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Journal of Virology, January 1999, p. 495-500, Vol. 73, No. 1
Fred Hutchinson Cancer Research Center,
Seattle, Washington 98109,1 and
Departments of Pathology,4
Pediatrics,2 and
Medicine,3 University of Washington,
Seattle, Washington 98195
Received 7 August 1998/Accepted 25 September 1998
Transduction by murine leukemia virus-based retrovirus vectors is
limited in certain cell types, particularly in nondividing cells. But
transduction can be inefficient even in cells that divide rapidly. For
example, exposure of 208F rat embryo fibroblasts to an excess of an
amphotropic retrovirus vector encoding alkaline phosphatase results in
a transduction efficiency of only about 10%, even though these cells
divide rapidly. Here we show that transduction of 208F cells is limited
by cell surface retrovirus receptor levels; overexpression of the
amphotropic retrovirus receptor Pit2 markedly improved the transduction
efficiency to 50%. To characterize receptor levels and binding
affinity, we synthesized a fusion protein that joins the amino terminus
of the amphotropic envelope protein to the Fc region of a human
immunoglobulin G1 molecule for use in binding assays. In comparison to
the parental cell line, the modified cell line showed an order of
magnitude increase in binding sites of from 18,000 to 150,000 per cell. Thus, efficient transduction by an amphotropic retrovirus vector requires high-level expression of the retrovirus receptor Pit2. These
results provide the rationale for further examination of the role of
receptor levels in inefficient transduction, especially with regard to
target cells for gene therapy, where a high transduction rate is often crucial.
The clinical application of
retrovirus-based gene therapy is limited by the poor efficiency of
transduction of target cells such as hematopoietic stem cells. This
cannot be resolved by simply increasing the amount of vector added. It
has been well documented that this is in part due to the quiescent
nature of these cells (14, 17), and evidence is emerging
that lentiviral vectors may help overcome this block to transduction
(29, 32, 34). Resistance of cells to retroviral infection
may also be the result of inhibitory factors in the medium of cell
cultures (19, 25), or it can be limited at the receptor
level (5, 28, 30, 35). Cells express multiple retrovirus
receptors, the abundance of which may vary from tissue to tissue
(15), and a correlation of receptor mRNA expression and the
susceptibility to infection has been proposed (16, 35).
We have previously found that 208F rat embryo fibroblasts are
inefficiently transduced by amphotropic retrovirus vectors, showing a
transduction rate between 10 and 20% (24). In contrast, primary human diploid fibroblasts exhibit rates of 50% or more (31). This result is puzzling because 208F cells divide
rapidly, while the human fibroblasts divide slowly and ultimately
undergo senescence and death with continued cultivation, indicating
that some block to transduction other than division rate is operative in the 208F cells. Thus, we have studied the basis for inefficient transduction of 208F cells as a model for the resistance of other cells
that are inefficiently transduced by retrovirus vectors.
In initial experiments we confirmed the low rate of 208F cell
transduction with an amphotropic vector encoding alkaline phosphatase, and we showed that inefficient transduction of 208F cells was not due
to the production of a soluble inhibitory factor by the cells. Next,
modified 208F cell lines that overexpress the rat amphotropic receptor
Pit2 were used to study effects of high-level receptor expression on
transduction efficiency, and the results showed an increased
susceptibility to infection compared to the parental cell line. In
further studies a fusion protein joining the amphotropic envelope SU
portion with the human immunoglobulin G1 (IgG) Fc domain (ASU-hFc) was
designed to study receptor expression and binding kinetics by Scatchard
analysis. The current study provides evidence in a fibroblast cell
culture model that efficient transduction of target cells requires
high-level receptor expression.
Cell lines and retrovirus vectors.
The 208F rat embryo
fibroblasts (33) and 293 human kidney fibroblasts (ATCC
CRL-1573) were maintained in Dulbecco's modified Eagle medium with a
high glucose concentration (4.5 g/liter) supplemented with 10%
heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, Utah), 100 U
of penicillin G per ml, and 100 µg of streptomycin sulfate per ml at
37°C in a 5% CO2-air atmosphere.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Efficient Transduction by an Amphotropic Retrovirus
Vector Is Dependent on High-Level Expression of the Cell Surface
Virus Receptor
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C.
Retroviral infections. All retroviral infections were performed in the presence of 4 µg of Polybrene (Sigma, St. Louis, Mo.) per ml. Target cells were plated at 5 × 104 cells per 6-cm-diameter dish 1 day prior to infection. Vectors were added at a multiplicities of infection (MOIs) of 4 for LAPSN(PA317), 4 for LAPSN(PE501), and 18 for LAPSN(PG13) unless otherwise noted.
Flow cytometry. Cells were analyzed for surface AP expression 2 days after vector exposure. The cells were trypsinized and incubated at 4°C for 30 min with a primary mouse anti-human AP monoclonal antibody (MAb) of the IgG2a isotype (DAKO, Carpenteria, Calif.) or an irrelevant mouse IgG2a MAb (DAKO) as a negative isotype control. Secondary antibody staining was performed by incubating the cells with fluorescein-isothiocyanate (FITC)-labeled anti-mouse IgG2a MAb (PharMingen, San Diego, Calif.) at 4°C for 30 min. Cells were washed twice with phosphate-buffered saline (PBS) containing 2% FBS, suspended in 2 µg of propidium iodide per ml, and analyzed by using a FACScan flow cytometer and Cellquest software (Becton Dickinson, San Jose, Calif.) with appropriate gating for cell size and viability.
For flow cytometric analysis of amphotropic SU-human Fc portion fusion protein (ASU-hFc) binding, cells were suspended by treatment with trypsin-EDTA or 0.5 mM EDTA only and then were incubated with either the amphotropic SU fusion protein or an irrelevant polyclonal human IgG antibody (Dako) for 60 min at 4°C. The cells were washed twice, incubated with an FITC-conjugated F(ab)2 fragment from a rabbit antibody directed against human Fc (Dako) for 30 min at 4°C, washed twice, and suspended in PBS containing 2 µg of propidium iodide per ml. Fluorescence-activated cell sorter analysis was carried out as described above.Fusion protein construction, purification, and analysis.
A
DNA fragment was constructed that encoded the SU portion of the
amphotropic 4070A Env protein, minus the carboxy-terminal 9 amino
acids, and was linked to a human IgG-Fc protein fragment lacking the
amino-terminal 3 amino acids after the Fc cleavage site (kindly
provided by David Cosman, Immunex, Seattle, Wash.). This DNA was cloned
in place of 
-galactosidase cDNA in the plasmid pCMV--gal
(Clontech, Palo Alto, Calif.), which contains a cytomegalovirus promoter upstream of the ASU-hFc gene, an SV40 intron, and an SV40
polyadenylation signal. The ASU-hFc expression plasmid was transfected
by CaPO4 coprecipitation (8) into 293 cells
plated at an ~70% confluence in 15-cm dishes 1 day earlier. The
fusion protein was then harvested in culture medium containing low-IgG serum (Gemini Bioproducts, Inc., Calabasas, Calif.) at 24 and 48 h
after transfection and filtered (0.45-µm pore size) to remove particles and debris. Subsequent purification was carried out by
affinity chromatography with a protein A column, elution in citrate-phosphate buffer at a pH of 2.8, and neutralization in 10× PBS
(pH 8.5). Stock solutions of concentrated fusion protein were stored at
20°C in PBS until used.
125I labeling of ASU-hFc and analysis of binding. Radioactive labeling was performed according to the method of Bolton and Hunter (6). Briefly, Na125I was converted to its active form by oxidation with chloramine T and bound to an N-hydroxysuccinimide ester of 3-(4-hydroxyphenyl) propionic acid to generate the Bolton-Hunter (B-H) reagent. Next, the fusion protein was incubated with the B-H reagent for 2 h on ice, leading to covalent binding of the labeled B-H reagent to lysine residues in the fusion protein. The labeled fusion protein was purified by chromatography by using a Sephadex G10 column (Boehringer Mannheim, Mannheim, Germany) to remove unincorporated isotope and yielded specific activities of labeled protein of 0.15 to 0.24 mCi/mg. Dilutions of this labeled fusion protein (0.045 to 4.5 nM) were prepared in RPMI 1640 containing 1% BSA.
Cells were grown to <50% confluence and were suspended by trypsinization, and 1 × 105 to 10 × 105 cells were incubated with labeled fusion protein at room temperature on a tube rotator revolving at ~30 rpm. After a thorough washing in RPMI 1640 containing 1% BSA, cell-associated (bound) radioactivity was measured by scintillation counting.Scatchard analysis. Analysis of ASU-hFc fusion protein binding to cells was performed as described by Badger et al. (3). In that study the authors modeled antibody binding to cell surface antigens, and for our analysis the virus receptor is the equivalent of the cell surface antigen, and the fusion protein is the equivalent of the antibody. Importantly, we have included the correction for the amount of fusion protein that is still able to bind receptors after radiolabeling of the fusion protein (3, 20), which is the equivalent of the immunoreactive antibody in the analysis by Badger et al. The mean value for the percentage of reactive fusion protein was 15% in several experiments, and this value was used for the calculations in all experiments.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Transduction of 208F cells with an amphotropic vector is inefficient. We exposed 208F cells to increasing amounts of an amphotropic vector encoding AP (LAPSN) in an attempt to maximize the transduction rate. The highest rate of transduction as measured by flow cytometry was 12% (Fig. 1A). No significant increase in transduction was seen at vector doses above 1 ml, which corresponds to an MOI of 4.
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208F fibroblasts divide rapidly. Given the requirement of cells to divide for successful transduction by murine leukemia virus-based retrovirus vectors, we calculated the cell division rate by plating a constant number of cells into multiple dishes and performing trypsinization at various time points for serial cell counts. The results confirmed that 208F cells are rapidly dividing, with a doubling time of ~18 h in their log phase of growth (data not shown). For comparison, human diploid fibroblasts have a doubling time of between 24 and 32 h (7, 12), and mouse fibroblasts have a doubling time between 15 and 16 h (13, 18).
Overexpression of the amphotropic retrovirus receptor in 208F cells promotes efficient transduction. We transduced 208F cells with the retrovirus vector LPit2SN, which contains the rat Pit2 cDNA under transcriptional control of an MoMLV long terminal repeat promoter and enhancer, and isolated several clonal lines that overexpressed Pit2 (26). We also transduced 208F cells with the empty parental vector LXSN and isolated several clonal lines that contained this vector. Exposure of two clonal lines of 208F/LPit2SN cells to 1 ml of LAPSN(PA317) virus gave a mean transduction rate of 52% as measured by flow cytometry, while infection of three 208F/LXSN clones gave a mean transduction rate of only 5.6% (Fig. 2). Exposure of 208F, 208F/LXSN, and 208F/LPit2SN cells to the LAPSN vector with a GALV (PG13) pseudotype resulted in similar transduction rates in all of these cell lines, as did exposure of the cell lines to the LAPSN vector with an ecotropic (PE501) pseudotype (Fig. 2). Thus, overexpression of Pit2 increased transduction specifically for an amphotropic vector and not for vectors with other pseudotypes.
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Production of an ASU-hFc fusion protein for receptor binding studies. For analysis of Pit2 levels and Pit2/Env binding properties, we used an envelope-antibody fusion protein (ASU-hFc), which consisted of the SU portion of the amphotropic envelope glycoprotein fused to the Fc domain of a human IgG heavy-chain molecule. The addition of the antibody constant region fragment allows simple purification of the fusion protein on a protein A column and provides an epitope for fusion protein detection that is recognized by commonly available antibodies. This protein was transiently expressed in 293 cells, harvested, and purified as a homodimer that separated in an acrylamide gel at ~220 kDa under nonreducing conditions and as a monomer of ~110 kDa under reducing conditions (Fig. 3).
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Amphotropic receptor quantitation and SU binding properties.
We used Scatchard analysis of iodinated ASU-hFc binding to cells to
quantitate receptor levels and SU binding affinity. In preliminary
experiments, we found that fusion protein binding to 208F/LPit2SN cells
stabilized after an hour at room temperature (Fig.
5A), and further binding studies were
performed by incubation with the fusion protein for 2 h.
Measurement of [125I]ASU-hFc binding to cells after
incubation of an identical number of cells with increasing
concentrations of the fusion protein revealed a binding curve with a
shape consistent with specific binding in the left-hand part of the
curve at lower concentrations of fusion protein and nonsaturable,
nonspecific binding in the right-hand part of the curve at higher
concentrations of the fusion protein (Fig. 5B). Addition of a 100-fold
excess of unlabeled fusion protein to the incubations performed with
0.045, 0.45, and 2.3 nM labeled fusion protein reduced the bound
radioactivity to 
5% of the binding measured in the absence of
unlabeled competitor, showing that nonspecific binding at these
concentrations of labeled ASU-hFc was a minor fraction of the total
(data not shown).
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1/(slope of the line). Mean values for the receptor number and
Kd for four independent experiments per cell
line are shown in Table 1. The number of
amphotropic SU binding sites detected on 208F/LXSN cells was equal to
that for 208F cells, showing that transduction with the control vector did not affect the receptor level. In contrast, 208F/LPit2SN cells showed an almost eightfold higher receptor level, which correlates well
with the 5- to 10-fold improvement in transduction efficiency observed
for 208F/LPit2SN cells compared to that observed for 208F or 208F/LXSN
cells (Fig. 2).
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Several groups have found that the ability of a retrovirus with a particular pseudotype to infect target cells correlates with the relative abundance of receptor RNA message (5, 16, 35). In contrast to the present study, these data provide only indirect evidence that the number of surface receptors expressed is crucial to the efficiency of retroviral infection.
Others have investigated receptor expression levels by using a sandwich assay involving antibody to the virus envelope glycoprotein gp70 (9, 27, 37). In this assay the binding of virus can be studied by the detection of antibody bound to the virions that in turn are bound to receptors on the cell surface. Studies show that gp70 binds in a saturable pattern that is dependent on the cell number and thus the receptor (11). However, the analysis of gp70 binding is not without pitfalls in that gp70 is shed from viral particles and from producer cell lines and is thus present in vector preparations (2). Free gp70 can therefore occupy receptors and compete with functional virus for binding sites.
Yu et al. (37) have systematically analyzed the binding kinetics of ecotropic pseudotype retrovirus to NIH 3T3 cells. In contrast to our study, they utilized an anti-gp70 antibody flow cytometric assay that did not allow the quantification of receptors. They found significant discrepancies between the dissociation constants measured directly in their experiments and those calculated from association and dissociation rates. Contamination of their virus preparations with free gp70, receptor-ligand internalization, and surface dissociation of the cell-bound SU portion from the virions were thought to account for these problems. This illustrates the shortcomings of this indirect, antibody-based assay.
Ganguly et al. (11) used radiolabeled gp70 antibody and estimated the number of ecotropic receptors on the surface of an NIH 3T3 cell to be about 5 × 105. While vectors of ecotropic pseudotype efficiently infect NIH 3T3 cells at up to 80% after one vector exposure (data not shown), the lower susceptibility of 208F fibroblasts to infection (9.5%) correlates with the lower number of amphotropic receptors (1.8 × 104) found in our study.
Another approach has been to directly label gp70 purified from virus
preparations. DeLarco and Todaro (10) used this approach to
study retrovirus receptor expression on NIH 3T3 cells. While they do
not provide data on binding kinetics, they were able to calculate the
number of receptors to be approximately 5.3 × 105 per
cell, a finding similar to the results Ganguly et al. (11). Interestingly, DeLarco and Todaro encountered some of the same problems
we did with their experimental design that were related to the
variation in the amount of labeled protein participating in binding.
They report a range of active protein from 7 to 41% between
experiments and also note some deterioration of the labeled preparation
over the time of storage prior to the individual experiment. This
parallels our observation regarding the variability in equilibrium dissociation constants (Kd) between experiments.
The Kd is affected by the fraction of labeled
fusion protein actually participating in binding (3), which
averaged 15% (range, 10 to 22%) in our series of experiments. This
average was applied to all calculations, but the reactive fraction of
fusion protein was not assessed prior to each avidity experiment. Some
variation over time due to degradation of the labeled protein is
conceivable, and this likely accounts for the magnitude of the standard
deviation for Kd in Table 1. DeLarco and Todaro
(10) also performed cold competition experiments and found
1.8% residual binding of labeled protein in the presence of 500-fold
excess of unlabeled protein. This is in agreement with the 5%
nonspecific binding of labeled protein we observed in our experiments.
Less data is available on the binding characteristics of amphotropic pseudotype retrovirus to its cellular receptor. Battini et al. (4) have purified a fragment containing the N-terminal binding domain of the amphotropic envelope SU portion. After radioactive labeling of the fragment they studied its binding characteristics to cells, performed Scatchard analysis, and estimated the number of binding sites to be 7 × 104 per cell for NIH 3T3 mouse fibroblasts. This again correlates well with the relatively lower number of amphotropic fusion protein binding sites (1.8 × 104) and more inefficient transduction (9.5% versus 20% on 3T3 cells [data not shown]) in 208F cells. A number of other points distinguish the present study from the one by Battini et al. First, the ability of our fusion protein to be labeled radioactively or by fluorescent secondary antibody permits us to perform Scatchard analysis or alternatively to measure the relative receptor expression levels on individual cells within a population. Second, we correlated the efficiency of transduction and receptor number by studying the effect of receptor overexpression on both parameters in the same cell line. Third, Battini et al. do not provide a correction for the reactive fraction of labeled protein (personal communication), resulting in an overestimate of free fusion protein in the calculations. Fourth, Battini et al. found a downward concave shape of the Scatchard, which contrasts with our more typical linear plots.
A question remains concerning the precise stoichiometry of binding of our fusion protein. The above results are based on the assumption that a fusion protein homodimer will bind to a single binding site, i.e., the receptor. If a single homodimer binds two receptors, this may lead to a two-fold underestimate of binding sites and might affect the Kd values.
Finally, the use of hematopoietic reconstituting cells in clinical gene therapy, with their apparently decreased propensity to enter cell cycle (1, 14, 17), will make it all the more important to find retrovirus vectors targeting an appropriately abundant receptor. We have previously provided evidence in the baboon competitive repopulation assay that transduction by a vector pseudotyped for the more abundantly available GALV receptor on hematopoietic CD34+ cells is preferentially detected over amphotropic pseudotype vector following reconstitution of the lethally irradiated animal (16). This data confirmed similar observations made in vitro (36). The fusion protein used in the present study will allow the direct quantification of retrovirus binding sites on the surface of such target cells.
In summary, we have systematically analyzed a 208F rat fibroblast culture model in which the block to efficient retrovirus transduction is primarily at the level of target cell surface receptor expression. Using this model, we provide direct evidence that efficient retrovirus transduction requires high-level expression of surface receptor as measured by fusion protein binding sites. These results have important implications for the maximization of retroviral infection in animal gene transfer studies and human gene therapy.
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ACKNOWLEDGMENTS |
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We acknowledge the contribution of David Cosman and Immunex Corporation in providing the human IgG Fc portion of the fusion protein and the technical assistance of Jennifer Smith.
This work was supported by grants DK47754, HL54881, and HL36444 from the National Institutes of Health. H.-P.K. is a Markey Molecular Medicine Investigator.
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FOOTNOTES |
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* Corresponding author. Mailing address: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, Room C2-023, Seattle, WA 98109-1024. Phone: (206) 667-2890. Fax: (206) 667-6523. E-mail: dmiller{at}fhcrc.org.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Journal of Virology, January 1999, p. 495-500, Vol. 73, No. 1
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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