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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MAIN TEXT |
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The ability of HCMV to perturb normal cellular control mechanisms has been studied intensively. Changes in cellular gene expression occur immediately after binding of virions to the cell (Boldogh et al., 1990; Simmen et al., 2001
; Yurochko & Huang, 1999
). Similarly, expression of the viral immediate-early (IE) and early (E) genes also results in physical and functional interactions between the viral gene products and cellular factors, resulting in perturbation of cellular transcription, cell cycle control and the secretion of chemokines and cytokines (Fortunato et al., 2000
).
The expression pattern of various cell surface proteins is also modulated during HCMV infection. It has been shown that HCMV leads to the upregulation of cellular adhesion molecules on endothelial cells as well as fibroblasts, such as the intercellular adhesion molecule 1 (ICAM-1). The increased expression of these adhesion molecules augments the adherence and infiltration of inflammatory cells that are capable of promoting vascular disease (Lemstrom et al., 1995; Steinhoff et al., 1995
; Yilmaz et al., 1996
; Martelius et al., 1998
; Waldman et al., 1998
; Lautenschlager et al., 1999
; Kronschnabl & Stamminger, 2003
). In contrast, killing of infected cells by cytotoxic T cells is inhibited by downregulating the cell surface expression of MHC class I receptors by a variety of specific mechanisms (Alcami & Koszinowski, 2000
; Warren et al., 1994
). Other cell surface proteins associated with peptide processing have also been reported to be downregulated during HCMV infection (Phillips et al., 1998
). Furthermore, a negative regulation of the epidermal growth factor receptor (EGFR) could be demonstrated. Interestingly, EGFR can serve as both a specific receptor for HCMV attachment and a mediator of HCMV-induced signal transduction (Fairley et al., 2002
; Wang et al., 2003
). Thus, downregulation of cell receptors, resulting in the abrogation of receptor-mediated cell signalling, may be a common occurrence during HCMV infection.
In this study, we investigated whether Thy-1 (CD90), a member of the group of adhesion molecules in the immunoglobulin superfamily, is similarly deregulated by HCMV infection. Thy-1 is a cell surface glycoprotein with a molecular mass of 35 kDa. In humans, constitutive Thy-1 expression is restricted to neuronal cells, fibroblasts and a subset of CD34+ blood stem cells (Craig et al., 1993). Fibroblasts possess a very high basal level of Thy-1 surface expression and therefore Thy-1 can be considered as a marker protein for this cell type. It is constitutively expressed in lipid rafts (Sharom & Lehto, 2002
) and is induced during the early phase of wound healing suggesting an activation of Thy-1 expression by inflammatory mediators (Saalbach et al., 1996
). Moreover, Thy-1 shows an inducible expression on endothelial cells and is detectable on microvascular endothelial cells during neoangiogenesis (Lee et al., 1998
). A so far unknown ligand of human Thy-1 has been postulated on monocytes and polymorphonuclear leukocytes and is suggested to mediate the binding to activated Thy-1-positive microvascular endothelial cells and fibroblasts (Saalbach et al., 2000
, 2002
). Additionally, the Thy-1 protein has been shown to be involved in the attachment of fibroblasts to extracellular matrix components, such as fibronectin and collagen I (Saalbach et al., 1998
).
In this report, we have demonstrated that HCMV leads to downregulation of Thy-1 protein levels after infection of human foreskin fibroblasts (HFFs). HFFs were infected with HCMV, strain AD169 (m.o.i. of 2) or mock infected. The cells were harvested at various time points post-infection (p.i.) for determination of Thy-1 cell surface expression by flow cytometry (FACS) analysis. In parallel, HCMV-mediated induction of ICAM-1 cell surface expression was monitored as a control. For FACS analysis, cells were fixed with formaldehyde (3 %, v/v, in PBS) and incubated with the Cohn-II fraction of human immunoglobulin (1 mg ml1) to avoid non-specific antibody binding. Thereafter, cells were stained with specific antibodies [fluorescein isothiocyanate (FITC)-conjugated anti-human ICAM-1 (Calbiochem); phycoerythrin (PE)-conjugated anti-human Thy-1; mouse IgG1 PE-conjugated and mouse IgG1 FITC-conjugated isotype control antibodies] and analysed with a Becton Dickinson FACScalibur with CELLQUEST software (BD Pharmingen). As a central finding, HCMV infection drastically decreased the expression of Thy-1 on the surface of HFFs (Fig. 1A). After 72 h, 77·3 % (Fig. 1A
; sum of cells present in lower-left and lower-right quadrants) of the infected cells showed a reduced level of Thy-1 surface protein. In contrast, the ICAM-1 cell surface expression was increased in 30·1 % of the cells at 72 h p.i. (Fig. 1A
; sum of upper-right and lower-right quadrants). Similarly, Thy-1 downregulation was also observed after infection with other HCMV strains, such as TB40E, Toledo and a clinical isolate (data not shown).
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The binding of HCMV virions to cellular receptors is sufficient for the activation of signalling cascades and the rapid upregulation of cellular transcription factors, such as Sp1 and NF-B (Yurochko et al., 1997
). Thus, the activated state of transcription factors is a hallmark of numerous regulatory effects induced by HCMV attachment. Therefore, we investigated whether the observed Thy-1 modulation was already affected by the physical interaction between the virus and the target cell. First, the human anti-HCMV monoclonal antibody (mAb) C23 was used to interfere with penetration, but not attachment of HCMV (Ohizumi et al., 1992
). Infection of HFFs with neutralized virions did not alter the Thy-1 cell surface expression indicating that virus attachment is not sufficient for Thy-1 modulation by HCMV (Fig. 2
A). To analyse whether penetration of the virions is able to induce Thy-1 downregulation, experiments with UV-inactivated virus were performed. Inoculation of HFFs with UV-inactivated HCMV resulted in a small decrease in Thy-1 cell surface expression; however, the effect was minor in comparison with infection with untreated virus (Fig. 2B
). Taken together, these data indicated that neither adsorption nor penetration of HCMV virions is sufficient for the downregulation of Thy-1 expression.
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In the next step, HFFs were infected with a recombinant GFP-expressing HCMV (AD169GFP) to distinguish between infected and non-infected cells by FACS analysis (Fig. 3A). Again, infected GFP-positive cells showed a clear decrease in Thy-1 cell surface expression. Interestingly, GFP-negative, non-infected cells also showed a reduced cell surface expression compared with mock infection. This finding suggested that a soluble factor might be induced after HCMV infection mediating Thy-1 downregulation. To confirm this result, co-cultivation experiments were performed. HFFs were infected with HCMV (AD169GFP) and thereafter co-cultivated with untreated cells in Transwell chambers. After 72 h, cells were harvested and the Thy-1 surface expression of both cell populations (infected and non-infected, co-cultivated cells) was determined by FACS analysis. Infected (Fig. 3B
, a and c) as well as co-cultivated cells (Fig. 3B
, b and d) showed a significantly decreased level of Thy-1 cell surface expression. Thus, these results excluded the influence of cell-to-cell contact on HCMV-mediated Thy-1 regulation and supported the conclusion that a soluble factor is involved.
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Perturbation of cellular functions appears to be essential for viruses to optimize the cell for productive infection. Thus, a number of cellular genes involved in immune modulation as well as chemokine and cytokine production have been described as being regulated by HCMV. The upregulation of cellular adhesion molecules such as ICAM-1, which augments the adherence and infiltration of leukocytes, directly contributes to the dissemination of the virus via the peripheral blood, thus playing an important role in the pathogenesis of acute infection (Grundy et al., 1998). In contrast, other cell surface markers that are involved in immune surveillance are counter-regulated by HCMV. The downregulation of MHC class I and other peptide-processing cell surface molecules has been studied in detail (Ahn et al., 1996
; Jun et al., 2000
; Phillips et al., 1998
). Here, we demonstrated that virus infection of fibroblasts, which constitutively express high amounts of Thy-1, led to a downregulation of this cellular adhesion molecule. Whether Thy-1 expression is also modulated in endothelial cells after HCMV infection, where Thy-1 has been described as a marker of neoangiogenesis (Lee et al., 1998
), needs further investigation. We have shown that virus attachment is not sufficient, and viral IE and/or E gene expression is necessary for this effect. Furthermore, evidence has been presented that Thy-1 was significantly downregulated in non-infected HFFs by a soluble factor induced after HCMV infection. Despite extensive investigation, the exact function of Thy-1 in various cell types remains unknown. It has been shown that Thy-1 acts as a cell adhesion molecule, which triggers the adhesion of polymorphonuclear leukocytes to activated endothelial cells and fibroblasts (Saalbach et al., 2000
). Furthermore, it has been demonstrated that Thy-1 mediates the adherence of fibroblasts to extracellular matrix components (Saalbach et al., 1998
). This might be in causal connection with the finding that Thy-1 is abundantly expressed in non-tumorigenic cells while expression could not be detected in several ovarian cancer cell lines with high migration capacity (Abeysinghe et al., 2003
). Besides Thy-1, several extracellular matrix transcripts are also downregulated during HCMV infection (Schaarschmidt et al., 1999
). Therefore, one molecular mechanism contributing to the cytopathic effects of HCMV infection may be the downregulation of proteins involved in cell structure and intercellular connection. Thus, we hypothesize that decreased Thy-1 surface expression after HCMV infection may promote the detachment of fibroblasts from extracellular matrix components, which may disturb the integrity of the vessel wall layer. Our findings suggest the possibility that Thy-1 is a regulated cellular factor possibly contributing to these HCMV-induced pathologies.
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ACKNOWLEDGEMENTS |
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Received 14 November 2003;
accepted 25 February 2004.