Human cytomegalovirus suppresses Fas expression and function

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.

Human cytomegalovirus (HCMV), the prototype member of the subfamily Betaherpesviridae, is ubiquitous in human populations worldwide. HCMV establishes a lifelong persistent infection that is normally controlled by continuous host immune surveillance. Although the vast majority of infections in the immunocompetent host appear to be benign, HCMV is a major cause of severe morbidity and mortality following congenital transmission, and in immunocompromised individuals. Studies using murine and rhesus cytomegaloviruses have shown that efficient infection, superinfection and long-term persistence in vivo are dependent on effective viral immune-evasion functions (Babić et al., 2011;Früh et al., 2013;Vidal et al., 2013). HCMV also possesses an impressive array of immunomodulatory functions that are instrumental in avoiding T cells, natural killer (NK) cells, the interferon response and apoptosis.
HCMV UL36 (vICA) and MCMV m36 are positional homologues (no overt amino acid sequence homology) that suppress death receptor (DR)-mediated apoptosis by inhibiting caspase-8 activation and promoting virulence in vivo, respectively (Ebermann et al., 2012;Skaletskaya et al., 2001). In HCMV, cellular DRs are also targeted directly during infection. The laboratory strain AD169 downregulates TNFR1 from the cell surface (Baillie et al., 2003) more efficiently than low-passage strains (Montag et al., 2006). This inconsistency was explained when UL138 was found to stimulate surface expression of TNFR1; strain AD169 has suffered a deletion of the 15 kb region UL/b', which encompasses UL138 (Le et al., 2011;Montag et al., 2011). HCMV thus appears to encode functions capable of acting post-transcriptionally to suppress and 'potentiate' TNFR1 expression. We recently demonstrated that HCMV also regulates expression of a second DR: TNF-related apoptosis-inducing ligand (TRAIL) receptor (Smith et al., 2013). Although HCMV infection stimulates expression of TRAIL receptor 2 (TR2) in fibroblasts, gpUL141 binds TR2 directly to sequester the DR in the endoplasmic reticulum, thereby protecting HCMV-infected cells against both soluble TRAIL and TRAIL-dependent NK cell-mediated killing (Nemčovičová et al., 2013;Smith et al., 2013).
Since HCMV exhibits an exceptionally high level of interstrain sequence variation (Dolan et al., 2004), we were interested in determining whether Fas regulation is a conserved function. The level of Fas downregulation was similar in cells infected with HCMV strains Merlin, AD169, FIX and TB40 (Fig. 1d). Comparable results were also obtained using HFFF cells and primary dermal fibroblasts (data not shown). Variation in the efficiency of MHC class-I downregulation is attributable to the fact that strains FIX (DUS2, DUS3 and DUS6) and TB40 (DUS3 and DUS6) are derived from BAC clones, and were deleted in the US segment to facilitate genome manipulation (Murphy et al., 2003;Sinzger et al., 2008).
The sensitivity of HCMV-infected cells to Fas-mediated apoptosis was ascertained by measuring the activation of effector caspases 3 and 7. Cells were infected with HCMV strains Merlin or AD169 or mock-infected and treated with FasL or a cross-linking Fas mAb, soluble TR2 or an IgM control antibody. Caspase 3/7 activity was then measured at 16 and 72 h p.i. by its capacity to cleave a luminogenic substrate in the presence of a recombinant luciferase (Fig.  2). At 16 h p.i., prior to Fas downregulation at the cell surface, there was no significant difference in caspase 3/7 activity between mock-infected and HCMV-infected cells in any of the treatment groups (Fig. 2a). However, at 72 h p.i., cells infected with strains Merlin or AD169 became less sensitive to Fas signalling induced by either FasL or Fas mAb (Fig. 2b). In addition, there was no significant difference in the level of protection imparted to cells by strains Merlin and AD169. This is interesting, since the AD169 variant that was used in this experiment carries a single amino acid substitution in the UL36 gene that abolishes the anti-apoptotic function of vICA (Skaletskaya et al., 2001). HCMV infection therefore renders cells less sensitive to Fas-mediated apoptosis. This function correlates with Fas downregulation from the surface of infected cells, and can occur independently of vICA function.
Fas mRNA levels, as assessed by quantitative reverse transcriptase PCR (qRT-PCR), were not significantly affected by HCMV infection at 24, 48 or 72 h p.i. (Fig. 3a). Nevertheless, levels of Fas in total cell lysates appeared moderately reduced following infection with HCMV strains Merlin, AD169, Fix or TB40 (Fig. 3b). HCMV is known to suppress the cell surface expression of specific proteins (e.g. CD112, CD155, MHC-I, MICB, TR2, ULBP2), often by sequestering them within the cell (Cosman et al., 2001;Jones et al., 1996;Nemčovičová et al., 2013;Prod'homme et al., 2010;Smith et al., 2013;Tomasec et al., 2005). N-linked glycoproteins acquire resistance to endoglycosidase-H (EndoH) during maturation in the Golgi apparatus. Fas was clearly heavily glycosylated, as evidenced by its sensitivity to peptide N-glycosidase-F (PNGaseF), and was resistant to EndoH treatment±HCMV infection (Fig. 3c). Consequently, HCMV does not appear to retain newly synthesized Fas in pre-Golgi compartments. Immunofluorescence showed Fas to illuminate the surface   of uninfected fibroblasts, in addition to a diffuse cytoplasmic staining pattern (Fig. 3d). In cells infected with HCMV, Fas appeared largely excluded from the plasma membrane; rather, the protein localized to extended membranous perinuclear structures (Fig. 3d).
HCMV infection induces resistance to Fas-mediated apoptosis, yet the extent to which this can be attributed to cell surface suppression of Fas will ultimately require the identification of the HCMV gene(s) responsible. Despite systematic screening of an expression library encoding the canonical HCMV genes, the function responsible has yet to be mapped (Seirafian, 2013). In this context, multiple HCMV genes can be expected to impact Fas signalling. The UL36 and UL37 gene products efficiently inhibit Fas-mediated apoptosis by inhibiting caspase-8 activation and cytochrome c release, respectively (Arnoult et al., 2004;Goldmacher et al., 1999;Skaletskaya et al., 2001). Moreover, IE2 is known to upregulate c-FLIP, a proteasedeficient procaspase-8 homologue (Chiou et al., 2006), whilst the tegument protein UL45 suppresses Fas-mediated killing in the context of HCMV infection by an uncharacterized mechanism (Patrone et al., 2003). These functions operate at or downstream of the DISC, and are thus likely to impact on both TRAIL and Fas-mediated signalling to similar degrees. In addition, since UL141 downregulation of TR2 had a marked impact on TRAIL-mediated cell death (Smith et al., 2013), it is likely that HCMV downregulation of Fas is also an important component of HCMV immune evasion.
Autoimmune lymphoproliferative syndrome (ALPS) is a rare disorder characterized by abnormal lymphocyte survival resulting from a defect in Fas function. A study of two brothers with ALPS experiencing HCMV disease following neonatal exposure documented the development of disseminated infections that were eventually controlled (Arkwright et al., 2000). That Fas-mediated apoptosis is not critical for the control of HCMV disease is consistent with the virus having evolved effective countermeasures to evade Fas-mediated killing. The immune-evasion functions of HCMV are a realistic target for therapeutic intervention.