Department of Medicine, University of Cambridge, Level 5, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, UK1
Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow, UK2
Author for correspondence: John Sinclair. Fax +44 1223 336846. e-mail js{at}mole.bio.cam.ac.uk
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Abstract |
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Introduction |
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Like other DNA viruses, the ability of HCMV to perturb normal cellular control mechanisms is well established. Virus-induced changes in cellular gene expression occur immediately on binding of virus to the cell (Boldogh et al., 1990 ; Simmen et al., 2001
; Yurochko & Huang, 1999
). Similarly, expression of the viral IE and 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 and expression of secreted chemokines and cytokines (Fortunato et al., 2000
). Perturbation of specific cellular transcription factors has been related to transcriptional activation of viral and cellular genes required during infection, and disruption of cell cycle control is believed to optimize the cellular environment for viral DNA replication.
HCMV infection is also known to inhibit killing by cytotoxic T cells, by down-regulating cell-surface expression of MHC Class I receptors by a variety of specific mechanisms (Alcami & Koszinowski, 2000 ; Barnes & Grundy, 1992
; Warren et al., 1994
). Similarly, other cell surface proteins associated with peptide processing have also been shown to be down-regulated during HCMV infection, although the relevance of this is not yet known (Phillips et al., 1998
).
Here, we now show that down-regulation of cell receptors that mediate a variety of cell signals, resulting in the abrogation of receptor-mediated cell signalling, may be a common occurrence during HCMV infection in that HCMV infection also results in the perturbation of the receptor for EGF.
EGF is a key mediator of lung maturation in the foetus (Klein et al., 2000 ) and is known to stimulate production of surfactant proteins such as SP-A (Klein et al., 1995
) which play an important role in host innate defence in the lung (Crouch & Wright, 2001
) a major site of HCMV-mediated disease.
EGF is also known to be a general mitogenic stimulator of fibroblast and monocytic cell types (Carpenter & Cohen, 1976 ; Higashiyama et al., 1991
), but it can also inhibit growth in cells with high levels of EGFRs and has been suggested to be important in differential cell cycle control (Baker & Yu, 2001
; Bromberg et al., 1998
). Cellular response to EGF is mediated via EGFR and EGF ligand binding to the receptor results in receptor dimerization and stimulation of intrinsic tyrosine kinase activity which, in turn, results in receptor autophosphorylation (Prigent & Lemoine, 1992
; Lemmon & Schlessinger, 1994
) and initiaton of an intracellular signal transduction cascade involving MAPK and ERK (Daub et al., 1996
; Holt et al., 1996
). EGF has also been shown to act as an inhibitor of cytokine mediated apoptosis in some cell types (Garcia-Lloret et al., 1996
) and to affect cell motility through the MAP kinase pathway (Glading et al., 2000
).
Clearly, EGF has profound effects on cells. We believe that in order for a virus to efficiently hi-jack normal cellular functions it is essential for the virus to prevent the cell from responding to external cellular signals which might conflict with the virally induced signals required to optimize the cellular mileau for productive infection.
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Methods |
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FACS analysis.
For FACS analyses, unfixed infected or unfixed control cells were stained with a mouse monoclonal anti-EGFR antibody directly conjugated to PE (R+D Systems) or isotype-matched control antibody (Ig2B) directly conjugated to PE (R+D Systems). Similarly, MHC Class I expression was determined using a FITC-conjugated mouse anti-HLA Class I A, B and C or FITC-conjugated isotype matched control antibody (IgG1) (R+D Systems). Alternatively, ethanol-fixed infected or control cells were stained with a goat anti-EGFR (Santa Cruz) antibody or goat immunoglobulins as control antibody and detected with PE-conjugated donkey anti-goat immunoglobulins. An aliquot of each cell population was also fixed, as above, and stained with an FITC-conjugated mouse anti-IE72/IE86 antibody (Biosys) to determine levels of HCMV infection. Cells were analysed using a Becton Dickinson FACsort.
EGFR expression was also analysed at specific times of infection as follows. For IE expression, cells were pre-treated with cycloheximide (50 µg/ml) for 3 h prior to, and for 3 h during, virus adherence. After this adherence period, virus and cycloheximide were washed off the cells and infection was allowed to progress for 3 h in the absence of cycloheximide. After this time, actinomycin D (20 µg/ml) was added for a further 14 h. Cells were then harvested and stained for EGFR as above. To inhibit viral late gene expression, cells were infected in the continual presence of the viral DNA polymerase inhibitor phosphonoformate (100 µg/ml).
Detection of EGFR and its autophosphorylation.
Cells (1x107) were infected with HCMV at approximately 5 p.f.u. per cell. Control or HCMV-infected cells were labelled with [35S]methionine, harvested 24 h post-infection, lysed in EBC buffer and immunoprecipitated with anti-EGFR or anti-IE72 antibody as described previously (Hagemeier et al., 1994 ). Immunoprecipitates were analysed by SDSPAGE and autoradiography.
To detect EGF-mediated phosphorylation of the EGFR, cells were mock infected or infected with HCMV at 5 p.f.u. per cell. At various times after infection, recombinant EGF (10 ng/ml) was added for 1 min and cells were harvested and immunoprecipitated using an anti-EGFR antibody as described above. Immunoprecipitated complexes were then analysed by SDSPAGE and Western blotting using an HRP-conjugated anti-phosphotyrosine antibody (Santa Cruz), followed by detection using the ECL system (Amersham).
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Results |
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Consequently, we also tested whether RV798 (Jones et al., 1995 ), which is deleted for all gene products involved in MHC Class I down-regulation, was still capable of EGFR re-localization. Firstly, we confirmed that, in our hands, RV798 was incapable of down-regulating MHC Class I cell-surface expression, as has previously been shown (Jones et al., 1995
). Fig. 3
shows that, as expected, Class I expression on fibroblasts infected with HCMV AD169 is extensively down-regulated (panel b) whereas fibroblasts infected with RV798 show no such Class I down-regulation (panel c). Fig. 3
also shows that RV798 infection still resulted in down-regulation of EGFR (panel g). Consequently, it appears that inhibition of EGFR expression involves a novel function of HCMV not associated with MHC Class I down-regulation.
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Discussion |
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A number of DNA viruses also perturb cellular receptor function (Stewart et al., 1995 ) and, at least in certain cell types, this may result from receptor inhibition (Tollefson et al., 2001
). As HCMV is known to perturb cell-surface MHC Class I expression, we also looked for the ability of HCMV to perturb additional receptors. HCMV infection clearly resulted in a reduction of cell-surface EGFR as determined by FACS analysis and this virus-induced reduction in cell-surface expression of EGFR was also reflected in a lack of detectable total cellular EGFR as determined by protein immunoprecipitation and FACS.
Interestingly, similar down-regulation of EGFR is observed as a result of adenovirus infection (Stewart et al., 1995 ) and may be mediated at least in part by adenovirus E1A (Prudenziati et al., 2000
).
We also determined which HCMV gene products may be responsible for this effect on the EGF receptor. Firstly, UV-inactivated virus appeared not to have any effect on cell-surface expression of EGFR, implying that viral gene expression and not virus binding per se was required for this phenomenon. Secondly, restriction of virus infection to IE events also did not result in EGFR down-regulation. In contrast, treatment of cells with phosphonoformate, which prevents viral DNA replication and hence viral late gene expression, still resulted in receptor down-regulation. Consequently, we believe that HCMV early gene products are likely to be responsible for this virus-mediated EGFR perturbation.
As HCMV infection of fibroblasts has already been shown to result in down-regulation of cell-surface expression of at least one other cellular protein, MHC class I (Ahn et al., 1996 ; Jones et al., 1996a
; Jun et al., 2000
), and all the viral gene products associated with this phenomenon have been shown to be encoded in the US2-11 region of HCMV (Jones & Sun, 1997
; Jones et al., 1996b
; Jun et al., 2000
; Lehner et al., 1997
; Reusch et al., 1999
), we also tested a virus deleted of US2-11, RV798 (Jones et al., 1995
), for EGFR down-regulation. The observation that infection with RV798 still resulted in down-regulation of EGFR cell-surface expression suggests that a novel viral function, not associated with MHC Class I down-regulation, is responsible for this effect. Interestingly, the down-regulation of EGFR was also observed with myelotropic strains of HCMV such as TB40E (Sinzger et al., 2000
) as well as with other laboratory strains of HCMV such as Towne (data not shown).
Consistent with a lack of surface EGFR, HCMV-infected cells showed no EGF-mediated autophosphorylation of EGFR. Time-course analysis suggested that virus functions associated with viral gene expression were required for EGFR down-regulation as UV-inactivated virus showed no such receptor inhibition and this down-regulation did not occur prior to 6 h post-infection. This is, again, consistent with early and not IE viral gene products being responsible for EGFR perturbation.
EGF is known to be a general mitogenic stimulator of fibroblast cells (Carpenter & Cohen, 1976 ) but can also inhibit growth in cells expressing high levels of receptor (Gardner & Shimizu, 1994
). Consequently, it is possible that HCMV infection lowers EGFR on the infected cell to prevent the cell from responding to host EGF signalling, which might drive the cell into a state that conflicts with an optimal state for virus production. Also, more recently, it has been suggested that EGFR inactivity is required for progression though G1 into S phase (Baker & Yu, 2001
). Whilst conflicting data exist as to just how far through the cell cycle HCMV advances the infected cell (Bresnahan et al., 1996
; Dittmer & Mocarski, 1997
; Jault et al., 1995
; Lu & Shenk, 1996
; Murphy et al., 2000
; Salvant et al., 1998
; Sinclair et al., 2000
), it is clear that virus does induce cell functions associated with progression though G0/G1 into early S phase. Consequently, it is possible that the observed down-regulation of EGFR function by HCMV may be required in order for virus to mediate cell cycle advance in resting cells. However, it must be said that, at present, we are not certain of the specific effects of EGFR down-regulation on the infected cell.
Whilst we have not directly addressed whether the inhibition of steady-state levels of EGFR expression shown here result from transcriptional or post-transcriptional events, interestingly, inhibition of EGFR mRNA expression has also been observed on HCMV infection in human lung fibroblasts (Prosch et al., Abstracts of the 26th International Herpesvirus Workshop, abstr.12.22, 2001).
It is interesting to note that HCMV, as well as apparently inhibiting EGFR-mediated signalling in the cell, has been shown to activate certain arms of the extracellular signal-regulated kinase cascade (Johnson et al., 2000 ; Rodems & Spector, 1998
; Chen & Stinski, 2002
). It is therefore possible that HCMV infection results in the expression of viral factors that activate some, but not all, aspects of normal cellular EGF-mediated signalling as well as concomitantly inducing down-regulation of cellular EGFR to ensure that viral signals are dominant.
It is clear that HCMV infection results in the expression of a number of viral gene products which optimize the cell for virus productive infection by interdicting control of a number of normal cellular functions. We believe that an integral part of this is the ability of the virus to isolate the infected cell from host-specific signals forcing the cell to respond solely to virus signals and these specifically optimize the cellular environment for productive infection. An understanding of what mechanisms are used by the virus to hi-jack the cell and modify its response to cell signals will be important in fully understanding the biology and pathogenesis of HCMV.
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Acknowledgments |
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References |
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Received 22 May 2002;
accepted 17 July 2002.