HCMV-induced signaling through gB–EGFR engagement is required for viral trafficking and nuclear translocation in primary human monocytes

Significance

Human cytomegalovirus (HCMV) pathogenesis relies upon viral infection of monocytes, as this is the key cell type mediating the hematogenous dissemination of HCMV throughout the host. Here we report that HCMV glycoprotein B surprisingly remains bound to the epidermal growth factor receptor (EGFR) throughout the duration of viral postentry events to induce chronic signaling through EGFR. This chronic EGFR signaling is required to facilitate viral localization to correct trafficking vesicles, as well as promoting nuclear translocation of viral DNA into the monocyte nucleus, both of which are necessary for infection of monocytes. Our data highlight how HCMV regulates the EGFR kinase externally and, uniquely, internally to promote separate and distinct events required for productive infection, spread, and persistence.

Abstract

Previous analysis of postentry events revealed that human cytomegalovirus (HCMV) displays a unique, extended nuclear translocation pattern in monocytes. We determined that c-Src signaling through pentamer engagement of integrins is required upon HCMV entry to avoid sorting of the virus into late endosomes and subsequent degradation. To follow up on this previous study, we designed experiments to investigate how HCMV-induced signaling through the other major axis—the epidermal growth factor receptor (EGFR) kinase—regulates viral postentry events. Here we show that HCMV induces chronic and functional EGFR signaling that is distinct to the virus as compared to the natural EGFR ligand: EGF. This chronic EGFR kinase activity in infected monocytes is required for the proper subcellular localization of the viral particle during trafficking events, as well as for promoting translocation of viral DNA into the host nucleus. Our data indicate that HCMV glycoprotein B (gB) binds to EGFR at the monocyte surface, the virus and EGFR are internalized together, and gB remains bound to EGFR throughout viral postentry events until de-envelopment to promote the chronic EGFR kinase activity required for viral trafficking and nuclear translocation. These data highlight how initial EGFR signaling via viral binding is necessary for entry, but not sufficient to promote each viral trafficking event. HCMV appears to manipulate the EGFR kinase postentry, via gB–EGFR interaction, to be active at the critical points throughout the trafficking process that leads to nuclear translocation and productive infection of peripheral blood monocytes.

Human cytomegalovirus (HCMV) is a species-specific betaherpesvirus that infects 60 to 90% of the world’s population and is the leading cause of congenital viral infections. HCMV poses a severe health risk to immunocompromised individuals, such as AIDS patients and solid organ and bone marrow transplant recipients, where infection can result in a multiorgan disease (1, 2). This multiorgan disease is a direct result of the broad cellular tropism of HCMV as well as the ability of the virus to disseminate throughout the host. From the site of primary infection, HCMV moves into the bloodstream where the virus infects peripheral blood monocytes to establish a cell-associated viremia (2–4). Monocytes are the key cell type mediating hematogenous dissemination of HCMV to secondary organs, including the bone marrow, which is the site of viral latency and life-long viral persistence (5–7). Therefore, determining how HCMV productively infects monocytes is important to understanding viral spread and persistence within the host.

Productive HCMV infection depends upon the ability of the virus to attach to a cell, cross the cellular membrane, de-envelop, traffic the viral capsid to the host nucleus, translocate viral DNA into the nucleus, and initiate viral replication (2). We have previously documented in monocytes that nuclear translocation of HCMV DNA into the host nucleus begins 3 d postinfection (dpi). This delayed timing of nuclear translocation is distinct to HCMV infection of monocytes, as it occurs much faster in fibroblasts and endothelial cells (30 min postinfection, mpi) as well as in hematopoietic progenitor cells (HPCs) (4 to 8 h postinfection, hpi) (8, 9). Further investigation of the postentry events leading to HCMV nuclear translocation in monocytes revealed a unique intracellular trafficking route. In monocytes, HCMV traffics from EEA1⁺ vesicles (15 to 45 mpi) to the trans-Golgi network (TGN) (45 mpi to 1 dpi) to recycling endosomes (2 hpi to 5 dpi), where the virus then de-envelops (2 to 5 dpi), followed by trafficking of the capsid to the nucleus where viral DNA is translocated beginning 3 dpi (9). Because these trafficking events occur prior to nuclear translocation, HCMV must facilitate these events in the absence of de novo viral gene expression.

Over the years, our laboratory has effectively demonstrated that productive HCMV infection of monocytes depends upon a unique signalosome initiated through virus–host receptor–ligand interactions (7, 10). At the monocyte cell surface, viral glycoprotein B (gB) binds to the epidermal growth factor receptor (EGFR) and the viral pentamer complex (gH/gL/UL128-131) binds to β₁ and β₃ integrins, inducing signaling through the EGFR kinase and c-Src kinase, respectively. HCMV-induced signaling promotes biological changes in the monocyte and is required for steps of the viral lifecycle itself, such as entry and efficient transcription of the viral genome (10–18). Examination of how HCMV-induced signaling affects early viral postentry events in monocytes revealed that c-Src signaling is required for proper viral sorting. The absence of c-Src signaling resulted in viral particles trafficking to late endosomes by 15 mpi and subsequent degradation by 24 hpi (9). These data set a precedence that HCMV-induced signaling may play an important role in regulating viral postentry events leading to productive infection of monocytes.

To further explore how HCMV-induced signaling regulates viral postentry events in monocytes, we began an investigation of the EGFR signaling axis because: 1) HCMV regulates EGFR signaling to mediate important viral processes in multiple cell types and 2) we see HCMV-induced EGFR signaling long past the initial receptor–ligand interaction at the monocyte cell surface. For example, EGFR signaling is required for efficient entry as well as efficient viral transcription in monocytes, viral entry and the establishment and regulation of viral latency in CD34⁺ HPCs and EGFR is removed from the surface of fibroblasts during lytic infection and down-regulated at the level of transcription (8, 13, 19, 20). By proxy of downstream EGFR signaling molecules, we have seen EGFR signaling in monocytes as late as 4 dpi (12, 13, 16). Taken together, these data led us to hypothesize that HCMV-induced temporal signaling through EGFR promotes viral postentry events, thus leading to productive infection of monocytes.

Here we report that HCMV induces chronic activation of the EGFR kinase resulting in long-term functional EGFR signaling. The temporal regulation of the EGFR kinase activity following HCMV infection is different when compared to the natural EGFR ligand EGF, suggesting that HCMV acts as a unique EGFR ligand. We show that HCMV-induced EGFR kinase activity is required throughout the duration of the postentry events to promote viral localization into the correct trafficking vesicles as well as maintaining the proper timing of nuclear translocation. Our data highlight how the EGFR kinase activity initiated during viral binding is necessary for efficient entry into the monocyte, but is not sufficient to promote the subsequent postentry events. As the mechanism perpetuating EGFR kinase activity during postentry events, we excitedly report that HCMV envelope gB binds EGFR at the monocyte surface and, unexpectedly, remains engaged throughout the duration of postentry events. Overall, our findings demonstrate how HCMV temporally regulates the EGFR kinase, via gB–EGFR interaction, to promote viral localization and nuclear translocation, leading to the productive infection of monocytes necessary for viral spread and persistence within the host.

Results

HCMV-Induced EGFR Signaling Is Chronic, Functional, and Distinct from That Seen with the Natural EGFR Ligand EGF.

Regulation of EGFR signaling has been documented to play a role in various steps of the HCMV lifecycle in many different cell types. As mentioned in the introduction, HCMV regulates EGFR signaling to facilitate viral entry into CD34⁺ HPCs as well as regulating viral latency (8, 21, 22). During lytic infection of fibroblasts, EGFR is down-regulated at the transcriptional level (19, 20). In monocytes, we have shown that EGFR signaling is required for early events, such as viral entry, and for later events, such as viral transcription at 3 wk postinfection (8, 13). Upon HCMV infection of monocytes, we have documented the early phosphorylation of EGFR, as well as the long-term activation of downstream EGFR signaling molecules: AKT, PI3K, and STAT1 (12, 13, 16). These data suggest that HCMV induces EGFR signaling to accomplish early and late events of viral infection of monocytes. To further characterize HCMV as an EGFR ligand in primary human monocytes, we determined whether HCMV infection led to chronic phosphorylation of EGFR distinct to that induced by EGF. To assess EGFR activation over time, monocytes were infected with HCMV or treated with EGF and phosphorylated EGFR (p-EGFR) was examined at 0 mpi, 15 mpi, and 24 hpi. To evaluate the differences in EGFR activation between the two ligands, we wanted to saturate surface EGFR with each ligand to ensure that differences detected were not due to varying amounts of EGFR being engaged. Previous work from our laboratory has shown that the amount of EGFR expressed on monocytes is 2 logs lower than that of a breast cancer cell line (MDA-MB-468). We also demonstrated that HCMV infection at a multiplicity of infection (MOI) of 5 is sufficient to saturate the amount of EGFR on monocytes (13). In breast cancer cells, others have shown that EGF at a concentration of about 150 ng/mL saturates surface EGFR (23). Since monocytes have less EGFR than the breast cancer cells, 200 ng/mL of EGF should result in complete engagement of EGFR on monocytes. Both HCMV infection and EGF treatment led to rapid phosphorylation of EGFR at 15 min; however, only HCMV infection resulted in prolonged phosphorylation of EGFR at 24 h. HCMV infection of monocytes resulted in about a threefold increase of p-EGFR as compared to EGF at 24 h (Fig. 1A).

Fig. 1.

HCMV-induced EGFR signaling is chronic, functional, and distinct from that seen with the natural EGFR ligand EGF. Primary human monocytes were serum-starved overnight. The monocytes were then infected with TB40/E or treated with EGF (200 ng/mL). (A) Protein was harvested at 0 mpi, 15 mpi, and 24 hpi. Western blot analyses were performed to detect p-EGFR and actin. Data were quantitated by normalizing to actin and setting the baseline to 0-min mock. Data are representative of four biological replicates. (B and C) Cells were fixed at 30 mpi and 24 hpi. In-cell Westerns were performed to detect actin+p-AKT or actin+p-STAT1. Data are presented as a ratio of target fluorescence intensity over actin fluorescence intensity. The above experiment is combined data from at least two biological replicates and each replicate was performed in technical triplicates. GraphPad Prism 8 software was utilized to perform t tests to determine the statistical significance between mock/HCMV and HCMV/EGF conditions. The error bars represent the SEM. ****P < 0.0001, **P < 0.005, *P < 0.05; n.s., not significant.

We next wanted to determine whether HCMV-induced EGFR activation resulted in functional signaling distinct to the virus as compared to EGF. Monocytes were infected with HCMV or treated with EGF and the activation of downstream EGFR signaling molecules AKT and STAT1 was examined at 30 min and 24 h. At 30 min, both HCMV and EGF induced the phosphorylation of AKT and STAT1 (Fig. 1B). At 24 h, HCMV-induced phosphorylation of both AKT and STAT1 was significantly increased over that observed in EGF-treated cells (Fig. 1C). At 24 h, HCMV infection led to a 3-fold induction of p-EGFR, resulting in a 3-fold increase in p-AKT and a 10-fold increase in p-STAT1 over mock and EGF-treated levels of these phosphorylated proteins. Together, these data demonstrate that HCMV-induced EGFR signaling is functionally distinct from EGF-mediated signaling and that the magnitude of viral-induced EGFR activation results in equivalent or even greater induction of specific downstream signaling molecules.

Chronic EGFR Kinase Activity Promotes HCMV Nuclear Translocation.

We have previously shown that EGFR signaling is required for HCMV entry into monocytes; however, the role of chronic EGFR signaling during postentry events has not been explored. We first wanted to investigate how EGFR signaling regulates HCMV nuclear translocation in monocytes, as this is a critical step in the viral lifecycle. We used the specific EGFR tyrosine kinase inhibitor AG1478, as previously published, to block EGFR kinase activity over a time course in HCMV-infected monocytes (13, 16). AG1478 is membrane permeable and inhibits EGFR kinase activity within 30 min postaddition (24). The inhibitor was added at specific time points that would affect EGFR kinase activity between important transitions during viral trafficking. To ensure that variations in the amount of virus detected in the following studies were due to the inhibition of the EGFR kinase and not variations in the amount of input virus, we examined viral binding of HCMV to monocytes treated with AG1478 or the DMSO vehicle control via end point PCR and immunofluorescence microscopy. These binding studies were performed on ice, which is the viral binding step of our infection protocol further detailed in SI Appendix, SI Material and Methods. We found there was no difference in viral binding when monocytes were treated with AG1478 (SI Appendix, Fig. S1).

To assess the presence of the viral genome in the host cell nucleus, monocytes were harvested 3 dpi, cells were fractionated to isolate nuclei, DNA was harvested from the nuclei, and qPCR was performed to detect the viral genome. When EGFR kinase activity was inhibited at each time point, there was a significant reduction in the amount of viral genome present within the monocyte nucleus at 3 dpi (Fig. 2A). EGFR kinase inhibition during viral transitions from EEA1⁺ vesicles to the TGN (30 mpi) and from the TGN to recycling endosomes (2 hpi) resulted in a decrease of viral genomes within the monocyte nucleus. EGFR kinase inhibition during viral de-envelopment (24 hpi and 48 hpi) also led to a decrease in nuclear translocation at 3 dpi. These data suggest that EGFR kinase activity is required not only upon viral binding to facilitate entry, but throughout the duration of viral postentry events to promote efficient nuclear translocation of the viral genome into the monocyte nucleus.

Fig. 2.

Chronic EGFR kinase activity promotes HCMV nuclear translocation. Primary human monocytes, where indicated, were pretreated with the specific EGFR tyrosine kinase inhibitor AG1478 (1 μM) for 1 h prior to infection. Cells were infected with TB40/E followed by AG1478 (1 μM) addition over a time course: 30 mpi, 2 hpi, 24 hpi, and 48 hpi. AG1478 was supplemented every 24 h after the initial addition to maintain EGFR kinase inhibition. (A) At 3 dpi cells were harvested and the nuclei were extracted. DNA was isolated from the nuclei and qPCR was performed using specific HCMV UL123 genomic or cellular 18s rRNA primers (n = 3). The graph was generated using Adobe Illustrator. Statistical significance was calculated using GraphPad Prism 8 software via t tests comparing the DMSO control to each treated sample. Error bars represent the SEM. *P < 0.001; ns, not significant. (B) TB40/E-infected monocytes were harvested 24 hpi or 3 dpi. DNA was extracted from whole-cell lysates and qPCR was performed using specific primers to detect the viral genome (UL123) or cellular 18s rRNA (n = 3). GraphPad Prism 8 software was used to generate the graphs and to perform the statistical analyses. Statistical significance was calculated via t tests comparing treated samples to the DMSO control. Error bars represent the SEM. (C) Monocytes were infected with TB40/E (GFP fused to UL32) and treated with AG1478 (1 μM) 2 hpi and the cells were fixed at 3 dpi. Cells were stained with an antibody to gB (red), GFP (UL32) (green), and Hoechst to stain the DNA (blue). Yellow arrows indicate areas of colocalization between gB and GFP (UL32) and white arrows point to GFP (UL32) that does not colocalize with gB. The 488 and 594 lasers of the superresolution system were used to image structures in the green and red channels. The images of the nuclei stained with Hoechst were captured using the widefield function of the Nikon, as the superresolution component did not possess a laser for the blue channel. The widefield image of the nucleus was merged into the corresponding superresolution image using the companion analysis software of the microscope. (Scale bars, 1 μm.) (D) The colocalization rate of gB and UL32 (GFP) was quantitated (n = 2).

We next wanted to know what happened to the viral genomes that were not translocated into the nucleus upon EGFR kinase inhibition. Were the viral genomes being degraded as we have previously documented when c-Src signaling is inhibited (9), or were they still present within the cytosol? Monocytes were infected with HCMV, treated with AG1478 over a time course, cells were harvested 24 hpi and 3 dpi, DNA was isolated from whole-cell lysates, and qPCR was performed to detect the viral genome. Inhibition of EGFR kinase activity at each time point did not lead to a significant decrease in the amount of viral genome present within the whole cell 24 hpi or 3 dpi (Fig. 2B). These data suggest that, in the absence of EGFR kinase activity, the viral genomes are not translocated into the nucleus and are likely still present within the cytosol. Based upon the aforementioned data, we began to investigate the nature of the viral particle upon EGFR kinase inhibition. We wanted to determine whether mature viral particles (viral capsid containing DNA within the viral envelope) were present in the cytosol. The presence of mature viral particles would suggest that the virus likely remains within trafficking vesicles when EGFR signaling is inhibited. The absence of mature viral particles would be indicative of premature de-envelopment, which could lead to a defect in trafficking of the viral capsid to the nucleus. To address this question, we utilized immunofluorescence microscopy. Monocytes were infected, AG1478 was added 2 hpi, and the cells were fixed 3 dpi. To identify mature viral particles, we analyzed the colocalization of gB (major envelope glycoprotein) with UL32 (inner tegument protein as a marker of the viral capsid). When EGFR kinase activity was inhibited, 60% of gB colocalized with UL32 as opposed to the untreated control, in which there was 0% colocalization (Fig. 2 C and D). These data demonstrate that the majority of viral particles did not de-envelop prematurely when EGFR kinase activity was inhibited. Together these data show that the inhibition of EGFR kinase activity results in a retention of mature viral particles in the cytosol, likely residing in trafficking vesicles, thus leading to a decrease in nuclear translocation of viral DNA.

HCMV Mislocalizes When EGFR Kinase Activity Is Inhibited.

When EGFR kinase activity is inhibited, the virus does not arrive at the nucleus, but remains intact as a mature viral particle within the cytoplasm. We therefore wanted to determine whether the virus localized to the correct subcellular compartment in the absence of EGFR kinase activity. We utilized AG1478 to block the EGFR kinase between two major transition points: 1) EEA1⁺ vesicles to the TGN and 2) the TGN to recycling endosomes. Immunofluorescence microscopy was used to determine whether HCMV localized to the TGN (TGN46⁺ structures) or recycling endosomes (Rab11⁺ structures). Images were acquired using a Nikon N-SIM E Super Resolution microscope system, for which the limit of resolution is ∼100 nm. When viewing viral particles in relation to a subcellular structure, we saw complete colocalization, partial colocalization (partial overlap of two fluorophore signals), particles touching a structure, nearby a structure, and far away from a structure. Therefore, to quantitate EGFR-dependent viral events, we measured the distance of the viral particle to the subcellular structure by taking advantage of the powerful resolution of the microscope. As monocytes have a larger nuclear-to-cytoplasmic ratio and recycling endosomes are found throughout the cytosol, we validated our quantitation method via 3D image analysis (SI Appendix, Fig. S2 and Movies S1 and S2).

When EGFR kinase activity was inhibited during the viral trafficking transition from EEA1⁺ vesicles to the TGN, there was a 98% increase in the average distance from the viral particle to the TGN; there was a significant reduction in the number of viral particles localized to the TGN as compared to the untreated control (Fig. 3A). Blocking EGFR kinase activity from the TGN to the recycling endosome led to a 130% increase in the average distance from the viral particle to the recycling endosome, thus resulting in a significant decrease in the number of viral particles localizing to recycling endosomes (Fig. 3B). Because primary peripheral blood monocytes are short-lived and nondividing cells, we are limited in our approaches in regard to genetic modifications. To demonstrate that the observed requirement of EGFR kinase to promote the progression of viral trafficking is not an off-target effect of AG1478, we made use of a second specific EGFR kinase inhibitor, Erlotinib. EGFR kinase activity was inhibited via AG1478 or Erlotinib during the viral trafficking transition from EEA1⁺ vesicles to the TGN. The addition of Erlotinib resulted in the same phenotype as AG1478; there was a significant increase in the distance from the viral particle to the TGN, indicating a decrease in colocalization of HCMV and the TGN as compared to the DMSO-treated control (SI Appendix, Fig. S3). Additionally, there was no significant difference between the AG1478 and Erlotinib-treated conditions. Together, these data suggest that HCMV-induced EGFR kinase activity is required for the proper subcellular localization of viral particles during postentry trafficking events and the absence of kinase activity acts as a brake during viral trafficking.

Fig. 3.

HCMV mislocalizes when EGFR kinase activity is inhibited. (A) Monocytes were infected with TB40/E (MOI 10) and treated with AG1478 (1 μM) at 30 mpi and fixed at 2 hpi. Cells were stained with an antibody to TGN46 to visualize the TGN (red), gB (green), and Hoechst to stain DNA (blue). The distances between gB and the TGN were quantified. Data are combined from three biological replicates. Each data point represents an individual viral particle to TGN measurement and each condition includes at least 50 data points. (B) TB40/E-infected monocytes were treated with AG1478 (1 μM) at 2 hpi and fixed at 24 hpi. Cells were stained with antibody to Rab11 to visualize recycling endosomes (red), gB (green), and Hoechst to stain DNA (blue). The distances between gB and recycling endosomes were quantified. Data are combined from three biological replicates. Each data point represents an individual viral particle to recycling endosome measurement and each condition includes at least 50 data points. Yellow arrows indicate areas of HCMV and target structure colocalization. White arrows point to HCMV that is not colocalized with the target structure. The dotted lines on the quantitation graphs indicate the limit of resolution for the microscope (∼100 nm). (C) Monocytes were infected with TB40/E and treated with AG1478 (1 μM) 2 hpi. The inhibitor was removed 3, 4, and 6 hpi. Cells were fixed at 24 hpi and stained with antibodies to detect gB (green) and Rab11 (red). The distances between gB (HCMV particles) and Rab11 were quantified. Data are combined from two biological replicates. Each data point represents an individual viral particle to recycling endosome measurement and each condition includes at least 45 data points. GraphPad Prism 8 software was used to generate the graphs and to perform the statistical analyses. Statistical significance was calculated via (A and B) t test, or (C) ANOVA, *P < 0.0001. For all images, the 488 and 594 lasers of the superresolution system were used to image structures in the green and red channels. The images of the nuclei stained with Hoechst were captured using the widefield function of the Nikon, as the superresolution component did not possess a laser for the blue channel. The widefield image of the nucleus was merged into the corresponding superresolution image using the companion analysis software of the microscope.

We next wanted to determine whether or not viral trafficking could be restored once the EGFR kinase inhibitor was removed. If viral trafficking can be restored, it would indicate that the viral particle remains within a trafficking vesicle in the absence of EGFR kinase activity as suggested by Fig. 2 C and D. Inability of the virus to resume trafficking once the inhibitor is removed could suggest that the viral particle is no longer present within a trafficking vesicle or that EGFR is no longer able to recruit the proper adaptor molecules required to promote viral trafficking. To address this question, EGFR kinase activity was inhibited during the viral transition from the TGN to recycling endosome, as described above. The EGFR kinase inhibitor was removed 1, 2, or 4 h postaddition. AG1478 is a reversible EGFR kinase inhibitor; once AG1478 is removed, the EGFR kinase can become phosphorylated in about 30 min (24). We then examined whether or not the viral particle localized to recycling endosomes 24 hpi via immunofluorescence microscopy. As shown in Fig. 3 B and C, the constant inhibition of EGFR kinase activity via prolonged treatment with AG1478 starting 2 hpi resulted in a significant decrease of viral localization to recycling endosomes 24 hpi. Removal of AG1478 1, 2, or 4 h posttreatment resulted in the majority of viral particles localizing to recycling endosomes similar to the DMSO-treated control within 18 h (Fig. 3C). These data show that removal of the EGFR kinase inhibitor led to a recovery of viral trafficking to recycling endosomes. Together, these data suggest that temporal EGFR kinase activity is required to maintain proper viral trafficking and the absence of kinase activity during this time frame leads to a reversible stalling of the viral particle within a trafficking vesicle.

HCMV gB Binds to EGFR at the Monocyte Surface and throughout Postentry Events to Promote EGFR Kinase Activity.

Others have demonstrated that gB directly binds to and activates EGFR as an entry receptor in fibroblasts and our laboratory has demonstrated that HCMV-induced EGFR signaling is required for entry into monocytes (13, 25). To assess the mechanism potentiating EGFR kinase activity in monocytes, we examined gB and its propensity to bind and chronically activate the EGFR kinase during postentry events. We first wanted to determine whether gB and EGFR cotrafficked during HCMV postentry events in monocytes. Cells were infected, fixed over a time course (0 mpi, 15 mpi, 2 hpi, and 24 hpi), and stained to detect gB and EGFR. At each time point the virus closely associated with EGFR itself; as seen in the quantitation of Fig. 4A, the majority of the distances from gB to EGFR were at the limit of resolution for our microscope. At 24 h there is some gB that is further away from EGFR, likely due to some viral particles beginning to de-envelop. These data suggest that HCMV cotraffics with EGFR in the same vesicle during viral postentry events.

Fig. 4.

HCMV gB binds to EGFR at the monocyte surface and throughout postentry events to promote EGFR kinase activity. Monocytes were infected with TB40/E (MOI 10) and fixed over the following time course: 0 mpi, 15 mpi, 2 hpi, and 24 hpi. (A) Cells were stained with primary antibodies to detect gB (green), EGFR (red), and Hoechst to stain DNA (blue). (B) Cells were stained with primary antibodies to detect gB (green), p-EGFR (red), and Hoechst to stain DNA (blue). Yellow arrows indicate areas of colocalization between gB and EGFR or p-EGFR. The distances between gB and EGFR or p-EGFR were quantified. These data are combined from three biological replicates. Each data point represents an individual viral particle to EGFR or p-EGFR measurement and each time point includes at least 45 data points. GraphPad Prism 8 software was used to generate the graphs. The dotted lines on the quantitation graphs indicate the limit of resolution of the microscope (∼100 nm). For all images, the 488 and 594 lasers of the superresolution system were used to image structures in the green and red channels. The images of the nuclei stained with Hoechst were captured using the widefield function of the Nikon, as the superresolution component did not possess a laser for the blue channel. The widefield image of the nucleus was merged into the corresponding superresolution image using the companion analysis software of the microscope. (C) Protein was harvested from monocytes infected with TB40/E (MOI 10 to 15) at 0 mpi, 15 mpi, 2 hpi, 24 hpi, and 72 hpi. As indicated, HCMV gB or EGFR was immunoprecipitated. Western blot analyses were performed to detect gB, EGFR, p-EGFR, and β₁ integrin (negative control). Data are a representative image of three biological replicates.

We next determined whether gB cotrafficked with activated EGFR by performing the same experiment and stained the cells to detect gB and p-EGFR. For each time point examined, the majority of the distances between gB and p-EGFR were at the limit of resolution of our microscope, thus suggesting a very close association of gB and p-EGFR during viral trafficking events (Fig. 4B). At 0 mpi and 2 hpi there was some gB signal that was further away from p-EGFR. Because the samples harvested 0 mpi were kept on ice, it is possible that some of the bound EGFR monomers had not fully dimerized and were not capable of receptor autophosphorylation, resulting in the absence of colocalization between p-EGFR and gB. Another possibility for some gB being further away from p-EGFR at 0 mpi is that the viral particle may have tethered to heparan sulfate proteoglycans and attached to the β₁ and β₃ integrins, but gB might not have bound to EGFR before the samples were harvested. At 2 hpi, gB is closely associated with EGFR as shown in the quantitation of Fig. 4A; however, there is more gB that is further away from p-EGFR at this time point (Fig. 4B). Our laboratory has documented that the viral particles move through the TGN at 2 hpi. We observed the TGN is a likely “bottleneck” during viral trafficking: Small numbers of viral particles traffic through the TGN over a long period of time (45 min to 24 hpi) (9). During this phase of viral trafficking, there are likely vesicle fusion events taking place at the TGN (26, 27). Perhaps these fusion events or the exposure to different adaptor proteins in the TGN could affect the phosphorylation pattern on EGFR bound to virus. The phosphorylation site we examined (tyrosine 1068) may not be phosphorylated while the viral particle is transiting the TGN. Together, these data demonstrate that HCMV cotraffics with activated EGFR throughout the postentry events, suggesting that HCMV may be potentiating the EGFR kinase activity from within trafficking vesicles.

We next wanted to determine whether gB directly binds EGFR during trafficking events to perpetuate EGFR kinase activity. Coimmunoprecipitations were performed over a time course: 0 mpi, 15 mpi, 2 hpi, 24 hpi, and 72 hpi. During viral binding (0 mpi), EGFR coprecipitated with gB at the cell surface. Unexpectedly, EGFR and p-EGFR coprecipitated with gB at each subsequent time point tested: Through 72 hpi (Fig. 4C). We show that gB and EGFR interact throughout the duration of viral postentry events to promote chronic activation of the EGFR. Because the studies in Fig. 1 were designed and completed before the coimmunoprecipitation studies, the time course ended at 24 hpi. Based on the data from the coimmunoprecipitation experiments showing gB–EGFR engagement and EGFR phosphorylation out to 72 hpi, we wanted to go back and examine downstream EGFR signaling out to 72 hpi. Immunoblot data show that HCMV infection results in increased levels of AKT phosphorylation out to at least 48 hpi (SI Appendix, Fig. S4). Additionally, the reverse coimmunoprecipitation was performed and HCMV gB coprecipitated with EGFR at 0 mpi and 24 hpi. To demonstrate specificity of the interaction between gB and EGFR, immunoblots were performed to detect β₁ integrins as our laboratory has demonstrated that gH and UL128-131, but not gB, bind to β₁ integrins on monocytes (28). As seen in Fig. 4C, β₁ integrins did not coprecipitate with gB or EGFR. Together, these data demonstrate that HCMV gB binds EGFR at the cell surface and remains bound to EGFR throughout viral trafficking events. The continuous gB–EGFR engagement promotes the chronic EGFR kinase activity required for maintaining viral localization and the proper timing of nuclear translocation.

Discussion

HCMV pathogenesis relies upon the successful infection of peripheral blood monocytes, as this is the key cell type mediating hematogenous dissemination of the virus to secondary organs, such as the bone marrow compartment, the site of viral latency and life-long persistence within the host following primary infection. Therefore, delineating how HCMV successfully infects monocytes is important to understanding HCMV spread and persistence within the host. A key facet of the HCMV lifecycle is the translocation of viral DNA into the host cell nucleus, the site of viral replication. We have previously documented that HCMV viral trafficking and nuclear translocation is extended and unique in monocytes as compared to fibroblasts, endothelial cells, and CD34⁺ HPCs. We demonstrated that viral-induced signaling through the c-Src kinase is required early during infection of monocytes to avoid viral sorting into late endosomes (9). These data set a precedence that HCMV induces unique signaling via receptor–ligand interactions that plays a role in viral postentry events required for nuclear translocation, and thus productive infection. To expand upon this idea, we began to investigate how the other major signaling axis through the EGFR kinase regulates HCMV postentry events in primary human monocytes.

We report that HCMV chronically activates the EGFR kinase throughout viral postentry events resulting in functional EGFR signaling, as indicated by the activation of downstream signaling molecules AKT and STAT1. The HCMV-induced chronicity of EGFR signaling is distinct from that induced by EGF (natural EGFR ligand), suggesting that HCMV is recognized by EGFR as a unique ligand. To ensure that the observed differences were due to the ligand itself and not variations in the amount of EGFR engaged, we utilized concentrations of virus and EGF that would saturate surface EGFR on monocytes. We do acknowledge that the pentamer-induced signaling axis through the β₁ and β₃ integrins could influence EGFR activation, resulting in differences between the ligands; but regardless, the HCMV-induced EGFR signaling results in different EGFR-dependent outcomes as compared to EGF. Our observations support and add molecular detail to previous reports regarding the differential activation of EGFR by HCMV. In a cytotrophoblast cell line, EGF and HCMV induced different patterns of phosphorylation on EGFR itself early during infection (29). It is known from the literature that different EGFR ligands can induce differential activation of EGFR with distinct downstream consequences (30, 31). In the cytotrophoblast cell line, EGF and HCMV had opposing effects on migration and invasion. LaMarca et al. (29) posed that the differential EGFR activation could be a potential mechanism for these observed functional differences. Based upon these data—that virus signaling through EGFR creates a different outcome—we hypothesized that the chronic HCMV-induced signaling through EGFR is essential for viral postentry events in primary human monocytes.

Surprisingly, we found that HCMV-induced chronic EGFR kinase activity is required to facilitate the proper timing of nuclear translocation. When EGFR kinase activity was inhibited at different points during viral trafficking and de-envelopment events, there was a decrease in the amount of viral genome in the nucleus at 3 dpi. These data strongly demonstrate that although EGFR activation during HCMV binding is required for viral entry, it is not sufficient to promote each subsequent postentry event leading to nuclear translocation. We have shown previously that the absence of viral-induced c-Src signaling early during infection leads to degradation of viral particles by 24 hpi, we therefore wanted to know what happened to viral particles upon EGFR kinase inhibition. Opposite to c-Src inhibition, we detected mature viral particles in the cytosol without a significant decrease in the amount of viral genome present. These data suggest that both signaling pathways are required to promote nuclear translocation, but achieve this function through different molecular mechanisms and potentially at different steps. In support of this idea, our laboratory has previously documented that both c-Src and EGFR kinases function to increase monocyte motility, but each pathway relies upon a distinct protein: Paxillin and N-WASP, respectively, to promote enhanced motility in infected monocytes (13, 17).

The presence of mature viral particles in the cytosol upon EGFR kinase inhibition suggested that the virus was likely still residing within a trafficking vesicle. This raised the question: Is HCMV properly localized in the absence of EGFR kinase activity? We found that EGFR kinase activity is required to promote proper viral localization during critical transition points of the viral trafficking pathway: From EEA1⁺ vesicles to the TGN and from the TGN to recycling endosomes. Viral particles did not traffic to the appropriate destination when EGFR kinase activity was inhibited. The presence of mature viral particles in the absence of EGFR kinase activity suggests that while the virus is not properly localized, it did not de-envelop and likely remains “trapped” within a trafficking vesicle. Perhaps inactivity of the EGFR kinase prevented the recruitment of adaptor proteins required for proper trafficking. A study in hepatocellular carcinoma tissues demonstrated the EGFR adaptor protein, GOLM1, facilitates tethering of EGFR to the TGN and recycling of EGFR back to the plasma membrane. Knockdown of GOLM1 resulted in retention of EGFR in the TGN and a decrease in EGFR recycled back to the plasma membrane, while restoration of GOLM1 resulted in a recovery of EGFR recycled to the plasma membrane (32). We found that there was a near complete restoration of viral trafficking to recycling endosomes when the EGFR kinase inhibitor was removed 1, 2, or 4 h posttreatment, thus indicating that a loss of EGFR kinase activity leads to a reversible stalling of viral trafficking. Together, these data demonstrate that HCMV manipulates the EGFR kinase well past initial binding at the monocyte surface to be active at critical points during viral trafficking to achieve proper localization and timing of nuclear translocation.

To address the mechanism perpetuating HCMV-induced EGFR signaling in monocytes, we investigated the interaction between gB and EGFR during viral postentry events. We now show that gB cotraffics with activated EGFR from viral binding at the surface of the monocyte throughout the duration of intracellular viral trafficking events. This suggests that HCMV and EGFR are likely internalized together and remain within the same trafficking vesicles during viral postentry events. Strikingly, via coimmunoprecipitations, we found that gB remains bound to activated EGFR throughout the entirety of viral postentry events. As a control for the specificity of the gB–EGFR interaction, we determined whether β₁ integrins coprecipitated with gB or EGFR. In addition to gB on the envelope, HCMV has two glycoprotein complexes, the trimer (gH/gL/gO) and the pentamer (gH/gL/UL128-131), both of which can bind to β₁ integrins during viral attachment (28). It has been documented that integrins are internalized along with the viral particle and EGFR during viral entry, suggesting that the viral particle, EGFR, and the integrins reside within the same vesicle (33). Our data show that β₁ integrins do not coprecipitate with gB or EGFR at any time point examined during viral postentry events in monocytes. These data support the specificity of the gB and EGFR interaction. These data also support that the gB–EGFR engagement leads to activation of the EGFR.

It has been well documented in the literature that integrins can form complexes with and transactivate EGFR (34, 35). The absence of β₁ integrins in the gB and EGFR pulldowns suggests that the gB–EGFR interaction leads to activation of EGFR, not transactivation induced by a direct integrin-EGFR complex formation. Taken together, these data show that gB binds to EGFR at the surface of the monocyte, that HCMV and EGFR are internalized together, and that gB remains bound to EGFR throughout the duration of postentry events to promote chronic EGFR kinase activity. This EGFR kinase activity might also play a role in viral de-envelopment as well as in events occurring after de-envelopment. Our data showing that gB is still engaged with p-EGFR at 3 dpi and that cessation of EGFR kinase activity resulted in a stalling of trafficking, but not de-envelopment, suggest that there may be an EGFR-dependent signal within the recycling endosome that triggers viral de-envelopment. Perhaps HCMV is retained within the recycling endosome because gB is still bound to EGFR. Ligands that activate EGFR to recycle (TGF-α, epiregulin, and amphiregulin) dissociate with EGFR in the slightly acidic conditions of endosomes (31). Could there be an EGFR-dependent signaling event at 2 dpi that cues viral de-envelopment? As this is an interesting question in cell biology and HCMV pathogenesis, our laboratory plans to further investigate this process in the future. We have also shown that the EGFR kinase does play an important role after viral de-envelopment. We have documented that EGFR kinase activity at 7 and 10 dpi is required for efficient viral transcription occurring 3 wk postinfection (8).

In summary, we report that HCMV serves as a unique EGFR ligand inducing functional chronic EGFR kinase activity distinct from that of the natural ligand EGF. Our findings show that HCMV gB binds to EGFR at the cell surface and surprisingly remains bound to EGFR throughout the duration of viral postentry events to promote the chronic EGFR kinase activity seen in monocytes during HCMV infection. This chronic EGFR kinase activity is required to promote proper viral trafficking and nuclear translocation, leading to productive infection of primary human monocytes (Fig. 5). Interestingly, the inhibition of EGFR kinase activity led to a reversible stalling of viral trafficking, but not degradation of the viral genome as previously documented when signaling through c-Src was inhibited. These data indicate that both signaling pathways are required to promote productive infection in monocytes, but likely utilize distinct molecular mechanisms.

Fig. 5.

Model: HCMV-induced signaling regulates postentry events in primary human monocytes. HCMV gB engages EGFR at the monocyte surface and remains bound during viral postentry events to promote chronic and functional EGFR kinase activity. This HCMV-induced EGFR kinase activity is required for correct viral localization throughout the duration of infection as well as maintaining the proper timing of viral nuclear translocation in monocytes. Inhibition of EGFR kinase activity at 30 mpi blocks viral trafficking from early endosomes to the TGN. Addition of the EGFR kinase inhibitor at 2 hpi prevents viral localization to recycling endosomes from the TGN. Inhibition of EGFR kinase activity at 30 mpi, 2 hpi, 24 hpi, and 48 hpi results in a decrease of HCMV genome present in the nucleus 3 dpi. Unlike inhibition of c-Src kinase activity, which results in degradation of the viral DNA, inhibition of EGFR kinase activity does not result in a significant decrease in the amount of viral genome present in the cytosol at 24 hpi or 3 dpi.

Our data also highlight how HCMV regulates the EGFR kinase externally and internally to facilitate distinct steps of the viral lifecycle. The external signaling event is necessary to promote efficient entry (the ability of the virus to cross the plasma membrane), but is not sufficient to drive postentry events (trafficking, de-envelopment, and nuclear translocation). In the literature it is evident that the endocytic pathway spatially regulates EGFR activity and signaling. The trafficking of EGFR can be ligand-dependent and result in exposure of the EGFR to different subcellular adaptor and effector molecules (31, 36, 37). For example, EGF–EGFR complexes are typically sorted to the degradative pathway via late endosomes, while TGF-α–EGFR complexes are sorted toward the recycling pathway (30, 31). Generally, EGFR is recycled back to the plasma membrane without transit through the TGN; however, in some cancers, such as hepatocellular carcinoma, EGFR can traffic from early endosomes to the TGN before being recycled (32). Additionally, activated EGFR can traffic in a retrograde manner through the Golgi to the nucleus where EGFR functions as a transcriptional regulator (38–40). In our system, we see HCMV and EGFR transit through the TGN to recycling endosomes. Retrograde trafficking from early endosomes to the TGN requires specific trafficking proteins and complexes, such as the retromer complex (26, 27, 41). HCMV-induced signaling in monocytes must allow for the viral cargo in the EEA1⁺ endosomes to be recognized by retrograde trafficking machinery and sorted toward the TGN.

Ongoing studies in our laboratory are seeking to define the virus-induced host cell factors regulating retrograde trafficking through the TGN and determine the molecular events that occur between HCMV and host cell components at the TGN. Broadly, we propose that the subcellular localization and chronic activation of EGFR likely allows for the recruitment of distinct adaptor/effector proteins to promote each viral postentry event. The subcellular regulation of EGFR can directly impact the biological outcome of EGFR signaling. For example, one study demonstrated that endocytic EGFR signaling, as compared to EGFR signaling from the plasma membrane, resulted in differential activation of transcription factors c-fos and c-jun, leading to contrasting cell size and proliferation rate (42). Previous transcriptomic data from our laboratory also support this idea. We found that in the EGFR-dependent HCMV-infected monocyte transcriptome, more genes were differentially regulated due to the internal signaling activity of the EGFR kinase (∼70% of all genes regulated by HCMV infection) (10, 13). These data fit with the idea that greater than 43% of receptor tyrosine kinase signaling occurs from endosomes and that receptor signaling from endosomes can be distinct from receptor signaling at the plasma membrane (37, 43). It would appear that HCMV has evolved to take advantage of the unique cell biology of the EGFR/EGFR kinase to productively infect peripheral blood monocytes, thus promoting viral dissemination and life-long persistence within the host.

Materials and Methods

Human Peripheral Blood Monocyte Isolation.

As previously described, human peripheral blood was collected via venipuncture using an approved Institutional Review Board (H99-064). Specifically, this study was approved by the Louisiana State University Health Sciences Center-Shreveport Institutional Review Board (approval no. H99-064), and all Health Insurance Portability Accountability Act guidelines were followed. Informed consent was obtained prior to every blood donation. Human subjects were informed of the present study, potential risks involved in the venipuncture procedure, and the fact that their consent could be revoked at any point during their participation in the study. The peripheral blood was passaged sequentially through Histopaque-1077 (Sigma) and Percoll (Sigma) density gradients to isolate monocytes (44, 45). Peripheral blood monocytes were cultured in RPMI medium 1640 (Corning) supplemented with 1% or 10% human serum (Gemini), as indicated below.

Virus Preparation.

HCMV strains TB40/E and TB40/E (GFP fused to UL32) were cultured on human embryonic lung fibroblasts and harvested via centrifugation through a sorbitol cushion (20% [wt/vol]) as previously described (44, 46, 47). Viruses were resuspended in RPMI 1640 media and used to infect monocytes at an MOI of 5 for each experiment unless otherwise stated.

DNA Extraction from Nucleus.

The Nuclear Extraction Kit (Active Motif) was utilized to isolate nuclei from monocytes. DNA was extracted from nuclei using the EZNA Tissue DNA Kit (Omega Bio-Tek). Controls to assess the quality of fraction separation were performed as previously described (16).

qPCR.

Monocytes were infected with TB40/E, treated with inhibitors where indicated, and harvested at various time points listed in the text. Total cellular DNA was extracted using the EZNA Tissue DNA Kit (Omega Bio-Tek) and qPCR was performed as described previously (9, 13, 28). To examine nuclear DNA, cells were fractionated using a Nuclear Extract Kit (Active Motif), nuclei collected, and DNA was isolated from the nuclei. Primers to detect 18s rRNA and HCMV UL123 were used in qPCR.

Western Blot Analysis.

Monocytes were serum-starved overnight and infected with TB40/E or treated with EGF (200 ng/mL) as indicated in the text. Protein was harvested across indicated time courses, resolved via SDS/PAGE, transferred to PVDF membranes, blocked, incubated with primary antibodies as indicated in the text (actin [Bio-Rad], EGFR [Cell Signaling], p-EGFR [Y1068; Cell Signaling], β₁ integrin [Abcam], gB [US Biologicals]), incubated with HRP-conjugated secondary antibodies, and bands were detected using chemiluminescence detection reagents.

In-Cell Western.

Monocytes were plated into black-walled 96-well plates, serum-starved overnight, and infected with TB40/E or stimulated with EGF (200 ng/mL), as indicated. Cells were fixed in 4% paraformaldehyde across a time course described in the text. The cells were permeabilized and stained with primary antibodies to detect: Actin (Santa Cruz), AKT (Cell Signaling), p-AKT (S473) (Cell Signaling), STAT1 (Cell Signaling), and p-STAT1 (Y107) (Cell Signaling). Cells were then incubated with secondary antibodies conjugated to IR dyes. Fluorescence intensity was then measured via LI-COR Odyssey CLx infrared imaging system.

Immunofluorescence Microscopy.

Monocytes were plated onto fibronectin-coated glass coverslips and incubated overnight. Cells were infected with TB40/E or TB40/E (GFP fused to UL32) (MOI 10) the following day. At each time point designated in the text, monocytes were stained with specific primary antibodies to detect TGN46 (ThermoFisher Scientific), Rab11 (Abcam), gB (eBioscience), GFP-488 (Life Technologies), EGFR (eBioscience), or p-EGFR (Y1068) (Cell Signaling). High-resolution images were acquired using a Nikon N-SIM E Super Resolution microscope system using a 100× objective. Images were acquired in single slices (z-stacks). Each image appearing in the text is a representative single slice from a z-stack. Images were acquired using the same laser power settings. For all images, the 488 and 594 lasers of the superresolution system were used to image structures in the green and red channels. The images of the nuclei stained with Hoechst were captured using the widefield function of the Nikon, as the superresolution component did not possess a laser for the blue channel. The widefield image of the nucleus was merged into the corresponding superresolution image using the companion analysis software of the microscope.

Coimmunoprecipitation.

Monocytes were serum-starved overnight followed by infection with TB40/E (MOI 10 to 15). Protein was harvested over the indicated time course. Primary antibody (gB [US Biologicals] or EGFR [ThermoFisher Scientific]) was added to lysates overnight. Protein G Dynabeads (ThermoFisher Scientific) were incubated with the protein–antibody mixture. The protein was eluted off of the Dynabeads. Western blots were performed as described above.

Please see SI Appendix, SI Material and Methods for detailed protocols.

Data Availability Statement.

All relevant data, and materials and methods are included in the main text and SI Appendix. Requests for reagents, such as virus, should be directed to A.D.Y.