1 Infection and Immunity, Henry Wellcome Research Building, Wales College of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK
2 Virus Receptor and Immune Evasion Group, Henry Wellcome Research Building, Wales College of Medicine, Cardiff University, Heath Park, Cardiff, UK
3 Biomolecular Sciences Building, School of Biology, University of St Andrews, St Andrews, UK
Correspondence
Martin Rowe
RoweM{at}Cardiff.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Tumour cells of lymphoid origin have been reported to be quite resistant to adenovirus infection (Cantwell et al., 1996; Prince et al., 1998
; Silver & Anderson, 1988
). However, a study by Buttgereit et al. (2000)
demonstrated effective gene transfer into lymphoma cells, using recombinant adenoviruses combined with the transfection reagent lipofectamine. This study reported that susceptibility to adenovirus infection correlated with cell-surface CAR levels. Human CAR is a 46 kDa member of the immunoglobulin (Ig) CTX subfamily (Chretien et al., 1998
) and is composed of two extracellular Ig-like domains, a typical transmembrane domain and a long cytoplasmic domain (Bergelson et al., 1997
). The most distal extracellular Ig loop (V region or D1 domain) of CAR allows CAR aggregation by facilitating a homophilic interaction between CAR molecules (Cohen et al., 2001
; Honda et al., 2000
; van Raaij et al., 2000
). CAR localizes to the region of tight junctions in polarized epithelia, suggesting a role in cellular adhesion (Cohen et al., 2001
). In terms of recombinant adenovirus infection, CAR functions as a high-affinity receptor for the adenovirus fibre protein (Bergelson et al., 1997
). The fibre knob of adenovirus interacts with the D1 domain of CAR (Bewley et al., 1999
) and it has been proposed that CAR is largely responsible for susceptibility to adenovirus infection (Buttgereit et al., 2000
; Fuxe et al., 2003
; McDonald et al., 1999
).
This study investigated the susceptibility of B-lymphocyte cell lines to recombinant adenovirus infection and proposes a mechanism by which to enhance this susceptibility. We were particularly interested in EpsteinBarr virus (EBV)-transformed B-lymphoblastoid cell lines (LCLs), which express very high levels of MHC class I at the cell surface (Rowe et al., 1995) but demonstrate a very low rate of infection by recombinant adenovirus vectors (von Seggern et al., 2000
). We characterized a role for CAR and
v
5 integrin in adenoviral entry into lymphocytes and engineered an LCL that contains high levels of both of these proteins. This cell line expressed green fluorescent protein (GFP) from an adenoviral construct and could also be infected with a virus expressing the V protein of simian virus 5 (SV5), which effectively reduced STAT1 protein levels.
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METHODS |
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Generation of an anti-CAR monoclonal antibody.
The monoclonal anti-CAR antibody BRAD30 was generated in house. Briefly, recombinant cDNA was engineered that contained the two extracellular Ig regions (V and C2) of CAR followed, in frame, by cDNA encoding the Fc portion of human IgG1. This recombinant cDNA was subcloned into a eukaryotic expression vector containing a hygromycin resistance gene (Yanagawa et al., 2004) and was transfected into CHO cells (ECACC). Stable clones expressing CAR-Fc were generated by propagation in selection medium (RPMI 1640 with 10 % fetal calf serum) containing 400 µg hygromycin ml1. Soluble recombinant CAR-Fc was purified from the cell supernatant with protein A-conjugated Sepharose (Amersham Biosciences), dialysed into PBS (pH 7·4) and used to immunize BALB/c mice via intraperitoneal inoculation following the addition of Freund's incomplete adjuvant. Hybridoma cells were created using SP2/0 fusion partners (ECACC) using standard techniques and positive splenocytemyeloma fused cells were selected by flow cytometry. Specific anti-CAR monoclonal antibody-secreting cells were identified and subsequently subcloned to homogeneity through the production of antibody that bound CHO cells expressing full-length human CAR (Spiller et al., 2002
), as detected by phycoerythrin (PE)-conjugated rabbit anti-mouse Ig antibody (Dako). BRAD30 recognized both human and pig CAR-expressing CHO cells (Spiller et al., 2002
), but not empty-vector-transfected CHO cells, and was a murine IgG1 isotype. Pre-incubation of HeLa cells (ECACC) with 10 µg BRAD30 ml1 blocked infection by coxsackievirus B3 (strain CG), confirming its specificity for CAR.
Immunofluorescence staining of cell-surface proteins.
The expression of cell-surface proteins such as CAR and v
3/
v
5 integrins was detected using flow cytometry analysis. The following antibodies were utilized: mouse anti-CAR (
HCAR-BRAD30); mouse anti-
v
3 (MAB1976Z; Chemicon) and mouse anti-
v
5 (MAB1961X; Chemicon). Cells were harvested and washed twice in PBS. Approximately 2x1055x105 cells were incubated with the primary antibody, generally at a dilution of 1 : 100 in PBS containing 10 % normal rabbit serum (NRS/PBS), for 30 min at 4 °C. Cells were washed twice with PBS and bound antibodies were detected by incubation of cells for 30 min at 4 °C with a 1 : 30 dilution of PE-conjugated anti-mouse IgG (R0439; Dako) in NRS/PBS. Cells were given a further two washes in PBS and resuspended in FACS Flow (Becton Dickinson) or in 2 % paraformaldehyde in PBS and analysed on a FACScalibur flow cytometer (Becton Dickinson) with CellQuest Pro software (Becton Dickinson).
Generation of cell extracts, SDS-PAGE and Western blot analysis.
Cells were counted on a haemocytometer and resuspended in 50 µl PBS per 106 cells. An equal volume of 2x gel sample buffer (100 mM Tris/HCl pH 6·8, 20 % glycerol, 0·2 M DTT, 4 % SDS, 0·02 % bromophenol blue) was added and the cells were sonicated using a W385 sonicator (Heat Systems Ultrasonics). Following sonication, samples were heated at 100 °C for 5 min. The solubilized proteins were separated by SDS-PAGE and transferred to PVDF membrane (Amersham) for immunoblotting using an alkaline phosphatase chemiluminescent detection protocol (Rowe & Jones, 2001). Primary antibody incubations were for 80 min at room temperature and primary antibodies were used at the following concentrations: rabbit anti-STAT1 (sc-346; Santa Cruz Biotechnology), 0·2 µg ml1; rabbit anti-STAT2 (sc-476; Santa Cruz Biotechnology), 0·4 µg ml1; rabbit anti-actin (A2066; Sigma), 4 µg ml1, and rabbit anti-CAR (Spiller et al., 2002
), 1 : 500 dilution. Secondary antibody incubations using a 1 : 10 000 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG (170-6518; Bio-Rad), were for 80 min at room temperature. Specific antibodyprotein complexes were detected using CDP-Star (Tropix) chemiluminescence reagent.
Adenoviral infection of B lymphocytes.
A GFP-expressing recombinant adenovirus (AdGFP) was generated by cloning enhanced GFP (Cormack et al., 1996) under the control of the human cytomegalovirus major immediate-early (hCMV IE) promoter to generate pAL322 (adenovirus transfer vector) and adenovirus was generated by co-transfecting pAL322 and pJM17 (E1-deleted adenovirus type 5 backbone) into 293 cells as described previously (Wilkinson & Akrigg, 1992
). A recombinant adenovirus expressing the V protein (Didcock et al., 1999
) of SV5 (V Adv) was made in a similar fashion. B lymphocytes were washed and resuspended in growth medium at a typical concentration of 8x105 cells ml1. Aliquots of 2x105 cells in 250 µl were then added to the wells of a 24-well plate. Cells were infected with recombinant adenoviruses at m.o.i. of 15 or 30, as indicated in the text, by the addition of an appropriate amount of recombinant adenovirus to the wells. The plate was then incubated for 2·5 h at 37 °C on a rocking incubator (STR8 drive unit and platform and S.1.600 incubator, speed set at 25; Stuart Scientific). After incubation, 1 ml fresh growth medium was added to each well before placing the plate in a 37 °C incubator for 3 days.
Generation of glycosylphosphatidylinositol (GPI)-anchored CAR-expressing cells.
Recombinant cDNA encoding the N-terminal V region or D1 domain of CAR fused to the Ser/Thr spacer region and GPI addition signal from human CD55 was created by PCR. Restriction enzyme sites were added at the 5' end, the CARGPI junction and the 3' end to allow fusion of the cDNA fragments and subcloning into the expression vector listed above for CAR-Fc production. Integrity of the sequence was confirmed by sequencing (ABI), as well as the maintenance of reading frame for the GPI signal. For the generation of DG75 and IB4 stable cell lines expressing GPICAR, 1x107 cells were transfected with 8 µg GPICAR expression plasmid by electroporation (Bio-Rad Genepulser II electroporator set at 270 V/950 µF). Following electroporation, cells were reseeded in 9·5 ml fresh growth medium and 200 µl fractions were aliquoted into the wells of a 96-well plate. Twenty-four hours post-transfection, cells were treated with hygromycin B to select GPICAR transfectants. DG75 and IB4 cells were treated with hygromycin B (Boehringer Mannheim) at concentrations of 400 µg ml1 and 200 µg ml1, respectively.
Cell-surface receptor blocking studies.
Blocking studies were performed with purified azide-free antibodies and the following antibodies were utilized: rabbit anti-CAR (raised in house); mouse anti-v
3 (MAB1976Z; Chemicon) and mouse anti-
v
5 (MAB1961X; Chemicon). Typically, 2·4x105 cells per 600 µl growth medium were incubated with purified antibodies at concentrations ranging from 0·03 to 40 µg ml1 for 40 min at room temperature under sterile conditions. Aliquots of 250 µl of the cell suspension (1x105 cells) were subsequently transferred to a 24-well plate and the cells were infected with AdGFP at an m.o.i. of 30. Following recombinant adenovirus infection, 1·25 ml fresh growth medium was added to each well and the 24-well plate was placed in a 37 °C incubator. Forty-eight or 72 h post-infection (p.i.), cells were harvested, washed in PBS and fixed in 2 % paraformaldehyde in PBS. The percentage of GFP-positive cells was determined by flow cytometry on a FACScalibur flow cytometer with CellQuest Pro software.
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RESULTS |
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Increased CAR expression does not correlate with increased infection of B lymphocytes
For mouse T lymphocytes and human dendritic cells, the efficiency of infection by adenovirus has been increased by the engineered expression of CAR (Hurez et al., 2002; Stockwin et al., 2002
). To investigate this issue in B lymphocytes, stable lines of IB4 LCL and DG75 cells were generated with increased CAR expression. Initial experiments were performed with a plasmid vector encoding full-length CAR. However, expression levels of CAR invariably remained low or undetectable and therefore it was not possible to interpret the observed failure to obtain enhanced adenovirus infectivity (data not shown). Subsequently, we chose to use GPICAR, a truncated form of human CAR with the transmembrane and cytoplasmic domains replaced with a GPI anchor. GPICAR has a number of advantages compared with full-length human CAR. Importantly, deletion of the cytoplasmic tail of CAR limits the signalling potential of the protein and inhibits its degradation. The transmembrane and cytoplasmic domains are not required for adenovirus infection (Leon et al., 1998
; Wang & Bergelson, 1999
) and this was validated for our constructs prior to introduction of this plasmid into B lymphocytes (data not shown). IB4 and DG75 cells were transfected by electroporation with 8 µg GPICAR expression plasmid. Twenty-four hours post-transfection, cells were treated with hygromycin B to select GPICAR transfectants. Hygromycin B-resistant lines that grew out were analysed for CAR cell-surface expression and susceptibility to adenovirus infection.
Clones were generated with a wide range of CAR expression levels (Fig. 2). Data from one representative clone of DG75 and two clones of IB4 are shown in Fig. 2
(a). In each case, a large increase in CAR expression was detected (compare Figs 1a and 2a
). Twelve hygromycin B-resistant DG75 GPICAR lines were generated. Fig. 2
(b) shows the level of adenovirus infection of these cell lines as a function of the level of CAR expression. Susceptibility to adenovirus infection was monitored by infecting cells with AdGFP at an m.o.i. of 30 and analysing the percentage of GFP-positive cells at 72 h p.i. by flow cytometry. The graph revealed no correlation between the amount of CAR expressed at the cell surface and susceptibility to AdGFP infection. Infection rates were generally less than 8 % for all clones tested, although one clone did exhibit an infection rate of approximately 15 %. However, these infection rates fall short of the rates observed in T lymphocytes and dendritic cells expressing high levels of CAR (Hurez et al., 2002
; Stockwin et al., 2002
).
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Adenovirus infection of IB4 GPICAR 8 cells is dependent on CAR
Given the lack of correlation between CAR expression and adenovirus infection, it was important to determine whether this adenoviral entry into IB4 GPICAR 8 cells was dependent upon CAR. We performed antibody-blocking experiments in which IB4 GPICAR 8 cells were pre-incubated with purified anti-CAR antibody. A control population and the pre-incubated population were subsequently infected with AdGFP at an m.o.i. of 30 and GFP expression was determined at 72 h p.i. by flow cytometry. In the experiment illustrated in Fig. 3(a), pre-incubation of cells with anti-CAR antibody decreased the percentage of GFP-positive cells from 53 to 3 %. Similar results were obtained in two repeat experiments, and blocking of CAR resulted in a reduction in the receptiveness of the cell line to adenovirus infection of 92·3±0·6 % (mean±SEM, n=3). Furthermore, titration of the purified CAR antibody revealed that this degree of resistance to adenovirus infection was observed using the antibody at concentrations ranging from 20 down to 3·33 µg ml1 (Fig. 3b
). Thus, blocking of CAR resulted in the cell line becoming almost totally resistant to adenovirus infection. These findings showed that CAR is critical for mediating adenovirus infection of these B lymphocytes.
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DISCUSSION |
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In a number of studies, the susceptibility of human cells to adenovirus infection has been shown to correlate with cell-surface CAR expression (Fuxe et al., 2003; McDonald et al., 1999
; Stockwin et al., 2002
). The present study clearly shows that this is not the case for human B-lymphocyte lines. There was no correlation between CAR cell-surface expression and susceptibility to adenovirus infection in three model B-lymphocyte cell lines each exhibiting a different EBV status, despite the generation of cell lines overexpressing GPICAR. Interestingly, EBV-immortalized LCLs have been shown to express very high levels of MHC class I on the cell surface (Rowe et al., 1995
), which has also been proposed to mediate adenovirus infection of target cells (Hong et al., 1997
). Nevertheless, the IB4 LCL was not particularly susceptible to adenovirus infection (Fig. 1
). The IB4 GPICAR clone 8 cell line proved to be distinct from the other IB4 GPICAR clones generated in that it displayed a notably enhanced susceptibility to adenovirus infection (Fig. 2
). This increased level of adenovirus infection could be effectively blocked by an antibody to CAR. Together these data clearly show that CAR is required but not sufficient for adenovirus infection of B lymphocytes.
The present study identified v
5 as a co-factor that works in combination with CAR to mediate adenovirus infection of the IB4 GPICAR 8 cell line. The elevated
v
5 expression observed on the IB4 GPICAR 8 cell line (Fig. 4
) most likely arose as a result of the hygromycin B selection and cloning process, which resulted in the fortuitous isolation of both CAR- and
v
5-positive cells. The importance of
v
5 is highlighted by the fact that the adenovirus-resistant DG75, Akata, IB4 and IB4 GPICAR 3 cell lines were virtually
v
5 negative (Fig. 4
). Whilst numerous studies have implicated CAR and integrins in adenovirus binding and entry, the precise molecular requirement appears to depend on the cell type and the adenovirus serotype. To our knowledge, this study is the first to demonstrate the importance of both a primary (CAR) and a secondary (
v
5) receptor in mediating adenovirus type 5 infection of a B-lymphocyte cell line. Two previous studies have reported that the susceptibility of tumour cells to adenovirus infection correlates well with cell-surface CAR expression and that this susceptibility appears to be independent of
v
3 and
v
5 integrin expression (Buttgereit et al., 2000
; Fuxe et al., 2003
). However, our data are more in line with another study showing that the levels of
v
5 may predict the susceptibility of human lung cancer cells to adenovirus-mediated gene transfer independently of
v
3 levels (Takayama et al., 1998
).
It is pertinent to note that the results obtained in this study may not be applicable for all adenoviral serotypes. For example, adenovirus type 37 has been reported to use sialic acid instead of CAR for virus attachment (Arnberg et al., 2000). In addition, the adenovirus types 11 and 35 differ from adenovirus type 5 in showing high binding efficiencies to some committed haematopoietic cell lines (Segerman et al., 2000
). However, this increased binding efficiency does not necessarily translate into increased infectivity. Most notably, the B-cell line used in the study by Segerman et al. (2000)
, which was also examined in our study, remained refractory to infection by adenovirus types 11 and 35.
Whilst in the present study we chose to modify the target cell to increase the efficiency of adenovirus transduction, an alternative approach involves modification of the fibre protein of the adenovirus to alter its tropism. This is potentially an attractive approach for gene therapy, where retargeting the virus to a different tissue may be especially advantageous (reviewed by Wickham, 2003), and in cases where experimental modification of the target cell is not an option. Interestingly, a study by von Seggern et al. (2000)
demonstrated that the susceptibility of EBV-immortalized LCLs to adenovirus infection could be increased by replacing the fibre protein knob of an Ad5
gal recombinant adenovirus with the fibre protein from adenovirus type 3. In that study, the efficiency of infection varied considerably between different LCLs and inspection of the data suggests that this variability may correlate with expression of
v
5. However, while modification of the fibre protein clearly increased the susceptibility of LCL to adenovirus infection, it is notable that in the order of 50 000 virus particles per cell were required to achieve expression of the adenovirus-transduced gene in 2050 % of the cells. In our experiments with the CAR-expressing IB4 LCL, a similar percentage of cells was transduced with conventional adenovirus type 5 recombinant vector at an m.o.i. of 30.
The engineering of a B-cell line that is susceptible to adenovirus 5 infection provides a valuable tool for EBV research. However, the molecular characterization of the adenoviral entry provides a strategy for the generation of other cell lines to augment or enhance adenoviral entry. We have shown that the V protein of SV5 can reduce STAT1 protein levels, demonstrating that adenovirus can be used for protein knockout studies in these cells. Thus, the increased susceptibility of this cell line to adenovirus infection provides an opportunity by which the function of proteins of interest may be investigated in an LCL, using recombinant adenoviruses as the gene delivery vehicles. Historically, the resistance of LCLs to traditional transfection approaches (White et al., 2002) has limited protein functional studies in these cells. Notably, this effect was observed by infecting the IB4 GPICAR 8 cell line with adenovirus expressing SV5 V protein at an m.o.i. of 15. Such low m.o.i. of adenovirus are unlikely to exert any toxic effects on the target-cell population and thus the reliability of such functional studies is enhanced.
In summary, this study has demonstrated the feasibility of using recombinant adenoviruses as gene delivery vehicles into human B lymphocytes in vitro. It showed that both CAR and v
5 are involved in mediating adenovirus infection of IB4 lymphoblastoid cells. The identification of a mechanism for adenovirus infection of B lymphocytes provides an opportunity by which B lymphocytes may be modified to increase their susceptibility to recombinant adenovirus infection. In addition, cell-surface expression levels of CAR and
v
5 may be used to predict the susceptibility of malignant B lymphocytes to adenovirus infection and perhaps the likely efficiency of adenovirus-mediated gene therapy directed against B lymphocytes.
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ACKNOWLEDGEMENTS |
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Received 3 December 2004;
accepted 19 March 2005.
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