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. 2014 Apr;95(Pt 4):933-939.
doi: 10.1099/vir.0.058313-0. Epub 2014 Jan 6.

Human cytomegalovirus suppresses Fas expression and function

Sepehr Seirafian  1 , Virginie Prod'homme  1 , Daniel Sugrue  1 , James Davies  1 , Ceri Fielding  1 , Peter Tomasec  1 , Gavin W G Wilkinson  1
Affiliations

Human cytomegalovirus suppresses Fas expression and function

Sepehr Seirafian et al. J Gen Virol. 2014 Apr.

Abstract

Human cytomegalovirus (HCMV) is known to evade extrinsic pro-apoptotic pathways not only by downregulating cell surface expression of the death receptors TNFR1, TRAIL receptor 1 (TNFRSF10A) and TRAIL receptor 2 (TNFRSF10B), but also by impeding downstream signalling events. Fas (CD95/APO-1/TNFRSF6) also plays a prominent role in apoptotic clearance of virus-infected cells, so its fate in HCMV-infected cells needs to be addressed. Here, we show that cell surface expression of Fas was suppressed in HCMV-infected fibroblasts from 24 h onwards through the late phase of productive infection, and was dependent on de novo virus-encoded gene expression but not virus DNA replication. Significant levels of the fully glycosylated (endoglycosidase-H-resistant) Fas were retained within HCMV-infected cells throughout the infection within intracellular membranous structures. HCMV infection provided cells with a high level of protection against Fas-mediated apoptosis. Downregulation of Fas was observed with HCMV strains AD169, FIX, Merlin and TB40.

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Figures

Fig. 1.
Fig. 1.
Modulation of Fas cell surface expression in cells infected with HCMV. (a) HFFF-hTERTs were infected with HCMV strain Merlin (m.o.i. 10) or mock-infected, and analysed at indicated time points by flow cytometry for cell surface Fas expression [mAb142 (R&D Systems), n≥3). (b) HFFFs were infected with HCMV strain Merlin (m.o.i. 10, 72 h) in the presence (i) or absence (ii) of 100 µM ganciclovir and analysed by flow cytometry for cell surface Fas expression (n = 3). (c) HFFF-hTERTs were incubated with supernatants (sn) of strain Merlin-infected cells (m.o.i. 10, 72 h p.i.) from which virions had been removed using a 0.1 µm filter, or were infected with HCMV strain Merlin (m.o.i. 10, 72 h) or an equivalent γ-irradiated preparation (2500 Gy) and analysed by flow cytometry for cell surface Fas expression (n = 3). (d) HFFF-hTERTs were infected with HCMV strains Merlin, AD169, FIX or TB40 (m.o.i. 10, 72 h) and analysed by flow cytometry for cell surface Fas expression (n≥3). Control IgG staining is denoted by black and grey lines for mock-infected and HCMV-infected cells, respectively.
Fig. 2.
Fig. 2.
HCMV infection renders cells less sensitive to Fas-mediated apoptosis. HFFFs were infected with strain Merlin or AD169 (m.o.i. 10), or mock-infected. At 4 (a) or 60 (b) h p.i., cells were treated with cycloheximide (Sigma) at 10 µg ml−1 concentration and FasL (IBA-Lifesciences), Fas mAb (Beckman-Coulter), sTRAIL-R2 (control for Fas ligand) or IgM isotype control at 500 ng ml−1. Apoptosis was then measured at the indicated time points as caspase 3/7 activation using the Caspase-Glo 3/7 kit (Promega,). Results are presented as mean relative light units (RLU)±se (n = 4). P-values were calculated using a one-way ANOVA test and a Bonferroni post test.
Fig. 3.
Fig. 3.
Analysis of Fas expression in HCMV-infected cells. (a) HFFF cells were infected with strain Merlin (m.o.i. 10) or mock-infected, and Fas mRNA levels were analysed at indicated times by qRT-PCR: total cell RNA was extracted (Qiagen) followed by RT-PCR using random hexamer primers (Amersham Biosciences). Resulting cDNA was analysed by qPCR using primers specific to Fas or GAPDH and SYBR green dye (applied Biosciences). Relative quantity (RQ) values were calculated by the comparative CT method. Fas RQ values are shown normalized to GAPDH and relative to the ‘24 h p.i’. mock sample (±se, n = 3). P-values were calculated using a one-way ANOVA test and a Bonferroni post test. (b) HFFF-hTERT cells were infected with strains Merlin, AD169, FIX or TB40 (m.o.i. 10, 72 h), or mock-infected. Total cell lysates were analysed by Western blot (ERP5700, Abcam, n≥3). (c) Cell lysates equivalent to (b) were treated with EndoH (New England Biolabs) or PNGaseF (New England Biolabs) and analysed by Western blot (n≥3). (d) HFFF-hTERT cells were infected with strains Merlin or AD169 (m.o.i. 10, 72 h), or mock-infected, and Fas expression was visualized by immunofluorescence [mAb142 (R&D Systems), n≥3, shown in red in top panels]. In the lower panels, outlines of cells were visualized with phalloidin-AF488 (phall; Invitrogen) and overlaid with Fas staining. Scale bars, 10 µm.
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References

    1. Ahn K., Angulo A., Ghazal P., Peterson P. A., Yang Y., Früh K. (1996). Human cytomegalovirus inhibits antigen presentation by a sequential multistep process. Proc Natl Acad Sci U S A 93, 10990–10995 10.1073/pnas.93.20.10990 - DOI - PMC - PubMed
    1. Arkwright P. D., Rieux-Laucat F., Le Deist F., Stevens R. F., Angus B., Cant A. J. (2000). Cytomegalovirus infection in infants with autoimmune lymphoproliferative syndrome (ALPS). Clin Exp Immunol 121, 353–357 10.1046/j.1365-2249.2000.01304.x - DOI - PMC - PubMed
    1. Arnoult D., Bartle L. M., Skaletskaya A., Poncet D., Zamzami N., Park P. U., Sharpe J., Youle R. J., Goldmacher V. S. (2004). Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria. Proc Natl Acad Sci U S A 101, 7988–7993 10.1073/pnas.0401897101 - DOI - PMC - PubMed
    1. Babić M., Krmpotić A., Jonjić S. (2011). All is fair in virus-host interactions: NK cells and cytomegalovirus. Trends Mol Med 17, 677–685 10.1016/j.molmed.2011.07.003 - DOI - PMC - PubMed
    1. Baillie J., Sahlender D. A., Sinclair J. H. (2003). Human cytomegalovirus infection inhibits tumor necrosis factor α (TNF-α) signaling by targeting the 55-kilodalton TNF-α receptor. J Virol 77, 7007–7016 10.1128/JVI.77.12.7007-7016.2003 - DOI - PMC - PubMed

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