Delivery of US28 by incoming HCMV particles rapidly attenuates Akt activity to suppress HCMV lytic replication in monocytes

Abstract

Establishing a nonproductive, quiescent infection within monocytes is essential for the spread of human cytomegalovirus (HCMV). We investigated the mechanisms through which HCMV establishes a quiescent infection in monocytes. US28 is a virally encoded G protein–coupled receptor (GPCR) that is essential for silent infections within cells of the myeloid lineage. We found that preformed US28 was rapidly delivered to monocytes by HCMV viral particles, whereas the de novo synthesis of US28 was delayed for several days. A recombinant mutant virus lacking US28 (US28Δ) was unable to establish a quiescent infection, resulting in a fully productive lytic infection able to produce progeny virus. Infection with US28Δ HCMV resulted in the phosphorylation of the serine and threonine kinase Akt at Ser⁴⁷³ and Thr³⁰⁸, in contrast to the phosphorylation of Akt only at Ser⁴⁷³ after WT viral infection. Inhibiting the dual phosphorylation of Akt prevented the lytic replication of US28Δ, and ectopic expression of a constitutively phosphorylated Akt variant triggered lytic replication of wild-type HCMV. Mechanistically, we found that US28 was necessary and sufficient to attenuate epidermal growth factor receptor (EGFR) signaling induced during the entry of WT virus, which led to the site-specific phosphorylation of Akt at Ser⁴⁷³. Thus, particle-delivered US28 fine-tunes Akt activity by limiting HCMV-induced EGFR activation during viral entry, enabling quiescent infection in monocytes.

Introduction

Infection with human cytomegalovirus (HCMV) is highly prevalent, with seropositivity reaching 80% in developed countries and upwards of 100% in developing countries (1). HCMV infection results in lifelong latent infection, with more than 50% of individuals aged 6 years harboring HCMV, which rises to 90% by the age of 75 in the United States (2, 3). In healthy individuals, primary HCMV infection is generally asymptomatic, and latency is tightly controlled by the immune system. However, HCMV primary infection or reactivation is associated with increased morbidity and mortality in the immunonaïve, such as neonates, and the immunocompromised, such as HIV patients and transplant recipients (4-7). HCMV-associated inflammatory diseases within these patients are often widespread and can lead to multi-organ failure.

The myeloid compartment is central for HCMV viral dissemination and persistence. Following a primary exposure to HCMV, the virus enters oral epithelial cells and initiates the lytic replication cycle whereby all the viral gene products necessary to produce progeny virus are expressed in a highly controlled and timed manner. Progeny virus then spreads to peripheral blood monocytes where the virus establishes a quiescent infection, which is defined by the suppression of virus replication and spontaneous reactivation of the virus after 2-3 weeks of infection in the absence of external stimuli (8, 9). Infection directly stimulates monocytes to travel to distal end-organ sites and differentiate into long-lived macrophages permissive for viral replication. Infected monocytes that journey to the bone marrow ultimately transfer infection to CD34⁺ hematopoietic progenitor cells (HPCs) to establish a lifelong reservoir of latency. Based on this model of viral dissemination, primary infected monocytes are critical for viral dissemination by linking initial lytic infection to life-long latent infection in CD34⁺ cells. Suppression of initial lytic infection in primary infected monocytes is essential for the immune evasion and spread of HCMV within the host because viral mutants unable to establish latency or wild-type (WT) HCMV forced out of latency can be targeted by the host immune response (10, 11). However, little is known about the mechanisms underpinning the establishment of a quiescent infection within monocytes.

The virally encoded G protein-coupled receptor (GPCR) US28 is found within HCMV viral particles (12) and is expressed during both latent (10, 12-19) and lytic infection (20-22). US28 acts as a signaling molecule to enhance cellular proliferation, chemotactic and mitotic processes (23), and promotes cellular migration (24-26) by activating different migratory factors, including Pyk2 and RhoA (27, 28). US28 also modulates multiple cellular pathways during lytic infection, including Ca²⁺ signaling (20, 21), FAK/Src (29), PLC (20, 30), COX-2 (31), STAT3 (32), Akt, ERK1/2, eNOS (33), and β-catenin (34). US28 is essential for viral latency/quiescence in cells of the myeloid lineage, including CD34⁺ HPCs (12, 14, 15, 17), THP1 cells (10, 15, 35), Kasumi-3 cells (12, 14, 15), and monocytes (10, 36, 37). During latency, US28-mediated signaling silences the major immediate-early promoter (MIEP) (10, 12, 14, 15), a key promoter in the latent-to-lytic switch that regulates the expression of UL123 and UL122, which encode immediate-early 1 (IE1) and immediate-early 2 (IE2) viral proteins, respectively. In Kasumi-3 cells and CD34⁺ HPCs, US28 attenuates cellular fos (c-fos) to prevent AP-1 transcription factor-dependent activation of the MIEP (14, 15). Additionally, in THP-1 cells, US28 downregulates the MAPK and NF-κB pathways during latency (10, 36). Similar to latent infection, the induction of viral lytic proteins, such as IE1, is inhibited during quiescent infection of monocytes (8, 9, 36, 38, 39). The role of US28-mediated regulation of the MIEP during the establishment of a quiescent infection in monocytes remains to be elucidated.

In this study, we demonstrate that preformed US28 delivered from viral particles limits HCMV-induced Akt activity to allow a quiescent infection to be established in infected monocytes. Specifically, we found that infection of peripheral blood monocytes with recombinant mutant viruses lacking US28 (US28Δ) or that were defective in US28-mediated signaling resulted in the rapid induction of IE, early (E), and late (L) viral proteins. Monocytes infected with US28-complemented US28Δ virus, which can deliver preformed US28 during infection but cannot synthesize de novo US28 in the infected cells, had reduced expression of UL123, indicating particle-associated US28 is sufficient to inhibit IE gene expression during early infection. Mechanistically, we found US28 was necessary and sufficient to limit EGFR activation induced by infection with WT HCMV, which led to the phosphorylation of Akt only at Ser⁴⁷³. In contrast, monocytes infected with US28Δ HCMV exhibited increased EGFR activity relative to those infected with WT HCMV as well as Akt phosphorylation at both Ser⁴⁷³ and Thr³⁰⁸, which is required for full Akt activity (40, 41). Stimulation of Akt phosphorylation at both Ser⁴⁷³ and Thr³⁰⁸ during WT infection of monocytes initiated IE1 synthesis, whereas suppression of either Ser⁴⁷³ or Thr³⁰⁸ phosphorylation during US28Δ infection attenuated IE1 induction. We previously showed that the partial activity induced by phosphorylation of Akt only at Ser⁴⁷³ is required for the long-term survival of HCMV-infected monocytes (42, 43). We now demonstrate that preventing the full activation of Akt through US28-mediated reduction in phosphorylation of Akt at Thr³⁰⁸ is also crucial to ensuring that IE synthesis is not initiated during the establishment of a quiescent infection within monocytes.

Results

US28-mediated signaling promotes HCMV quiescent infection within monocytes.

US28 is essential for HCMV latency in CD34⁺ HPCs (12, 14, 15, 17), THP1 cells (10, 15, 35), Kasumi-3 cells (12, 14, 15), and monocytes (10, 36, 37). Accordingly, we found that infection of CD14⁺ primary peripheral blood monocytes with an HCMV mutant lacking US28 (US28Δ) did not result in quiescent infection, leading to rapid IE1 induction at 24 through 48 hours post-infection (hpi), similar to infection of replication-permissive fibroblasts with WT HCMV (Fig. 1A to 1C). In addition to IE induction, infection of monocytes with US28Δ also resulted in early (E) and late (L) gene expression (fig. S1A) and protein synthesis (fig. S1B, C), indicating progression through the late stages of the lytic replication cycle. These data are in line with our previous studies demonstrating that viral DNA is replicated and infectious progeny are produced during US28Δ infection (10, 36), a phenotype recapitulated in primary CD34⁺ cells and Kasumi-3 cells, a CD34⁺ line that supports HCMV latency and reactivation (12, 14, 15, 36). To determine the importance of US28 signaling in inhibiting IE1, we used two additional US28-recombinant viral strains with altered signaling capabilities (15). TB40/EmCherry-US28ΔN-3xF (ΔN) lacks a chemokine binding domain [amino acids (AA) 2-16], thereby preventing the interaction of US28 with many of its ligands (44-46). TB40/EmCherry-US28-R129A-3xF (R129A) harbors a point mutation in the ‘DRY’ motif to which G proteins couple, rendering R129A a G protein-coupling deficient mutant (47, 48). We confirmed that recombinant US28 was present on viral particles generated by both ΔN and R129A HCMV mutants (Fig. 1D). Similar to US28Δ HCMV, infection with either signaling mutant resulted in IE1 induction in monocytes (Fig. 1E, 1F), which is consistent with previous findings in THP-1, Kasumi-3 cells, and CD34⁺ HPCs (10, 15, 16, 36), suggesting these mutants fail to undergo quiescence in monocytes. Monocytes infected with R129A or ΔN HCMV showed greater induction of IE1 compared to those infected with US28Δ HCMV. Why IE1 induction was less robust in US28Δ-infected monocytes relative to the signaling mutants is unclear, although a possibility is that the signaling-deficient US28 may act as a scaffold to enhance the formation of signaling complexes during viral entry. Regardless, our data indicate both ligand binding and G protein-mediated signaling mediated by US28 are pivotal to preventing IE1 induction in monocytes.

Fig 1. US28 mediated signaling inhibits IE1 expression in monocytes.

(A to C) Primary human peripheral blood monocytes were mock-infected or infected (MOI = 1) with wild-type (WT) or US28Δ HCMV for 24 h (A, C) or 48 h (C). IE1 induction was detected by Western blot (A, C) and quantified (B). WT-infected primary human fibroblasts are shown as a positive control. (D) NuFF-1 cells were infected (MOI= 1) with US28-3xFLAG, R129A, or ΔN HCMV. After 100% cytopathic effect occurred, cell-free HCMV was purified from supernatants and cell lysates were collected. Incorporation of recombinant US28 into HCMV viral particles was detected by Western blotting for FLAG. IE1, pp65, and tubulin were used as controls. (E and F) Monocytes were mock-infected or infected with WT, US28Δ, ΔN or R129A HCMV (MOI = 1) for 24 h. IE1 induction was detected by Western blot (E) and quantified (F). β-actin was used as a loading control. Western blots and densitometry are representative of at least 3 biological replicates per group. *P<0.05, **P < 0.005, ****P < 0.0001, by one-way ANOVA with Tukey’s HSD post hoc test or Student’s t-test.

Although IE1 induction was restricted in monocytes infected with WT HCMV, IE1 protein in monocytes infected with US28Δ HCMV was detectable as early as 6 hpi (Fig. 2A), which was similar to the IE1 induction kinetics in both fibroblasts infected with either HCMV variant (Fig. 2B). These results further confirmed previous studies demonstrating US28 is dispensable for in vitro infection of fibroblasts (12, 20, 21, 49, 50). We next ensured that the absence of IE1 induction following infection with WT HCMV was not due to reduced viral binding or entry into monocytes. First, binding assays confirmed that both WT and US28Δ HCMV bound to monocytes to a similar extent (fig. S2A). Additionally, the absence of US28 had no effect on viral entry because both WT and US28Δ HCMV entered monocytes to a similar extent (fig. S2B). As a control, we used the EGFR inhibitor AG1478, which blocks HCMV entry into monocytes at high concentrations (51), and found that AG1478 reduced entry of both WT and US28Δ HCMV. Next, we confirmed that translocation of the viral genome to the nucleus was also not impaired by the deletion of US28. Flow cytometric analysis of mCherry expression driven from an independent SV40 promoter inserted in an intergenic region of the HCMV genome (52) did not reveal significant differences in the percentage of mCherry positive cells or in mCherry fluorescence intensity between WT- and US28Δ-infected monocytes (fig. S2C, S2D). Because differentiated monocytes support lytic HCMV infection (38), we next examined if loss of US28 enhanced the differentiation of the infected cells. The surface abundance of several macrophage-associated cell-surface markers were similar in monocytes infected with either WT and US28Δ HCMV at early (fig. S3A) and late (fig. S3B) stages of infection, suggesting lytic infection in cells infected with US28Δ HCMV was not due to an acceleration in the monocyte-to-macrophage differentiation process. Collectively, our results indicate US28 impedes IE1 induction upon entry into monocytes in a ligand- and G protein-coupling-dependent fashion.

Fig 2. The kinetics of IE1 induction in monocytes are similar to those in fibroblasts upon infection with WT or US28Δ HCMV.

(A and B) Monocytes (A) and fibroblasts (B) were mock-infected or infected (MOI = 1) with WT or US28Δ HCMV for the indicated times. IE1 induction was detected by Western blot. β-actin was used as a loading control. Western blots are representative of at least 3 biological replicates per group.

Viral particle-associated US28 blocks early IE1 induction in monocytes.

During a productive HCMV infection, infectious (virions) and non-infectious (dense bodies (DBs) and noninfectious enveloped particles (NIEPs)) particles are simultaneously produced from infected cells in vitro (53-55) and in vivo (56). Delivery of US28 from these HCMV particles is important for establishing latency in Kasumi-3 and CD34⁺ HPCs (15), and de novo synthesized US28 is required to maintain latency (10, 12, 14-17, 36). Thus, we next assessed the kinetics of US28 expression following infection of primary monocytes. To this end, we infected primary monocytes with TB40/EmCherry-US28-3xFLAG (US28-3xFLAG), a viral recombinant that contains a triple FLAG epitope tag inserted in-frame with the US28 ORF virus at the C-terminus (20). We also included lytic-infected primary fibroblasts as a control. Although US28 expression was observed in fibroblasts at 48 hpi, de novo synthesized US28 in monocytes was not detectable by Western blot (Fig. 3A) or RT-qPCR (Fig. 3B) analyses until 4 days post-infection (dpi) (fig. S4A, S4B). SV40-driven mCherry was detected in US28-3xFLAG-infected monocytes by 24 hpi using Western blot (Fig. 3A) and flow cytometry (fig. S5A, S5B), confirming delivery of the viral genome to the nucleus. These data suggest that de novo synthesized US28 is unlikely to be involved in the rapid suppression of IE1 induction during monocyte infection and that HCMV particle-delivered US28 is responsible for preventing IE1 induction. Accordingly, particle-associated US28 was delivered to infected monocytes as soon as within 15 min of infection (Fig. 3C), which was reduced over 48 h through proteasomal degradation (Fig. 3C; fig. S6). To determine if particle-associated US28 suppressed IE1 induction upon infection, we infected monocytes with US28-complemented US28Δ (US28comp) HCMV that lacks the US28 gene but expresses particle-associated US28 protein derived from the complementing cell line. We found that IE1 protein induction was suppressed in monocytes infected with US28comp HCMV (Fig. 3D, 3E), indicating that particle-delivered US28 is sufficient to attenuate IE1 induction, which is consistent with previous findings (12, 15). Together, our data suggest that particle-associated US28 suppresses IE1 induction to promote the establishment of HCMV quiescence within monocytes.

Fig 3. Particle-associated US28 inhibits the induction of IE1.

(A to C) Monocytes or fibroblasts were mocked-infected or infected at MOI 1 (A, B) or MOI 5 (monocytes) (C) with US28-3xFLAG HCMV for the indicated times. US28 protein and mRNA abundance were detected by Western blot (A, C) or RT-qPCR analysis (B), respectively. RT-qPCR data are means ± SEM from 4 biological replicates per group. SV40-driven mCherry expression in infected cells was assessed by Western blot for mCherry (A, C). US28-3xFLAG and US28Δ cell-free virus lysates were used as positive and negative controls for US28, respectively. The red pixels represent the over saturation of the positive control band from long exposure of the blot during image capture (C). (D, E) Monocytes were mock-infected or infected (MOI = 1) with WT, US28Δ, or US28-complemented US28Δ (US28comp) HCMV for 24 h. IE1 induction was detected by Western blot (D) and quantified (E). β-actin was used as a loading control. Western blots and densitometry are representative of at least 3 biological replicates per group. *P<0.05, ****P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test.

US28 reduces Akt activity to restrict UL123 transcription.

Activation of EGFR and its downstream pathways, such as phosphoinositide 3-kinase (PI3K) and Akt, are essential for the maintenance of HCMV latency because inhibition of these proteins results in lytic replication of HCMV in CD34⁺ HPCs (57, 58). To investigate if the activation of this pathway is regulated by US28 during the establishment of quiescent infection in monocytes and if inhibition leads to IE1 induction during WT infection, we treated monocytes infected with WT or US28Δ HCMV with small-molecule inhibitors targeting EGFR, PI3K, or Akt. However, we failed to detect IE1 protein in inhibitor-treated monocytes infected with WT HCMV, suggesting that signaling from the EGFR/PI3K/Akt pathway induced during viral entry may be crucial in the early establishment of a quiescent infection in monocytes compared to the long-term maintenance of latency with CD34⁺ HPCs (Fig. 4A to 4C). In contrast to infection with WT HCMV, monocytes infected with US28Δ HCMV that were treated with vehicle (DMSO) showed robust UL123 transcription and IE1 protein induction, which was significantly attenuated in the presence of each inhibitor (Fig. 4A to 4C). The concentrations of the inhibitors used did not affect cell viability (fig. S7A) or viral entry (fig. S7B), indicating signaling from the EGFR/PI3K/Akt pathway is necessary for early UL123 transcription in monocytes infected with US28Δ HCMV. Because the phosphorylation pattern controls the activity (40, 41) and therefore the substrate specificity of Akt (59-62), we next assessed the activation profile of Akt. Consistent with our previous reports (42, 43, 63), WT HCMV infection of monocytes preferentially induced phosphorylation of Akt only at Ser⁴⁷³ during HCMV entry into monocytes, whereas Thr³⁰⁸ phosphorylation was unchanged relative to mock-infected control cells (Fig. 4D, 4E). These changes to Akt phosphorylation following infection with WT HCMV were not due to changes in total protein abundance (Fig. 4F). In contrast, infection with US28Δ HCMV stimulated the phosphorylation of Akt at Ser⁴⁷³ and Thr³⁰⁸, both of which are required for the full activation of Akt and are observed after Akt activation by growth factors (40, 41). Moreover, the site-specific phosphorylation of Akt at Ser⁴⁷³ also occurred in monocytes infected with US28comp HCMV (fig. S8), indicating the importance of viral particle-associated US28 in dampening Akt activation during viral entry. Accordingly, the phosphorylation of proline-rich Akt substrate of 40 kDa (PRAS40), a Akt substrate (64), was greater in monocytes infected with US28comp HCMV than in those infected with WT HCMV (fig. S9). The multiprotein complex mTORC1 is another downstream target of Akt, and its activity appeared to be enhanced in monocytes infected with US28Δ HCMV relative to WT HCMV as evidenced by S6K phosphorylation (fig. S9), a marker for mTORC1 activation (65). In addition to Akt, pUL38 antagonizes the ability of the tumor suppressor protein complex to inhibit mTORC1 and was also detectable in monocytes infected with US28Δ HCMV (fig. S1A) (66). However, our data suggest that Akt activity is a regulator of mTORC1 in monocytes infected with US28Δ HCMV because loss of Akt activity prevented S6K phosphorylation (fig. S9) and IE1 induction (Fig. 4A). Overall, these data suggest particle-associated US28 redirects Akt signaling during early infection to prevent the initiation of UL123 transcription and the establishment of HCMV quiescence in monocytes.

Fig 4. US28 attenuates Akt signaling to establish quiescence.

(A to C) Monocytes were infected with WT or US28Δ HCMV (MOI = 1). After 30 min to allow uninterrupted viral entry, monocytes were treated with the EGFR inhibitor AG1478 (5 μM), the pan-PI3K inhibitor LY294002 (25 μM) or the Akt inhibitor MK2206 (10 μM). After 24 h, IE1 protein and UL123 transcript levels were measured by Western blot (A, B) and RT-qPCR analysis (C), respectively. RT-qPCR data are means ± SEM from at least 3 biological replicates per group. (D to F) Phosphorylation of Akt at Ser⁴⁷³ and Thr³⁰⁸ and total Akt were detected by Western blot in monocytes after 30 min of infection (D). β-actin was used as a loading control. Levels of phosphorylated or total Akt were normalized to actin (E, F). The phosphorylation ratios of Ser⁴⁷³ or Thr³⁰⁸ to total Akt were determined with WT infection set to 1 (E). Total Akt abundance is shown relative to WT infection, which was set to 1 (F). Western blots and densitometry are representative of at least 3 biological replicates per group. *P<0.05, **P < 0.005, ***P < 0.0005 by one-way ANOVA with Tukey’s HSD post hoc test.

US28 dampens Akt activity induced by HCMV entry into monocytes to prevent IE1 induction.

During canonical activation of Akt, receptor tyrosine kinases (RTKs) activate PI3K, which phosphorylates PI(4,5)P₂ to generate PI(3,4,5)P₃ (PIP₃). PIP₃ recruits Akt to the plasma membrane, leading to the phosphorylation of Akt at Thr³⁰⁸ and Ser⁴⁷³ by PDK1 and mTORC2, respectively (40, 41). We used several strategies to force the phosphorylation at both residues during WT HCMV infection. First, we pretreated monocytes with GM-CSF prior to infection to stimulate Akt phosphorylation at both Ser⁴⁷³ and Thr³⁰⁸ (67) (Fig. 5A), which was associated with IE1 induction during WT HCMV infection (Fig. 5B, 5C). Flow cytometry showed the abundance of macrophage-associated surface markers at 24 hpi were similar between untreated WT HCMV-infected monocytes and those treated with GM-CSF (fig. S10), suggesting the short duration of GM-CSF treatment was unlikely to promote sufficient monocyte differentiation that could result in IE1 induction in cells infected with WT HCMV. As a second approach, we added exogenous PIP₃ to induce phosphorylation of Akt at Ser⁴⁷³ and Thr³⁰⁸ (Fig. 5D) (40, 41), which was associated with increased IE1 abundance in WT HCMV-infected monocytes (Fig. 5E, 5F). The SH2 domain-containing inositol 5-phosphatase (SHIP) 1 product PI(3,4)P₂ (PIP₂), which promotes the site-specific phosphorylation of AKT at Ser⁴⁷³ during infection with WT HCMV (42, 43), also led to IE1 induction, albeit to a lesser extent than with PIP₃ treatment. However, IE1 induction caused by PIP₂ was likely due to the unexpected increase in Thr³⁰⁸ phosphorylation along with the expected increase in Ser⁴⁷³ phosphorylation (Fig. 5D). Finally, we transfected monocytes with an Akt-expressing construct in which the N-terminus of Akt is fused to a myristylation domain (myr-Akt), which results in the recruitment of Akt to the cell membrane (67, 68) and constitutive phosphorylation at both Ser⁴⁷³ and Thr³⁰⁸. As expected, monocytes expressing the myr-Akt construct and infected with WT HCMV had robust phosphorylation at both Ser⁴⁷³ and Thr³⁰⁸ (Fig. 5G), which was associated with robust IE1 induction in monocytes infected with WT HCMV (Fig. 5H, 5I). Additionally, we found that UL123 transcription was significantly increased in myr-Akt-expressing monocytes infected with WT HCMV (fig. S11), indicating IE1 induction in monocytes infected with WT HCMV with Akt phosphorylation at Ser⁴⁷³ and Thr³⁰⁸ was not due to enhanced Akt/mTORC1-mediated protein translation. Overall, our data suggest that phosphorylation of Akt at both Ser⁴⁷³ and Thr³⁰⁸ during HCMV entry leads to the initiation of UL123 transcription and subsequent IE1 induction.

Fig 5. Induction of phosphorylation of Akt at Thr³⁰⁸ results in IE1 induction during infection with WT HCMV.

(A to C) Monocytes were treated with PBS or GM-CSF (100 ng/ml) for 30 min and mock-infected or infected with WT or US28Δ HCMV (MOI = 1) for 30 min (A) or 24 h (B, C). (D to F) Monocytes were treated with 15 μM of indicated phosphatidylinositol (PIP) or empty lipid carrier (EC) for 50 min and mock-infected or infected with WT and US28Δ HCMV for 30 min (D) or 24 h (E, F). (G to I) Monocytes were transfected with myristyolated-Akt (myr-Akt) plasmid or empty vector (EV) for 48 h and were mock-infected or infected with WT, or US28Δ infected for 30 min (G) or 24 h (H, I). Phosphorylation of Akt at Ser⁴⁷³ and Thr³⁰⁸ and total Akt (A, D, G) and IE1 (B, E, H) were detected by Western blot and quantified (C, F, I). β-actin was used as a loading control. Western blots and densitometry are representative of at least 3 biological replicates per group. *P<0.05, **P < 0.005, ***P < 0.0005, **** P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test.

Akt activity and substrate specificity depends on the phosphorylation ratio between Ser⁴⁷³ and Thr³⁰⁸ (59-62); thus, we asked whether the US28-mediated dampening of Akt activity (by the loss of Thr³⁰⁸ phosphorylation) induced during viral entry was necessary to prevent IE1 induction. Knockdown of PDK1 with siRNAs (Fig. 6A), which reduced Akt phosphorylation at Thr³⁰⁸ (Fig. 6B, 6C) without affecting total Akt abundance (Fig. 6D), reduced IE1 induction in monocytes infected with US28Δ HCMV (Fig. 6E, 6F). Additionally, we transfected monocytes with siRNA targeting rictor (which is critical for the kinase activity of mTORC2) to assess if the full activation of Akt through phosphorylation of Ser⁴⁷³ and Thr³⁰⁸ was required for IE1 induction (Fig. 6A). Rictor-deficient monocytes infected with US28Δ HCMV exhibited decreased Ser⁴⁷³ phosphorylation relative to cells transfected with a control siRNA (Fig. 6B, 6C) without affecting total Akt abundance (Fig. 6D) and reduced IE1 induction (Fig. 6E, 6F). Collectively, our data indicates US28 dampens HCMV-induced Akt activity by preventing Thr³⁰⁸ phosphorylation. This partial Akt activity allows for the induction of select antiapoptotic proteins necessary for the survival of HCMV-infected monocytes without initiating IE gene expression associated with fully active Akt, which requires phosphorylation of both Ser⁴⁷³ and Thr³⁰⁸.

Fig 6. Akt phosphorylation at both Ser⁴⁷³ and Thr³⁰⁸ is required for IE1 induction in monocytes infected with US28Δ HCMV.

(A to D) Monocytes were transfected with scrambled (NC), PDK1, or rictor siRNA for 48 h and infected with WT or US28Δ HCMV for 30 min (A, B) or 24 h (C, D). PDK1, rictor, total Akt, phosphorylation of Akt at Ser⁴⁷³, phosphorylation of Akt at Thr³⁰⁸, and IE1 were determined by Western blot (A and B) and quantified (C and D). β-actin was used as a loading control. Western blots and densitometry are representative of at least 3 biological replicates per group. **P < 0.005, ***P < 0.0005, **** P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test.

US28 restricts EGFR activation to reduce Akt phosphorylation.

Next, we investigated the mechanism by which US28 suppresses Akt phosphorylation. During HCMV-mediated non-canonical activation of Akt, SHIP1 is activated and converts PIP₃ to PIP₂, which leads to the phosphorylation of Akt at Ser⁴⁷³ (42, 43). Thus, we hypothesized that US28 promotes SHIP1 activity to ensure phosphorylation of Akt at Ser⁴⁷³ during infection with WT HCMV. However, SHIP1 activity (Fig. 7A, 7B) and abundance (Fig. 7C) was similar between monocytes infected with WT or US28Δ HCMV. Further, inhibition of SHIP1 using 3AC at concentrations that attenuated HCMV-induced Ser⁴⁷³ phosphorylation (Fig. 7D), but did not affect cell survival (fig. S12A) or viral entry (fig. S12B), did not allow for UL123 mRNA and IE1 induction during WT infection (Fig. 7E to 7G). Inhibition of SHIP1 during infection with US28Δ HCMV increased IE1 abundance (Fig. 7E, 7F), which was likely due to the increased accumulation of PIP₃ and subsequent enhanced phosphorylation of Akt at both Thr³⁰⁸ and Ser⁴⁷³ phosphorylation. Together, these data argue that US28 may regulate factors upstream of SHIP1, such as EGFR. Indeed, we found that EGFR phosphorylation was greater in monocytes infected with US28Δ HCMV than in those infected with WT HCMV (Fig. 8A, 8B). Infection with either WT or US28Δ HCMV had no effect on EGFR abundance (Fig. 8C). Additionally, inhibiting EGFR activity with AG1478 after infection with US28Δ HCMV reduced Akt phosphorylation at both Ser⁴⁷³ and Thr³⁰⁸ (Fig. 8D) without affecting monocyte viability (fig. S7A) or viral entry (fig. S7B) at the concentrations used. Similarly, inhibition of downstream PI3K resulted in decreased Akt phosphorylation (Fig. 8D), suggesting that US28 attenuates EGFR activation to reduce Akt activity induced during viral entry. Moreover, infection with the US28-recombinant ΔN and R129A HCMV mutants with defective signaling capabilities also resulted in increased EGFR activation and Akt phosphorylation at Ser⁴⁷³ and Thr³⁰⁸ (Fig. 8E). To assess if US28 was sufficient to limit EGFR activity, we transduced THP-1 cells, a monocytic cell line permissive for HCMV latency, with a lentivirus construct (pSLIK-US28-3xFLAG) that allows for the doxycycline (DOX)-inducible expression of US28 (15). Indeed, we found that expression of US28 alone attenuated EGF-induced EGFR activity (Fig. 8F, 8G). Together, these data indicate US28 is necessary and sufficient to restrict EGFR activation to limit HCMV-induced Akt activity during the establishment of HCMV quiescence in monocytes.

Fig 7. US28 does not regulate SHIP1 activity.

(A to G) Monocytes were mock-infected or infected with WT or US28Δ HCMV (MOI = 1) for 30 min and incubated with ethanol (EtOH; carrier control) or 3AC (5 μM; SHIP1 inhibitor) for 30 min (A to C) or 24 h (D to F). Total SHIP1, p-SHIP1, total Akt, phosphorylation of Akt at Ser⁴⁷³, and phosphorylation of Akt at Thr³⁰⁸ were detected by Western blot (A to D). IE1 protein and UL123 transcript levels were detected by Western blot (E, F) or RT-qPCR analysis (G), respectively. Western blots and densitometry are representative of at least 3 biological replicates per group. RT-qPCR data are means ± SEM from at least 3 biological replicates per group. ns= not significant, *P<0.05, ***P < 0.0005, ****, P < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test.

Fig 8. US28 limits HCMV-induced EGFR activation during viral entry.

(A to C) Monocytes were mock-infected or infected with WT or US28Δ HCMV (MOI = 1) for 30 min. Total EGFR and phosphorylated EGFR were detected by Western blot (A) and quantified (B and C). (D) Monocytes were mock-infected or infected with WT or US28Δ HCMV (MOI = 1) for 30 min. After 30 min of uninterrupted binding and entry, monocytes were treated with AG1478 (5 μM), LY294002 (25 μM), or MK2206 (10 μM) for an additional 30 min. Phosphorylation of Akt at Ser⁴⁷³ and Thr³⁰⁸ and total Akt were detected by Western blot. (E) Monocytes were mock-infected or infected with WT, US28Δ, ΔN, or R129A HCMV (MOI = 1) for 30 min. Phosphorylated EGFR, total Akt, total Akt, phosphorylation of Akt at Ser⁴⁷³, and phosphorylation of Akt at Thr³⁰⁸ were detected by Western blot. (F and G) pSILK empty vector or pSLIK-US28-3xF-expressing THP-1 cells were treated with DOX for 24 hours and then with EGF (200 ng/mL) for 30 min. Total EGFR and phosphorylated EGFR, and FLAG (to detect US28) were detected by Western blot (F) and quantified (G). β-actin was used as a loading control. Western blots and densitometry are representative of at least 3 biological replicates per group. ns= not significant, *P<0.05, **P < 0.005 by one-way ANOVA with Tukey’s HSD post hoc test.

Discussion

Silent infections within the myeloid compartment play a critical role in HCMV’s dissemination (mediated by monocytes) and persistence (mediated by CD34⁺ cells) strategies. A considerable effort has been made to elucidate the mechanisms regulating viral latency in myeloid cells (69-71). However, mechanisms by which HCMV promotes the establishment of a quiescent infection within monocytes remain unclear. Thus, we sought to determine the mechanism through which US28 promotes the establishment of a quiescent infection within monocytes. Here, we demonstrated that particle-associated US28 was both necessary and sufficient to dampen EGFR signaling to limit Akt activity triggered by HCMV entry. The reduced non-canonical activation was associated with phosphorylation of Akt only at Ser⁴⁷³ (Fig. 9), which increases the expression a select subset of pro-survival proteins required for the long-term survival of normally short-lived monocytes (42, 43, 63, 72). In this study, we showed that the preferential phosphorylation at Ser⁴⁷³, but not at Thr³⁰⁸, was also critical for the establishment of a quiescent infection because the full activation of Akt by phosphorylation at both sites leads to the initiation of IE gene expression and subsequent lytic replication cycle (Fig. 9). These results highlight particle-delivered US28 as a critical player to the viral dissemination strategy because it rapidly modulates cellular signaling pathways triggered during viral entry to produce a cellular environment conducive to both the long-term survival of infected monocytes and the establishment of a quiescent infection.

Fig 9. Proposed model for US28-mediated regulation of quiescence during HCMV infection.

During infection of monocytes with WT HCMV, US28 limits HCMV-induced EGFR phosphorylation. Downstream of EGFR, PI3K phosphorylates PI(4,5)P₂ to PIP₃. Additionally, HCMV infection results in increased SHIP1 activity, which dephosphorylates PIP₃ to PIP₂. Recruitment of Akt to PIP₂ leads to preferential phosphorylation at Ser⁴⁷³ and establishment of a quiescent infection during infection with WT HCMV. In monocytes infected with US28Δ HCMV, EGFR is robustly phosphorylated without changes in SHIP1 activity. Thus, in the absence of US28, an accumulation of PIP₃ leads to Akt phosphorylation at both Ser⁴⁷³ and Thr³⁰⁸, which is required for IE1 induction and lytic HCMV replication. Created with Biorender (biorender.com).

Akt is responsible for translating receptor-mediated signaling events into different cellular functional outcomes. However, the role of Akt during lytic infection of fibroblasts has remained controversial due to contradictory findings from different groups. Initial studies found that HCMV entry into permissive fibroblasts triggered early activation of Akt, which was maintained throughout lytic replication to support viral entry, gene expression, and DNA replication (73-76). In contrast, emerging evidence indicate that Akt activity is suppressed as the lytic infection cycle progresses, allowing for maximal virus production (58, 77-79). The Akt phosphorylation pattern was not defined in these studies; thus, we speculate that HCMV infection exerts differential effects on the Akt activation profile depending on the stage of the replication cycle, which could explain these opposing results. Here, we demonstrated that the phosphorylation at Akt Thr³⁰⁸ and Ser⁴⁷³ initiated lytic replication in myeloid cells, consistent with previous studies showing that the early activation of Akt triggered by viral entry supports lytic replication in fibroblasts. Another possibility is that lytic infection of highly permissive fibroblasts and lytic infection following reactivation in myeloid cells are not equivalent, and that the signaling and/or factors required to complete the lytic cycle are cell-type-specific. Nonetheless, these findings underscore the complex relationship between Akt and HCMV lytic infection, whereby Akt appears to exert either positive or negative effects depending upon the stage of the lytic replication cycle.

During latent infection, PI3K/Akt signaling is also rapidly activated during the initial establishment of latency within CD34⁺ stem cells to promote viral entry (80). Following the establishment of latency, EGFR/PI3K/Akt signaling is required to maintain latency because inhibition of this pathway enhances viral reactivation from latently infected CD34⁺ cells (58). During quiescent infection of monocytes, HCMV rapidly induces noncanonical, partial activation of Akt during entry of monocytes to promote the long-term survival of HCMV-infected monocytes (Fig. 4D, 4E) (43, 81-83). In contrast to latently infected CD34⁺ HPCs, inhibition of Akt did not lead to IE expression, but rather stimulating the full activation of Akt promoted the rapid expression of IE following viral entry into monocytes (Fig. 5A to 5I). The discrepancy between the positive role of Akt on IE induction during the establishment of quiescence in monocytes versus the negative role of Akt during the maintenance of latency in CD34⁺ HPCs is unclear. The Thr³⁰⁸/Ser⁴⁷³ phosphorylation ratio of Akt was not examined in CD34⁺ HPCs and thus, it is possible that Akt has a distinct activation profile during viral entry into monocytes. Alternatively, Akt activity could change as silent infections transition from establishment to maintenance. Regardless, how HCMV modulates the EGFR/PI3K/Akt signaling pathway and the subsequent functional outcomes appears to be cell specific and timing dependent.

Monocytes are short-lived cells programmed to undergo apoptosis 48 h after their release from the bone marrow in the absence of differentiation stimuli (84-86). Following stimulation with normal myeloid growth factors, Akt is activated by phosphorylation at both Ser⁴⁷³ and Thr³⁰⁸ to mediate the survival of short-lived monocytes (87-89). In contrast, HCMV promotes the survival of the infected monocytes by an atypical activation of Akt phosphorylated only at Ser⁴⁷³ (42, 43). The phosphorylation ratio between Ser⁴⁷³ and Thr³⁰⁸ dictates Akt activity as well as substrate specificity (59-62), suggesting that HCMV-activated Akt exhibits a distinct biological output compared to growth factor-activated Akt. Indeed, during quiescent infection of monocytes, HCMV increases the synthesis of a subset of Akt-dependent antiapoptotic proteins, including Mcl1, HSP27, and XIAP (72, 90). In this current study, we found that WT HCMV-mediated Akt phosphorylation at Ser⁴⁷³ did not result in IE induction, whereas US28Δ-mediated Akt phosphorylation at Ser⁴⁷³ and Thr³⁰⁸ led to IE1 production (Fig. 5A to 5I; Fig. 6B to 6F; Fig. 7D to 7F) and the subsequent induction of viral early and late proteins (fig. S1A to S1C). Because MIEP is differentially regulated by multiple transcription factors during lytic and latent infections (70), we speculate that Akt phosphorylated at Ser⁴⁷³ does not activate the same subset of transcription factors as Akt phosphorylated at both Ser⁴⁷³ and Thr³⁰⁸. Our data suggests that US28 guides the precise activation of Akt to promote the survival of infected monocytes without triggering Akt-dependent transcription factors necessary for the initiation of IE gene expression. In support of this, US28 inhibits several transcription factors that bind to the MIEP, including AP-1 and NF-κB, which are required for attenuating IE gene expression and establishing latency within CD34⁺ (14), Kasumi-3 (14), and THP1 cells (10).

It is unclear how US28 modulates the phosphorylation profile of Akt to generate a signaling network that is both essential to the survival of infected monocytes and beneficial to the establishment of quiescent infection. During viral entry, the HCMV viral glycoprotein gB directly binds and activates EGFR (42, 43), which then activates downstream PI3K to phosphorylate PI(4,5)P₂ into PIP₃. During canonical EGFR/PI3K signaling, PIP₃ directs Akt phosphorylation at both Ser⁴⁷³ and Thr³⁰⁸. In naïve uninfected cells, PIP₃ can be dephosphorylated to PIP₂ by SHIP1 to attenuate Akt activity. However, in certain cancer cells, including leukemia cells and HCMV-infected monocytes, the recruitment of Akt to PIP₂ leads to a site-specific phosphorylation at Ser⁴⁷³ (43, 91-93). Thus, we hypothesized US28 enhances Ser⁴⁷³ phosphorylation by either increasing SHIP1 activity to promote the conversion of PIP₃ into PIP₂ or decreasing EGFR activity to reduce accumulation of PIP₃ within HCMV-infected monocytes. Our study demonstrated that particle-associated US28 expression attenuates EGFR activity (Fig. 8A, 8B) and does not affect SHIP1 activity (Fig. 7A, 7B). Because the conversion of PIP₃ to PIP₂ by SHIP1 appears to be the rate limiting step, we accordingly found the addition of exogenous PIP₃ during infection with WT HCMV resulted in canonical phosphorylation of Ser⁴⁷³ and Thr³⁰⁸ (Fig. 5D). These data suggest that HCMV prevents the accumulation of PIP₃ by restricting EGFR activity, allowing PIP₂ to be the predominant signaling lipid responsible for mediating Akt phosphorylation.

Many biological effects of GPCRs occur through crosstalk with EGFR (94, 95). The exact mechanism by which particle-associated US28 limits EGFR activity remains to be determined. One possibility is that US28 functions similar to GPRC5A by binding and sequestrating EGFR, leading to its recycling/degradation through endocytic pathway (96). Another possibility is through the proteolytic cleavage and subsequent release of EGFR ligands by US28. GCPRs stimulate the release of EGFR ligands (97-100), which induce a conformational change in EGFR, resulting in dimerization and auto-phosphorylation of the tyrosine kinase domain (101-103). However, the stability of dimerization is dictated by ligand specificity, which affects the phosphorylation and signaling properties of EGFR (104). US28 may attenuate EGFR activity by desensitization through proteolytic cleavage of EGFR ligands such as epiregulin and epigen, which are found on the monocyte surface and promote EGFR activation to a lesser extent relative to EGF (104, 105). Although elucidating the mechanism through which US28 limits Akt activity requires additional research, our study demonstrates that US28-mediated suppression of EGFR signaling plays a pivotal role in shaping the Akt signaling network to promote the establishment of quiescent HCMV infection within monocytes.

Our study demonstrates the effect of differential Akt phosphorylation status in the regulation of IE gene expression during the establishment of a quiescent infection within primary monocytes. Suppression of the initial lytic infection in monocytes is critical for long-term viral persistence within monocytes and HCMV’s dissemination. Together, our study demonstrates that particle-associated US28 attenuates EGFR to dampen and redirect Akt activity into generating a cellular environment simultaneously conducive to both monocyte survival and quiescent infection. Unraveling the interplay between viral and cellular signaling factors could identify new antiviral targets aimed at preventing reactivation of HCMV from the myeloid compartment.

Materials and Methods

Human Peripheral Blood Monocyte Isolation.

Peripheral blood was drawn from random deidentified donors by venipuncture, diluted in Roswell Park Memorial Institute medium (RPMI) 1640 (ATCC, Product #30-2001), and centrifuged through Histopaque 1077 (MilliporeSigma) to remove red blood cells and neutrophils. Mononuclear cells were collected, washed with saline to remove the platelets, and separated by centrifugation through a Percoll (GE Healthcare) gradient (40.48% and 47.70%). More than 90% of isolated peripheral blood mononuclear cells were monocytes, as determined by CD14-positive staining (106). Cells were washed with saline, resuspended in RPMI 1640 (ATCC, Product # 30-2001), and counted. All experiments were performed in the absence of human serum (unless mentioned otherwise) at 37°C in a 5% CO₂ incubator. SUNY Upstate Medical University’s 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 (IRB#: 262458-19). Isolation of human peripheral blood monocytes was performed as previously described (9, 81).

Virus Preparation and Infection.

Bacterial artificial chromosome (BAC)-derived TB40/EmCherry (wild-type; WT) (52) , TB40/EmCherry-US28-3xF (US28-3xF) (20), TB40/EmCherry-US28Δ (US28Δ) (20), TB40/EmCherry-US28ΔN-3xF (ΔN) (15), TB40/EmCherry-US28-R129A-3xF (R129A) (15), and US28-complemented US28Δ (US28comp) (15) were previously described. All virus stocks were propagated on human embryonic lung (HEL) 299 fibroblasts (CCL-137, ATCC) of low passage (P7-15) in Dulbecco's Modified Eagle medium (DMEM) (Lonza) with 2.5 μg/ml plasmocin (Invivogen) and 10% fetal bovine serum (FBS) (MilliporeSigma). When a 100% cytopathic effect was observed, virus was purified from the supernatant by ultracentrifugation (115000 x g, 65 minutes (min), 22°C) through a 20% sorbitol cushion to remove cellular contaminants and resuspended in RPMI 1640 medium (ATCC, Product # 30-2001). A multiplicity of infection (MOI) of 1 genome copy/cell was used for each experiment unless otherwise stated. Mock infection was performed by adding an equivalent volume of RPMI 1640 medium to monocytes.

To determine the incorporation of US28 into HCMV particles (virions, DBs, or NIEPs), primary newborn human fibroblasts (NuFF-1; GlobalStem) were infected with TB40/EmCherry-US28-3xFLAG (US28-3xFLAG), TB40/EmCherry-US28-R129A-3xF (R129A), or TB40/EmCherry-US28ΔN-3xF (ΔN) (MOI = 1.0 TCID₅₀/cell). Once a 100% cytopathic effect was observed, the media was collected and precleared two times by low-speed centrifugation (500 x g) and viral particles were purified through a 20% sorbitol cushion (51,610 x g, 90 min, room temperature). Viral particles were concentrated 100X in 10 mM Tris-Cl, pH8.0, 400 mM NaCl, and 10 mM EDTA, and one-fifth of this was used for Western blot analyses. As controls for expression of US28 and its mutants, whole cell lysates were also collected, lysed, denatured (at 42°C), separated by SDS-PAGE, and assessed by Western blot. US28 abundance was assessed by probing for the FLAG epitope tag.

Virus-Binding and Entry Assay.

To assess viral binding, monocytes were incubated on ice for 1 hour (h) and infected with WT or US28Δ for 2 h on ice to allow for viral binding. Monocytes were washed twice with ice-cold 1X PBS to remove any unbound viral particles. As a negative control, another set of infected monocytes were treated with proteinase K (1.0 mg/ml, 30 m on ice) after the 2 h incubation and washed twice with ice-cold 1X PBS to remove bound virus. DNA was extracted with QIAamp DNA mini kit (Qiagen) according to the manufacturer’s recommendations.

To quantify viral entry, monocytes were pre-treated with the EGFR inhibitor AG1478 (10 μM) for 1 h at 37°C to block efficient viral entry into monocytes or with vehicle control (DMSO). Cells were incubated on ice for 1 h to allow for particle binding and infected with WT or US28Δ HCMV for an additional hour on ice. Infected cultures were shifted to 37°C for 2 h to allow for viral entry into monocytes. Finally, monocytes were treated with proteinase K (1.0 mg/ml, 5 min at 37°C) and washed twice with 1X PBS to remove the viral particles that remained outside of the cell membrane and were unable to enter monocytes. RNeasy mini kit (Qiagen) was used per the manufacturer’s recommendation to extract mRNA. Any contaminating DNA from the samples was removed using TURBO DNA free kit (ThermoFisher), according to manufacturer’s protocols.

Compound Treatments.

Where indicated, the following reagents were used to treat the cells at concentrations noted in the text: the lysosomal inhibitor bafilomycin (Baf (107)), the proteasomal inhibitor MG132 (MG (108)), the EGFR inhibitor AG-1478 (109), the SHIP1 inhibitor 3-α-aminocholestane (3AC (93)) and the pan-PI3K inhibitor LY294002 (110), the AKT Inhibitor MK-2206 (111) and the broad spectrum protein kinase inhibitor/inducer of apoptosis staurosporine (ST (112)). All compounds were from Calbiochem, except for MK and ST, which were from Selleckchem. To induce canonical Akt activation, monocytes were treated with PBS or GM-CSF (100 ng/ml) for 30 min before infection. Additionally, where indicated, samples were treated with 15 μM of phosphatidylinositol lipids (PIP₃, Echelon Biosciences, Product #P-3908; PIP₂, Echelon Biosciences, Product #P-3416) or empty lipid carrier (EC; Echelon Biosciences, Product #P-9C1) for 50 min before infection according to the manufacturer’s recommendations.

Western Blot Analysis.

Cells were harvested in modified radioimmunoprecipitation assay (RIPA) 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 (MilliporeSigma) and phosphatase inhibitor cocktails 2 and 3 (MilliporeSigma) for 30 min on ice. The lysates were cleared of cell debris by centrifugation at 4°C (5 min, 21000 x g) and stored at −20°C until further analysis. Protein samples were solubilized in Laemmli SDS-sample non-reducing (6x) buffer (Boston Bioproducts), supplemented with β-mercaptoethanol (Amresco) by incubation at 95°C for 5 min, unless otherwise stated. To detect EGFR, phospho-EGFR and US28, protein samples were incubated at room temperature for 30 min after solubilizing in Laemmli SDS-sample nonreducing (6x) buffer (113). Equal amounts of total protein were loaded, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes (Bio-Rad). Blots were blocked in 5% bovine serum albumin (BSA; Fisher Scientific) for 1 h at room temperature andincubated with primary antibodies overnight at 4°C. The blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 30 min at room temperature, and chemiluminescence was detected using the Clarity Western ECL substrate (Bio-Rad). The antibodies used in this study are described in Table S1. Densitometry was performed using Image Lab software (Bio-Rad).

Semiquantitative and Quantitative PCR.

Total DNA and mRNA were isolated using QIAamp DNA mini kit and RNeasy mini kit (Qiagen, Germantown, MD), per the manufacturer’s recommendation. TURBO DNA free kit (ThermoFisher, Waltham, MA) was used to remove the contaminating DNA from the extracted mRNA samples. Semiquantitative PCR was used to detect the expression of UL122 (sense, 5’-CGCCTTCGTTACAAGCATCG-3’; antisense, 5’-AAGAGCAAACGCATCTCCGA-3’), UL54 (sense, 5’-AAGGACAGGCATCGATAGCG-3’; antisense, 5’-CACCTACGATCAGACGGACG-3’), UL83 (sense, 5’-TGCATAAAGAGCTTGCCGGA-3’; antisense, 5’-CAGACGGGTATCCACGTACG-3’), GAPDH (sense, 5’-ACCCACTCCTCCACCTTTGAC-3’; antisense, 5’-CTGTTGCTGTAGCCAAATTCGT-3’) using Green GoTaq master mix (Promega, Madison, WI) with C1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA). For quantitative PCR, UL123 (sense, 5’-AGTGACCGAGGATTGCAACG-3’; antisense, 5’-CCTTGATTCTATGCCGCACC-3’), US28 (sense, 5’-CCAGAATCGTTGCGGTGTCTCAGT-3’; antisense, 5’-CGTGTCCACAAACAGCGTCAGGT-3’) and GAPDH were detected with CFX Connect Real Time System (Bio-Rad) using iTaq Universal SYBR Green Supermix (Bio-Rad). For quantitative real time PCR (RT-PCR), iTaq Universal SYBR Green One-Step Kit was used to detect the expression of UL123, US28 and 18S rRNA (sense, 5’-GCAATTATTCCCCATGAACG-3’; antisense, 5’-GGGACTTAATCAACGCAAGC-3’). For quantitative PCR, samples were analyzed in technical duplicate and normalized to GAPDH (for DNA) or 18S rRNA (for mRNA).

Flow Cytometry.

Monocytes were washed in 1X phosphate-buffered saline (PBS) and incubated in blocking solution, consisting of fluorescence-activated cells sorting (FACs) buffer (1X PBS, 2mM EDTA, 0.5% BSA), 5% bovine serum albumin (BSA), and human Fc receptor (FcR) binding inhibitor (eBioscience). Cells were stained with an allophycocyanin (APC)-anti-CD14 or APC-anti-mouse IgG1 isotype control antibody (BioLegend) on ice, washed, and stained with FITC-annexin V and propidium iodide (PI; ThermoFisher Scientific) to detect dead and dying cells. For differentiation studies, cells were stained with anti-CD86 and anti-CD163. The antibodies used in this study are described in Table S1. After staining, cells were analyzed by flow cytometry using an LSRFortessa cell analyzer and BD FACSDiva software (BD Biosciences). To measure mCherry fluorescence, monocytes were washed in 1X PBS and resuspended in FACs buffer before flow cytometry analysis.

Plasmid and siRNA Transfection.

Primary monocytes (3 × 10⁶ cells/transfection) were washed with 1X PBS and resuspended in 100 μl of P3 Primary Cell Nucleofector solution (Lonza) containing either 1000 ng empty vector (EV) (Addgene plasmid # 10841 (114)), or myristylated-Akt (myr-Akt) plasmid (Addgene plasmid # 26453 (68)). To knockdown PDK1 and Rictor, 1000 nM validated Silencer Select siRNAs against PDK1 (Invitrogen-Themo Fisher Scientific; siRNA ID # s10274) or Rictor (Invitrogen-Themo Fisher Scientific; siRNA ID # s48410) or Silencer negative-control (NC) siRNAs (Themo Fisher Scientific; catalog # AM4642) were mixed in the nucleofector solution before transfection. Plasmids and siRNAs were transfected with a 4D-Nucleofector (Lonza) using program EI-100. After transfection, monocytes were incubated in RPMI 1640 supplemented with 2% human AB serum at 37°C for 48 hours, after which transfected monocytes were infected (multiplicity of infection MOI = 1) with mock, WT, or US28Δ for 24 hours. Whole cell lysates were collected and subjected to Western blot analyses.

Statistical analyses.

All experiments were performed with a minimum of 3 biological replicates per group using primary monocytes isolated from different blood donors. Data were analyzed using one-way ANOVA with Tukey’s honest significant difference (HSD) post hoc test or Student’s t test comparison with GraphPad Prism software, and p-values less than 0.05 were considered statistically significant.