Distinct early role of PTEN regulation during HCMV infection of monocytes.

Human cytomegalovirus (HCMV) infection of monocytes is essential for viral dissemination and persistence. We previously identified that HCMV entry/internalization and subsequent productive infection of this clinically relevant cell type is distinct when compared to other infected cells. We showed that internalization and productive infection required activation of epidermal growth factor receptor (EGFR) and integrin/c-Src, via binding of viral glycoprotein B to EGFR, and the pentamer complex to ß1/ß3 integrins. To understand how virus attachment drives entry, we compared infection of monocytes with viruses containing the pentamer vs. those without the pentamer and then used a phosphoproteomic screen to identify potential phosphorylated proteins that influence HCMV entry and trafficking. The screen revealed that the most prominent pentamer-biased phosphorylated protein was the lipid- and protein-phosphatase phosphatase and tensin homolog (PTEN). PTEN knockdown with siRNA or PTEN inhibition with a PTEN inhibitor decreased pentamer-mediated HCMV entry, without affecting trimer-mediated entry. Inhibition of PTEN activity affected lipid metabolism and interfered with the onset of the endocytic processes required for HCMV entry. PTEN inactivation was sufficient to rescue pentamer-null HCMV from lysosomal degradation. We next examined dephosphorylation of a PTEN substrate Rab7, a regulator of endosomal maturation. Inhibition of PTEN activity prevented dephosphorylation of Rab7. Phosphorylated Rab7, in turn, blocked early endosome to late endosome maturation and promoted nuclear localization of the virus and productive infection.

Highly socially vulnerable communities exhibit disproportionately increased viral loads as measured in community wastewater.

Wastewater surveillance has proven to be a useful tool for evidence-based epidemiology in the fight against the SARS-CoV-2 virus. It is particularly useful at the population level where acquisition of individual test samples may be time or cost-prohibitive. Wastewater surveillance for SARS-CoV-2 has typically been performed at wastewater treatment plants; however, this study was designed to sample on a local level to monitor the spread of the virus among three communities with distinct social vulnerability indices in Shreveport, Louisiana, located in a socially vulnerable region of the United States. Twice-monthly grab samples were collected from September 30, 2020, to March 23, 2021, during the Beta wave of the pandemic. The goals of the study were to examine whether: 1) concentrations of SARS-CoV-2 RNA in wastewater varied with social vulnerability indices and, 2) the time lag of spikes differed during wastewater monitoring in the distinct communities. The size of the population contributing to each sample was assessed via the quantification of the pepper mild mottle virus (PMMoV), which was significantly higher in the less socially vulnerable community. We found that the communities with higher social vulnerability exhibited greater viral loads as assessed by wastewater when normalized with PMMoV (Kruskal-Wallis, p < 0.05). The timing of the spread of the virus through the three communities appeared to be similar. These results suggest that interconnected communities within a municipality experienced the spread of the SARS-CoV-2 virus at similar times, but areas of high social vulnerability experienced more intense wastewater viral loads.

Human Cytomegalovirus Manipulates Syntaxin 6 for Successful Trafficking and Subsequent Infection of Monocytes.

Human cytomegalovirus (HCMV) exhibits a complex host-pathogen interaction with peripheral blood monocytes. We have identified a unique, cell-type specific retrograde-like intracellular trafficking pattern that HCMV utilizes to gain access to the monocyte nucleus and for productive infection. We show that infection of primary human monocytes, epithelial cells, and fibroblasts leads to an increase in the amount of the trafficking protein Syntaxin 6 (Stx6). However, only knockdown (KD) of Stx6 in monocytes inhibited viral trafficking to the trans-Golgi network (TGN), a requisite step for nuclear translocation in monocytes. Conversely, KD of Stx6 in epithelial cells and fibroblasts did not change the kinetics of nuclear translocation and productive infection. Stx6 predominantly functions at the level of the TGN where it facilitates retrograde transport, a trafficking pathway used by only a few cellular proteins and seldom by pathogens. We also newly identify that in monocytes, Stx6 exhibits an irregular vesicular localization rather than being concentrated at the TGN as seen in other cell-types. Lastly, we implicate that viral particles that associate with both Stx6 and EEA1 early in infection are the viral population that successfully traffics to the TGN at later time points and undergo nuclear translocation. Additionally, we show for the first time that HCMV enters the TGN, and that lack of Stx6 prevents viral trafficking to this organelle. We argue that we have identified an essential cell-type specific regulator that controls early steps in efficient productive infection of a cell-type required for viral persistence and disease. IMPORTANCE Human cytomegalovirus (HCMV) infection causes severe and often fatal disease in the immunocompromised. It is one of the leading infectious causes of birth defects and causes severe complications in transplant recipients. By uncovering the unique pathways used by the virus to infect key cells, such as monocytes, responsible for dissemination and persistence, we provide new potential targets for therapeutic intervention.

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

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.

Overview of Human Cytomegalovirus Pathogenesis.

Human cytomegalovirus (HCMV) is a betaherpesvirus with a global seroprevalence of 60-90%. HCMV is the leading cause of congenital infections and poses a great health risk to immunocompromised individuals. Although HCMV infection is typically asymptomatic in the immunocompetent population, infection can result in mononucleosis and has also been associated with the development of certain cancers, as well as chronic inflammatory diseases such as various cardiovascular diseases. In immunocompromised patients, including AIDS patients, transplant recipients, and developing fetuses, HCMV infection is associated with increased rates of morbidity and mortality. Currently there is no vaccine for HCMV and there is a need for new pharmacological treatments. Ongoing research seeks to further define the complex aspects of HCMV pathogenesis, which could potentially lead to the generation of new therapeutics to mitigate the disease states associated with HCMV infection. The following chapter reviews the advancements in our understanding of HCMV pathogenesis in the immunocompetent and immunocompromised hosts.

Collection and Isolation of CD14+ Primary Human Monocytes Via Dual Density Gradient Centrifugation as a Model System to Study Human Cytomegalovirus Infection and Pathogenesis.

Human cytomegalovirus (HCMV) can cause severe disease in the immunocompromised. One of the hallmarks of HCMV infection of a human host is the targeted infection of peripheral blood monocytes (but not other leukocyte populations) that in turn serve as the key cell type for hematogenous dissemination and the establishment of persistence following primary infection. Monocytes are also a key cell type associated with viral reactivation and spread following viral reactivation. Because of their importance in the HCMV-host infection cycle and lifelong infection, it is critical to be able to study their infection in controlled in vitro systems in the laboratory. In this chapter, we discuss a viable protocol for harvesting fresh ex vivo blood monocytes from human donors that are pure and unactivated cells and that can be used in a research setting.

Using a Phosphoproteomic Screen to Profile Early Changes During HCMV Infection of Human Monocytes.

During the binding and infection of monocytes, HCMV binds to at least two major cell surface receptors/receptor families: the epidermal growth factor receptor (EGFR) to initiate downstream signaling through the EGFR-PI3K pathway, and to ß1- and ß3-integrins to initiate downstream signaling through the integrin-c-Src pathway (Nogalski et al. PLoS Pathog 9:e1003463, 2013; Chan et al. Proc Natl Acad Sci U S A 106:22369-22374, 2009; Kim et al. Proc Natl Acad Sci U S A 113:8819-8824, 2016; Wang et al. Nature 424:456-461, 2003; Wang et al. Nat Med 11:515-521, 2005; Yurochko et al. Proc Natl Acad Sci U S A 89:9034-9038, 1992). Signaling through these receptors can occur rapidly with phosphorylation observed as early as 15 s after EGF-EGFR interaction, for example (Alvarez-Salamero et al. Front Immunol 8:938, 2017). The ability to detect signaling and the consequences of that signaling are critical for our understanding of how HCMV-receptor engagement promotes infection and modulates the biology of different target cells. In this chapter we describe how we used an ELISA-based antibody platform to perform an assessment of the rapid phosphorylation events that occur in monocytes following infection. This assay can be adapted to other infection systems, time points and cell types as needed. Together, we examined via an ELISA-based antibody array a phosphoproteomic screen to search for potential phosphorylated proteins that might influence HCMV infection.

CD34+ Hematopoietic Progenitor Cell Subsets Exhibit Differential Ability To Maintain Human Cytomegalovirus Latency and Persistence.

In human cytomegalovirus (HCMV)-seropositive patients, CD34+ hematopoietic progenitor cells (HPCs) provide an important source of latent virus that reactivates following cellular differentiation into tissue macrophages. Multiple groups have used primary CD34+ HPCs to investigate mechanisms of viral latency. However, analyses of mechanisms of HCMV latency have been hampered by the genetic variability of CD34+ HPCs from different donors, availability of cells, and low frequency of reactivation. In addition, multiple progenitor cell types express surface CD34, and the frequencies of these populations differ depending on the tissue source of the cells and culture conditions in vitro In this study, we generated CD34+ progenitor cells from two different embryonic stem cell (ESC) lines, WA01 and WA09, to circumvent limitations associated with primary CD34+ HPCs. HCMV infection of CD34+ HPCs derived from either WA01 or WA09 ESCs supported HCMV latency and induced myelosuppression similar to infection of primary CD34+ HPCs. Analysis of HCMV-infected primary or ESC-derived CD34+ HPC subpopulations indicated that HCMV was able to establish latency and reactivate in CD38+ CD90+ and CD38+/low CD90- HPCs but persistently infected CD38- CD90+ cells to produce infectious virus. These results indicate that ESC-derived CD34+ HPCs can be used as a model for HCMV latency and that the virus either latently or persistently infects specific subpopulations of CD34+ cells.IMPORTANCE Human cytomegalovirus infection is associated with severe disease in transplant patients and understanding how latency and reactivation occur in stem cell populations is essential to understand disease. CD34+ hematopoietic progenitor cells (HPCs) are a critical viral reservoir; however, these cells are a heterogeneous pool with donor-to-donor variation in functional, genetic, and phenotypic characteristics. We generated a novel system using embryonic stem cell lines to model HCMV latency and reactivation in HPCs with a consistent cellular background. Our study defined three key stem cell subsets with differentially regulated latent and replicative states, which provide cellular candidates for isolation and treatment of transplant-mediated disease. This work provides a direction toward developing strategies to control the switch between latency and reactivation.

The Differentiation of Human Cytomegalovirus Infected-Monocytes Is Required for Viral Replication.

Viral dissemination is a key mechanism responsible for persistence and disease following human cytomegalovirus (HCMV) infection. Monocytes play a pivotal role in viral dissemination to organ tissue during primary infection and following reactivation from latency. For example, during primary infection, infected monocytes migrate into tissues and differentiate into macrophages, which then become a source of viral replication. In addition, because differentiated macrophages can survive for months to years, they provide a potential persistent infection source in various organ systems. We broadly note that there are three phases to infection and differentiation of HCMV-infected monocytes: (1) Virus enters and traffics to the nucleus through a virus receptor ligand engagement event that activates a unique signalsome that initiates the monocyte-to-macrophage differentiation process. (2) Following initial infection, HCMV undergoes a "quiescence-like state" in monocytes lasting for several weeks and promotes monocyte differentiation into macrophages. While, the initial event is triggered by the receptor-ligand engagement, the long-term cellular activation is maintained by chronic viral-mediated signaling events. (3) Once HCMV infected monocytes differentiate into macrophages, the expression of immediate early viral (IE) genes is detectable, followed by viral replication and long term infectious viral particles release. Herein, we review the detailed mechanisms of each phase during infection and differentiation into macrophages and discuss the biological significance of the differentiation of monocytes in the pathogenesis of HCMV.

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

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 Infection Suppresses CD34+ Progenitor Cell Engraftment in Humanized Mice.

Human cytomegalovirus (HCMV) infection is a serious complication in hematopoietic stem cell transplant (HSCT) recipients due to virus-induced myelosuppression and impairment of stem cell engraftment. Despite the clear clinical link between myelosuppression and HCMV infection, little is known about the mechanism(s) by which the virus inhibits normal hematopoiesis because of the strict species specificity and the lack of surrogate animal models. In this study, we developed a novel humanized mouse model system that recapitulates the HCMV-mediated engraftment failure after hematopoietic cell transplantation. We observed significant alterations in the hematopoietic populations in peripheral lymphoid tissues following engraftment of a subset of HCMV+ CD34+ hematopoietic progenitor cells (HPCs) within the transplant, suggesting that a small proportion of HCMV-infected CD34+ HPCs can profoundly affect HPC differentiation in the bone marrow microenvironment. This model will be instrumental to gain insight into the fundamental mechanisms of HCMV myelosuppression after HSCT and provides a platform to assess novel treatment strategies.

Human Cytomegalovirus miRNAs Regulate TGF-ß to Mediate Myelosuppression while Maintaining Viral Latency in CD34+ Hematopoietic Progenitor Cells.

Infection with human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality following hematopoietic stem cell transplant (HSCT) because of various hematologic problems, including myelosuppression. Here, we demonstrate that latently expressed HCMV miR-US5-2 downregulates the transcriptional repressor NGFI-A binding protein (NAB1) to induce myelosuppression of uninfected CD34+ hematopoietic progenitor cells (HPCs) through an increase in TGF-ß production. Infection of HPCs with an HCMVΔmiR-US5-2 mutant resulted in decreased TGF-ß expression and restoration of myelopoiesis. In contrast, we show that infected HPCs are refractory to TGF-ß signaling as another HCMV miRNA, miR-UL22A, downregulates SMAD3, which is required for maintenance of latency. Our data suggest that latently expressed viral miRNAs manipulate stem cell homeostasis by inducing secretion of TGF-ß while protecting infected HPCs from TGF-ß-mediated effects on viral latency and reactivation. These observations provide a mechanism through which HCMV induces global myelosuppression following HSCT while maintaining lifelong infection in myeloid lineage cells.

Human Cytomegalovirus US28 Ligand Binding Activity Is Required for Latency in CD34+ Hematopoietic Progenitor Cells and Humanized NSG Mice.

Human cytomegalovirus (HCMV) infection of CD34+ hematopoietic progenitor cells (CD34+ HPCs) provides a critical reservoir of virus in stem cell transplant patients, and viral reactivation remains a significant cause of morbidity and mortality. The HCMV chemokine receptor US28 is implicated in the regulation of viral latency and reactivation. To explore the role of US28 signaling in latency and reactivation, we analyzed protein tyrosine kinase signaling in CD34+ HPCs expressing US28. US28-ligand signaling in CD34+ HPCs induced changes in key regulators of cellular activation and differentiation. In vitro latency and reactivation assays utilizing CD34+ HPCs indicated that US28 was required for viral reactivation but not latency establishment or maintenance. Similarly, humanized NSG mice (huNSG) infected with TB40E-GFP-US28stop failed to reactivate upon treatment with granulocyte-colony-stimulating factor, but viral genome levels were maintained. Interestingly, HCMV-mediated changes in hematopoiesis during latency in vivo and in vitro was also dependent upon US28, as US28 directly promoted differentiation toward the myeloid lineage. To determine whether US28 constitutive activity and/or ligand-binding activity were required for latency and reactivation, we infected both huNSG mice and CD34+ HPCs in vitro with HCMV TB40E-GFP containing the US28-R129A mutation (no CA) or Y16F mutation (no ligand binding). TB40E-GFP-US28-R129A was maintained during latency and exhibited normal reactivation kinetics. In contrast, TB40E-GFP-US28-Y16F exhibited high levels of viral genome during latency and reactivation, indicating that the virus did not establish latency. These data indicate that US28 is necessary for viral reactivation and ligand binding activity is required for viral latency, highlighting the complex role of US28 during HCMV latency and reactivation.IMPORTANCE Human cytomegalovirus (HCMV) can establish latency following infection of CD34+ hematopoietic progenitor cells (HPCs), and reactivation from latency is a significant cause of viral disease and accelerated graft failure in bone marrow and solid-organ transplant patients. The precise molecular mechanisms of HCMV infection in HPCs are not well defined; however, select viral gene products are known to regulate aspects of latency and reactivation. The HCMV-encoded chemokine receptor US28, which binds multiple CC chemokines as well as CX3CR1, is expressed both during latent and lytic phases of the virus life cycle and plays a role in latency and reactivation. However, the specific timing of US28 expression and the role of ligand binding in these processes are not well defined. In this report, we determined that US28 is required for reactivation but not for maintaining latency. However, when present during latency, US28 ligand binding activity is critical to maintaining the virus in a quiescent state. We attribute the regulation of both latency and reactivation to the role of US28 in promoting myeloid lineage cell differentiation. These data highlight the dynamic and multifunctional nature of US28 during HCMV latency and reactivation.

OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism.

A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH-gL-UL128-UL130-UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT-mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.

HCMV Infection and Apoptosis: How Do Monocytes Survive HCMV Infection?

Human cytomegalovirus (HCMV) infection of peripheral blood monocytes plays a key role in the hematogenous dissemination of the virus to multiple organ systems following primary infection or reactivation of latent virus in the bone marrow. Monocytes have a short life span of 1⁻3 days in circulation; thus, HCMV must alter their survival and differentiation to utilize these cells and their differentiated counterparts-macrophages-for dissemination and long term viral persistence. Because monocytes are not initially permissive for viral gene expression and replication, HCMV must control host-derived factors early during infection to prevent apoptosis or programmed cell death prior to viral induced differentiation into naturally long-lived macrophages. This review provides a short overview of HCMV infection of monocytes and describes how HCMV has evolved to utilize host cell anti-apoptotic pathways to allow infected monocytes to bridge the 48⁻72 h viability gate so that differentiation into a long term stable mature cell can occur. Because viral gene expression is delayed in monocytes following initial infection and only occurs (begins around two to three weeks post infection in our model) following what appears to be complete differentiation into mature macrophages or dendritic cells, or both; virally-encoded anti-apoptotic gene products cannot initially control long term infected cell survival. Anti-apoptotic viral genes are discussed in the second section of this review and we argue they would play an important role in long term macrophage or dendritic cell survival following infection-induced differentiation.

Human Cytomegalovirus Encodes a Novel FLT3 Receptor Ligand Necessary for Hematopoietic Cell Differentiation and Viral Reactivation.

The ability of human cytomegalovirus (HCMV) to reactivate from latent infection of hematopoietic progenitor cells (HPCs) is intimately linked to cellular differentiation. HCMV encodes UL7 that our group has shown is secreted from infected cells and induces angiogenesis. In this study, we show that UL7 is a ligand for Fms-like tyrosine kinase 3 receptor (Flt-3R), a well-known critical factor in HPC differentiation. We observed that UL7 directly binds Flt-3R and induces downstream signaling cascades, including phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. Importantly, we show that UL7 protein induces differentiation of both CD34+ HPCs and CD14+ monocytes. Last, we show that an HCMV mutant lacking UL7 fails to reactivate in CD34+ HPCs in vitro as well as in humanized mice. These observations define the first virally encoded differentiation factor with significant implications not only for HCMV reactivation but also for alteration of the hematopoietic compartment in transplant patients.IMPORTANCE Human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant recipients. CD34+ hematopoietic progenitor cells (HPCs) represent a critical reservoir of latent HCMV in the transplant population, thereby providing a source of virus for dissemination to visceral organs. HCMV reactivation has been linked to HPC/myeloid cellular differentiation; however, the mechanisms involved in these events are poorly understood at the molecular level. In this study, we show that a viral protein is a ligand for Fms-like tyrosine kinase 3 receptor (Flt-3R) and that the binding of HCMV UL7 to the Flt-3R triggers HPC and monocyte differentiation. Moreover, the loss of UL7 prevents viral reactivation in HPCs in vitro as well as in humanized mice. These observations define the first virally encoded differentiation factor with significant implications not only for HCMV reactivation but also for alteration of the hematopoietic compartment in transplant patients.

Human Cytomegalovirus Utilizes a Nontraditional Signal Transducer and Activator of Transcription 1 Activation Cascade via Signaling through Epidermal Growth Factor Receptor and Integrins To Efficiently Promote the Motility, Differentiation, and Polarization of Infected Monocytes.

Human cytomegalovirus (HCMV) infects peripheral blood monocytes and triggers biological changes that promote viral dissemination and persistence. We have shown that HCMV induces a proinflammatory state in infected monocytes, resulting in enhanced monocyte motility and transendothelial migration, prolonged monocyte survival, and differentiation toward a long-lived M1-like macrophage phenotype. Our data indicate that HCMV triggers these changes, in the absence of de novo viral gene expression and replication, through engagement and activation of epidermal growth factor receptor (EGFR) and integrins on the surface of monocytes. We previously identified that HCMV induces the upregulation of multiple proinflammatory gene ontologies, with the interferon-associated gene ontology exhibiting the highest percentage of upregulated genes. However, the function of the HCMV-induced interferon (IFN)-stimulated genes (ISGs) in infected monocytes remained unclear. We now show that HCMV induces the enhanced expression and activation of a key ISG transcriptional regulator, signal transducer and activator of transcription (STAT1), via an IFN-independent but EGFR- and integrin-dependent signaling pathway. Furthermore, we identified a biphasic activation of STAT1 that likely promotes two distinct phases of STAT1-mediated transcriptional activity. Moreover, our data show that STAT1 is required for efficient early HCMV-induced enhanced monocyte motility and later for HCMV-induced monocyte-to-macrophage differentiation and for the regulation of macrophage polarization, suggesting that STAT1 may serve as a molecular convergence point linking the biological changes that occur at early and later times postinfection. Taken together, our results suggest that HCMV reroutes the biphasic activation of a traditionally antiviral gene product through an EGFR- and integrin-dependent pathway in order to help promote the proviral activation and polarization of infected monocytes.IMPORTANCE HCMV promotes multiple functional changes in infected monocytes that are required for viral spread and persistence, including their enhanced motility and differentiation/polarization toward a proinflammatory M1 macrophage. We now show that HCMV utilizes the traditionally IFN-associated gene product, STAT1, to promote these changes. Our data suggest that HCMV utilizes EGFR- and integrin-dependent (but IFN-independent) signaling pathways to induce STAT1 activation, which may allow the virus to specifically dictate the biological activity of STAT1 during infection. Our data indicate that HCMV utilizes two phases of STAT1 activation, which we argue molecularly links the biological changes that occur following initial binding to those that continue to occur days to weeks following infection. Furthermore, our findings may highlight a unique mechanism for how HCMV avoids the antiviral response during infection by hijacking the function of a critical component of the IFN response pathway.

New Mechanism by Which Human Cytomegalovirus MicroRNAs Negate the Proinflammatory Response to Infection.

Viruses have evolved many novel mechanisms to promote infection and to mitigate the host cell response to that infection. In the article by M. H. Hancock et al. (mBio 8:e00109-17, 2017, https://doi.org/10.1128/mBio.00109-17), the authors describe a new mechanism by which human cytomegalovirus (HCMV) microRNAs (miRNAs; miR-US5-1 and miR-UL112-3p) negate the proinflammatory response to infection. The authors document that these two viral miRNAs downregulate the NF-κB response through direct targeting of the IKKα and IKKß mRNAs, which in turn, through diminished IκB kinases (IKKs), block production of proinflammatory cytokines (interleukin-6 [IL-6], CCL5, and tumor necrosis factor alpha [TNF-α]). Because most signaling pathways that promote NF-κB activation and nuclear translocation ultimately converge on the activation of the IKK complex, this new study documents that HCMV can strongly dictate how infected cells respond to internal and/or external stimuli and thus positively influence the outcome of both lytic and latent infection.

Human Cytomegalovirus Requires Epidermal Growth Factor Receptor Signaling To Enter and Initiate the Early Steps in the Establishment of Latency in CD34+ Human Progenitor Cells.

The establishment of human cytomegalovirus (HCMV) latency and persistence relies on the successful infection of hematopoietic cells, which serve as sites of viral persistence and contribute to viral spread. Here, using blocking antibodies and pharmacological inhibitors, we document that HCMV activation of the epidermal growth factor receptor (EGFR) and downstream phosphatidylinositol 3-kinase (PI3K) mediates viral entry into CD34+ human progenitor cells (HPCs), resulting in distinct cellular trafficking and nuclear translocation of the virus compared to that in other immune cells, such as we have documented in monocytes. We argue that the EGFR allows HCMV to regulate the cellular functions of these replication-restricted cells via its signaling activity following viral binding. In addition to regulating HCMV entry/trafficking, EGFR signaling may also shape the early steps required for the successful establishment of viral latency in CD34+ cells, as pharmacological inhibition of EGFR increases the transcription of lytic IE1/IE2 mRNA while curbing the expression of latency-associated UL138 mRNA. EGFR signaling following infection of CD34+ HPCs may also contribute to changes in hematopoietic potential, as treatment with the EGFR kinase (EGFRK) inhibitor AG1478 alters the expression of the cellular hematopoietic cytokine interleukin 12 (IL-12) in HCMV-infected cells but not in mock-infected cells. These findings, along with our previous work with monocytes, suggest that EGFR likely serves as an important determinant of HCMV tropism for select subsets of hematopoietic cells. Moreover, our new data suggest that EGFR is a key receptor for efficient viral entry and that the ensuing signaling regulates important early events required for successful infection of CD34+ HPCs by HCMV.IMPORTANCE HCMV establishes lifelong persistence within the majority of the human population without causing overt pathogenesis in healthy individuals. Despite this, reactivation of HCMV from its latent reservoir in the bone marrow causes significant morbidity and mortality in immunologically compromised individuals, such as bone marrow and solid organ transplant patients. Lifelong persistent infection has also been linked with the development of various cardiovascular diseases in otherwise healthy individuals. Current HCMV therapeutics target lytic replication, but not the latent viral reservoir; thus, an understanding of the molecular basis for viral latency and persistence is paramount to controlling or eliminating HCMV infection. Here, we show that the viral signalosome activated by HCMV binding to its entry receptor, EGFR, in CD34+ HPCs initiates early events necessary for successful latent infection of this cell type. EGFR and associated signaling players may therefore represent promising targets for mitigating HCMV persistence.

Integrins as Herpesvirus Receptors and Mediators of the Host Signalosome.

The repertoire of herpesvirus receptors consists of nonintegrin and integrin molecules. Integrins interact with the conserved glycoproteins gH/gL or gB. This interaction is a conserved biology across the Herpesviridae family, likely directed to promote virus entry and endocytosis. Herpesviruses exploit this interaction to execute a range of critical functions that include (a) relocation of nonintegrin receptors (e.g., herpes simplex virus nectin1 and Kaposi's sarcoma-associated herpesvirus EphA2), or association with nonintegrin receptors (i.e., human cytomegalovirus EGFR), to dictate species-specific entry pathways; (b) activation of multiple signaling pathways (e.g., Ca2+ release, c-Src, FAK, MAPK, and PI3K); and (c) association with Rho GTPases, tyrosine kinase receptors, Toll-like receptors, which result in cytoskeletal remodeling, differential cell type targeting, and innate responses. In turn, integrins can be modulated by viral proteins (e.g., Epstein-Barr virus LMPs) to favor spread of transformed cells. We propose that herpesviruses evolved a multipartite entry system to allow interaction with multiple receptors, including integrins, required for their sophisticated life cycle.