PI(3)K-Dependent Upregulation of Mcl-1 by Human Cytomegalovirus is Mediated by Epidermal Growth Factor Receptor and Inhibits Apoptosis in Short-Lived Monocytes

Abstract

Monocytes are a primary target for HCMV infection and are a key cell type responsible for hematogenous dissemination of the virus. Biologically these cells have a short life span of 1–3 days in the circulation, yet infected cells remain viable for weeks despite the lack of viral anti-apoptotic gene expression during this time period. To understand the mechanism by which HCMV inhibits the initial phase of monocyte apoptosis, we focused on the viral modulation of early pro-survival cell signalling events following infection. We demonstrate here that the viral upregulation of the phosphatidylinositol 3-kinase [PI(3)K] pathway promotes an early block in apoptosis following infection. Temporal transcriptome and protein analyses revealed Mcl-1, a member of the Bcl-2 family, was transiently induced in a PI(3)K-dependent manner during the early stages of HCMV infection. In accord with the survival studies, virally induced levels of Mcl-1 expression dissipated to mock levels by 72 hours post infection. Through the use of Mcl-1 specific siRNA, we confirmed the functional role that Mcl-1 plays as a key early regulator of apoptosis in monocytes. Lastly, we showed that HCMV engagement and activation of the epidermal growth factor receptor (EGFR) during viral binding triggered the upregulation of Mcl-1. Overall, our data indicates that activation of the EGFR/PI(3)K signalling pathway, via the PI(3)K-dependent upregulation of Mcl-1, is required to circumvent apoptosis in naturally short-lived monocytes during the early stages of HCMV infection, thus ensuring the early steps in the viral persistence strategy.

Introduction

Human cytomegalovirus (HCMV), a member of the Herpesviridae family, is endemic throughout the world with 50–90% of the adult population infected (1). In immunocompetent individuals, HCMV infection is generally asymptomatic, although infection can lead to monocucleosis (2) and is associated with several chronic inflammatory diseases such as atherosclerosis and inflammatory bowel disease (3, 4). In contrast, infection of immunocompromised hosts, including neonates, AIDS patients and transplant recipients, causes significant morbidity and mortality (5–7).

HCMV pathogenesis is a direct result of systemic viral spread to and infection of multiple organ sites that occurs during either asymptomatic or symptomatic infections (8–10). Viral dissemination to different organ systems occurs via a hematogenous route since a cell associated viremia is a prerequisite for viral spread (11, 12). Monocytes are principal in vivo targets of HCMV (12) and are the most abundant infiltrating cell type found in infected organs during primary infection (13, 14), suggesting these blood-borne immunological cells are responsible for mediating hematogenous dissemination of the virus. We have previously provided evidence that following infection with HCMV monocytes acquire a distinctive M1 pro-inflammatory phenotype (15), which we propose is necessary to mediate viral spread. Our data showed unique HCMV-induced functional changes, including increased production of M1 pro-inflammatory chemokines alongside an increase in select M2 anti-inflammatory chemokines and enhanced motility relative to monocytes activated by alternative agents such as LPS and PMA (10, 15, 16). Moreover, HCMV infection of monocytes induced their differentiation into macrophages, which, to our knowledge, is the only identified pathogen that can directly induce the monocyte-to-macrophage differentiation process (16, 17). For the virus, monocyte-to-macrophage differentiation appears to be an essential step in the viral survival strategy, because, in contrast to monocytes, which are non-permissive for viral replication, macrophages can support viral replication and the production of viral particles.

Although the biological changes in HCMV-infected monocytes discussed above provide the virus with the necessary tools to mediate spread to multiple host organ systems and to establish life-long viral persistence, infection of monocytes is not without significant biological hurdles. HCMV must subvert the intrinsic biological programming of monocytes to undergo rapid cell death within 1–3 days of entering the circulation (18). In addition to counteracting the short-life span of monocytes, HCMV must also neutralize the cellular anti-viral pro-apoptotic cascades. In replication permissive model cell lines, such as fibroblasts, control of the apoptotic cascade is mediated by immediate-early (IE) viral proteins, which are induced within a few hours after infection (19). In monocytes however, because viral gene expression/replication is not observed until 3–4 weeks post infection (16), HCMV must also have evolved a strategy to inhibit the pro-apoptotic cellular signalling pathways in the absence of de novo viral gene expression and replication. Deciphering the pro-survival mechanism(s) utilized by HCMV during primary infection of non-replication permissive monocytes, in order to bridge the gap between short-lived monocytes and long-lived macrophages, is critical to the understanding of viral dissemination and persistence within the infected host.

The rapid HCMV-induced functional changes that take place in monocytes during the initial non-replication permissive stages of infection occur in a temporal manner consistent with a receptor-ligand mediated process. We have previously shown that challenge with UV-inactivated HCMV or purified glycoprotein B (gB) induced rapid functional changes in monocytes similar to that seen with replication competent virus (16, 17). Consistent with a direct role of viral binding in cellular activation, we recently showed that activation of the cellular epidermal growth factor receptor (EGFR) during viral binding (20) and the subsequent activation of the downstream phosphatidylinositol 3-kinase [PI(3)K]/Akt signalling cascade was required for the viral induction of monocyte motility, adhesion to endothelial cells and tranendothelial migration (21, 22). Transcriptome analyses of 4 hour infected monocytes also revealed the PI(3)K-dependent upregulation of several transcripts encoding anti-apoptotic proteins (23). This regulation of multiple anti-apoptotic proteins by PI(3)K activity also suggests a direct role for this pathway in the early survival of monocytes.

Constitutive activation of the PI(3)K/Akt-1 pathway is known to promote the survival of cells differentiating along the monocyte/macrophage lineage through the expression of the downstream target Mcl-1 (24), a potent anti-apoptotic protein of the Bcl-2 family (25). During the early phase of myelopoiesis, Mcl-1 plays an obligate role in ensuring the survival of myeloid progenitors (26). Similar to myeloid progenitors, monocytes initially express high levels of Mcl-1, which rapidly diminishes during the short life-span of these cells (25). In vivo studies have demonstrated that monocytes have a life-span of 1–3 days upon entering the circulation (18); thus, we hypothesize that the declining Mcl-1 levels serve as a biological “clock” to ensure a controlled population of these pro-inflammatory immune cells. In contrast, Bcl-2 appears to be directly involved in both the differentiation of monocytes into and the long-term survival of macrophages upon initiation of the differentiation programming in short-lived monocytes (27). These data indicate the existence of two distinct viability strategies occurring prior to and after 48–72 h of monocytes entering the peripheral blood and that Bcl-2 family members may act as temporal viability “gates” along the myelopoiesis differentiation continuum. In vitro, adherent-monocytes exhibit low level activation and differentiation leading to survival through the early cell fate decision checkpoints (16, 28). However, cultured monocytes differentiating along the M1 continuum are sensitive to IL-10-induced apoptosis, via the downregulation of Bcl-2 family members, during the first 48 h of the differentiation process, but become resistant to the pro-apoptotic effects of IL-10 after 48 h (29), thus indicating the presence of a 48–72 h viability “gate” in vitro. Because HCMV-infected monocytes successfully navigate the 48–72 h viability “gate” in the absence of viral anti-apoptotic proteins, we focused our study on the early stages of monocyte survival following infection and the induced cellular mechanism(s) responsible for this survival.

In this study, we examined the mechanism utilized by HCMV to ensure the short-term survival of non-replication permissive monocytes, until the subsequent HCMV-induced macrophage differentiation reprogramming can occur. We demonstrate here that HCMV infection inhibited cell death of monocytes independent of anti-apoptotic viral IE protein expression and that the induction of PI(3)K activity following infection is critical to the rapid conversion of the infected monocyte into a pro-survival state. Under normal regulatory conditions the homeostatic levels of Mcl-1 rapidly declined; however, HCMV infection was able to slow the loss of Mcl-1, and thus decelerate the internal viability “clock” of short-lived monocytes. More specifically, we identified that the EGFR/PI(3)K signalling cascade initiates the activation of the early HCMV-infected monocyte signalosome required for the upregulation of Mcl-1 and the acquisition of apoptotic resistance during infection. Taken together, our data indicates that HCMV binding to cellular receptors during viral entry plays a pivotal role in the pathogenic induction of host anti-apoptotic pathways, thus circumventing apoptosis in naturally short-lived monocytes and promoting early events in the viral dissemination and persistence strategy.

Materials and Methods

Virus Preparation

HCMV (Towne/E strain; passages 35–45) was cultured in human embryonic lung (HEL) fibroblasts (16, 17). Virus was purified on a 0.5 M sucrose cushion, resuspended in RPMI 1640 media (Cellgro, Mediatech, Herndon, VA), and used to infect monocytes at a multiplicity of infection (MOI) of 5 for each experiment. We have previously shown that infection with a MOI 5 results in 100% of monocytes being infected with HCMV at 4 hpi (16). Monocytes were mock infected using equivalent volumes of RPMI 1640 media alone.

Human Peripheral Blood Monocyte Isolation

Isolation of human peripheral blood monocytes was performed as previously described (16, 17, 21, 30). Briefly, blood was drawn by venipuncture and centrifuged through a Ficoll Histopaque 1077 gradient (Sigma, St. Louis, MO) at 200 × g for 30 min at room temperature (RT). Mononuclear cells were collected and washed 2X with PBS + 1 mM EDTA to remove platelets at 150 × g for 10 min at RT. Monocytes were then layered on top of a 45% and 52.5% iso-osmotic Percoll gradient and centrifuged for 30 min at 400 × g at RT yielding a population >90% monocyte. Cells were washed twice with saline at 150 × g for 10 min at RT to remove residual Percoll and suspended in RPMI 1640 (Cellgro, Mediatech, Herndon, VA) supplemented with 10% human serum (Sigma). University Institutional Review Board and Health Insurance Portability and Accountability Act guidelines were followed for all experimental protocols.

Cell Viability Assays

Isolated monocytes (30,000 cells/experimental arm) were plated onto fibronectin coated microtiter plates for 1 h at 37°C. Following incubation, cells were mock infected or HCMV infected for varying times, after which monocytes were washed with warm media and varying concentrations of LY294002 added for an additional 24 h at 37°C. MTT [3-(4,5-dimethulthiazol-2-yl)-2,5-diphenyltetrazolium] cleavage was performed as directed by the manufacturer’s instructions (Zymed/Intermedico, Markham, CA) to assess cell viability.

To determine the frequency of apoptosis, TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin DNA-nick end labelling (35)] was performed as previously described (31). Briefly, after acetone:methanol fixation and PBS washing, the fraction of nuclei with nicked DNA was determined by TUNEL reaction. After the reaction was terminated by adding 300 mM sodium chloride plus 30 mM sodium citrate, the cells were washed with distilled water, and endogenous peroxidase activitywas neutralized by a 30 min incubation at RT with3% H2O2. Non-specific binding was blocked with 10% nonimmune goat serum at RT (Zymed/Intermedico) and a secondary biotinylated goat anti-mouse antibody and streptavidin-peroxidaseconjugate (Streptavidin Biotin System, Histostain-SP Kit; Zymed)added according to the manufacturer’s instructions. Nickel-diaminobenzidine substrate was added to culture wells to yield a dark brown precipitate localized to apoptotic nuclei. Monocytes were counterstained with hematoxylin (Sigma)to visualize total nuclei.

Western blot analysis

Monocytes were harvested in RIPA buffer [50 mM Tris-HCl (pH 7.5), 5mM EDTA, 100 mM NaCl, 1% Triton X 100, 0.1% sodium dodecyl sulfate (SDS) and 10% glycerol] containing 1X protease inhibitor cocktail (Sigma), 1X phosphatase inhibitor cocktail I (Sigma), and 1X phosphatase inhibitor cocktail II (Sigma) for 30 min on ice. The lysates were cleared by centrifugation at 4°C (2 min, 16000×g) and stored at −20°C until analyzed. Sample protein concentrations were determined in duplicate using the DC protein assay (Bio-Rad; Hercules, CA). Sample protein (15–20 μg) was solubilized in 6x sample buffer (Sigma) by boiling for 5 min and stored until electrophoresis. Equal amounts of total cellular protein from each sample were separated using SDS-PAGE followed by immunoblotting. Blots were blocked in a 5% milk – Tris-buffered saline –Tween 20 solution, followed by incubation overnight at 4°C with an anti-Bcl-2 antibody (Santa Cruz), an anti-Mcl-1 antibody (Santa Cruz), an anti-caspase 3 antibody (Calbiochem; San Diego, CA), an anti-pro-caspase 9 antibody (Calbiochem), an anti-cleaved caspase 9 antibody (Calbiochem) or an anti-β-actin antibody. Blots were then incubated with diluted horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences; Piscataway, NJ) for 1 h at RT, washed extensively and developed using Enhanced Chemiluminescence Plus (Amersham Biosciences) following the manufacturer’s protocol.

Affymetrix gene array and analysis

Ontology reanalysis of affymetrix gene data obtained from our previous studies were utilized to perform a global transcriptional evaluation of the HCMV-infected monocyte transcriptome at 4 (15), 24 (20) and 48 hpi (21). Briefly the following criteria were used for compilation of the data from all experiments. ANOVA tests were performed on the HCMV-infected versus mock-infected replicates and p-values were calculated for genes upregulated or downregulated. A p-value of ≤ 0.05 was used as the criteria for statistically significant genes among replicates. These criteria allowed us to generate a pool of genes that were statistically significant in all of the replicates following infection. An average fold-change of ≥1.5 HCMV-infected versus mock-infected samples were considered to be significantly regulated by infection. For a detailed description of the experimental procedures see the Materials and Methods section from our previous studies (15, 20, 21). The GEO accession number for these data are GSE11408 (15), GSE17948 (20) and GSE19772 (21).

siRNA silencing of Mcl-1

Monocytes (3 × 10⁶ cells/experimental arm) were resuspended in 100 μl Amaxa nucleofection solution for monocytes (Human Monocyte Nucleofector Kit; Amaxa Biosystems, Cologne, Germany) containing 300 μg Mcl-1 siRNA (Invitrogen) or control siRNA (Invitrogen) and transfected in an Amaxa nucleofector electroporator in accordance with the manufacturer’s instructions. The cells were immediately mixed with 500 μl of prewarmed human monocyte nucleofector medium, transferred into 1 ml of medium, and incubated at 37°C for 24 h. Following incubation, TUNEL and Western blot analysis were performed as described above.

Results

HCMV promotes survival of monocytes by inducing an anti-apoptotic state via the upregulation of PI(3)K activity

PI(3)K activity is essential to the survival of cells differentiating in the monocyte/macrophage lineage (24, 32); however, the extent to which PI(3)K activity functions to enhance monocyte survival prior to and after the 48–72 h viability “gate” is unclear. To determine if PI(3)K activity plays a prominent role in short-term and/or long-term viability, adherent monocytes, which exhibit low level activation and differentiation (16, 28), were treated with LY294002 [PI(3)K inhibitor] before and after the 48–72 h cell fate decision checkpoint. Monocytes treated with 25 μM LY294002 at time points prior to 72 h exhibited similar hypersensitivity kinetics to the cytotoxic effects of the PI(3)K inhibitor (Figure 1). However, although still sensitive to the cell death inducing properties of LY294002, monocytes treated at 72 h post isolation displayed significantly higher rates of cell survival than those treated with LY294002 at earlier time points. These data demonstrate the critical role of PI(3)K activity in the short-term survival of monocytes prior to the 48–72 h viability “gate”.

Figure 1. PI(3)K activity is critical for the survival of monocytes prior to the 48–72 h viability “gate”.

Monocytes were treated with DMSO or 25 μM of LY294002 at 1, 4, 24, 48 or 72 h following isolation from human peripherial blood. Cells were then incubated for an additional 0, 24 or 48 h following LY294002 treatment and the percent cell survival determined by MTT viability assay. Results are from 3 independent experiments from different donors. Lanes marked with an asterisk denote significance (P≤0.05).

We have previously shown that monocyte PI(3)K activity is stimulated within 10 min following infection with HCMV (20) and now show that the increased kinase activity is maintained through 96 hpi (Figure 2A). This rapid and chronic induction of PI(3)K activity suggested to us that PI(3)K activity may an important role in the protection of HCMV-infected monocytes from cell death prior the 48–72 h viability “gate”. To examine this possibility, cell viability analysis of mock-infected and HCMV-infected monocytes treated with varying concentrations of LY294002 at 24 hpi revealed a hypersensitivity of mock-infected monocytes to the inhibition of PI(3)K activity (Figure 2B). Uninfected and infected monocytes were resistant to the cell death inducing effects of LY294002 at 5 μM. In contrast, at 25 μM and 100 μM, HCMV-infected cells were protected from LY294002 treatment, while the viability of mock-infected cells decreased by 40% and 55% following treatment, respectively. Next, we examined the length of time that HCMV-infected monocytes exhibited a pro-survival state following infection. By 1 hpi HCMV-infected monocytes were partially resistance to LY294002 at 25 μM. A 20% decrease in cell viability was observed when compared to mock-infected cells which showed a 33% decrease (Figure 2C). The resistance of HCMV-infected monocytes to LY294002 increased with time until complete resistant was reached at 24 hpi. Infected cells exhibited resistance through 48 hpi. However, at 72 hpi HCMV-infected monocytes did not display enhanced protection from the effects of LY294002 treatment (Figure 2C), despite the high levels of PI(3)K activity at this time point (Figure 2A).

Figure 2. HCMV infection induces a rapid and transient PI(3)K-dependent pro-survival state in monocytes.

(A) Monocytes were mock infected or HCMV infected for 48, 72 and 96 h. Total lysate was harvested and phosphorylated PI(3)K [pPI(3)K], total PI(3)K, phosphorylated Akt (pAkt) and total Akt determined by immunoblotting. Membranes were reprobed with antibody against β-actin. (B) Monocytes were mock infected, HCMV infected (MOI 5) or UV-HCMV (at an equivalent MOI 5) infected for 24 h. Cells were then incubated with 5, 25, 50, 100 or 500 μM of the PI(3)K inhibitor, LY294002, for an additional 24 h. (C) Monocytes were mock infected, HCMV infected (MOI 5) or UV-HCMV infected (at an equivalent MOI 5) for 1–72 h, as indicated. Following the specified infection time, cells were treated with 25 μM of LY294002 for 24 h. (B, C) Percent cell survival was determined by MTT viability assay. Results are from 3 independent experiments from different donors. Lanes marked with an asterisk denote significance (P≤0.05).

Congruent with the MTT viability assays (Figure 2C), examination of DNA fragmentation associated with apoptosis utilizing TUNEL analysis found that HCMV-infected monocytes exhibited resistance to apoptosis induced by LY294002 (Figure 3A). Partial resistance from LY294002-induced apoptosis was observed at 4 hpi and complete resistance was observed by 24 hpi, which was then lost by 72 hpi. It should also be noted that no significant decrease in cell survival of HCMV-infected/LY-treated monocytes were observed at 4 hpi (Figure 2C), despite the induction of apoptosis (Figure 3A). Based on our analyses, in mock-infected monocytes, a 6-fold increase in the frequency of apoptosis was required to induce a 30% decrease in cell survival 24 hours after LY294002 treatment. Since HCMV infection of monocytes decreased the rate of LY294002-induced apoptosis to 2.5-fold, the threshold frequency required to detect any concurrent decrease beyond the standard deviation in monocyte survival is unlikely to be achieved. Monocytes challenged with UV-inactivated viral particles (UV-HCMV) displayed sensitivity kinetics to LY294002 treatment similar to that observed for “live” HCMV-infected cells, indicating newly synthesized viral gene products were not responsible for the induction of the monocyte pro-survival state following infection (Figure 2B, C and 3A). The lack of long-term protection of HCMV-infected monocytes to LY294002 treatment, despite the high levels of PI(3)K activity, suggest a complex signalling profile involving the simultaneous activation of multiple pathways during HCMV bind/entry is required for the induction of survival factors. Nevertheless, it appears that the rapid activation of the PI(3)K pathway is central to the favourable outcome during the early critical cell fate decision period that infected monocytes must navigate.

Figure 3. HCMV-infected monocytes exhibit maximum resistance to the induction of apoptosis following inhibition of PI(3)K activity at 24 hpi.

(A) Monocytes were mock infected, HCMV infected (MOI 5) or UV-HCMV infected (at an equivalent MOI 5) for 1–72 h, as indicated. Following the specified infection time, cells were incubated with 25 μM of LY294002 for an additional 24 h. Frequency of apoptosis was determined by TUNEL analysis. Results are from 3 independent experiments from different donors. Lanes marked with an asterisk denote significance (P≤0.05). (B) Monocytes were mock infected or HCMV infected (MOI 5) for 1, 24 or 72 h. Cells were then incubated with 25 μM of LY294002 for 30 min, 1, 2, 4, or 6 h. After treatment with LY294002, monocytes were harvested and Western blot analysis performed to examine pan levels and cleavage of pro-caspase 9 and pro-caspase 3. Results are representative of 3 independent experiments from different donors.

To expand our understanding of the early resistance to LY294002-induced apoptosis in monocytes following infection with HCMV, we next examined the cleavage and subsequent activation of caspase 3 and 9, which occur early in the apoptotic process, prior to DNA fragmentation. Caspase 3 and 9 are synthesized as inactive pro-caspase precursors of 32-kDa and 46-kDa, respectively. Caspase 3 is proteolytically cleaved into a 20-kDa and a 12-kDa subunit that together forms an intermediary protease with partial activity, which then undergoes a second cleavage event to generate the fully active 17-kDa/12-kDa protease (33). Pro-caspase 9 is processed into an active heterodimer consisting of a 35-kDa and a 10-kDa subunit. At 1 hpi and 72 hpi, no significant difference in caspase 3 and 9 cleavage was observed between mock-infected and HCMV-infected monocytes following LY294002 treatment (Figure 3B). In contrast, when LY294002 was added at 24 hpi there was no stimulation of the cleavage of pro-caspase 3 or pro-caspase 9 in HCMV-infected monocytes, while both caspases were efficiently proteolytically cleaved in mock-infected cells. In accord, on Western blots where HCMV-infected and mock-infected samples were run simultaneously to allow for a direct comparison, the basal levels of cleaved caspase 9 in HCMV-infected/LY294002-treated cells were similar to the basal levels observed in mock-infected/mock-treated monocytes (data not shown). At 24 hpi, the presence of the fully active 17-kDa activated subunit was not observed in HCMV-infected monocytes following treatment with LY294002, although the presence of the intermediary 20-kDa subunit of caspase 3 was observed. Our data indicate that PI(3)K is rapidly activated in monocytes following infection with HCMV and that this activated signalling pathway results in the initiation of a cellular anti-apoptotic reprogramming of short-lived monocytes.

Mcl-1 is rapidly induced in HCMV-infected monocytes in a PI(3)K-dependent manner

Viral IE anti-apoptotic genes, including IE1-72, IE2-86, UL36 [viral inhibitor of caspase-8 activation (v-ICA)] and UL37 [viral mitochondria-localized inhibitor of apoptosis (v-MIA)] are some of the first gene products produced during productive HCMV infection (34). We have previously shown that viral gene expression and replication does not occur in infected monocytes until ~3 weeks post-infection (16), thus the involvement of these viral anti-apoptotic proteins in the acquisition of a pro-survival state within 24 hpi in HCMV-infected monocytes is not likely. We confirmed that IE1-72, IE2-86, UL36 and UL37 were not transcribed in monocytes during the first 72 hours of infection with HCMV (Figure 4), despite internalization of the viral particle within 1 hpi (16, 20). Infection of a control HEL fibroblast cell line showed IE1-72, IE2-86 and UL36 gene expression by 24 hpi and UL37 transcription by 48 hpi. The absence of these IE gene transcripts during the initial 72 hours of monocyte infection indicates that new viral gene products are unlikely to regulate the early events in HCMV-infected monocytes.

Figure 4. Anti-apoptotic HCMV IE genes are not expressed in monocytes during the first 72 h of infection.

Monocytes or HEL fibroblasts were infected with HCMV at a MOI of 5. At 24, 48 or 72 hpi, RNA was harvested and RT-PCR analysis performed to detect anti-apoptotic HCMV IE gene expression (IE1-72, IE2-86, UL-36 and UL-37). GAPDH expression is shown as a control.

Because our cell viability assays provided evidence that a cellular anti-apoptotic reprogramming of HCMV-infected monocytes occurred within the first 48 hpi, we analyzed our transcriptome data bases from our previously published studies to create a temporal analysis of HCMV-infected monocytes at 4 hpi, 24 hpi and 48 hpi (15, 20, 21). Ontology examination of cellular anti-apoptotic genes revealed 35, 18 and 7 anti-apoptotic genes were upregulated ≥1.5-fold at 4 hpi, 24 hpi and 48 hpi, respectively (Table 1), indicating that HCMV infection transcriptionally induces a pro-survival cellular environment in monocytes following infection. The induction of multiple anti-apoptotic genes likely counteracts the pro-apoptotic anti-viral processes activated upon viral entry and the intrinsic programming of monocytes to undergo cell death within 3 days of entering the circulation (18, 35). The decline in the number of upregulated anti-apoptotic genes over 48 hours hints that the remaining elevated transcripts are critical to the long-term survival of infected monocytes. We identified two members of the Bcl-2 family, Mcl-1 and Bcl-2, which were upregulated at 24 hpi and 48 hpi, respectively. These factors have been shown to be involved in the survival of macrophages (24, 27) and to regulate mitochondrial mediated apoptosis. Since our data showed the inhibition of LY294002-induced caspase-9 cleavage, which occurs upon activation of the mitochondrial stress pathway, in HCMV-infected monocytes, we focused our examination on the potential role mitochondrial membrane permeabilization regulators, Mcl-1 and Bcl-2, had in the survival of infected monocytes.

Table I.

Select statistically significant anti-apoptotic mRNAs that increase ≥1.5-fold following infection with HCMV

Full Gene Name	Probe Set	Gene Title	Fold Change
			4 hpia	24 hpib	48 hpic
Activating transcription factor 5	39158_at	ATF5		12.1
Annexin A4	37374_at	ANXA4		3.0
Baculoviral IAP repeat-containing 3	1717_s_at	BIRC3	2.4
B-cell CLL/lymphoma 2	1909_at	BCL2	2.1		2.2
B-cell CLL/lymphoma 6	40091_at	BCL6	1.6
BCL2-associated athanogene	34798_at	BAG1		4.5
BCL2-like 1	34742_at	BCL2L1	1.7
BCL2-related protein A1	2002_s_at	BCL2A1	4.8
BRCA1 associated RING domain 1	1801_at	BARD1		4.0
CASP8 and FADD-like apoptosis regulator	1868_g_at	CFLAR	4.0	5.2	3.0
Caspase 3, apoptosis-related cysteine peptidase	36143_at	CASP3		2.6
Catenin, alpha 1, 102kDa	2085_s_at	CTNNA1		2.1
Cell division cycle 2	33324_s_at	CDC2	3.2
Chemokine (C-C motif) ligand 2	34375_at	CCL2	1.8	34.4
Cofilin 1	33659_at	CFL1		1.8
Cyclin-dependent kinase inhibitor 1A (p21, Cip1)	2031_s_at	CDKN1A		6.1	2.3
Fas (TNF receptor superfamily, member 6)	1440_s_at	FAS	2.6	2.9
Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide	36889_at	FCER1G		2.4
Glutamate-cysteine ligase, catalytic subunit	31850_at	GCLC	2.8
Glutamate-cysteine ligase, modifier subunit	33163_r_at	GCLM	3.0		3.9
Heat shock 70kDa protein 1B	32965_f_at	HSPA1B	2.4
Heat shock 70kDa protein 9	41510_s_at	HSPA9	1.8
Immediate early response 3	1237_at	IER3	2.2
Interleukin 1, beta	39402_at	IL1B	2.7
Interleukin 10	1548_s_at	IL10	4.4		3.4
Interleukin 6 (interferon, beta 2)	38299_at	IL6	291.4		192.9
Interleukin 7	1159_at	IL7	4.6
Myeloid cell leukemia sequence 1	277_at	MCL1		1.6
Neuregulin 2	35089_at	NRG2	2.0
Neurogenic differentiation 1	36768_at	NEUROD1	4.2
Non-metastatic cells 5	36859_at	NME5	3.8
Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105)	1378_g_at	NFKB1	3.2
Nucleoporin 62kDa	39274_at	NUP62		4.9
Phosphoprotein enriched in astrocytes 15	32260_at	PEA15		1.7
Pim-1 oncogene	883_s_at	PIM1	3.7
Pim-2 oncogene	1633_g_at	PIM2	3.6
Presenilin 1	35658_at	PSEN1		8.3
Prolactin receptor	1078_at	PRLR	5.5
PROP paired-like homeobox 1	33086_at	PROP1	2.4
Serpin peptidase inhibitor member 2	37185_at	SERPINB2	2.2
Serpin peptidase inhibitor member 9	34438_at	SERPINB9	3.4
Signal transducer and activator of transcription 5A	506_s_at	STAT5A	3.0
Superoxide dismutase 2, mitochondrial	34666_at	SOD2	3.2
Suppressor of cytokine signaling 3	40968_at	SOCS3	6.4
Tumor necrosis factor	1852_at	TNF	14
Tumor necrosis factor receptor superfamily, member 6b, decoy	35381_at	TNFRSF6B		2.7
Tumor necrosis factor, alpha-induced protein 3	595_at	TNFAIP3	2.5
Tumor necrosis factor, alpha-induced protein 8	33243_at	TNFAIP8	3.7		2.9
Tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide	1235_at	YWHAZ		1.6
V-rel reticuloendotheliosis viral oncogene homolog A	1295_at	RELA	1.6

Anti-apoptotic genes were acquired from the following transcriptional profiling analyses:

^a(15),

^b(20) and

^c(21)

Analysis of protein expression levels confirmed the upregulation of Mcl-1 and Bcl-2 protein in HCMV-infected monocytes at 24 hpi and 48 hpi, respectively (Figure 5A). By 72 hpi Mcl-1 protein expression in HCMV-infected monocytes had returned to mock levels, while Bcl-2 protein levels remained elevated. High levels of Mcl-1 protein expression were initially observed in newly isolated mock-infected monocytes at 1 h post isolation and the levels rapidly dissipated over 72 h. In contrast, elevated levels of Mcl-1 was maintained in infected monocytes over the same time course, demonstrating that HCMV infection extended the period in which monocytes maximally express Mcl-1. Moreover, a detailed analysis revealed that, following isolation of peripheral blood monocytes, an induction of Mcl-1 protein in both mock-infected and HCMV-infected monocytes had occurred at 2 hpi. However, Mcl-1 expression declined by 4 h in mock-infected cells, while a sustained high level of Mcl-1 protein remained through 24 h in HCMV-infected cells (Figure 5B). Our data suggests that the decrease in the rate of Mcl-1 loss in HCMV-infected monocytes could be a central mechanism by which infected monocytes survive through the 48–72 h viability “gate”.

Figure 5. Mcl-1 is rapidly upregulated in HCMV-infected monocytes in a PI(3)K dependent manner.

Monocytes were mock infected or HCMV infected (MOI 5) for (A) 1, 24, 48 and 72 h or (B) 1, 2, 3, 4, 6, 8, 10, and 24 h. (C) Monocytes were mocked infected or HCMV infected (MOI 5) for 24 h. HCMV-infected monocytes were then treated with 0–200 μM of LY294002 for an additional 24 h. (D) Monocytes were mock infected or HCMV infected (MOI 5) for 1, 24, 48, and 72 h. Following the indicated infection times, cells were treated with 25 μM of LY294002 for 24 h. (A, B, C, D) Monocytes were harvested and Western blot analysis performed to examine Mcl-1 and Bcl-2 expression. Membranes were reprobed with antibody against β-actin to verify equal loading. Results are representative of 3 independent experiments from different donors.

Viability assays indicated that the expression of the cellular factor responsible for the increase in apoptotic resistance at 24 hpi was dependent on the PI(3)K signalling pathway (Figure 2A); thus, we examined whether Mcl-1 and/or Bcl-2 expression were regulated by PI(3)K activity. Monocytes infected for 24 h exhibited a dose dependent decrease in Mcl-1 levels in response to LY294002 treatment, whereas Bcl-2 expression was unaffected by LY294002 treatment (Figure 5C). Concurrent with data indicating that maximum protection from LY294002-induced apoptosis in HCMV-infected monocytes occurs at 24 hpi and 48 hpi, Mcl-1 protein expression remained high at these time points following LY294002 treatment (Figure 5D). In accord with the temporal apoptotic resistant state of HCMV-infected monocytes, our data suggest that the early signalling events following infection lead to the upregulation of Mcl-1 and that the chronic activation of PI(3)K alone is unable to sustain the increase in Mcl-1 expression. This correlation between Mcl-1 protein levels and resistance to LY294002-induced apoptosis suggest that elevated Mcl-1 expression may be the molecular link to the rapid generation of the anti-apoptotic state during the early stages of HCMV infection.

Mcl-1 expression is required for survival of HCMV-infected monocytes

To investigate if Mcl-1 is critical to the early survival of monocytes, we utilized siRNA specifically targeted against Mcl-1 and examined the relative rates of apoptosis. At 24 hpi, mock-infected and HCMV-infected monocytes were transfected with Mcl-1 siRNA for 24 h and examined for Mcl-1 expression. The knockdown of Mcl-1 levels was efficient as demonstrated by the ≥90% downregulation of Mcl-1 protein expression in transfected mock-infected (Figure 6A) and HCMV-infected monocytes (Figure 6B). Bcl-2 protein levels were unaffected by transfection of the Mcl-1 siRNA and transfection of the non-specific control siRNA had no effect on Mcl-1 expression levels. We found a 3-and 5-fold increase in the percentage of Mcl-1 deficient mock-infected and HCMV-infected monocytes undergoing apoptosis, respectively (Figure 6C). The elevated levels of apoptosis observed with Mcl-1 deficient HCMV-infected monocytes suggests an initiation of pro-apoptotic signalling pathways following infection and that Mcl-1 plays a central role in counteracting this early cellular anti-viral response. Moreover, the rapid induction of apoptosis in mock-infected monocytes following knockdown with Mcl-1 siRNA provide the first documented evidence of a direct role that Mcl-1 plays in normal monocyte survival. Overall, these data imply that, despite the increased expression of a multitude of cellular anti-apoptotic factors in monocytes following HCMV infection (Table 1), Mcl-1 is essential for the early steps of monocyte survival.

Figure 6. Mcl-1 is responsible for mediating apoptotic resistance in HCMV-infected monocytes.

(A) Monocytes were nucleofected with control siRNA or Mcl-1 siRNA and incubated for 24 h. (B, C) Monocytes were mock infected or HCMV infected (MOI 5) for 24 h. Following infection, HCMV-infected monocytes were nucleofected with control siRNA or Mcl-1 siRNA and incubated for an additional 24 h. (A, B) Monocytes were harvested and Western blot analysis performed to determine Mcl-1 and Bcl-2 expression. Membranes were reprobed with antibody against β-actin to verify equal loading. Results are representative of 3 independent experiments from different donors. (C) TUNEL analysis was performed to determine the level of apoptosis. Results are from 3 independent experiments with different donors. Lanes marked with an asterisk denote significance (P≤0.05).

HCMV engages EGFR to facilitate the upregulation of Mcl-1 and the acquisition of the apoptotic resistant phenotype in infected monocytes

To investigate the cellular trigger responsible for the activation of the PI(3)K signalling branch within the HCMV-infected monocyte signalosome and the subsequent upregulation of Mcl-1, we examined the potential role of EGFR engagement during viral binding. We have new data showing that EGFR is expressed on human peripheral blood monocytes and that activation of EGFR kinase activity following infection results in a rapid induction of PI(3)K activity (20). Consequently, we next examined if the PI(3)K-dependent upregulation of Mcl-1 in HCMV-infected monocytes required the activation of EGFR. Reanalysis of our previous transcriptome study (20), with a focus on Mcl-1 gene expression, revealed a 50% reduction in Mcl-1 expression in HCMV-infected monocytes pre-treated with functional blocking anti-EGFR antibody, while the presence of AG1478 (a pharmalogical inhibitor of EGFR) completely abrogated Mcl-1 upregulation in HCMV-infected monocytes (Figure 7A). Similarly, analysis of Mcl-1 protein levels from HCMV-infected monocytes pre-treated with anti-EGFR antibody or AG1478 paralleled the transcriptional data (Figure 7B). Densitometic analysis revealed a 50% and 95% decrease in Mcl-1 protein expression in anti-EGFR antibody and AG1478 pre-treated HCMV-infected monocytes, respectively. Treatment with anti-EGFR antibody or AG1478 did not affect Mcl-1 protein levels in (Figure 7C), virion binding to (20) or cell viability of (data not shown) mock-infected cells, suggesting that inhibition EGFR signal does not affect basal Mcl-1 expression. These data indicated that Mcl-1 is rapidly upregulated following infection with HCMV at the transcriptional level in a manner dependent on the activation of EGFR during viral binding.

Figure 7. HCMV stimulates the anti-apoptotic phenotype in infected monocytes via an EGFR-dependent upregulation of Mcl-1.

(A, B, C, D) Monocytes were treated with 10 μg/mL anti-EGFR antibody or 1 μM AG1478. (A, B, D) Following pre-treatment, monocytes were infected with HCMV. (A) Mcl-1 gene expression at 24 hpi was determined from a previous transcriptome study (20). At 24 hpi, (B) HCMV-infected or (C) mock-infected monocytes were harvested and total Mcl-1 and Bcl-2 expression was detected by immunoblotting. Membranes were reprobed with antibody against β-actin to verify equal loading. Results are representative of 3 independent experiments from different donors. (D) Following infection, monocytes were treated with 25 μM of LY294002 for 24 h. Frequency of apoptosis was determined by TUNEL analysis. Lanes marked with an asterisk denote significance (P≤0.05).

Since HCMV-infected monocytes pretreated with EGFR inhibitors displayed lower levels of Mcl-1, we next asked if these cells were hypersensitive to LY294002-induced apoptosis. Mock-infected monocytes treated with LY294002 for 24 h exhibited a 5-fold induction of apoptosis, while monocytes infected for 24 h prior to treatment with LY294002 exhibited similar levels of apoptosis to that of the untreated control cells (Figure 7D). However, inhibition of EGFR signalling prior to 24 h infection significantly reduced the HCMV mediated protection from LY294002-induced apoptosis. HCMV-infected monocytes pre-treated with anti-EGFR antibody and AG1478 exhibited a 3-fold and 6-fold increase in apoptosis in response to LY294002 treatment, respectively. The heightened sensitivity of AG1478 pre-treated HCMV-infected monocytes to LY294002 treatment is consistent with the lower levels of Mcl-1 expression observed in AG1478 pre-treated infected cells (Figure 7B). Overall, we propose that engagement and activation of EGFR by HCMV during viral binding/entry rapidly upregulates Mcl-1, which generates an anti-apoptotic microcellular environment. Biologically enhanced survival of infected monocytes would support viral spread by allowing the infected monocyte to survive until differentiation into virus replication permissive macrophages can occur.

Discussion

We have previously shown that HCMV infection of monocytes stimulates the polarization of infected cells towards a unique M1 pro-inflammatory phenotype, exhibiting secretion of M1 and M2 chemokines, “hyper” cell motility and monocyte-to-macrophage differentiation (15, 16). This HCMV-directed differentiation of infected monocytes into M1 macrophages is likely a critical step for viral persistence since HCMV requires macrophages for viral replication (16). However, in order for the monocyte-to-macrophage differentiation process to occur, HCMV-infected monocytes must be reprogrammed to survive past the 48–72 hr viability “gate.” The lack of anti-apoptotic viral IE proteins during this critical early cell fate decision period [(16), Figure 3] suggests that HCMV has evolved a specific mechanism to utilize available cellular factors to promote the survival of monocytes during the early stages of infection. In the current study, we provide functional evidence that HCMV usurps the EGFR/PI(3)K signalling pathway to rapidly upregulate the anti-apoptotic Mcl-1 protein following infection, thereby stimulating the acquisition of a pro-survival phenotype, a necessary step for the virally induced differentiation of monocytes to take place.

We speculate that Mcl-1 acts as a rapidly inducible, short-term effector of cell viability, allowing for the initiation of monocyte-to-macrophage differentiation, at least when maximally expressed. Indeed, Mcl-1 is rapidly and transiently upregulated in human myeloblastic leukemia cells upon exposure to several differentiation-inducing agents and is believed to prevent cell death, thereby allowing cellular differentiation (36). Similarly, the transient induction of Mcl-1 in HCMV-infected monocytes suggest that once the intrinsic monocyte programming to undergo cell death has been circumvented by Mcl-1, high levels are no longer required for the survival of the differentiating monocyte. Our data indicate that an apoptotic regulatory “switch” occurs at ~48–72 hpi when another anti-apoptotic factor, such as Bcl-2, becomes the predominant survival factor (over Mcl-1) in the HCMV-infected differentiating monocyte. The effects of Mcl-1 on cell viability are not as prolonged as the effects of Bcl-2 (37), and Bcl-2 expression is critical to the long-term survival of cells differentiating along the myeloid lineage (27). We found the PI(3)K-independent induction of Bcl-2 protein expression in HCMV-infected monocytes occurs after 48 hpi. Studies are currently underway in our laboratory to determine if Bcl-2 activity is critical for the long-term survival and differentiation of HCMV-infected monocytes. Together, these data suggest that HCMV may exploit the temporal differences in Bcl-2 family members to control short-term and long-term cell survival.

Although our data suggests that Mcl-1 and Bcl-2 play an essential role in the early and late phases of HCMV-infected monocyte survival, temporal transcriptome analysis of the infected monocyte provided evidence that several anti-apoptotic factors are involved in the complex regulation of apoptosis during HCMV-induce monocyte differentiation. Ontology analysis revealed that 35 (0.27% of the total genes examined), 18 (0.14%) and 7 (0.06%) anti-apoptotic genes were upregulated ≥1.5-fold at 4 hpi, 24 hpi and 48 hpi, respectively (Table 1), indicating a potentially complex regulation of the induction of a pro-survival state in HCMV-infected monocytes. The gradual decline in the number of upregulated anti-apoptotic genes following initial infection indicated that those remaining elevated at later time post infection including Bcl-2 are likely involved in the long-term survival of the infected differentiating monocyte. Overall, the overwhelming upregulation of anti-apoptotic gene expression during early infection suggest that HCMV evolved a mechanism to coerce the cellular signalling pathways into initiating a rapid conversion to an apoptotic resistant state, which we advocate is necessary for directing a favourable outcome when the critical cell fate decision is determined early in the monocyte-to-macrophage differentiation process.

The rapid degradation of Mcl-1 suggests that the anti-apoptotic activity of this pro-survival factor is tightly controlled and crucial to the early regulation of the short lifespan of monocytes (38, 39). In accord with other studies, we show that the homeostatic expression of Mcl-1 rapidly declines during the initial 24–72 hours of monocytes entering the peripheral circulation, suggesting to us that Mcl-1 may act as a biological “clock” to regulate the lifespan of unstimulated monocytes. We now show for the first time that Mcl-1 is directly involved in the survival of primary human monocytes and that HCMV infection is able to transcriptionally induce Mcl-1 expression, thus decreasing the rate of Mcl-1 loss and slowing the intrinsic monocyte pro-apoptotic “clock.” Similarly, transformation of normal monocytes into long-lived leukemia cell lines has been shown to require Mcl-1 (24, 40, 41). In addition, our data also indicate that post-transcriptional regulation of Mcl-1 occurs in monocytes following HCMV infection, since infected monocytes lacked elevated levels of Mcl-1 transcript at later times post infection (Table 1), but continued to exhibit high levels of Mcl-1 protein expression (Figure 5A). Our data indicate that the aberrant induction of Mcl-1 in HCMV-infected monocytes provides the rapid pro-survival signal necessary for the positive outcome of the early 48–72 h cell fate decision period that infected monocytes must navigate prior to differentiation into long-lived macrophages.

Several monocyte/macrophage tropic pathogens appear to utilize the anti-apoptotic properties of Mcl-1 to enhance host cell survival by inducing apoptotic resistance during infection (42–44). M. tuberculosis required the expression of a bacterial gene product in order to induce Mcl-1 upregulation following infection (42), and HIV indirectly upregulated Mcl-1 expression via the secretion of macrophage colony-stimulating factor following infection (44). This convergent evolution displayed by biologically distinct pathogens in the regulation of Mcl-1 demonstrates the essential function that this anti-apoptotic protein must have in regulating the viability of cells in the myeloid lineage. We now show for the first time a pathogen that has evolved a strategy to directly regulate the cellular signalling pathways responsible for modulating Mcl-1 expression. The unique EGFR-activating properties of HCMV provide the virus with a distinct survival advantage by immediately inducing Mcl-1 expression without the need of an intermediary signalling pathway or molecule. Unlike the aforementioned pathogens, the lack of HCMV replication in monocytes necessitates the rapid regulation of the host cellular anti-apoptotic pathways to ensure differentiation into viral replication-permissive macrophages.

In addition to the EGFR/PI(3)K signalling cascade, our data also suggest the involvement of other receptors and signalling pathways in the HCMV-mediated induction of Mcl-1. First, inhibition of EGFR by anti-EGFR antibody was 50% less effective than inhibition with AG1478. Integrins are activated during HCMV entry (45, 46) and are able to phosphorylate the cytoplasmic kinase domain of EGFR (47); thus, unlike anti-EGFR antibody, AG1478 is able to block “crosstalk” activation of EGFR by other receptors (Figure 8). Second, although we observed increasing levels of PI(3)K through 96 hpi (Figure 1A), the elevated levels of Mcl-1 message and protein dissipated by 72 hpi, suggesting a distinct combination of signalling events during HCMV infection is necessary to modulate Mcl-1 expression and function (Figure 8). Nonetheless, our data indicates that the EGFR/PI(3)K signalling cascade is a necessary component of the early monocyte signalosome required for the increased production of Mcl-1 and the acquisition of a pro-survival phenotype following infection with HCMV.

Figure 8. Model of the regulation of Mcl-1 expression in monocytes following HCMV infection.

HCMV virion particles bind to the surface of peripheral blood monocytes and engage multiple cellular receptors leading to the simultaneous activation of several signalling pathways. The EGFR/PI(3)K signalling axis is a central component of the early HCMV-infected signalosome necessary for the upregulation of Mcl-1. Activation of other receptors, such as integrins, during viral entry can indirectly lead to cross activation of EGFR/PI(3)K pathway and/or directly to the upregulation of Mcl-1.

HCMV engagement of EGFR can mediate multiple early events necessary for hematogenous dissemination including viral entry [independent of PI(3)K activity] and cellular motility [dependent on PI(3)K activity] (20). We now provide evidence of an additional biological function occurring in HCMV-infected monocytes that is mediated by the EGFR/PI(3)K cascade: the rapid acquisition of a pro-survival state. Our current study indicates that HCMV activates the EGFR signalling pathway and the downstream PI(3)K activity during viral binding/entry to induce Mcl-1. Overall, we suggest that a unique combination of receptor-ligand events during binding/viral entry is responsible for mediating the early anti-apoptotic state necessary for the survival monocytes through the 48–78 h viability “gate”; thus, allowing for the virally induced monocyte-to-macrophage differentiation and viral spread to occur. By deciphering how HCMV modulates these signal transduction pathways in order to promote viral dissemination and persistence, we hope to provide insight to the immunopathogenesis of HCMV and, thus, potentially identify novel therapeutic targets.