ABSTRACT
Human cytomegalovirus (HCMV) is a pervasive herpesvirus responsible for significant morbidity and mortality among immunodeficient/naive hosts. Following a primary HCMV infection, circulating blood monocytes mediate the systemic spread of the virus. Extending the short 48-h life span of monocytes is critical to the viral dissemination process, as these blood-borne cells are nonpermissive for virus replication until they are fully differentiated into macrophages. Here, we show that HCMV glycoprotein gB binding to cellular epidermal growth factor receptor (EGFR) during HCMV entry initiated a rapid (within 15 min) activation of the apoptosis suppressor Akt, which was maintained through 72 h. The virus-induced activation of Akt was more robust than that with the normal myeloid growth factor macrophage colony-stimulating factor (M-CSF) and was essential for infected monocytes to bypass the 48-h viability checkpoint. Activation of phosphoinositide 3-kinase (PI3K) following EGFR engagement by HCMV mediated the phosphorylation of Akt. Moreover, HCMV entry drove a switch away from the PI3K p110δ isoform, which was required for the viability of uninfected monocytes, to the p110β isoform in order to facilitate the Akt-dependent prosurvival state within infected cells. Simultaneously, in contrast to M-CSF, HCMV promoted a rapid increase in SH2 domain-containing inositol 5-phosphatase 1 (SHIP1) expression, leading to signaling through a noncanonical Akt activation pathway. To ensure maximum Akt activity, HCMV also induced an early phosphorylation-dependent inactivation of the negative regulator phosphatase and tensin homolog. Overall, our data indicate that HCMV hijacks the upstream Akt signaling network to induce a nontraditional activation of Akt and subsequently a prosurvival decision at the 48-h cell fate checkpoint, a vital step for HCMV's dissemination and persistence strategy.
IMPORTANCE HCMV is found throughout the world with a prevalence of 55 to 100% within the human population. HCMV infection is generally asymptomatic in immunocompetent or naive individuals but is a significant cause of morbidity and mortality among the immunocompromised. Widespread organ inflammation is associated with symptomatic infections, which is a direct consequence of the viral dissemination strategy. Inflammatory peripheral blood monocytes facilitate the spread of HCMV. However, HCMV must subvert the naturally short life span of monocytes. In this work, we demonstrate that HCMV induces the activation of Akt, an antiapoptotic protein, in a manner distinct from that of normal myeloid growth factors. Moreover, we decipher how HCMV dysregulates the upstream Akt signaling network during viral entry to promote an Akt-dependent prosurvival state following infection. Delineation of the virus-specific mechanisms that regulate cellular prosurvival pathways in order to drive the survival of HCMV-infected monocytes is important to identifying new anti-HCMV therapeutic targets.
INTRODUCTION
Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus, with the rate of seropositivity for HCMV among adults in the United States reaching 60 to 90% (1). HCMV infection is generally asymptomatic in immunocompetent individuals but has been associated with several chronic inflammatory diseases, such as atherosclerosis and inflammatory bowel disease (2, 3), and cancers, such as glioblastoma and colon cancer (4, 5). In immunodeficient hosts, including neonates, AIDS patients, transplant recipients, and patients undergoing chemotherapy, HCMV is a significant cause of morbidity and mortality (6–8). HCMV disease is characterized by systemic multiorgan inflammation, which can lead to end-organ dysfunction (9, 10). HCMV pathogenesis is a direct result of the virus's ability to disseminate throughout the body and establish a persistent infection.
Monocytes are the primary cell type in blood infected by HCMV during an acute infection and the main cell type found in the infected organs of transplant recipients, indicating a central role for these blood sentinels in disseminating the virus (11–14). However, in the absence of myeloid growth factors, monocytes are naturally short-lived with a life span of ∼48 h after release from the bone marrow into the circulation (15). Further complicating matters, monocytes do not support viral replication unless they are differentiated into macrophages (16–19). We have previously shown that HCMV circumvents these biological hurdles by driving monocytes to survive past the 48-h viability gate and to differentiate into replication-permissive macrophages (19–22). Thus, HCMV-induced monocyte survival bridges the process of viral dissemination with the establishment of viral persistence.
In quiescently infected monocytes, no viral antiapoptotic proteins are expressed to inhibit cell death (20), and challenge with UV-inactivated HCMV (UV-HCMV) or purified glycoproteins results in rapid cellular survival changes similar to those induced by live virus (19, 23), suggesting that HCMV stimulates monocyte survival through the entry process. Indeed, we found that virus engagement with cellular epidermal growth factor receptor (EGFR) upon viral binding to the monocyte cell surface induced an antiapoptotic state dependent on the activation of phosphoinositide 3-kinase (PI3K) (20, 24). Directly downstream of PI3K, Akt is known to act as a central hub responsible for interpreting and converting upstream signals into the appropriate biological output; i.e., Akt activation has multiple outcomes, depending on the input signal (25). Although we have shown the virus to rapidly activate Akt (20, 26, 27), its role in directly mediating HCMV-induced monocyte survival is unknown.
Akt mediates the survival of short-lived monocytes after stimulation with myeloid growth factors, such as macrophage colony-stimulating factor (M-CSF) (28–30). Upstream signaling controls the activation status of Akt in order to allow the selective targeting of the plethora of downstream Akt substrates (25). Specifically, Akt activation signals modulate the phosphorylation ratio between two sites on Akt, serine 473 (S473) and threonine 308 (T308), which regulate Akt substrate specificity (31, 32). Our recent data showed that HCMV-activated Akt and M-CSF-activated Akt target different substrates within monocytes (33). Specifically, HCMV rapidly stimulated mTOR phosphorylation, while M-CSF did not. In addition, glycogen synthase kinase 3β (GSK3β), another known target of Akt, was not phosphorylated in HCMV-infected cells, further demonstrating a unique substrate specificity exhibited by HCMV-activated Akt. Consequently, we also found an Akt-dependent increase in the expression of select survival factors only in HCMV-infected cells and not in growth factor-treated cells. The targeting of a distinct subset of Akt substrates within HCMV-infected monocytes suggests that a virus-specific regulation of upstream signals controlling Akt activity is responsible for the unique survival changes occurring within infected monocytes.
A complex network of cellular positive and negative regulators controls Akt activity. Activation of receptor tyrosine kinases (RTKs), such as EGFR, initiates the PI3K/Akt signaling pathway through recruitment of class 1A PI3Ks, which are comprised of p110α, p110β, and p110δ isoforms (34). Activated PI3K phosphorylates the 3′ position of the inositol ring of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a signaling lipid on the internal leaflet of the plasma membrane, to form phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], which allows Akt recruitment and phosphorylation at the cell membrane (34). However, although they are highly homologous, the different PI3K isoforms have divergent, nonredundant biological functions and differential effects on Akt activity (35). PI3K activity is directly reversed by the phosphatase and tensin homolog (PTEN), which dephosphorylates PI(3,4,5)P3 back into PI(4,5)P2 (34). Several cancers have been shown to harbor inactivation mutations of PTEN (36), which lead to an aberrant activation of Akt and an enhanced phosphorylation of select downstream targets (37). Alternatively, PI3K activity is also opposed by SH2 domain-containing inositol 5-phosphatase 1 (SHIP1), which hydrolyzes PI(3,4,5)P3 into phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] (34). SHIP1-deficient macrophages exhibit enhanced Akt phosphorylation and increased cell viability under homeostatic conditions following M-CSF treatment (38). However, SHIP1 has also been shown to act as a proto-oncogene (39–41). Under conditions reflective of a tumor microenvironment, PI(3,4)P2 appears to recruit and activate Akt more efficiently than PI(3,4,5)P3 does, leading to different functional outcomes following Akt activation (42). Overall, the mechanisms modulating Akt phosphorylation are highly context specific and critical to the biological output of Akt activation. How HCMV controls the regulators of Akt to fine-tune its activity within infected monocytes and stimulate survival is unclear.
In this study, we report that HCMV infection rapidly activates Akt similarly to M-CSF to allow infected monocytes to bypass the 48-h viability gate. However, HCMV induced an early activation more robust than that induced by M-CSF and a temporal profile of Akt phosphorylation distinct from that induced by M-CSF; therefore, we hypothesized HCMV uniquely regulates Akt activity by modulating its positive (PI3K) and negative (PTEN and SHIP1) regulators to induce monocyte survival. Although PI3K p110δ is the primary isoform responsible for monocyte viability under normal conditions, we found that HCMV induces a switch to the PI3K p110β isoform to mediate the Akt-dependent survival of infected monocytes. Concomitantly with the activation of PI3K p110β, HCMV entry triggers a phosphorylation-mediated inactivation of PTEN allowing enhanced Akt signaling. Finally, similar to the reversed activity of SHIP1 observed within cancer cells, we found this normally negative regulator of Akt to have a positive effect during HCMV infection and to contribute to the survival of infected monocytes. Taken together, these data indicate that HCMV usurps the upstream Akt signaling network in order to rapidly stimulate Akt activity and allow infected monocytes to successfully navigate the 48-h viability checkpoint and disseminate the virus.
MATERIALS AND METHODS
Human peripheral blood monocyte isolation and culture.Isolation of human peripheral blood monocytes was performed as previously described (19, 20, 23). Briefly, blood was drawn from random donors by venipuncture, diluted in RPMI 1640 medium, and centrifuged through Histopaque 1077 cell separation medium (Sigma-Aldrich, St. Louis, MO) to remove red blood cells and neutrophils. Mononuclear cells were collected and washed with saline to remove the platelets and then separated by centrifugation through a Percoll (GE Healthcare, Wilkes-Barre, PA) gradient (40.48% and 47.7%). More than 95% of isolated peripheral blood mononuclear cells were monocytes, as determined by CD14-positive staining (22). The cells were washed with saline, resuspended in RPMI 1640 medium (Lonza, Walkersville, MD) supplemented with 1% human type AB serum (Lonza), and counted. All experiments were performed in 1 to 2% human serum at 37°C in a 5% CO2 incubator, unless otherwise stated. SUNY Upstate Medical University Institutional Review Board and Health Insurance Portability and Accountability Act guidelines for the use of human subjects were followed for all experimental protocols in our study.
For the inhibitor studies, the following reagents were used: MK-2206 2HCl (MK; an Akt inhibitor), BYL-719 (BYL; a p110α inhibitor), TGX-221 (TGX; a p110β inhibitor), and CAL-101 (CAL; a p110δ inhibitor) from Selleckchem (Houston, TX); AG-1478 (AG; an EGFR inhibitor) and LY-294002 (LY; a pan-PI3K inhibitor) from Calbiochem (Billerica, MA); and SF-1670 (SF; a PTEN inhibitor), 3-α-aminocholestane (3AC; a SHIP1 inhibitor), and PI(3,4)P2 from Echelon Biosciences (Salt Lake City, UT).
Virus preparation and infection.Human embryonic lung (HEL) 299 fibroblasts (CCL-137; American Type Culture Collection, Manassas, VA) from a low passage number (passage 7 [P7] to P15) were subcultured in Dulbecco modified Eagle medium (DMEM; Lonza) with 2.5 μg/ml Plasmocin (InvivoGen, San Diego, CA) and 10% fetal bovine serum (FBS; Sigma). When the culture reached confluence, the cells were infected with HCMV (strain Towne/E; between passages 35 and 39) in DMEM with 4% FBS. Virus was purified from the supernatant on a 20% sorbitol cushion to remove cellular contaminants and resuspended in RPMI 1640 medium. A multiplicity of infection (MOI) of 5 was used for each experiment, as >99% of monocytes were infected with strain Towne/E (26). Mock infection was performed by adding an equivalent volume of RPMI 1640 medium to monocytes, while M-CSF treatment was performed by adding an equivalent volume of RPMI 1640 medium with recombinant human M-CSF at 100 ng/ml (R&D Systems, Minneapolis, MN). In some experiments, HCMV was pretreated for 1 h with blocking antibodies to glycoprotein B (gB; clone 10B2664; United States Biological, Salem, MA) or an isotype control (Abcam, Cambridge, MA), both of which were used at 5 μg/ml. UV-inactivated virus was prepared by incubating virus under a 30-W germicidal (UV type C wavelength, 254 nm) UV lamp (G30 T8; GE Lighting, East Cleveland, OH) for 20 min on ice and was used in the same manner as live virus. The UV-inactivated virus did not replicate or produce any detectable levels of immediate early (IE) gene products (23).
Western blot analysis.Monocytes were harvested in modified radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 5 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 10% glycerol) supplemented with protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktails 2 and 3 (Sigma) for 15 min on ice. The lysates were cleared from the cell debris by centrifugation at 4°C (5 min, 21,130 × g) and stored at −20°C until further analysis. Protein samples were solubilized in Laemmli SDS-sample nonreducing (6×) buffer (Boston Bioproducts, Boston, MA) supplemented with β-mercaptoethanol (Amresco, Solon, OH) by incubation at 100°C for 10 min. Equal amounts of total protein from each sample were loaded in each well, separated by SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). Blots were blocked in 5% bovine serum albumin (BSA; Fisher Scientific, Waltham, MA) for 1 h at room temperature (RT) and then incubated with primary antibodies overnight at 4°C. The following antibodies were purchased from the indicated companies: anti-Akt, anti-phospho-Akt (anti-p-Akt; Ser473), anti-SHIP1, and anti-phospho-PTEN (anti-p-PTEN; Ser380) antibodies were from Cell Signaling (Danvers, MA), and anti-PTEN and anti-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz), and chemiluminescence was detected using the Amersham ECL Prime Western blotting detection reagent (GE Healthcare).
Flow cytometry.Monocytes were washed in phosphate-buffered saline and incubated in blocking solution consisting of fluorescence-activated cells sorting buffer, 5% BSA, and human FcR binding inhibitor (eBioscience, San Diego, CA), followed by staining with an allophycocyanin (APC)–anti-CD14 or APC–anti-mouse IgG1 isotype control antibody (BioLegend, San Diego, CA) on ice. The cells were then washed and stained with phycoerythrin-annexin V (BD Pharmingen, Franklin Lakes, NJ) and Sytox Blue dead cell stain (Life Technologies, Carlsbad, CA) to detect dead and dying cells. After staining, the cells were analyzed by flow cytometry using an LSRFortessa cell analyzer and BD FACSDiva software (BD Biosciences, Franklin Lakes, NJ). Our gating strategy on forward scatter (FSC)/side scatter (SSC) was set to include both cells in the early stages of apoptosis (decreased FSC and increased SSC compared to those for viable cells) and cells in the late stages of apoptosis (decreased FSC and decreased SSC compared to those of viable cells).
RESULTS
HCMV rapidly activates Akt to promote monocyte survival.M-CSF activates Akt to stop the molecular clock of monocytes and allow survival past the 48-h viability gate (28–30). In accord with our previous findings (26), HCMV infection also rapidly stimulated Akt phosphorylation at 30 min postinfection (mpi) (Fig. 1A). However, we found that the early activation of Akt (i.e., p-Akt levels) induced by HCMV infection was more robust than that induced by M-CSF treatment (Fig. 1A). Elevated p-Akt levels were maintained through 72 h postinfection (hpi) relative to those for mock-infected cells, although the amount of p-Akt began to diminish to levels below those in M-CSF-treated cells at 48 hpi (Fig. 1B). Nonetheless, the robust early activation of Akt suggests a pivotal role for Akt in allowing infected monocytes to successfully bypass the 48-h viability gate. In support of this, HCMV triggers an antiapoptotic state during viral entry into monocytes dependent on the activation of PI3K, which is directly upstream of Akt (20). To test if the rapid activation of Akt during the viral entry process is required for the HCMV-induced prosurvival state, monocytes were pretreated prior to infection with MK-2206 (MK), a highly selective Akt inhibitor with no activity against 256 other protein kinases (43). Pretreatment with 0.25, 2.5, and 10 μM MK resulted in 1.2-, 1.4-, and 2.5-fold decreases in cell viability, respectively, indicating that the rapid triggering of Akt activity during viral entry is necessary for monocyte survival (Fig. 1C). Next, we addressed if a continued increase of Akt activity following entry is necessary to maintain the survival of infected monocytes or if an initial burst alone is sufficient to induce the long-term survival changes within infected cells. We found that treatment of HCMV-infected monocytes with MK at 24 hpi led to the apoptosis of infected cells (Fig. 1D), suggesting that the chronically elevated level of Akt activity is required to maintain monocyte survival through 48 h. Moreover, at 0.25 μM the cytotoxic effect of MK was limited in HCMV-infected cells compared with that in mock-infected cells (1.9% versus 16.6% decreased cell viability, respectively), indicating a shift in the inhibitor 50% lethal dose (LD50) following HCMV infection (Fig. 1D). Indeed, the LD50 of MK was ∼0.25 μM for mock-infected monocytes and ∼5 μM for HCMV-infected monocytes. At concentrations above the LD50, HCMV-infected cells were completely sensitive to the loss of Akt, as the cell viability of HCMV-infected monocytes was reduced to the level for mock-infected cells (Fig. 1D). Overall, these data demonstrate that HCMV rapidly activates Akt to induce and maintain the survival of short-lived monocytes through the 48-h viability checkpoint. In addition, the distinct temporal phosphorylation kinetics of Akt activation induced by HCMV infection compared to that induced by M-CSF treatment suggests a unique virus-specific mechanism for regulating Akt activity.
HCMV rapidly activates Akt in order to promote monocyte survival. (A and B) Human peripheral blood monocytes were mock or HCMV infected or treated with M-CSF for 30 min (A) or 24, 48, or 72 h (B). Levels of p-Akt (S473), total Akt, and actin were detected by immunoblotting from whole-cell lysates. (C) Monocytes were pretreated with MK (an Akt inhibitor) at 0.25, 2.5, or 10 μM or with the vehicle control for 1 h and then mock or HCMV infected for 24 h. (D) Monocytes were mock or HCMV infected for 24 h and then treated with MK at 0.25, 2.5, or 10 μM or with the vehicle control for 24 h. (C and D) Viability was measured by flow cytometry using Sytox (Syt) and annexin V (Ann) staining. (A to D) Results are representative of those from 3 to 8 independent experiments using monocytes from different donors.
HCMV entry stimulates an Akt prosurvival state by inducing a switch in the PI3K isoform utilized.UV-inactivated HCMV (UV-HCMV) and purified glycoproteins induce functional survival changes in monocytes similarly to replication-competent virus (19, 23). Accordingly, UV-HCMV phosphorylated Akt comparably to HCMV (Fig. 2A). Internalization of HCMV into monocytes occurs via virus engagement of EGFR on the monocyte cell surface and the subsequent triggering of the PI3K/Akt signaling pathway (26). Consistent with our previous findings (20, 26, 27), treatment with either an EGFR inhibitor (AG1478 [AG]) or a pan-PI3K inhibitor (LY-294002 [LY]) prevented Akt phosphorylation by HCMV (Fig. 2B and C). However, to date the viral glycoprotein responsible for initiating the EGFR/PI3K/Akt cascade following infection of monocytes is unknown. HCMV glycoprotein B (gB) has been shown to bind EGFR to allow entry into fibroblasts (44, 45). Indeed, we found that infection of monocytes with HCMV particles coated with anti-gB neutralizing antibodies reduced Akt phosphorylation (Fig. 2D), demonstrating that gB-initiated signaling from EGFR contributes to Akt activation during HCMV entry into monocytes.
HCMV gB binding to EGFR during viral entry activates downstream PI3K to phosphorylate Akt. (A) Monocytes were mock, HCMV, or UV-HCMV infected for 24 h. (B and C) Monocytes were pretreated for 1 h with dimethyl sulfoxide or increasing concentrations (10, 20, or 40 μM) of AG (an EGFR inhibitor) (B) or 50 μM LY (a pan-PI3K inhibitor) (C) for 1 h and then mock or HCMV infected for 30 min (B) or 1 h (C). (D) Monocytes were mock or HCMV infected or infected with HCMV pretreated with an anti-gB antibody or an isotype control antibody at 5 μg/ml for 30 min. (A to D) The levels of p-Akt and actin were measured from whole-cell lysates using immunoblotting. Results are representative of those from at least 3 independent experiments using monocytes from different donors.
RTKs such as EGFR regulate Akt activity through the activation of PI3K. We found that inhibition of PI3K with a pan-PI3K inhibitor (LY) prior to infection or at 24 hpi completely abolished the ability of HCMV to facilitate a monocyte prosurvival state (Fig. 3A and B). However, RTKs are able to recruit different isoforms of PI3K, including p110α, p110β, and p110δ, that exhibit nonredundant activity (34). The p110α and p110β isoforms are ubiquitously expressed, while p110δ is enriched in the hematopoietic system and selectively controls Akt activity in primary macrophages (34, 46). In accord with p110δ being the primary PI3K isoform found in leukocytes, uninfected monocytes were sensitive to only CAL-101 (CAL), a p110δ-specific inhibitor, which induced a 2.1-fold reduction in cellular survival (Fig. 3C). Surprisingly, the loss of p110δ activity had little effect on the viability of HCMV-infected monocytes, suggesting that a potential switch in the PI3K isoform drives the survival of infected versus uninfected monocytes. Indeed, HCMV-infected cells were the most sensitive to pretreatment with TGX-221 (TGX), a p110β inhibitor, which resulted in a 2.1-fold reduction in cell viability. Similarly, inhibition of p110β activity at 24 hpi resulted in apoptosis of infected cells, while inhibition of the other PI3K isoforms had no effect on cell viability, indicating that persistent signaling from p110β was needed to maintain the survival of HCMV-infected monocytes (Fig. 3D). In contrast, a loss of p110δ activity did not induce the death of infected monocytes, while it induced the death of mock-infected cells. Consistent with p110β being responsible for mediating the Akt-dependent survival of infected monocytes, we found that inhibition of p110β prior to infection prevented Akt activation at 1 and 24 hpi (Fig. 3E). These data indicate that HCMV entry triggers a switch in the PI3K isoform from p110δ to p110β in order to stimulate Akt activity and allow the survival of infected monocytes past the 48-h cell fate decision checkpoint.
HCMV preferentially uses the PI3K p110β isoform to mediate survival of infected monocytes. (A) Monocytes were treated for 1 h with dimethyl sulfoxide or 50 μM LY and then mock or HCMV infected for 24 h. (B) Monocytes were mock or HCMV infected for 24 h and then treated with dimethyl sulfoxide or 50 μM LY for 24 h. (C and D) The PI3K isoform-specific inhibitor BYL (a p110α inhibitor), TGX (a p110β inhibitor), or CAL (a p110δ inhibitor) was added to mock- or HCMV-infected monocytes at 50 μM for 1 h prior to a 24-h infection (C) or at 24 hpi for 24 h (D). (A to D) Monocyte viability was measured by Sytox and annexin V staining using flow cytometry. (E) TGX was added to mock- or HCMV-infected monocytes at 50 μM for 1 h prior to a 1-h or 24-h infection. The levels of p-Akt (S473) and actin were detected by immunoblotting. (A to E) Results are representative of those from 3 to 5 independent experiments using monocytes from different donors.
HCMV inactivates PTEN in monocytes.The enhanced and sustained activation status of Akt in HCMV-infected cells through 48 h suggests that HCMV may also restrict the activity of Akt negative regulators, in addition to inducing PI3K (Fig. 1A and B). PTEN is a critical negative regulator of cell survival by directly reversing the activity of PI3K (34). Upon reexamination of the results of our previous microarray analyses (20, 47), we unexpectedly found that HCMV-infected monocytes had increased PTEN transcript levels. We confirmed the elevated levels of PTEN protein within infected cells, which was maintained through 72 h (Fig. 4A). Furthermore, on the basis of the protein expression levels over the 72-h time course, HCMV appears to decrease the rate of PTEN loss within infected monocytes compared with that in uninfected monocytes rather than directly simulate the upregulation of protein expression. However, PTEN phosphorylation at serine 380 (S380) is known to inactivate PTEN (48, 49) and is associated with increased p-Akt levels (50). We found that both HCMV infection and M-CSF treatment increased phosphorylated PTEN (p-PTEN) levels to a greater extent than total PTEN protein levels at 24 h posttreatment (Fig. 4B and C), suggesting that HCMV act similarly to M-CSF by negatively regulating PTEN. Indeed, pretreatment with SF1670 (SF), a highly selective small-molecule compound that inhibits PTEN′s cellular enzymatic activity (51), for 1 h prior to infection did not further increase Akt phosphorylation at 24 hpi, confirming the absence of PTEN activity (Fig. 4D). In contrast, infected cells pretreated with SF for 1 h exhibited elevated levels of p-Akt compared to the levels in uninfected cells at 1 hpi (Fig. 4E), indicating that PTEN inactivation likely occurs through a postentry event. Regardless of the mechanism of inhibition, the inactivation of PTEN by 24 hpi allows increased levels of Akt to be maintained through the 48-h viability gate.
HCMV inactivates PTEN through phosphorylation at S380. (A) Monocytes were mock or HCMV infected or treated with M-CSF for 24, 48, or 72 h, and PTEN levels were measured by immunoblotting. (B) Monocytes were mock or HCMV infected or treated with M-CSF for 24 h, and p-PTEN (S380) was detected by immunoblotting. (C) Representative ratios of increased p-PTEN levels relative to those in mock-infected cells over increased total PTEN levels relative to those in mock-infected cells were determined by densitometry at 24 h. (D and E) Monocytes were pretreated for 1 h with dimethyl sulfoxide or SF (a PTEN inhibitor) at 250 nM and then mock or HCMV infected for 24 h (D) or 1 h (E). The levels of p-Akt and actin were determined by immunoblotting. (A to E) Results are representative of those from at least 3 independent experiments using monocytes from different donors.
HCMV uses SHIP1 as a positive regulator of Akt to promote survival of monocytes.SHIP1 functions as a second negative regulator of the PI3K/Akt pathway by hydrolyzing PI(3,4,5)P3 into PI(3,4)P2 (52). Similarly to the upregulation of PTEN, SHIP1 is upregulated by HCMV at 24 hpi and its upregulation is sustained through 72 hpi (Fig. 5A). Unlike with PTEN, the early increase of SHIP1 occurred only with HCMV infection, while M-CSF treatment induced a less robust upregulation of SHIP1 with delayed kinetics (Fig. 5A). This early-targeted stimulation of SHIP1 activity by HCMV appears to be in conflict with the need for HCMV-infected monocytes to exhibit high levels of activated Akt prior to the 48-h viability checkpoint. However, despite the downregulation of PI3K/Akt activity under homeostatic conditions, recent reports have demonstrated that SHIP1 has positive effects on Akt activation under pathological conditions, such as a tumor microenvironment (39–41). Therefore, we assessed SHIP1 activity in HCMV-infected cells. Pretreatment with a SHIP1-selective inhibitor, 3-α-aminocholestane (3AC) (39), resulted in decreased p-Akt levels in HCMV-infected cells at both 15 mpi (Fig. 5B) and 24 hpi (Fig. 5C), indicating that SHIP1 has a positive effect on Akt activity. Accordingly, the addition of PI(3,4)P2 back to HCMV-infected cells treated with 3AC rescued the loss of p-Akt in a dose-dependent manner (Fig. 5D), suggesting that SHIP1 may play a positive role during HCMV-induced monocyte survival. Indeed, pretreatment of cells with 3AC prior to infection blocked the ability of HCMV to stimulate a prosurvival state within infected monocytes (Fig. 6A). Next, we tested if continued SHIP1 activity was required for the maintenance of monocyte viability following the initial infection, since elevated levels of SHIP1 persisted for 72 hpi. The loss of SHIP1 activity at 24 hpi resulted in a 4-fold reduction in the viability of infected cells to levels similar to those for uninfected cells (Fig. 6B). Together, these data suggest that HCMV utilizes SHIP1 as an additional positive regulator of Akt to drive monocyte survival, a necessary step in the viral dissemination process.
HCMV activates Akt through a noncanonical SHIP1-dependent pathway. (A) Monocytes were mock or HCMV infected or treated with M-CSF for 24, 48, or 72 h. SHIP1 and actin levels were detected by immunoblotting. (B) Monocytes were pretreated with 3AC (a SHIP1 inhibitor) at 20 μM for 1 h and then mock or HCMV infected for 15 min. (C) Monocytes were pretreated with 3AC at 15 μM for 1 h and then mock or HCMV infected for 24 h. (D) Monocytes were pretreated with 5, 10, or 20 μM PI(3,4)P2 for 1 h and then treated for 1 h with 15 μM 3AC or vehicle control, followed by a 24-h infection. (B to D) The levels of p-Akt and actin were measured from whole-cell lysates by immunoblotting. (A to D) Results are representative of those from at least 3 independent experiments using monocytes from different donors.
HCMV-activated SHIP1 is required for the survival of infected monocytes. (A) Monocytes were pretreated for 1 h with the vehicle control or 3AC at 20 μM and then mock or HCMV infected for 24 h. (B) Monocytes were mock or HCMV infected for 24 h and then treated with 3AC at 20 μM or the vehicle control for 24 h. (A and B) Monocyte viability was measured by Sytox and annexin V staining using flow cytometry. Results are representative of those from 3 to 5 independent experiments using monocytes from different donors.
DISCUSSION
Peripheral blood monocytes are a primary target of HCMV in vivo and are believed to be responsible for the hematogenous dissemination of the virus to organ sites and bone marrow (11–14). Monocyte differentiation into macrophages is an essential step in the process of HCMV dissemination in the body, as virus replication and progeny production cannot occur in monocytes until their differentiation into tissue macrophages is complete (16–19). A key step in the monocyte-to-macrophage differentiation process is the survival of monocytes through 48 h, a critical cell fate checkpoint where monocytes are programmed to undergo apoptosis in the absence of survival stimuli (15). Previously, we have shown that HCMV infection allows short-lived monocytes to successfully navigate the 48-h viability gate and differentiate into long-lived macrophages (19, 20, 22). Despite the repertoire of antiapoptotic proteins encoded within the HCMV genome, these viral proteins are known not to be expressed within the first 48 h of infection of monocytes (20), indicating a mechanism whereby HCMV exploits the cellular antiapoptotic machinery to promote monocyte survival. We and others have shown that triggering of prosurvival signaling pathways during viral entry is critical for the survival of cells that support quiescent HCMV infection (20–22, 53, 54). In the current study, we present the mechanisms by which HCMV stimulates Akt activity in a virus-specific manner in order to promote the long-term survival of infected monocytes. A proposed model for HCMV regulation of Akt-dependent survival of infected monocytes is shown in Fig. 7.
Proposed model for HCMV regulation of Akt-dependent survival of infected monocytes. HCMV entry into monocytes induces a robust activation of Akt via the direct modulation of the upstream regulators PI3K, PTEN, and SHIP1 in order to drive the survival of infected monocytes. Initiation of the Akt signaling cascade is triggered by gB binding to EGFR, which preferentially utilizes the PI3K p110β isoform to promote cell survival. Concurrently, in contrast to normal myeloid growth factors, HCMV stimulates a rapid increase in SHIP1 expression leading to a noncanonical activation of Akt. To ensure maximum Akt activity, HCMV also induces an early phosphorylation-dependent inactivation of the negative regulator PTEN. This nontraditional activation of Akt by HCMV allows the increased expression of a select subset of Akt-dependent antiapoptotic proteins specifically required for the survival of infected monocytes past the critical 48-h cell fate checkpoint (20, 22, 33).
Elevated Akt activity is essential to the survival of monocytes through the 48-h cell fate checkpoint (28–30). We found that HCMV entry rapidly activates Akt and that the activation is more robust than that obtained with treatment with normal myeloid growth factors. The reasons for the enhanced Akt activation are unclear. Transcriptome studies have shown the increased expression of several antiviral proapoptotic genes following HCMV infection but not M-CSF treatment (24, 47, 55), leading us to speculate that the higher levels of Akt may be necessary to overcome both the intrinsic biological programming of monocytes to undergo cell death and the proapoptotic antiviral host response triggered by HCMV. The phosphorylation signature of Akt regulates substrate specificity, and higher levels of phosphorylated S473 correspond with increases in the expression of specific Akt-dependent antiapoptotic proteins (31, 32). Thus, the enhanced phosphorylation of Akt at S473 induced during HCMV entry may allow the increased expression of specific prosurvival factors necessary to abrogate the heightened levels of death signals found within infected monocytes. In support of this, we previously found that HCMV-activated Akt induces a distinct subset of antiapoptotic proteins within infected monocytes not upregulated by myeloid growth factor-activated Akt (33). Although we are currently investigating how the phosphorylation profile of Akt regulates substrate specificity within infected monocytes, our data indicate that a virus-specific mechanism leads to a unique activation of Akt and the expression of select antiapoptotic proteins required for the survival of infected monocytes.
Several herpesviruses have convergently evolved mechanisms to regulate Akt activity in order to promote the survival of carrier cells. During latent infection, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) express viral proteins that activate Akt, resulting in cell survival (56, 57). In contrast to the direct modulation of Akt activity by viral proteins, HCMV appears to have evolved a different Akt regulatory strategy in cells supportive of a quiescent infection involving the modification of Akt activity through the viral entry process (20, 26, 27, 58). Initiation of the upstream Akt signaling cascade is triggered by gB activation of the EGFR/PI3K signaling axis upon engagement of the virion with the monocyte cell surface (26). Targeting of EGFR by HCMV appears to induce a switch from p110δ, the predominant regulator of Akt and the main PI3K isoform recruited to the activated M-CSF receptor in leukocytes (34, 46), to p110β as the primary PI3K isoform responsible for the survival of HCMV-infected monocytes. Biologically, we speculate that the preferential usage of the p110β isoform is due to the lack of negative self-regulatory activity by p110β. Autophosphorylation of p110δ in a time-dependent manner completely abrogates kinase activity (59), and hence, the targeted use of a PI3K isoform that lacks autoinhibitory activity would allow the longer transmission of survival signals within infected monocytes. Moreover, p110δ exhibits strong antiviral activity upon activation, unlike p110β (60); thus, a switch away from p110δ may represent a viral strategy to limit antiviral responses following HCMV infection. Regardless, to the best of our knowledge, our study provides the first documentation of a virus inducing a specific switch in the PI3K isoform needed for the survival of carrier cells.
Concomitantly with inducing PI3K, HCMV must also inhibit the counteracting activity of PTEN and SHIP1 to ensure maximum Akt activity during the first 48 h of infection. Surprisingly, HCMV promotes elevated levels of the PTEN protein within infected monocytes; however, HCMV concurrently abrogates PTEN activity through the phosphorylation of PTEN at S380. Our data further suggest that inhibition of PTEN may be not a virus-specific event but may be a general monocyte response to survival stimuli, as M-CSF treatment also induces S380 phosphorylation. In contrast, SHIP1 expression is rapidly upregulated following HCMV infection but not following treatment with myeloid growth factors. The targeting of SHIP1 activity by HCMV suggests an important role for the biological function of SHIP1 during the infection of monocytes. Although SHIP1 is typically viewed to be a negative regulator of the PI3K signaling cascade by reducing PI(3,4,5)P3 levels, SHIP1 removes the phosphate from the D5 phosphate position of the inositol ring, while PTEN removes the D3 phosphate, enabling SHIP1 and PTEN to have very different effects on Akt signaling (40, 41, 61). Indeed, SHIP1 levels are increased in acute myeloid leukemia cells and positively regulate Akt activity (39, 42, 62). The SHIP1 product, PI(3,4)P2, binds with greater affinity to Akt, resulting in a greater phosphorylation of Akt compared to the level of phosphorylation achieved with PI(3,4,5)P3 (42). Thus, the overexpression of SHIP1 likely leads to the aberrant accumulation of PI(3,4)P2 within cells, promoting a malignant state due to a more potent activation of Akt (41). Similarly, HCMV infection increases SHIP1 expression through 72 hpi and the loss of SHIP1 activity prevents Akt phosphorylation following infection, which is recued by the addition of PI(3,4)P2 back to HCMV-infected cells. How SHIP1 acts as a positive regulator in HCMV-infected monocytes is unclear. We speculate that HCMV may dysregulate downstream players responsible for the dephosphorylation of PI(3,4)P2, leading to the accumulation of PI(3,4)P2 within infected monocytes but not uninfected cells. Regardless, our data indicate HCMV utilizes a two-pronged approach to stimulate a more robust activation of Akt via the actions of both phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate (PIP3), which to date has not been observed in noncancerous cells. Furthermore, the double activation of Akt through both PI3K-generated PI(3,4,5)P3 and SHIP1-generated PI(3,4)P2 may in part account for the substrate specificity difference between Akt activated by HCMV and Akt activated by M-CSF. The noncanonical activation of Akt with PI(3,4)P2 preferentially phosphorylates Akt at S473 (63), while PIP3-mediated activation leads to S473 and T308 phosphorylation (34). Recent studies showing that the ratio of phosphorylated S473 to T308 modulates Akt target specificity hint at the possibility that the unique biological output of HCMV-activated Akt is the result of a specific virus-induced phosphorylation pattern (31–33).
In summary, we demonstrate that HCMV infection of monocytes rapidly induces a unique temporal profile of Akt phosphorylation with early activation that is more robust than that achieved with normal myeloid growth factors. The enhanced activation of Akt is essential to the survival of infected monocytes through the 48-h viability checkpoint, as the loss of Akt activity completely abrogated the ability of HCMV to subvert cell death. We found that the rapid peak of Akt activity was mediated by gB triggering of EGFR and the subsequent recruitment of the PI3K p110β isoform to facilitate the Akt-dependent prosurvival state. Concomitantly, a rapid phosphorylation-mediated inactivation of PTEN by HCMV likely ensures that maximum p-Akt levels are maintained during the crucial 48-h cell fate decision period. Finally, we show that SHIP1 activity positively regulates Akt within infected monocytes as it does in tumor cells and that targeting of SHIP1 appears to be specific to HCMV-induced monocyte survival, since M-CSF does not upregulate SHIP1. Elucidation of the unique molecular mechanisms leading to the survival of HCMV-infected monocytes may provide new therapeutic targets to specifically eliminate infected monocytes, thereby preventing viral dissemination in transplant patients at high risk for acquiring an acute infection.
ACKNOWLEDGMENTS
We thank Christine Burrer in the Department of Microbiology and Immunology at SUNY Upstate Medical University for technical support and maintenance of viral stocks.
We have no conflicting financial interests.
The funders had no role in the study design, data collection, and interpretation, or the decision to submit the work for publication.
FOOTNOTES
- Received 2 February 2016.
- Accepted 26 April 2016.
- Accepted manuscript posted online 4 May 2016.
- Address correspondence to Gary C. Chan, chang{at}upstate.edu.
Citation Cojohari O, Peppenelli MA, Chan GC. 2016. Human Cytomegalovirus induces an atypical activation of Akt to stimulate the survival of short-lived monocytes. J Virol 90:6443–6452. doi:10.1128/JVI.00214-16.
REFERENCES
- Copyright © 2016, American Society for Microbiology. All Rights Reserved.
