Human Cytomegalovirus Host Interactions: EGFR and Host Cell Signaling Is a Point of Convergence Between Viral Infection and Functional Changes in Infected Cells

Abstract

Viruses have evolved diverse strategies to manipulate cellular signaling pathways in order to promote infection and/or persistence. Human cytomegalovirus (HCMV) possesses a number of unique properties that allow the virus to alter cellular events required for infection of a diverse array of host cell types and long-term persistence. Of specific importance is infection of bone marrow derived and myeloid lineage cells, such as peripheral blood monocytes and CD34⁺ hematopoietic progenitor cells (HPCs) because of their essential role in dissemination of the virus and for the establishment of latency. Viral induced signaling through the Epidermal Growth Factor Receptor (EGFR) and other receptors such as integrins are key control points for viral-induced cellular changes and productive and latent infection in host organ systems. This review will explore the current understanding of HCMV strategies utilized to hijack cellular signaling pathways, such as EGFR, to promote the wide-spread dissemination and the classic life-long herpesvirus persistence.

Keywords: cell signaling; differentiation; epidermal growth factor receptor (EGFR); glycoproteins; human cytomegalovirus (HCMV); latency; monocytes; progenitor cells.

FIGURE 1

Schematic of the HCMV glycoprotein complexes and their cognate receptors. HCMV attachment and entry into monocytes depends on several envelope glycoprotein complexes including gM/gN, gB, and the gH complexes. gM/gN mediates initial attachment via glycosaminoglycans. gB is known to interact with the epidermal growth factor receptor (EGFR) on monocytes. gB may also interact with integrins on monocytes, although this is unresolved. The trimeric gH/gL/gO complex interacts with integrins and the platelet-derived growth factor receptor alpha (PDGFRα) on fibroblasts; at present the trimer only has been reported to interact with integrins on monocytes. The pentameric gH/gL/UL128-131 complex interacts with β1- and β3-integrins on monocytes. Several other receptors, such as OR14I1 and Nrp2, have been reported to bind to the pentamer; this engagement remains unresolved during infection of monocytes. The membrane of fibroblasts is illustrated on the right and the membrane of epithelial cells is illustrated on the left and they include the multiple receptors that have been reported to engage the HCMV glycoprotein complexes during infection of fibroblasts and epithelial cells, respectively.

FIGURE 2

Human cytomegalovirus (HCMV) glycoprotein binding to cognate receptors controls viral signaling, trafficking and the key cellular functions required for productive infection of monocytes. The HCMV glycoproteins bind several cellular receptors, including EGFR and the β1- and β3-integrins. Following binding, these receptor engagements trigger a variety of signal transduction pathways in the infected monocyte. For example, gB binding to EGFR results in activation of the intrinsic EGFR tyrosine kinase and down stream signaling that is required for entry, for the unique multi-vesicular nuclear translocation process seen in monocytes, as well as for motility, and the survival of the monocytes and their differentiation into macrophages. The engagement of the pentameric complex, gH/gL/UL128-131 with β1- and β3-integrins on the surface of monocytes activates c-Src to drive the required signaling pathways needed for entry, nuclear translocation, motility, survival and differentiation. More specifically, this downstream signaling following EGFR and integrin engagement acts in concert to activate numerous down stream signaling pathways, including the NF-κB pathway, PI(3)K, and the MAPK pathway, which culminates in enhanced transactivation of many host cell promoters via increases in transcriptional regulators, altercations in actin regulatory proteins that modulate motility (such as N-WASP and paxillin) and changes in Mcl-1 and Bcl-2 to promote survival of short-lived monocytes.

FIGURE 3

Human cytomegalovirus infection of monocytes drives a number of key molecular mechanisms required to enhance survival of normally short-lived monocytes and for the promotion of the differentiation of these infected monocytes into long-lived productively infected macrophaqes. It is important to note that monocytes, although they can be infected, they are not permissive for viral replication; only upon differentiation is de novo viral gene expression observed. HCMV-infected monocytes show enhanced expression of Mcl-1 early after infection (<48 h post infection) to prolong survival through the key 48-h viability gate. Mcl-1 levels then gradually decrease through this 48 h viability gate. Enhanced expression of Bcl-2 is then observed, as well as partial activation of caspase-3, which together seem to promote both long term survival and differentiation of these infected monocytes toward viral replication-permissive tissue macrophages that begin to produce mature virus around 3 weeks after infection.

FIGURE 4

Many HCMV-expressed proteins and miRNAs regulate the signaling pathways known to be important for the establishment, maintanence and reactivation from latency. The initial infection of CD34⁺ HPCs lays the foundation for the establishment of latency. Initially, gB engagement of EGFR and the ensuing signaling is required for HCMV entry into CD34⁺ HPCs. This initial ligand-dependent event prepares the cell for the establishment of latency through the manipulation of host signaling pathways and regulation of viral latent gene expression. These latency gene products in turn are critical for the establishment, the maintenance of and/or reactivation from latency. For example, gene products such as UL138 and UL135 regulate viral latency and host interactions by regulating various signaling pathways [such as Egr-1 activity or PI(3)K signaling] during the establishment of and exit from latency. Other products such as the viral miRNAs miR-US5-1 and miR-UL112-3p target and down regulate IKKα and IKKβ to control inflammatory cytokine regulation (such as IL-6 and TNFα) and likely to mitigate NF-KB dependent signaling that might interfere with latency programming. Other products such as soluble UL7 are released during reactivation and through binding to the Flt-3R induces activation of the downstream PI3K/AKT and MAPK/ERK signaling pathways that in turn triggers the HPC and monocyte differentiation required for complete viral reactivation. US28, another critical latency modulator, is involved in the MEK/ERK and EGFR/PI3K signaling required for HCMV reactivation from latency, as well as playing a key role in monocyte to macrophage differentiation. In addition, a variety of viral miRNAs (such as miR-US5-1, miR-US5-2, miR-US22, and miR-UL112-3p) target cellular processes and regulate virus-mediated signaling. As examples, miR-US5-2 blocks NAB1 activity, a represser of TGFβ resulting in increased production of the TGFβ that suppresses hematopoietic events; miR-US5-2 also down regulates GAB1, thus lowering MEK/ERK signaling, as well as regulates EGR1 and UL138 expression supporting a role for miR US5-2 during reactivation. Overall these viral miRNAs, UL7, US28, UL138/135 control vital signaling pathways during the establishment, maintenance of and during reactivation from latency and are intimately involved in the differentiation of the infected CD34⁺ HPCs into monocytes and then into productive tissue macrophages.