taken with caution because MPtV has been less characterized than MPyV (Greenlee & Law, 1985; Kanda & Takemoto, 1984). In vitro studies on MPtV have been hampered, since the virus has been very difficult to replicate in vitro and, moreover, less interest has been focused on MPtV because it is non-tumourigenic (Greenlee & Law, 1985; Kanda & Takemoto, 1984). Nevertheless, it has been shown recently that the MPtV regulatory region influences cellular tropism, since a substitution of the MPtV regulatory region with a segment containing the MPyV transcriptional enhancer had a positive effect on viral DNA replication in cells that are normally non-permissive for MPtV (Zhang & Magnusson, 2001). Furthermore, MPtV DNA could replicate in NIH 3T3 cells when MPtV large T antigen was expressed from a co-transfected plasmid using a cytomegalovirus (CMV) promoter (Zhang & Magnusson, 2001). Moreover, in vivo-derived mutations in the MPtV enhancer could also increase DNA replication (Zhang & Magnusson, 2003).

Still, the virus receptor is particularly important for cellular tropism and is yet to be determined for MPtV. Most polyomavirus receptors, however, such as those of MPyV, BK polyomavirus, JC polyomavirus (JCPyV) and lymphotropic polyomavirus (LPyV), have a sialic acid component and are thus neuraminidase sensitive (Fried et al., 1981; Haun et al., 1993; Keppler et al., 1995; Liu et al., 1998; Sinibaldi et al., 1990). Furthermore, in contrast to all polyomaviruses described above, SV40 is the only known polyomavirus with a sialidase-resistant receptor (Clayson & Compans, 1989) and MHC class I molecules have been reported to be one receptor for SV40 (Atwood & Norkin, 1989).

Recently, the major capsid proteins (VP1) of some polyomaviruses, e.g. MPyV, JCPyV, SV40 and LPyV, have been produced in baculovirus or bacterial protein expression systems and have been shown to spontaneously form capsid-like complexes (Goldmann et al., 1999; Montross et al., 1991; Pawlita et al., 1996; Salunke et al., 1986; Sandalon & Oppenheim, 1997). These so-called virus-like particles (VLPs), devoid of viral DNA, are able to bind eukaryotic DNA, attach to cell surface receptors (Goldmann et al., 1999) and transduce DNA into cells both in vitro and in vivo (Forstova et al., 1995; Goldmann et al., 1999; Heidari et al., 2000). Hence, there is great interest to use such VLPs for gene therapy, particularly the murine counterparts, towards which pre-existing immunity is absent in humans. However, similar to other vectors, immune responses can occur after only one attempt of gene transfer in vivo (Heidari et al., 2000).

The present study was initiated to investigate the possibility of using MPtV-VLPs to study the potential of MPtV to bind to different cell types and to acquire some information regarding the receptor-binding qualities of MPtV. Furthermore, as a first step to evaluate if MPtV-VLPs and MPyV-VLPs could potentially be used for prime–boost gene therapy, the ability of MPtV-VLPs to serologically cross react with MPyV-VLPs and to transduce cells was examined. The capability of MPtV VP1, obtained in a recombinant baculovirus-infected insect (Spodoptera frugiperda) cell line (Sf9), to form MPtV-VLPs and to bind DNA was established first. Thereafter, the capacity of MPtV-VLPs to haemagglutinate red blood cells was tested as an indicator of sialic acid content in the MPtV receptor. Fluorescent MPtV-VLPs were then used to study binding to different mouse, green monkey and human cells and to study the sensitivity of binding to treatment with neuraminidase, trypsin and papain. To assess the feasibility to interchange MPtV-VLPs and MPyV-VLPs as carriers of DNA in gene therapy, an ELISA, a haemagglutination inhibition (HAI) test and an in vivo neutralization assay were used to examine if the two types of VLPs were serologically cross-reactive. Finally, we analysed if MPtV-VLPs could transduce foreign DNA into cells in vitro and into mice in vivo.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of baculoviruses expressing the MPtV and MPyV VP1 genes.
The MPtV VP1 gene was excised from the recombinant plasmid pKV19, carrying MPtV DNA in the XbaI site of pUC12 (Mayer & Dorries, 1991), by digestion with BglII (MPtV nt 3748) and XbaI (nt 2490), excising a 1258 bp fragment. This fragment was then inserted into the XbaI/BglII sites of the baculovirus transfer vector pVL1392 (Pharmingen). The MPyV VP1 gene was cloned into the baculovirus transfer vector pVL1393, according to Forstova et al. (1993). Recombinant baculoviruses were acquired by co-transfection with BaculoGold-linearized Autographa californica nucleopolyhedrovirus baculovirus DNA in Sf9 cells, according to the manufacturer's instructions (Pharmingen).

Expression and purification of MPtV-VLPs and MPyV-VLPs.
Both MPtV and MPyV VP1 genes were expressed by recombinant baculoviruses in Sf9 cells and VLPs were purified by an equilibrium-density CsCl gradient (Clark et al., 2001). CsCl fractions of 0·4 ml were collected from the top and investigated for density by refractometry. All fractions were analysed for the presence and relative quantity of VP1, using SDS-PAGE on 10 % polyacrylamide gels and by an anti-VLP ELISA (Vlastos et al., 2003). The presence of Sf9 DNA was analysed on a 0·7 % agarose gel with ethidium bromide staining. Fractions containing empty capsids, defined by the absence of DNA, were pooled and dialysed against PBS supplemented with 0·1 mM CaCl2. Total protein from the VP1 preparations was quantified by spectrophotometry at 280 nm.

DNA gel retardation assays.
For evaluation of DNA–VLP binding, 1 µg MPtV-VLPs or MPyV-VLPs (or as negative control, 1 µg BSA) were incubated with 100 ng linearized pVAXlacZ DNA in PBS supplemented with 0·1 mM CaCl2 in a total volume of 10 µl for 45 min at room temperature. DNA–VLP complexes (or DNA incubated with BSA) were then run on a 0·7 % agarose gel and thereafter stained with ethidium bromide.

Electron microscopy of MPtV-VLPs.
MPtV-VLPs were diluted 10 times in cell culture medium containing 0·1 % FCS to facilitate staining of capsids. A droplet of the preparation was incubated for 1 min on a Formvar carbon-coated grid and subsequently stained by 2 % tungstophosphoric acid (pH 6). The preparations were studied under an electron microscope (Philips CM 100) at a magnification of 46 000 or via a CCD camera (Gatan Wide Angle TV System) at a magnification of 920 000. Video prints were recorded at a final magnification of 300 000.

Haemagglutination (HA) and HAI assays
HA assay.
MPtV-VLPs and MPyV-VLPs were tested for their ability to haemagglutinate guinea pig or sheep erythrocytes by incubating 0·1 ml of serially diluted VLPs (5·0–0·002 µg) with 0·1 ml of a 0·4 % guinea pig or sheep erythrocyte suspension at 4 °C for 4 h.

HAI assay.
Sera (0·1 ml) diluted 1 : 10 to 1 : 10 240 from mice immunized with MPyV-VLPs or MPtV-VLPs were incubated at room temperature with 0·1 ml MPyV-VLP (concentration corresponding to 24 HAU) before incubation with guinea pig or sheep erythrocytes (as described above).

Generation of VP1-specific sera.
C57BL/6 mice were immunized subcutaneously with 2 µg VLPs in Freund's adjuvant once a week for 4 consecutive weeks and bled 10 days after the last challenge.

ELISA.
Microtitre plates (Maxisorp, Life Technologies) were coated with 500 ng of either MPtV-VLPs or MPyV-VLPs per well and the ELISA was performed with sera obtained from MPtV-VLP- or MPyV-VLP-immunized mice, according to Vlastos et al. (2003).

In vivo neutralization assay.
ACA mice were immunized subcutaneously, weekly, three consecutive times, with 5 µg MPtV-VLPs or MPyV-VLPs and then challenged together with non-immunized controls with 50 HAU of polyoma A2 virus. Mice were sacrificed 2 weeks later and the presence of polyomavirus DNA from different organs was assessed by PCR (Vlastos et al., 2003).

Fluorescent labelling of VLPs and BSA.
Purified VLPs were labelled with Alexa Fluor 488 succinimidyl ester (AF), according to the manufacturer's protocol (Molecular Probes). Labelled VLPs were separated from free dye on a Sephadex G-25 column. The ratio of AF to VP1 (degree of labelling) was estimated by measuring absorbance at 495 and 280 nm. As a negative control for unspecific binding, BSA was labelled as above.

Cells and cell lines
Murine cells.
MAE is an endothelial cell line (Bastaki et al., 1997) and B16 is a melanoma cell line (Fidler, 1974). RMA and its TAP-2-mutated derivative RMA-S, which does not express MHC class I, are T cell lymphoma lines (Ljunggren & Karre, 1985; Ljunggren et al., 1990).

Human cells.
SK-N-AS is a neuroblastoma cell line (Sugimoto et al., 1984), RCC is a renal cell carcinoma, RD is a rhabdomyosarcoma, DFW is a melanoma, HeLa is a cervical adenocarcinoma (Gey et al., 1952) and 293 is an embryonic kidney cell line. HEF are embryo fibroblast cells. Daudi is a B lymphoblastoid cell line devoid of surface MHC class I molecules because of a {beta}2-microglobulin gene mutation (Rosa et al., 1983).

Green monkey cells.
GMK and COS-1 are kidney cell lines, the latter containing the early region of SV40.

Monolayers of cells were maintained in Iscove's modified Dulbecco's medium (Gibco) and suspension cultures were maintained in RPMI 1640 (Gibco) supplemented with 10 % FCS and penicillin and streptomycin. Human dendritic cells (DCs) were prepared from PBMCs and were maintained in X-vivo medium (BioWhittaker) supplemented with 50 ng human granulocyte macrophage-colony stimulating factor (GM-CSF) ml-1 and 40 ng human interleukin 4 (IL-4) ml-1 and assayed after 5–7 days of culture. Murine DCs prepared from bone marrow from newborn mice were maintained in X-vivo medium supplemented with {beta}-mercaptoethanol, mouse GM-CSF (10 ng ml-1) and IL-4 (10 ng ml-1). All cells were grown at 37 °C in 10 % CO2.

Flow cytometry
VLP binding to cells.
Adherent (detached by 2 mM EDTA for 5 min) and non-adherent cells were collected, centrifuged at 200 g for 5 min and re-suspended in serum-free Iscove's modified Dulbecco's medium. Cells (5x105) were incubated with 5 µg labelled VLPs in 0·5 ml medium for 1 h at 4 °C and washed three times in ice-cold PBS before FACS analysis, which was performed on a FACScalibur (Becton Dickinson) with cells being gated on forward and side scatter. Controls were cells alone and cells incubated at 4 °C with labelled BSA. Data analysis was performed on 10 000 gated events per sample using the CELLQUEST program.

Trypsin, neuraminidase and papain treatment.
For studies on the sensitivity of the MPtV and MPyV receptor to neuraminidase or papain, cells were detached with 2 mM EDTA, washed in PBS and treated with neuraminidase (24 mU in PBS) for 1 h at 37 °C or with papain (1 mg ml-1 in PBS) for 5 min at 37 °C before incubation with VLPs for 1 h at 4 °C. The cells were then washed and analysed as described above. For studies on trypsin sensitivity, cells were treated with trypsin (0·05 % trypsin and 0·5 mM EDTA) for 5 min at 37 °C before incubation with VLPs, as described above.

Kinetics of virus entry.
MAE cells were incubated with VLPs at 4 °C for 1 h, as described above, and then transferred to 37 °C for 5, 10, 20, 30 or 60 min. After each time point, half of the cells were washed in PBS immediately and the other half of the cells were treated with trypsin for 5 min at 37 °C and then washed. The percentage of internalized VLPs was evaluated by FACS analysis and calculated as mean labelling of cells after trypsin treatment divided by the mean labelling at the same time point without trypsin treatment.

Transduction efficiency measured by fluorescence microscopy.
293 and COS-1 cells were grown on coverslips in 6-well plates at a density of 1x105 per well 24 h prior to transduction. For VLP transduction, sonicated VLPs (10–30 µg) were incubated with 2 µg pEGFP-C1 (Clontech) for 1 h before transduction. As a positive control, 5 µl lipofectin was incubated for 45 min with 2 µg pEGFP-C1 in 400 µl serum-free medium. Before transduction, serum-containing medium was removed and the cells were washed with PBS. After 5 h of incubation of the cells with pEGFP-C1 alone (negative control), pEGFP-C1–VLPs or pEGFP-C1–lipofectin in serum-free medium, serum-containing medium was added. Transduction efficiency was analysed after 66–72 h. At this time point, the medium was removed and the cells were washed twice with PBS. Cells on coverslips were fixed in 3 % paraformaldehyde for 10 min at room temperature, washed with PBS, incubated with 5 µg Hoechst 33342 ml-1 (Sigma) for 30 min in a humid box to stain the nuclei and then mounted on microscope slides with Dako fluorescent mounting media. GFP expression was detected by fluorescence microscopy (Zeiss Axioplan2).

Inoculation of MPtV-VLP–DNA complexes into mice.
pEGFP-C1 was used as target DNA. This plasmid (5 µg) alone or mixed with 150 µg sonicated MPtV-VLPs was introduced intraperitoneally into C57BL/6 mice. Mice were sacrificed 3 weeks later and total cellular DNA was extracted from several organs (see Table 2). For amplification of pEGFP-C1 DNA, a forward primer representing nt 561–580 on the CMV promoter (5'-GCAGAGCTGGTTTAGTGAAC-3') and a reverse primer representing nt 171–152 on the GFP gene (5'-TGGCCATGGAACAGGTAGTT-3') were used to generate an amplified fragment of 550 bp. Each PCR mixture contained 500 ng template DNA, 1 µM primers, 3 mM MgCl2, 0·5 mM dNTP and 1·5 units Taq polymerase (PE Biosystems) in a final volume of 50 µl. The target gene was amplified using the following profile: 30 cycles with steps at 95 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min. During the last cycle, the 72 °C step was extended to 6 min.


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Table 2. Detection of pEGFP-C1 by PCR in organs of normal C57BL/6 mice inoculated with complexes of MPtV-VLP and pEGFP-C1 or pEGFP-C1 alone

 

   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression and purification of MPtV VP1 VLPs (MPtV-VLPs)
The MPtV VP1 gene was cloned into a recombinant baculovirus and expressed in Sf9 cells. Recombinant baculoviruses were thereafter analysed for expression of MPtV VP1 by SDS-PAGE followed by Coomassie blue staining and viruses expressing a strong MPtV VP1 band of around 43 kDa (Fig. 1a, lane 2) were selected for further analysis and production of MPtV VP1. Baculovirus-infected Sf9 cells were grown for 72 h and not longer in order to minimize the number of capsids that had internalized cellular DNA (Gillock et al., 1997). MPtV VP1 was extracted from Sf9 cells in the form of self-assembled MPtV-VLPs, which were further purified using sucrose gradient followed by CsCl gradient centrifugation (Fig. 1a, lane 3). CsCl gradient fractions of 0·4 ml were collected from the top and investigated for density by refractometry. The relative amount of MPtV-VLPs in the fractions was assayed by an anti-MPtV-VLP ELISA and the peak antigenic activity was detected in the CsCl fractions corresponding to densities of 1·28–1·29 g ml-1 (Fig. 1b). Fractions were also analysed for the presence of Sf9 DNA on a 0·7 % agarose gel, followed by ethidium bromide staining (data not shown). Fractions that contained DNA had a higher density (>1·29–1·30 g ml-1). Fractions containing empty capsids, defined by the absence of DNA, were pooled and were estimated to have >99 % purity with regard to other proteins, as evaluated by SDS-PAGE (data not shown). Purified MPtV-VLPs appeared to be homogeneous and had an icosahedral shape with a diameter of around 45 nm, which is typical for polyomaviruses, as shown in Fig. 1(c).



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Fig. 1. MPtV-VLPs have a high purity and bind DNA readily. (a) Coomassie blue gel. Lanes: 1, extract of Sf9 cells alone; 2, extract of Sf9 cells expressing MPtV-VLPs; 3, CsCl-purified MPtV-VLPs. (b) Purification of MPtV-VLPs by CsCl equilibrium-density gradient centrifugation. Density and relative content of VLPs, as determined by ELISA, are shown for individual fractions collected from top to bottom. (c) Morphology of purified MPtV-VLPs by electron microscopy. (d) MPyV-VLPs and MPtV-VLPs binding to linearized plasmid DNA (pVAXlacZ), illustrated by gel retardation on a 0·7 % agarose gel. Lanes: 1, MPyV-VLPs and DNA; 2, MPtV-VLPs and DNA; 3, DNA alone.

 
DNA-binding ability of MPtV-VLPs
MPtV-VLPs were tested in parallel to MPyV-VLPs for their ability to bind to non-viral, linearized, plasmid DNA by incubation of 1 µg MPtV-VLPs or MPyV-VLPs to 100 ng plasmid DNA for 45 min at room temperature and then run on a gel retardation assay. The presence of MPtV-VLPs and MPyV-VLPs strongly retarded the migration of the DNA following agarose gel electrophoresis, with most DNA remaining in the wells in comparison to plasmid DNA alone (Fig. 1d). The addition of BSA to the plasmid DNA did not affect gel retardation (data not shown).

MPtV-VLPs do not haemagglutinate guinea pig or sheep erythrocytes
MPtV-VLPs did not haemagglutinate guinea pig or sheep red blood cells when using 10 ng VLPs µl-1, while MPyV-VLPs induced HA down to a concentration of 5 pg µl-1 (data not shown).

Fluorescent labelling and binding of MPtV-VLPs to different cell types, with and without MHC class I antigen expression
MPtV-VLPs and MPyV-VLPs were labelled with a fluorescent dye, AF, to study the binding and internalization of VLPs to cells. Labelling did not affect the shape of the VLPs, as analysed by electron microscopy (data not shown). The AF to VP1 ratio (degree of labelling) varied between 0·5 and 1·5 in different experiments, which corresponds to 180–540 molecules of AF per VLP. As negative control for unspecific binding, BSA was labelled in the same way. The relative amount of VLP binding per cell was evaluated by FACS analysis, by comparing the mean fluorescent intensity obtained for different cells before and after different treatments. Initial experiments were performed with the murine endothelial cell line MAE, since endothelial cells have been shown to be permissive to MPtV in vivo. Labelled MPtV-VLPs and MPyV-VLPs were incubated 1 h at 4 °C with MAE, a mouse endothelial cell line, before FACS analysis, and both VLPs gave a strong signal on 100 % of the cells (Fig. 2). As expected, labelled BSA, the negative control, gave a very weak signal (Fig. 2). The ability of MPtV-VLPs to bind cells of non-endothelial origin and from different species was then tested (Table 1). Independent of MHC class I expression, all murine (MAE, RMA, RMA-S and DC) and human (HeLa, DFW, Daudi, HEF, RCC, RD, 293, SK-N-AS and DC), as well as green monkey (GMK, COS-1) cells were able to bind MPtV-VLPs (Table 1).



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Fig. 2. MAE cells bind to AF-labelled MPtV-VLPs and MPyV-VLPs, but not BSA, as analysed by FACS. Plots represent the cell number (y-axis) versus fluorescent intensity (x-axis). In this particular experiment, MAE alone and MAE with BSA both have a mean fluorescent intensity (MFI) of 8, MAE with MPtV-VLPs have a MFI of 254 and MAE with MPyV-VLPs have a MFI of 622. The data represent one of three independent experiments.

 

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Table 1. MPtV-VLP binding to different cell types before and after different enzyme treatment

 
MPtV-VLP receptor is resistant to treatment with neuramindase but can be sensitive to treatment with trypsin and papain
The fact that MPtV-VLPs did not induce HA indicated that MPtV, similarly to SV40, does not bind to sialic acid residues. This possibility was further investigated by FACS analysis by comparing the binding of labelled MPtV-VLPs and MPyV-VLPs to neuraminidase-treated and non-treated MAE. Neuraminidase treatment of MAE cells increased MPtV-VLPs binding by two- to fourfold, while it reduced MPyV-VLP binding, as expected, almost completely (Fig. 3a). This result and similar results obtained from a human, as well as a green monkey, cell line (Table 1) further support the possibility that sialic acid is not a component of the MPtV receptor. In addition, it seems likely that sialic acid residues block some of the binding sites of MPtV-VLPs (Fig. 3a).



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Fig. 3. MPtV-VLP and MPyV-VLP binding to non-treated and enzyme pre-treated MAE cells. (a) Non-treated and neuraminidase (NA) pre-treated MAE cells. (b) Non-treated and trypsin (try) pre-treated MAE cells. (c) Non-treated and papain (pap) pre-treated MAE cells. The data represent one of three independent experiments. MFI, Mean fluorescent intensity.

 
Treatment of MAE, RMA, RMA-S, HEF, RCC and GMK cells with trypsin or papain (non-cleaving and cleaving surface MHC class I, respectively) before incubation with VLPs was also performed. Trypsin and papain reduced the ability of MAE cells to bind MPtV-VLPs considerably and the binding efficiency was decreased with 80–90 % if compared to non-enzyme-treated MAE cells (Fig. 3b, c). Similar results were obtained for all cell lines tested, except GMK cells (Table 1). In contrast, the binding efficiency of MPyV-VLPs to MAE cells increased by around 50 % after trypsin treatment (Fig. 3b) and was not influenced by papain treatment (Fig. 3c).

Hence, the data suggest that the MPtV receptor is neuraminidase resistant and trypsin and papain sensitive for most cell lines.

MPtV-VLPs are rapidly internalized by MAE cells at 37 °C
Labelled MPtV-VLPs were incubated with MAE cells at 4 °C for 1 h and then treated with trypsin for 5 min at 37 °C. This procedure completely abolished all bound MPtV-VLPs as assayed by FACS analysis (data not shown). However, when MPtV-VLPs were incubated with MAE cells at 4 °C for 1 h, followed by an additional 1 h of incubation at 37 °C, the majority of the fluorescent signal was retained even after the cells were treated with trypsin, indicating that most of the bound VLPs had been internalized (data not shown). Hence, at 4 °C, MPtV-VLPs are not internalized and remain on the cell surface, while at 1 h at 37 °C, most MPtV-VLPs have been internalized.

Trypsin treatment of MPtV-VLPs bound to MAE cells was then used as a method for analysing the relative amount of internalized MPtV-VLPs at different time points. MAE cells were incubated with MPtV-VLPs at 4 °C for 1 h and then transferred to 37 °C for 5, 10, 20, 30 and 60 min. After each time point, half of the cells were washed immediately, while the other half were treated with trypsin for 5 min at 37 °C and then washed. By comparing relative fluorescent intensity (measured by FACS analysis) before and after trypsin treatment, the percentage of uptake at different time points was calculated. As shown in Fig. 4, the internalization of MPtV-VLPs was rapid. Already after 10 min a significant uptake was observed and within 40 min approximately 50 % of all membrane-bound capsids had been internalized.



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Fig. 4. MPtV-VLPs are internalized rapidly by MAE cells at 37 °C, as evaluated by FACS. The y-axis denotes percentage of internalized MPtV-VLPs and the x-axis denotes incubation time at 37 °C. The data represent one of three independent experiments.

 
MPtV-VLPs and MPyV-VLPs do not serologically cross react by ELISA, HAI or in vivo neutralization tests
One condition for using MPtV-VLPs and MPyV-VLPs in repeated gene therapy is that they do not serologically cross react and this was investigated by ELISA and HAI. Sera obtained from mice immunized with either MPtV-VLPs or MPyV-VLPs were tested for immune reactivity against both MPtV-VLPs and MPyV-VLPs. In microtitre plates coated with MPtV-VLPs, only sera from MPtV-VLP-immunized mice and not from MPyV-VLP-immunized mice were immune-reactive (Fig. 5a). The reverse situation was observed when the same sera were tested against MPyV-VLPs (Fig. 5b). In addition, only antiserum induced against MPyV-VLPs, but not against MPtV-VLPs, induced HAI and blocked HA induced by MPyV-VLPs (data not shown). Finally, after immunization in vivo, followed by MPyV challenge, all MPtV-VLP-immunized (4/4) and non-immunized (4/4), but no MPyV-VLP-immunized (0/4), mice exhibited the presence of MPyV DNA in different organs (as assessed by PCR), indicating that MPtV-VLPs do not cross-immunize against MPyV. Hence, we concluded that MPtV-VLPs and MPyV-VLPs do not cross react serologically.



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Fig. 5. MPtV-VLPs and MPyV-VLPs do not cross react serologically, as demonstrated by ELISA. y-axis, A405; x-axis, serum dilution. Serum from MPtV-VLP-immunized mice ({square}), serum from MPyV-VLP-immunized mice ({triangleup}), and serum from control mice ({circ}). (a) Microtitre plates coated with MPtV-VLPs. MPtV-VLP immune sera are seropositive to MPtV-VLPs, while MPyV-VLP immune sera and control sera are non-reactive. (b) Microtitre plates coated with MPyV-VLPs. MPyV-VLP immune sera are seropositive to MPyV-VLPs, while MPtV-VLPs immune sera and control sera are non-reactive. The data represent one of three independent experiments.

 
MPtV-VLPs transduce DNA into cells in vitro
293 and COS-1 cells grown on coverslips in 6-well plates for 24 h were incubated in serum-free medium for 5 h with pEGFP-C1 (2 µg) and VLPs (25 µg), with pEGFP-C1 (2 µg) alone as a negative control or with lipofectin and pEGFP-C1 (2 µg) as a positive control. Thereafter, serum-containing medium was added to the cells and 66–72 h later, a time point when approximately 1x105 cells were observed on each coverslip, the efficiency of GFP transduction was examined by fluorescence microscopy. MPtV-VLPs with plasmid DNA induced GFP expression in both 293 and COS-1 cells (data not shown) with a mean of 30 transduced cells per coverslip, which gives a transduction efficiency of 0·03 %. None of the cells incubated with plasmid alone exhibited GFP expression, while approximately half of the cells incubated with lipofectin together with plasmid expressed GFP (data not shown).

MPtV-VLPs introduces DNA into different tissues in mice
Two groups of C57BL/6 mice, with two animals per group, were used for these experiments. The first group received an intraperitoneal inoculation of 2 µg plasmid pEGFP-C1 DNA in complex with 150 µg MPtV-VLPs and the second group received 2 µg plasmid DNA alone. After 3 weeks, mice were sacrificed and the presence of pEGFP-C1 DNA was analysed by PCR. pEGFP-C1 DNA was detected in the lung, bone, heart, kidney, lymph node and gonads of mice inoculated with pEGFP-C1 together with MPtV-VLPs, but not in mice inoculated with plasmid alone (Table 2).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, MPtV-VLPs, which can bind DNA, were easily obtained in a baculovirus system and their morphology was similar to that of other polyomaviruses. However, unlike most polyomaviruses, but like SV40, they did not haemagglutinate red blood cells. MPtV-VLPs could nonetheless bind to several cell types, independent of MHC class I expression, and their uptake was rapid. Their receptor binding was neuraminidase resistant in all cells tested and was trypsin and papain sensitive for most cells. Finally, MPtV-VLPs did not serologically cross react with MPyV-VLPs and could transduce DNA into cells both in vitro and in vivo.

The fact that MPtV-VLPs could be produced at >99 % purity in a baculovirus system and could bind DNA was as expected and analogous to that reported for other polyomaviruses (Goldmann et al., 1999; Montross et al., 1991; Pawlita et al., 1996; Sandalon & Oppenheim, 1997). However, similar to SV40, but in contrast to most polyomaviruses, MPtV-VLPs did not haemagglutinate erythrocytes (Clayson & Compans, 1989). Our findings are in line with the data of Greenlee & Dodd (1987), where MPtV passaged in mouse embryo cells in vitro did not induce HA, but differ from previous MPtV in vivo results (Kilham, 1961). The reason for the discrepancy between the in vitro and the in vivo data has not been pursued so far; however, one possibility is that low-grade contamination of MPtV in vivo by a haemagglutinating agent could have occurred.

MPtV-VLPs were then tested for their ability to bind to different cells by FACS analysis. Furthermore, since MPtV-VLPs, similar to SV40, did not haemagglutinate red blood cells, and it is known that SV40 binding to cells is commonly dependent on MHC class I expression (Atwood & Norkin, 1989), we studied if MPtV-VLP binding to cells was influenced by MHC class I expression. MPtV-VLPs were shown to bind to a variety of cells from different species, including cells that lacked MHC class I expression.

It has long been known that MPtV has a more specific cell tropism than MPyV in vivo. Originally, MPtV, causing a severe, interstitial pneumonia in newborn mice, was believed to be restricted to growth in pulmonary endothelial cells (Margolis et al., 1976). However, other reports have indicated a somewhat broader host range, including endothelial cells of the lungs' systemic vascular endothelial cells and scattered lymphoid cells within the spleen and renal epithelium (Greenlee et al., 1991). Our results support the concept that MPtV has the potential to attach to an even broader range of cell targets and suggest that the reported specific cell tropism could be due mainly to the fact that the MPtV-enhancer requires a more cell-specific regulation. This possibility is highlighted by the report of Zhang & Magnusson (2001), where part of the MPtV enhancer region was substituted with the corresponding region from MPyV and where the expression of the late genes encoding the viral structural proteins was increased in NIH 3T3 cells.

To characterize the receptor-binding qualities of MPtV-VLPs further, the MAE cell line was incubated with neuraminidase, papain or trypsin, and in this way we could show that the binding of MPtV-VLPs was neuraminidase resistant but trypsin and papain sensitive. These results suggest that the MPtV-VLP receptor is proteinaceous. Furthermore, these results confirmed that MPtV binding is independent of sialic acid residues and MHC class I binding. This first conclusion was based on the findings that MPtV-VLPs do not haemagglutinate red blood cells and that the binding of MPtV-VLPs is insensitive to neuraminidase treatment. The second conclusion was based on the fact that MPtV-VLPs bound to Daudi and RMA-S cells, both lacking MHC class I antigen expression, and that binding to MAE was trypsin and papain sensitive. The latter finding contrasts to what has been demonstrated for SV40, where MHC class I binding was trypsin resistant but papain sensitive (Clayson & Compans, 1989). Nonetheless, since binding of MPtV-VLPs to one cell line (GMK) was insensitive to trypsin and papain, it is possible that MPtV has the potential to bind to more than one receptor. In our experiments with the use of trypsin, we could show that the internalization of MPtV-VLPs was a very rapid process with a significant uptake already after 5–10 min. These data are similar to the rapid uptake of SV40, MPyV and adenovirus capsids (Leopold et al., 1998; Pelkmans et al., 2001; Richterova et al., 2001), possibly indicating a specific mechanism of uptake.

MPtV-VLPs and MPyV-VLPs did not cross react serologically, as measured by ELISA. Furthermore, only antisera against MPyV-VLPs sera and not antisera to MPtV-VLPs could induce HAI towards MPyV-VLPs or protect against MPyV infection in vivo. The lack of cross-reactivity between the two virus VLPs was in agreement with the data of Greenlee & Dodd (1987) and is probably due to that the induced antibody response is directed against surface epitopes and not to linear epitopes, where VP1 of MPyV and MPtV have a 44 % identity. Moreover, the regions with the strongest identity are mainly situated within the non-exposed areas of the capsid. These results suggest that it should be possible to use MPtV-VLP and MPyV-VLP for prime–boost gene therapy.

Finally, we tested if MPtV-VLPs could transduce DNA in vitro and transfer target DNA into different cell tissues in vivo. The transduction efficiency of MPtV-VLPs in vitro was low in our present experiments but comparable to that obtained by MPyV-VLPs when using a similar protocol (Clark et al., 2001; Krauzewicz et al., 2000; unpublished data). The in vivo data with MPtV-VLPs were similar to our previously published results for MPyV (Heidari et al., 2000) and suggest that MPtV-VLPs similar to MPyV-VLPs can attach and transfer DNA into different organs in vivo.

In conclusion, the data presented suggest that MPtV-VLPs, and probably also MPtV, can bind to several different cell types, that binding is neuraminidase resistant and that MPtV-VLPs may have the potential to complement MPyV-VLPs in repeated gene therapy. However, for both types of VLP, the transduction efficiency using the present protocol was low and needs to be improved further.


   ACKNOWLEDGEMENTS
 
DCs were kindly obtained from Andreas Lundqvist and Jan-Alvar Lindencrona at the Department of Oncology-Pathology, Karolinska Institute. We thank Beverly Griffin, Jitka Forstova and Nina Krauzewicz for valuable scientific discussions. We thank the Swedish Cancer Foundation, the Swedish Medical Research Council (VR), the Gustav Vth Jubileum Society, the Stockholm Cancer Society, the Stockholm City Council and the Karolinska Institute for financial support.


   REFERENCES
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METHODS
RESULTS
DISCUSSION
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Received 19 June 2003; accepted 28 August 2003.