Institut für Klinische und Molekulare Virologie, Schlossgarten 4, 91054 Erlangen, Germany
Correspondence
Thomas Stamminger
thomas.stamminger{at}viro.med.uni-erlangen.de
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ABSTRACT |
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Introduction |
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Recent evidence suggests that HCMV not only causes acute infection in transplanted patients but may also be associated with extensive transplant vasculopathy, ultimately leading to chronic allograft rejection (Grattan et al., 1989; Skowronski et al., 1993
; Koskinen et al., 1993
; Lautenschlager et al., 1997a
; Schnitzler et al., 1997
; Borchers et al., 1999
). One of the molecular mechanisms underlying the acceleration of vascular disease processes by HCMV infection may be enhancement of transplant immunogenicity. Several studies indicate that this involves the HCMV-induced upregulation of cellular adhesion molecules, which augments the adherance and infiltration of inflammatory cells that are capable of promoting vascular disease (Einsele et al., 1994
; Lemstrom et al., 1995
; Steinhoff et al., 1995
; Koskinen et al., 1996
; Yilmaz et al., 1996
; Martelius et al., 1998
; Waldman et al., 1998
; Lautenschlager et al., 1999
; The et al., 2001
). For instance, cell culture experiments have demonstrated that HCMV-infection of endothelial cells as well as fibroblasts leads to increased cell-surface expression of intercellular adhesion molecule-1 (ICAM-1, CD54) (Sedmak et al., 1994
; Grundy & Downes, 1993
; Ito et al., 1995
; Knight et al., 1999
). In vivo evidence for a pathogenetic role of CMV-induced upregulation of ICAM-1 is provided by animal models: rat cytomegalovirus infection in kidney allograft recipients has been linked to increased expression of ICAM-1 and its ligand leukocyte function antigen-1 (LFA-1) in the vascular endothelium and this correlated with enhanced histological changes of chronic allograft rejection (Kloover et al., 2000
).
ICAM-1 is an inducible cell-surface glycoprotein and a member of the immunoglobulin supergene family. It is expressed in both haematopoietic and non-haematopoietic cells (such as endothelial cells and fibroblasts) and mediates cellular adhesive interactions by binding to its receptors on leukocytes, LFA-1 and Mac-1. Therefore, increased ICAM-1 cell-surface expression is accompanied by consequential leukocyte recruitment as well as activation and plays a central role in a wide range of inflammatory and immune responses (van de Stolpe & van der Saag, 1996; Roebuck & Finnegan, 1999
). Induction of high levels of ICAM-1 occurs in response to various mediators of inflammation, including bacterial lipopolysaccharides, phorbol esters, oxidant stress and pro-inflammatory cytokines as well as viral infections [e.g. infections with rhinovirus (Papi & Johnston, 1999
), respiratory syncytial virus (Stark et al., 1996
), hepatitis B virus (Hu et al., 1992
), human immunodeficiency virus type 1 (Dhawan et al., 1997
), EpsteinBarr virus (Mehl et al., 2001
) and HCMV (Sedmak et al., 1994
; Ito et al., 1995
)]. So far, the mechanism by which HCMV upregulates ICAM-1 cell-surface expression has been poorly understood.
Productive infection with HCMV results in a regulated cascade of immediate-early (IE), early (E) and late (L) viral gene expression (Wathen et al., 1981; DeMarchi, 1981
; McDonough & Spector, 1983
). For the onset of the IE phase, structural protein components of the incoming virion play an important role (Baldick et al., 1997
; Stamminger et al., 2002
). These proteins are localized in the so-called tegument, which separates the nucleocapsid from the viral envelope. One tegument component is the pp71 phosphoprotein, which activates IE gene expression in infected cells via stimulation of the HCMV major IE enhancer/promoter (MIEP) (Bresnahan & Shenk, 2000
). During the IE phase, viral regulatory factors are synthesized, in particular the major IE proteins IE1p72 and IE2p86, which act as strong transactivators of viral early promoters and are therefore required for efficient productive infection (Stenberg et al., 1989
; Pizzorno et al., 1991
; Mocarski et al., 1996
; Marchini et al., 2001
). In transient transfection analysis, IE2p86 acts as a transcriptional regulator of cellular promoters such as the hsp70 and cyclin E promoters, as well as of heterologous viral control elements (e.g. human immunodeficiency virus long terminal repeat, LTR) (Biegalke & Geballe, 1991
; Ghazal et al., 1991
; Hagemeier et al., 1992b
; Bresnahan et al., 1998
). Therefore, IE2p86 seems to act as a transcription factor that exerts a broad activation pattern. Since IE2p86 interacts with the basal transcription factors TBP (Hagemeier et al., 1992a
; Sommer et al., 1994
) and TFIIB (Caswell et al., 1993
) and with distinct cellular transcription factors such as CREB, Ap-1, Egr-1, Spi-1/PU.1 or Sp1, protein interactions are believed to be essential for transactivation (Lukac et al., 1994
; Lang et al., 1995
; Scully et al., 1995
; Yoo et al., 1996
; Yurochko et al., 1997
; Wara-Aswapati et al., 1999
).
In this report we provide evidence for the mechanism by which HCMV upregulates ICAM-1 on the cell surface of endothelial cells as well as fibroblasts. We show that the regulatory proteins IE2p86 and pp71 act as strong synergistic inducers of the ICAM-1 promoter. The Sp1 site at -53 bp upstream of the ICAM-1 transcriptional start site was identified as a major response element for HCMV transactivation. Importantly, the synergistic action of IE2p86 and pp71 on endogenous ICAM-1 gene expression could be confirmed by baculovirus-mediated gene transfer followed by FACS analysis. Thus, we identified IE2p86 and pp71 as important mediators of HCMV-induced ICAM-1 upregulation.
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Methods |
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Cell culture.
Spodoptera frugiperda (Sf) 158 insect cells, primary human foreskin fibroblasts (HFF) and U373MG cells were cultivated as described previously (Kronschnabl et al., 2002). Primary human umbilical vein endothelial cells (HUVEC) were obtained by chymotrypsin treatment of single umbilical vein cords and were cultivated as described previously (Kronschnabl et al., 2002
).
Generation of recombinant baculoviruses.
Recombinant baculoviruses were generated by using the Bac-to-Bac system, as described by the manufacturer (Invitrogen). Construction of the IE2p86-expressing baculovirus baculo-IE2p86 and the baculo-control virus have been described previously (Kronschnabl et al., 2002). The pp71-expressing baculovirus (baculo-pp71) was generated by transformation of the shuttle vector pHM1587 into the baculoviral genome that is maintained as a BACmid in E. coli cells (bMON14272). BACmid-DNA isolated from positive colonies was used for transfection of Sf158 cells in order to reconstitute infectious baculoviruses. After further amplification by passaging in Sf158 cells, virus titres were determined by plaque assay.
Western blotting.
Samples were subjected to SDS-PAGE, and the proteins were blotted on to nitrocellulose membranes (Schleicher and Schuell). Immunoblot analysis was carried out as described previously (Hofmann et al., 2000). For detection of the ICAM-1 protein, the monoclonal antibody anti-CD54 (mAb1379; Chemicon) was used; the polyclonal anti-serum anti-pHM178 (Hofmann et al., 2000
) was used for the detection of IE2p86, and the polyclonal anti-serum SA1718 (kindly provided by B. Plachter, Mainz, Germany) for detection of pp71. Anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies were obtained from Dianova.
RT-PCR.
Total RNA from HFF cells was isolated with the High Pure RNA Isolation Kit (Roche). The reverse transcription reaction was performed according to the manufacturer's protocol using the Titan One Tube RT-PCR System (Roche). GAPDH mRNA was amplified as an internal control by using a specific primer pair creating a 698 bp fragment [5' primer (698GAP-5) 5'-GTACGTCGTGGAGTCCACTG-3'; 3' primer (698GAP-3) 5'-TCCACCACCCTGTTGCTGTA-3']. The specific primer pair for the amplification of the ICAM-1 mRNA generated a 380 bp fragment [5' primer (ICAMmRNA5) 5'-ACATGCAGCACCTCCTGTG-3'; 3' primer (ICAMmRNA3) 5'-CACCGTGGTCGTGACCTCAG-3']. PCR cycling parameters were chosen as follows: 10 cycles of 94 °C for 10 s, 55 °C for 45 s and 68 °C for 45 s, followed by an additional 25 cycles with 5 s elongation of each polymerization step at 68 °C per cycle.
Transfection and reporter assays.
Plasmid transfection in U373MGs was performed by the DEAEdextran method as described previously (Arlt et al., 1994). Routinely, 1 µg luciferase target and 2·3 µg transactivator plasmid were used. In co-transfection experiments with two transactivator plasmids, 1·15 µg of each were transfected. The total amount of transfected DNA was kept constant by using the cloning vector pcDNA3 (Invitrogen). In superinfection experiments with HCMV or baculovirus, U373MG cells were transfected with 1 µg of the luciferase target vector alone and superinfected at 24 h after transfection. At 48 h after transfection, cells were harvested and luciferase assays were performed as described previously (Kronschnabl et al., 2002
). Each transfection was determined in triplicate and was repeated at least three times.
Flow cytometry (FACS).
HFFs or HUVECs were infected with baculo-IE2p86, baculo-pp71 or baculo-control at an m.o.i. of 50. In co-infection experiments, the total amount of virus was kept constant (m.o.i. of 50) by co-infection with baculo-control. Cells were fixed with 3 % formaldehyde for 15 min at room temperature. For antibody staining, cells were first Fc-blocked with 0·5 mg Cohn-II fraction for 15 min on ice. Cells were then stained with a mouse monoclonal anti-CD54 R-Phycoerythrin (PE) conjugate at a 1 : 50 dilution. ICAM-1 cell-surface expression was measured by FACS analysis using a Becton Dickinson FACScan.
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Results |
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Discussion |
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Furthermore, upregulation of ICAM-1 directly contributes to the dissemination of HCMV via the peripheral blood, and thus this adhesion molecule also plays an important role in the pathogenesis of acute infections (Fish et al., 1995; Waldman et al., 1995
; Grundy et al., 1998
). Recent reports have demonstrated that ICAM-1-mediated cell interactions are essential for microfusion events taking place between polymorphonuclear leukocytes (PMNLs) and infected endothelial cells (Gerna et al., 2000
, 2002
). Virus is delivered via these fusion events from the endothelium to transiently adherent PMNLs, which are implicated in the subsequent haematogenous spreading of HCMV. Transmission could be prevented by inhibition of the contact between the two cell populations and by the use of monoclonal antibodies to CD18 and ICAM-1. Therefore, ICAM-1 upregulation by HCMV is a crucial determinant of viral dissemination and is even proposed as a surrogate marker of pathogenicity as well as attenuation of HCMV strains (Gerna et al., 2000
; Knight et al., 2000
).
The aim of this study was to characterize in more detail the mechanism by which HCMV activates ICAM-1 gene expression. Since we and others have shown that ICAM-1 protein and mRNA levels are increased after HCMV infection, this suggested that regulation occurs at the level of promoter activation (Burns et al., 1999; Knight et al., 2000
). Consistent with this, we observed strong ICAM-1 promoter activation after superinfection of transfected cells with HCMV. Deletion analysis revealed that 135 bp upstream of the transcriptional start site of the ICAM-1 promoter were sufficient for strong activation by HCMV. Interestingly, although the NF-
B element at -140 bp upstream of the transcriptional start site has been shown to be responsible for the induction of ICAM-1 by several mediators of inflammation such as IFN-
, TNF-
or IL-1
(van de Stolpe & van der Saag, 1996
; Roebuck & Finnegan, 1999
), this transcription factor binding site was not influential on the ICAM-1 stimulation by HCMV, since an internal deletion of the NF-
B binding site did not change ICAM-1 promoter activation. Instead, the Sp1-binding site at -53 bp upstream of the transcriptional start site was identified, both by deletion analysis and by site-directed mutagenesis, as a response element for HCMV-mediated ICAM-1 promoter stimulation. This result is surprising in view of several previous publications indicating that HCMV infection induces NF-
B and that NF-
B binding sites constitute important control elements for virus-regulated gene expression (Sambucetti et al., 1989
; Kowalik et al., 1993
; Yurochko et al., 1995
; Sun et al., 2001
).
In transient transfection experiments we identified the IE2p86 protein as a potent transcriptional activator of the ICAM-1 promoter. In addition, the tegument protein pp71 was found to have a strong synergistic effect on promoter activation by IE2p86, while pp71 alone had no effect. One potential explanation for the observed synergistic stimulation could be enhanced IE2p86 expression due to pp71-mediated transactivation of the MIEP, since IE2p86 expression in plasmid pHM134 is driven by this promoter (Liu & Stinski, 1992). This possibility, however, was excluded by transfection experiments using an IE2p86-expressing plasmid under the control of the RSV LTR. With this construct, a comparable synergistic induction of ICAM-1 promoter activity by IE2p86 and pp71 was measurable while pp71 was not able to enhance IE2p86 protein expression (see Fig. 3
). Furthermore, the synergy between IE2p86 and pp71 seems not to be confined to the ICAM-1 promoter, since this has previously been reported for the US11 promoter of HCMV (Chau et al., 1999
). Thus, the results obtained by transient transfection analysis suggested that the viral tegument protein pp71, in conjunction with the IE protein IE2p86, is able to stimulate ICAM-1 gene expression.
In order to confirm that these two viral regulatory proteins are capable of stimulating the endogenous ICAM-1 gene, we generated recombinant baculoviruses expressing pp71 or IE2p86 under the control of the HCMV MIEP. Previously, it has been shown that recombinant baculoviruses can efficiently transduce a variety of mammalian cells, and we observed in Western blot experiments that IE2p86 and pp71 were easily detectable after infection of HFF cells. The functionality of the baculovirus-expressed proteins was assessed by transfection of an ICAM-1 promoterluciferase construct followed by superinfection with baculoviruses. This revealed synergistic promoter activation by co-infection with baculoviruses expressing pp71 and IE2p86. Most importantly, we were also able to detect an increase in endogenous ICAM-1 on the surface of baculovirus-infected HFF or HUVEC cells, as measured by FACS analysis. Again, pp71 was able to enhance the IE2p86-mediated ICAM-1 induction. Interestingly, however, there was a difference between the effects of pp71 on ICAM-1 promoter activation and cell-surface expression: while no significant promoter activation could be detected, we observed an increase in ICAM-1 surface expression after infection with baculo-pp71 alone. This could be due to the pleiotropic effects of this viral regulatory protein, which may not be confined to transcriptional regulation, as it has recently been proposed that pp71 may exert a function comparable with the ubiquitin E3 ligase ICP0 of herpes simplex virus (Marshall et al., 2002). The overall induction levels in the presence of both viral regulators correlated well with those observed after HCMV infection (see Fig. 1A
). This strongly suggests that IE2p86 and pp71 are mainly responsible for HCMV-induced ICAM-1 upregulation, also supported by recent results demonstrating an inhibition of ICAM-1 stimulation by an antisense oligonucleotide complementary to the IE2 RNA (Cinatl et al., 2000
).
In accordance with our results, Burns et al. (1999) also identified the IE2p86 protein of HCMV as a transactivator of the ICAM-1 promoter in transient transfection experiments. In their assays, a minimum of 370 bp of 5' flanking sequences of the ICAM-1 gene were required for maximal transactivation by IE gene products. In contrast, our investigations revealed that 135 bp of 5' sequences were sufficient for stimulation of the ICAM-1 promoter by IE2p86. The reason for this discrepancy is unclear at present, but could be due to the different cell types used for transfection experiments. Moreover, we identified the Sp1-binding site at -53 bp as a target site for strong IE2p86 and pp71 activation. Even a promoter construct with a single Sp1-binding site upstream of the ICAM-1 minimal promoter was as strongly activated as the wild-type promoter. However, the synergistic action of pp71 was not strictly dependent on the Sp1-binding site, since pp71 also enhanced IE2p86 transactivation of the -277Sp1mut construct of the ICAM-1 promoter, although to a lesser degree (data not shown).
Previous studies have demonstrated that Sp1-binding sites are also crucial for maximal IE2p86-mediated activation of the HCMV DNA polymerase promoter and the cellular promoters of the NF-B subunits p65 and p105/p50, respectively (Yurochko et al., 1995
, 1997; Luu & Flores, 1997
; Wu et al., 1998
). In these reports, IE2p86 was shown to increase the Sp1 DNA-binding activity, which could be responsible for promoter activation. Results from other studies have suggested a direct interaction of the IE2p86 protein with the Sp1 transcription factor (Lukac et al., 1994
; Yurochko et al., 1997
). Furthermore, several recent publications have suggested that Sp1-mediated transcriptional activation could also be modulated by phosphorylation events (Fojas et al., 2001
; Milanini-Mongiat et al., 2002
). Thus, there are various possibilities as to how IE2p86, in conjunction with pp71, transactivates the ICAM-1 promoter via the Sp1-binding site. So far, we have not detected elevated protein levels of Sp1 after infection of HFF cells with baculo-IE2p86 in Western blot analysis (data not shown). In conclusion, we hypothesize that IE2p86 may either use Sp1 as a bridging factor to facilitate increased binding of the basal transcription apparatus or may modulate the phosphorylation status of Sp1. A further detailed analysis of the exact mechanism of transactivation will be important in order to develop strategies that can interfere with HCMV-mediated upregulation of ICAM-1 and might thus be able to inhibit viral dissemination as well as the development of vascular pathology.
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ACKNOWLEDGEMENTS |
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Received 10 July 2002;
accepted 28 August 2002.