Edited by Thomas E. Shenk, Princeton University, Princeton, NJ, and approved October 16, 2009 (received for review August 10, 2009)
Human cytomegalovirus (HCMV) rapidly induces a mobile and functionally unique proinflammatory monocyte following infection that is proposed to mediate viral spread. The cellular pathways used by HCMV to initiate these biological changes remain unknown. Here, we document the expression of the epidermal growth factor receptor (EGFR) on the surface of human peripheral blood monocytes but not on other blood leukocyte populations. Inhibition of EGFR signaling abrogated viral entry into monocytes, indicating that EGFR can serve as a cellular tropism receptor. Moreover, HCMV-activated EGFR was required for the induction of monocyte motility and transendothelial migration, two biological events required for monocyte extravasation into peripheral tissue, and thus viral spread. Transcriptome analysis revealed that HCMV-mediated EGFR signaling up-regulated neural Wiskott–Aldrich syndrome protein (N-WASP), an actin nucleator whose expression and function are normally limited in leukocytes. Knockdown of N-WASP expression blocked HCMV-induced but not phorbol 12-myristate 13-acetate (PMA)-induced monocyte motility, suggesting that a switch to and/or the distinct use of a new actin nucleator controlling motility occurs during HCMV infection of monocytes. Together, these data provide evidence that EGFR plays an essential role in the immunopathobiology of HCMV by mediating viral entry into monocytes and stimulating the aberrant biological activity that promotes hematogenous dissemination.
The wide range of pathological complications associated with human cytomegalovirus (HCMV) infection is a direct consequence of viral spread to peripheral organ sites and the broad cellular tropism of the virus (1). Monocytes are primary in vivo targets for HCMV and are believed to be responsible for hematogenous dissemination of HCMV to multiple organ systems (2). We previously showed that HCMV infection of monocytes polarized the infected cell toward a distinct proinflammatory phenotype that possessed the distinct biological changes necessary to promote viral spread (3–5). Specifically, HCMV infection induced polarization of infected monocytes toward an M1 proinflammatory cell type that simultaneously exhibited characteristics associated with an M2 antiinflammatory macrophage (6). The infected monocytes also displayed a high level of chemokinesis when compared with monocytes activated by LPS or phorbol 12-myristate 13-acetate (PMA) (4, 5). Moreover, an accelerated rate of differentiation from short-lived monocytes (nonpermissive for HCMV replication) into long-lived macrophages (permissive for HCMV replication) was observed following infection with HCMV (4). Based on our studies, we suggest that during primary infection, newly infected peripheral monocytes acquire a motile phenotype that promotes exit of the infected cell from the circulating blood into multiple organ tissue despite the absence of a chemotactic gradient. Once in the surrounding tissue, differentiation into HCMV replication-competent macrophages occurs, resulting in viral spread and lifelong persistence in the organs that serve as portals of viral exit.
The biological changes induced in HCMV-infected monocytes occurred within a time frame in which no new viral gene expression was observed (4), suggesting that receptor–ligand interactions triggered the rapid activation of infected monocytes. Although viral glycoproteins have been documented to stimulate proinflammatory cellular signaling pathways in infected monocytes (4, 7), the host cell receptors used by HCMV to modulate the cellular signaling network, and thus the unique functional changes in monocytes, remain unknown. The major HCMV glycoprotein B has been shown to bind to and directly activate epidermal growth factor receptor (EGFR), leading to the induction of PI(3)K activity in model cell types (8). In support, rapid phosphorylation of EGFR was demonstrated in endothelial cells (ECs) (9) and trophoblasts (10) following infection with HCMV. However, unlike the transient activation observed in fibroblasts (8) and trophoblasts (10), HCMV infection stimulated chronic activation of EGFR in ECs (9), suggesting that distinct signaling profiles originating from the same cellular receptor can occur in different cell types. EGFR expression was required for viral entry/infection of breast cancer cells and trophoblasts (8, 11). However, the role that EGFR plays during viral infection remains controversial, because it has been reported that EGFR does not mediate entry into fibroblasts (12) and others have shown that PDGF receptor (PDGFR)-α rather than EGFR was required for efficient viral entry (13). Deciphering which cellular receptor is used during viral entry is critical to unraveling how HCMV infection modulates monocyte biology and consequently dictates viral spread and disease.
The presence of EGFR on the surface of human peripheral blood monocytes is unclear. Examination of mixed populations of peripheral blood mononuclear cells (PBMCs) did not detect EGFR expression (14). However, functional EGFR was detected on some monocytic leukemic cell lines and macrophage subpopulations (15). Here, we report the expression and functionality of EGFR on human peripheral blood monocytes. Rapid activation of the EGFR signaling pathway following HCMV infection was necessary for viral entry into monocytes and viral modulation of monocyte motility. Mechanistically, viral activation of EGFR on monocytes stimulates cell movement via the specific up-regulation of a highly active actin nucleator, neural Wiskott-Aldrich syndrome protein (N-WASP), that is normally expressed at near-undetectable levels in leukocytes (16). Because N-WASP possesses a significantly greater actin nucleating potential than WASP (17), the actin nucleator normally thought to control actin growth in leukocytes (18), our data suggest that HCMV has evolved a strategy involving a switch in key actin nucleators following infection to generate monocytes exhibiting high levels of chemokinesis. Overall, these findings indicate that EGFR plays a dual role in the immunopathobiology of HCMV by mediating viral entry into monocytes and the induction of a unique proinflammatory motile phenotype, thus promoting viral spread into distinct organ sites.
The acquisition of a motile monocyte phenotype within 1 h postinfection (hpi) suggests that the direct activation of a cellular receptor during viral binding/entry mediates the PI(3)K-dependent induction of infected monocyte motility (3). EGFR and PDGFR-α have both been shown to stimulate the PI(3)K signaling pathway and to be engaged during HCMV entry into some cell types (8, 13). PDGFR-α is not expressed, however, in cells of the myeloid lineage (19); we confirmed this lack of expression on human primary monocytes (Fig. S1). Similarly, it has been suggested that EGFR is not a major HCMV entry receptor, because monocytes are reported to lack this receptor (12, 20), although some myeloid leukemic cells express EGFR (15). To date, EGFR expression on primary monocytes remains unclear, because previous studies were performed on PBMCs (14), in which the monocyte subpopulation constitutes only 5% of the total PBMC population (21). Consequently, we next focused on determining whether EGFR could be responsible for HCMV-induced PI(3)K activity in infected monocytes. We tested EGFR expression on different blood leukocyte populations by flow cytometry. We failed to detect EGFR expression on T cells (CD3+) and B cells (CD20+); however, EGFR was expressed on monocytes (CD14+), albeit at lower levels than in the breast cancer cell line MDA-MB-468 (Fig. 1A).
HCMV and EGF activate EGFR found on primary human peripheral blood monocytes. (A) PBMCs were stained with anti-CD3-PE-Cy5 (T cells), anti-CD14-APC-Cy7 (monocytes), and anti-CD20-PE (B cells) antibodies and were then costained with either an anti-EGFR-GFP antibody (green) or a GFP isotype-matched control antibody (blue). The red line indicates unlabeled cells. MDA-MB-468 breast cancer cells were used as a positive control cell line. Examination of EGFR expression was performed by flow cytometry. (B) Isolated monocytes were treated with EGF or infected with HCMV (Towne/E or TB40/E) for 0, 10, 30, 45, and 60 min. Phosphorylated EGFR (pEGFR), total EGFR, phosphorylated Akt (pAkt), and total Akt were detected by immunoblotting. (C) Monocytes were treated with an anti-EGFR antibody or AG1478 before treatment with EGF or infection with Towne/E or TB40E. Total lysate was harvested, and pEGFR, total EGFR, phosphorylated PI(3)K [pPI(3)K], total PI(3)K, pAkt, and total Akt were determined by immunoblotting. Membranes were reprobed with antibody against β-actin (B and C).
Next, we examined whether EGFR engagement by EGF, HCMV Towne/E, or HCMV TB40/E resulted in receptor activation. Consistent with receptor–ligand kinetics, EGFR was rapidly activated within 10 min of treatment with EGF or infection with either HCMV strain (Fig. 1B). We found concomitant phosphorylation of the downstream PI(3)K target, Akt, within 10 min of treatment with EGF or infection with HCMV. Treatment with a potent EGFR-specific kinase inhibitor, AG1478, or a neutralizing anti-EGFR antibody before culture with EGF or infection with HCMV diminished the rapid phosphorylation of EGFR and downstream targets PI(3)K and Akt (Fig. 1C). This slight induction of PI(3)K and Akt in the presence of either EGFR inhibitor following infection suggests that other HCMV entry receptors, such as integrins (22, 23), may also be involved in the activation of the PI(3)K pathway (24). Nonetheless, our results indicate that peripheral blood monocytes express bona fide functional cell surface EGFR.
With the controversy surrounding the role that EGFR plays in viral entry (8, 12) and the lack of an identifiable cell-specific receptor on blood leukocytes, we next assessed whether the expression of EGFR on monocytes was a determinant factor required for HCMV binding/entry to monocytes. TB40/E, a recombinant HCMV strain with GFP fused to the C-terminal end of the capsid-bound tegument protein pUL32 (25, 26), was used to infect PBMCs, and flow cytometric analyses were performed to detect the presence of bound GFP-labeled viral particles associated with CD3+ (T cells), CD20+ (B cells), and CD14+ (monocytes) leukocyte subpopulations (Fig. 2 A–C). We found high levels of GFP-labeled virus associated with CD14+ cells (Fig. 2B) but not with CD3+ or CD20+ cells (Fig. 2 A and C).
EGFR acts as a tropism receptor directing HCMV internalization. PBMCs were mock-infected (red) or infected with a GFP tegument-tagged TB40/E virus (blue) and then labeled with anti-CD3-PE-Cy5 (T cells) (A), anti-CD14-APC-Cy7 (monocytes) (B), or anti-CD20-PE (B cells) (C) antibody and examined by flow cytometry to detect GFP-positive viral particles. (D) Purified CD14+ monocytes were mock infected (red), TB40/E infected (blue), or TB40/E infected in the presence of anti-EGFR antibody (orange) or soluble heparin (green) and then analyzed by flow cytometry. (E) Monocytes were pretreated with an anti-EGFR antibody or AG1478, cooled to 4 °C, and infected with TB40/E. Cells were then immediately fixed or temperature-shifted to 37 °C for 1 h and then fixed. Red fluorescence represents the labeled EGFR staining, green fluorescence represents TB40/E-GFP staining, blue fluorescence represents DAPI-stained nuclei, and the white dashed line represents the outer monocyte cell membrane. Images are representative of three independent experiments from different donors. (F and G) Monocytes were pretreated with an anti-EGFR antibody, an anti-IgG control antibody, AG1478, or LY294002; were cooled to 4 °C; and were infected with Towne/E. Cells were then treated with PK or incubated at 37 °C for 1 h before PK treatment. The presence of genomic HCMV DNA was determined by RT-PCR (F) and qRT-PCR (G) analysis using HCMV genomic immediate early-specific primers.
Is expression of EGFR responsible for the selective binding of virus to different populations of blood leukocytes, or does it play a different role in viral tropism, such as in the regulation of viral fusion? The presence of soluble heparin, a known inhibitor of HCMV attachment (20), significantly decreased the presence of bound GFP-tagged TB40/E with monocytes, as determined by flow cytometric analysis, indicating that viral binding was blocked (Fig. 2D). However, the presence of a blocking EGFR antibody did not significantly affect the levels of GFP-tagged virus associated with monocytes, indicating that EGFR expression alone does not dictate viral attachment to monocytes.
Because flow cytometric analysis cannot differentiate between surface-bound and internalized virus, we next used immunofluorescent microscopy to examine whether GFP-labeled HCMV particles bound to the monocyte cell surface required EGFR activity to be internalized into the host cell cytoplasm. Infection of monocytes with TB40/E carried out at 4 °C showed viral particles localized at the cell surface (Fig. 2E). Following a temperature shift to 37 °C to initiate internalization, we found TB40E in the cytoplasm of the infected cell. Pretreatment of monocytes with AG1478 or a neutralizing EGFR antibody resulted in the inhibition of internalization of the viral capsid and the clustering of viral particles at the cell surface, which was also observed in EGFR-negative cytotrophoblasts (11). To assess the contribution of EGFR in the process of viral entry further, cells were examined for the presence of internalized HCMV genomic DNA. We detected low levels of viral DNA in samples infected at 4 °C following removal of surface-bound viral particles by proteinase K (PK) treatment, whereas high levels of viral DNA were detected in samples with the temperature shifted to 37 °C before the addition of PK, demonstrating that viral entry had occurred (Fig. 2F). Consistent with the immunofluorescent analysis, PK treatment of HCMV-infected monocytes demonstrated a significant reduction in HCMV entry in the absence of EGFR activation, although the lack of EGFR signaling did not completely eliminate viral entry. LY294002, an inhibitor of PI(3)K activity, did not block HCMV entry, suggesting that although PI(3)K is directly downstream of EGFR and is rapidly activated following infection, a divergent signaling pathway initiated from the EGFR kinase must mediate viral entry. All samples from infected monocytes not treated with PK exhibited similar amounts of detectable HCMV genomic DNA, indicating that viral binding was not affected by the different pretreatments (Fig. S2). qRT-PCR analysis confirmed EGFR-mediated viral entry (Fig. 2G) and revealed a 41% and 59% decrease in viral entry in the presence of neutralizing-EGFR antibody and AG1478, respectively, which is consistent with the 63% reduction in infectivity observed in trophoblasts treated with an anti-EGFR antibody before HCMV infection (11). Other growth factor-like receptor tyrosine kinases, including PDGFR-α, VEGF receptor 1 (VEGFR1), and VEGFR2, are not involved in mediating viral entry into monocytes (Fig. S3). Taken together, these results suggest that EGFR-mediated signaling is initiated following viral binding to the receptor and that this signaling dictates monocyte tropism by directing efficient internalization of the viral particle.
Because our early data showed that viral binding triggers functional changes in monocytes (4, 7), we hypothesized that HCMV engagement of EGFR during viral binding/entry was responsible for the HCMV-induced polarization of infected monocytes. Infection of monocytes by HCMV induced lamellipodium and tail formation, which are hallmarks of cell motility (Fig. 3A). However, monocytes pretreated with EGFR inhibitors before infection exhibited morphological characteristics similar to those of quiescent mock-infected cells. Quantitative evaluation of motility by phagokinetic motility track assays revealed that treatment of monocytes with AG1478 or an anti-EGFR antibody before infection abrogated virus-induced chemokinesis (Fig. 3B). Pretreatment with the DMSO solvent control (3, 5) or the IgG isotype-matched control did not affect HCMV-induced motility. Similar results were observed when EGF was used as the activating ligand, further supporting the role that EGFR plays in directly modulating monocyte motility (Fig. 3B). In agreement with the viral entry assays, the presence of neutralizing PDGFR-α, VEGFR1, or VEGFR2 antibodies during infection was not able to block HCMV-induced monocyte chemokinesis (Fig. S4). Overall, our data suggest that EGFR plays two essential roles during HCMV infection of monocytes. First, EGFR activity is required for efficient viral entry, although other receptors may partially compensate for the lack of EGFR signaling, as indicated by only a 50% inhibition of viral entry in the presence of EGFR inhibitors. Second, EGFR activation during viral entry mediates HCMV-induced motility, because monocyte chemokinesis was inhibited nearly 100% when EGFR signaling was abrogated.
HCMV stimulates monocyte motility and transendothelial migration in an EGFR-dependent manner. Monocytes were pretreated with an anti-EGFR antibody, an IgG isotype-matched control antibody, or AG1478. (A) Monocytes were infected with Towne/E, and photomicrographs were captured at 6 hpi. Images are representative of three independent experiments from different donors. (B) Cells were plated on colloidal gold-coated coverslips and infected with Towne/E or treated with EGF for 24 h. The average area of colloidal gold cleared per monocyte was determined. (C) Monocytes were mock infected, Towne/E infected, or EGF treated for 6 h nonadherently, and they were subsequently labeled with CellTracker Green. Labeled monocytes were added to cell-culture inserts containing confluent monolayers of ECs and incubated for 24 h. Following incubation, the percentage of monocytes that underwent diapedesis was determined by fluorescence microscopy. (B and C) Results are from three independent experiments from different donors. Statistical significance between experimental means (P value) was determined using the Student's t test. *, P < 0.05.
Increased cell motility stimulates diapedesis (transendothelial migration) by promoting monocytes to push between tight junctions of adjacent ECs (27). At 24 hpi of EGF treatment, we found ≈50% of monocytes undergoing diapedesis vs. only 20% of mock-infected cells (Fig. 3C). The 2.5-fold induction in transendothelial migration by HCMV or EGF was abrogated by inhibition of EGFR signaling. Pretreatment with the DMSO solvent control (3, 5) or the IgG isotype-matched control antibody did not affect HCMV-induced monocyte transendothelial migration. Together, these data show that EGFR possesses biological functions in human peripheral blood monocytes and are consistent with our hypothesis that EGFR engagement by the virus triggers changes in monocytes to promote hematogenous dissemination.
To identify the EGFR-dependent mechanism by which HCMV stimulates monocyte chemokinesis, we examined the HCMV-infected EGFR-dependent monocyte transcriptome at 24 hpi. Because HCMV-induced monocyte motility was abrogated by pretreatment with either the anti-EGFR antibody (inhibition of the extracellular activation domain of EGFR) or AG1478 (inhibition of the intracellular kinase activity), we focused on HCMV-induced genes that were dependent on direct binding to and signaling through EGFR. Ontology analyses of cellular genes known to be involved in cell motility identified N-WASP as a potential mediator of the changes observed in HCMV-infected monocytes (Table S1). N-WASP is an actin nucleator that mediates endocytosis, lamellipodia formation, and motility in EGF-stimulated carcinoma cells (28–30); however, because N-WASP is generally expressed at near-undetectable levels in monocytes (16), we wanted to confirm the induction of N-WASP protein expression in monocytes following HCMV infection (Fig. 4A). We also found that inhibition of the EGFR downstream target PI(3)K blocked HCMV-induced expression of N-WASP.
HCMV up-regulation of N-WASP occurs in an EGFR-dependent manner and is required for HCMV-induced monocyte motility. (A) Monocytes were treated with an anti-EGFR antibody, AG1478, or LY294002 before infection with HCMV. At 24 hpi, total N-WASP expression was detected by immunoblotting. (B) Cells were transfected with N-WASP siRNA or control siRNA, and N-WASP protein levels were analyzed by immunoblotting. (C) Monocytes were mock-transfected (lanes 1–3), N-WASP siRNA-transfected (lane 4), or control siRNA-transfected (lane 5) and incubated for 24 h. Cells were then cooled to 4 °C for 30 min and mock-infected (lane 1) or Towne/E-infected (lanes 2–5) for 90 min at 4 °C. Cells were either immediately treated with PK (lane 3) or incubated for 1 h at 37 °C (lanes 1, 2, 4, and 5) before treatment with PK. The presence of genomic HCMV DNA was determined by RT-PCR using HCMV genomic immediate early-specific primers. RT-PCR analysis of GAPDH was done as a loading control. (D) N-WASP siRNA, control siRNA, or untransfected monocytes were infected with Towne/E, and photomicrographs captured at 6 hpi. Images are representative of three independent experiments from different donors. (E) N-WASP siRNA, control siRNA, or untransfected monocytes were plated on colloidal gold-coated coverslips and infected with Towne/E (multiplicity of infection of 5), treated with PMA (10 ng/mL), or treated with EGF (100 ng/mL). The average area of colloidal gold cleared per monocyte was determined. Results are from three independent experiments from different donors. Statistical significance between experimental means (P value) was determined using the Student's t test. *, P < 0.05.
To dissect the biological consequence(s) of N-WASP up-regulation during HCMV infection, siRNA knockdown was used. We observed a 90% reduction in N-WASP protein levels following densitometric analyses in N-WASP siRNA-transfected monocytes but not in control siRNA-transfected monocytes when compared with mock-transfected cells by 24 h posttransfection (Fig. 4B). The down-regulation continued for up to 72 h and had no effect on monocyte viability. Because N-WASP deficiency impairs EGFR-mediated endocytosis (28), we first tested whether HCMV entry into monocytes was reduced in N-WASP-deficient monocytes. Similar levels of internalized viral genomes in monocytes expressing low and high levels of N-WASP were observed (Fig. 4C), suggesting that N-WASP was not required for efficient viral entry.
Because EGF stimulation can target N-WASP to the leading membrane edge of a motile cell to mediate movement (31), we examined whether N-WASP plays a functional role in the “hyper”-motility observed in HCMV-infected monocytes. Examination of cellular morphology revealed that, unlike HCMV-infected WT monocytes, N-WASP-deficient monocytes lacked distinct tail and lamellipodia formation (Fig. 4D). Quantitative phagokinetic motility track assays demonstrated a 6-fold increase in monocyte chemokinesis following HCMV infection, whereas only a 4-fold and 3-fold induction in motility was observed following PMA and EGF treatment, respectively (Fig. 4E). An 80% reduction in motility was seen in N-WASP-deficient HCMV-infected monocytes. Similarly, N-WASP-deficient monocytes derived from a different N-WASP-specific siRNA exhibited analogous decreases in HCMV-induced chemokinesis (Fig. S5), demonstrating that the decrease in the viral induction of monocyte motility was not likely attributable to off-target effects. Because 100% inhibition of EGF-induced monocyte motility was observed in the absence of N-WASP activity, our data suggest that low-level EGFR-independent cell motility may also be generated in HCMV-infected monocytes. The utilization of N-WASP to induce hypermotility in cells appeared to be specific to HCMV infection, because the less proficient PMA induction of monocyte motility was not affected by the decrease in N-WASP expression. Overall, these data suggest that HCMV specifically stimulates the rapid and aberrant up-regulation of N-WASP activity in monocytes (dependent on the EGFR/PI(3)K signaling cascade) to induce a distinct multidirectional motility.
Hematogenous dissemination of HCMV to multiple host organ sites is critical to viral persistence within the infected host following primary infection. Viral spread occurs in a cell-dependent manner, with monocytes serving as direct mediators of viral spread to peripheral tissue (2, 32, 33). In our investigation of the mechanism for how infected monocytes promote the simultaneous spread of HCMV to biologically distinct tissue, we reported that monocytes acquire a unique chemokinetic phenotype via receptor–ligand interactions during viral binding/entry (4, 7). In this study, we demonstrate the expression of EGFR on the surface of primary peripheral blood monocytes and that HCMV engagement of EGFR results in rapid autophosphorylation and subsequent phosphorylation of downstream targets. The activation of the EGFR signaling pathway was required for efficient viral entry and monocyte chemokinesis, thus providing a link between viral entry and the pathogenic motility required for viral spread during primary infection despite the absence of a chemotactic gradient.
The relation between EGFR expression and HCMV infection of the monocyte subpopulation in PBMCs indicated that EGFR could be an integral player determining monocyte tropism for HCMV. Because the presence of neutralizing anti-EGFR antibodies significantly reduced virus internalization into monocytes but did not affect attachment of HCMV, we suggest that EGFR is a viral tropism receptor that functions at the stage of internalization. Integrins and PDGFR-α have also been reported to be required for efficient viral entry (13, 22, 23), but because of ubiquitous integrin expression on PBMCs and the lack of PDGFR-α expression on monocytes and macrophages (in this study and ref. 19), it is unlikely that these receptors direct HCMV infection toward the monocyte subpopulation. EGFR has been shown to act as a viral tropism receptor for targeting HCMV entry into biologically distinct populations of trophoblast cells (11). Our data now provide evidence that EGFR expression and the ensuing downstream signaling following viral engagement on monocytes provide cell type specificity for viral internalization.
Several studies have reported the rapid activation of EGFR to occur in various cell lines following HCMV infection (8–10) and that the activation of EGFR signaling was required for efficient infection (8, 11). We similarly found that EGFR activity was necessary for HCMV entry into primary monocytes, although in contrast to model cell types (23), activation of the downstream target PI(3)K was not required for HCMV entry. This difference highlights how unique pathways originating from the same cellular receptor in biologically distinct cell types are used by HCMV to ensure self-survival in multiple cell types. To add to the complexity of HCMV entry, other studies report that EGFR expression is not required for viral entry (12, 34). The reasons for these conflicting results remain unresolved. In contrast to endotheliotropic/clinical HCMV strains, such as Towne/E and TB40/E, laboratory/fibroblast-adapted strains, such as AD169, Towne, and TB40/F, have reduced infectivity of endothelial/epithelial cells and monocytes because of losses or mutations in the ULB′ region of the viral genome (35–37). Expression of distinct viral glycoproteins from the ULB′ region allows viral infection of endothelial/epithelial cells but not of fibroblasts, indicating the existence of cell type-specific receptors for different strains of HCMV (38). Although more work is needed to address whether strain variation can account for the requirement of EGFR signaling during entry, our data demonstrate that endotheliotropic strains Towne/E and TB40/E signal and enter through an EGFR-dependent mechanism on monocytes.
Analysis of the EGFR-dependent HCMV-infected monocyte transcriptome revealed a complex regulation of monocyte gene expression that originates from engagement of and signaling through EGFR. More genes were regulated by the intracellular signaling of EGFR when compared with genes regulated by the extracellular signaling of EGFR (Fig. S6; R = 0.78 vs. 0.93), which is likely attributable to the cross-talk phosphorylation of the cytoplasmic domain of EGFR by integrins activated during HCMV entry (22, 23, 39). We suggest that HCMV initiates a multireceptor activation process during viral entry that depends on the expression of cell-specific HCMV receptors on different cell types.
Our global transcriptional and functional analysis identified the actin nucleator N-WASP as a potential mediator of EGFR-dependent HCMV-induced monocyte chemokinesis. Although N-WASP is expressed in many different cell types, homeostatic expression of N-WASP is low in hematopoietic cells (16), in which the related WASP is the dominant actin nucleator (18); thus, the role that N-WASP plays in HCMV-infected monocytes is unclear. Perhaps specific targeting of N-WASP by HCMV is based on the 20 times higher actin-polymerizing potential of N-WASP when compared with WASP activity (17). Indeed, it has been observed that actin-based spreading of Shigella to adjacent cells is dependent on the exclusive recruitment of N-WASP (16). Because of low N-WASP expression in hematopoietic cells, spread of Shigella was not observed from infected macrophages and was only restored by ectopic expression of N-WASP (16). Our data indicate that HCMV has devised a specific strategy to up-regulate the expression of N-WASP rapidly, thus circumventing the normally low levels of this protein in monocytes. This requirement of N-WASP to mediate HCMV-induced chemokinetic monocyte motility is in contrast to chemokine-induced chemotactic motility, which requires WASP activity (40). Our data also indicate that PMA induction of monocyte motility was not affected by N-WASP deficiency in monocytes, thus providing further evidence for the select utilization of N-WASP during HCMV infection to induce hypermotility in cells. These observations provide a potential mechanism by which the N-WASP-dependent chemokinetic movement of monocytes favors the hematogenous dissemination of HCMV to multiple organ sites during primary infection.
Although the heightened motility induced following HCMV infection is dependent on EGFR activity, our data indicate that divergent signaling pathways are generated from EGF- and HCMV-stimulated EGFR. The inhibition of EGFR by the presence an anti-EGFR antibody or AG1478 completely abrogated HCMV- and EGF-induced monocyte motility. However, unlike EGF treatment, HCMV infection was able to induce motility partially in N-WASP-deficient monocytes, indicating that HCMV induction of EGFR leads to cellular signals different from those of EGF-induced EGFR activation. Indeed, other studies have also shown that HCMV infection stimulates a phosphorylation pattern on the cytoplasmic tail of EGFR different from the phosphorylation signature induced by EGF treatment (10), indicating that HCMV acts as a unique ligand able to initiate an aberrant EGFR cellular signaling profile not observed with EGF treatment.
In summary, we have documented the expression of biologically functional EGFR on primary human peripheral blood monocytes and suggest that HCMV has evolved to use this cellular receptor to initiate the multiple early events required for viral dissemination. EGFR acts as a tropism receptor by mediating efficient HCMV entry in a PI(3)K-independent manner to the monocyte subpopulation of PBMCs. In addition, the activation of EGFR induces the pathogenic chemokinetic movement of monocytes following HCMV infection, which is dependent on PI(3)K activity and is essential for the migration of infected cells into peripheral tissue in which differentiation into replication-permissive macrophages can occur. Our identification of EGFR on primary human monocytes provides a possible mechanistic link between HCMV infection and the development of cardiovascular diseases, such as atherosclerosis, in which pathogenesis is coupled to the migration of proinflammatory monocytes into atherosclerotic plaques (41).
Blood was drawn by venipuncture and centrifuged through a Ficoll Histopaque 1077 gradient (Sigma). Monocytes were then isolated by centrifugation through a Percoll (Amersham Pharmacia) gradient and suspended in RPMI 1640 (Cellgro; Mediatech) supplemented with 1% human serum (Sigma).
HCMV strains Towne/E (passages 35–45) (7) and GFP-labeled TB40/E-UL32 (TB40/E) (25, 26) were cultured in human embryonic lung fibroblasts. Virus was purified on a 0.5-M sucrose cushion, resuspended in RPMI 1640 media (Cellgro), and used to infect monocytes at a multiplicity of infection of 5 for each experiment unless otherwise stated.
For complete details of all protocols, see SI Materials and Methods.
This work was supported by a Malcolm Feist Cardiovascular Research Fellowship and the National Institutes of Health (Grants AI56077, HD051998, and 1-P20-RR018724).
Author contributions: G.C., M.T.N., and A.D.Y. designed research; G.C. and M.T.N. performed research; G.C., M.T.N., and A.D.Y. analyzed data; and G.C. and A.D.Y. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE17948).
This article contains supporting information online at www.pnas.org/cgi/content/full/0908787106/DCSupplemental.
