We have established that human cytomegalovirus (HCMV) infection modulates the biology of target primary peripheral blood monocytes, allowing HCMV to use monocytes as "vehicles" for its systemic spread. HCMV infection of monocytes results in rapid induction of phosphatidylinositol-3-kinase [PI(3)K] and NF-B activities. Integrins, which are upstream of the PI(3)K and NF-B pathways, were shown to be involved in HCMV binding to and entry into fibroblasts, suggesting that receptor ligand-mediated signaling following viral binding to integrins on monocytes could trigger the functional changes seen in infected monocytes. We now show that integrin engagement and the activation of the integrin/Src signaling pathway are essential for the induction of HCMV-infected monocyte motility. To investigate how integrin engagement by HCMV triggers monocyte motility, we examined the infected-monocyte transcriptome and found that the integrin/Src signaling pathway regulates the expression of paxillin, which is an important signal transducer in the regulation of actin rearrangement during cell adhesion and movement. Functionally, we observed that paxillin is activated via the integrin/Src signaling pathway and is required for monocyte motility. Because motility is intimately connected to cellular cytoskeletal organization, a process that is also important in viral entry, we investigated the role paxillin regulation plays in the process of viral entry into monocytes. New results confirmed that HCMV entry into target monocytes was significantly reduced in cells deficient in paxillin expression or the integrin/Src/ paxillin signaling pathway. From our data, HCMV-cell interactions emerge as an essential trigger for the cellular changes that allow for HCMV entry and hematogenous dissemination.Human cytomegalovirus (HCMV), a betaherpesvirus, is a prevalent infectious agent, with seropositivity reaching 50 to 80% among adults in the United States (15). In immunocompromised individuals, viral infection can lead to significant morbidity and mortality (19,35). HCMV is the leading cause of congenital central nervous system damage and a leading opportunistic pathogen in AIDS and transplant patients (19,35). In immunocompetent individuals, HCMV infection is usually mild or asymptomatic, although results now show that HCMV is a strong risk factor in the development of cardiovascular diseases (CVDs) (25,(42)(43)(44)47).After primary infection, HCMV establishes a lifelong persistent infection, with frequent reactivations and active infection of new target cells. The virus can be shed in nearly all body fluids, illustrating HCMV's broad cellular tropism and capacity to spread to and infect most organ systems (5, 27, 54). It is postulated that effective multiorgan viral spread is critical for HCMV survival and persistence within infected hosts (35). HCMV has been shown to infect circulating cells in the blood (30), such as monocytes, and to use these cells as viral carriers, allowing dissemination to target tissues (37). In support, HCMV infection is c...

The wide range of disease pathologies seen in multiple organ sites associated with human cytomegalovirus (HCMV) infection results from the systemic hematogenous dissemination of the virus, which is mediated predominately by infected monocytes. In addition to their role in viral spread, infected monocytes are also known to play a key role in viral latency and life-long persistence. However, in order to utilize infected monocytes for viral spread and persistence, HCMV must overcome a number of monocyte biological hurdles, including their naturally short lifespan and their inability to support viral gene expression and replication. Our laboratory has shown that HCMV is able to manipulate the biology of infected monocytes in order to overcome these biological hurdles by inducing the survival and differentiation of infected monocytes into long-lived macrophages capable of supporting viral gene expression and replication. In this current review, we describe the unique aspects of how HCMV promotes monocyte survival and differentiation by inducing a “finely-tuned” macrophage cell type following infection. Specifically, we describe the induction of a uniquely polarized macrophage subset from infected monocytes, which we argue is the ideal cellular environment for the initiation of viral gene expression and replication and, ultimately, viral spread and persistence within the infected host.

We have established that HCMV acts as a specific ligand engaging and activating cellular integrins on monocytes. As a result, integrin signaling via Src activation leads to the functional activation of paxillin required for efficient viral entry and for the biological changes in monocytes needed for viral dissemination. These biological/molecular changes allow HCMV to use monocytes as “vehicles” for systemic spread and the establishment of lifelong persistence. However, it remains unresolved how HCMV specifically induces this observed monocyte activation. It was previously demonstrated that the HCMV gH/gL/UL128-131 glycoprotein complex facilitates viral entry into biologically relevant cell types. Nevertheless, the mechanism by which the gH/gL/UL128-131 complex promotes this process is unknown. We now show that only HCMV virions possessing the gH/gL/UL128-131 complex are capable of activating integrin/Src/paxillin-signaling in monocytes. In fibroblasts, this signaling is reversed, such that virus lacking the gH/gL/UL128-131 complex is the only virus able to induce the paxillin activation cascade. The presence of the gH/gL/UL128-131 complex also may have an inhibitory effect on integrin-mediated signaling pathway in fibroblasts. Furthermore, we demonstrate that the presence of the gH/gL/UL128-131 complex on the viral envelope, through its activation of the integrin/Src/paxillin pathway, is necessary for efficient HCMV internalization into monocytes and that appropriate actin and dynamin regulation is critical for this entry process. Importantly, productive infection in monocyte-derived macrophages was seen only in cells exposed to HCMV expressing the gH/gL/UL128-131 complex. From our data, the HCMV gH/gL/U128-131 complex emerges as the specific ligand driving the activation of the receptor-mediated signaling required for the regulation of the actin cytoskeleton and, consequently, for efficient and productive internalization of HCMV into monocytes. To our knowledge, our studies demonstrate a possible molecular mechanism for why the gH/gL/UL128-131 complex dictates HCMV tropism and why the complex is lost as clinical isolates are passaged in the laboratory.

Review on the viral entry process of HCMV and the potential role of receptor-ligand interactions in modulating monocyte biology. HCMV pathogenesis is a direct consequence of the hematogenous dissemination of the virus to multiple host organ sites. The presence of infected monocytes in the peripheral blood and organs of individuals exhibiting primary HCMV infection have long suggested that these blood sentinels are responsible for mediating viral spread. Despite monocytes being “at the right place at the right time”, their short lifespan and the lack of productive viral infection in these cells complicate this scenario of a monocyte-driven approach to viral dissemination by HCMV. However, our laboratory has provided evidence that HCMV infection is able to induce a highly controlled polarization of monocytes toward a unique and long-lived proinflammatory macrophage, which we have demonstrated to be permissive for viral replication. These observations suggest that HCMV has evolved as a distinct mechanism to induce select proinflammatory characteristics that provide infected monocytes with the necessary tools to mediate viral spread following a primary infection. In the absence of viral gene products during the early stages of infection, the process by which HCMV “tunes” the inflammatory response in infected monocytes to promote viral spread and subsequently, viral persistence remains unclear. In this current review, we focus on the viral entry process of HCMV and the potential role of receptor-ligand interactions in modulating monocyte biology. Specifically, we examine the signaling pathways initiated by the distinct combination of cellular receptors simultaneously engaged and activated by HCMV during viral entry and how the acquisition of this distinct signalsome results in a nontraditional activation of monocytes leading to the induction of the unique, functional attributes observed in monocytes following HCMV infection.

Monocytes IMPORTANCEHematogenous dissemination of HCMV via infected monocytes is a crucial component of the viral survival strategy and is required for the establishment of persistent infection and for viral spread to additional hosts. Our system of infected primary human blood monocytes provides us with an opportunity to answer specific questions about viral spread and persistence in in vivo-relevant myeloid cells that cannot be addressed with the more traditionally used replication-permissive cells. Our goal in examining the mechanisms whereby HCMV reprograms infected monocytes to promote viral dissemination is to uncover new targets for therapeutic intervention that would disrupt key viral survival and persistence strategies. Because of this important role in maintaining survival of HCMV-infected monocytes, our new data on the role of Bcl-2 regulation during viral infection represents a promising molecular target for mitigating viral spread and persistence. H uman cytomegalovirus (HCMV) is a ubiquitous host-restricted betaherpesvirus that infects 60 to 90% of the population and persists for the lifetime of the infected individual (1). HCMV infection results in a wide range of pathogenic outcomes dependent upon the age and immune status of the host (2). Infection of immunocompetent hosts is usually asymptomatic or only mildly symptomatic (3, 4); however, it can cause infectious mononucleosis, is a risk factor for the development of cardiovascular disease (5-9), and has been linked to certain types of cancers in otherwise healthy individuals (10-15). In contrast, HCMV infection leads to significant morbidity and mortality in the immunocompromised. HCMV is an important opportunistic pathogen in AIDS patients (16)(17)(18)(19)(20), is a leading infectious cause of complications in transplant recipients (21-28), and causes severe neurological disease in congenitally infected neonates (29)(30)(31)(32)(33)(34).HCMV pathogenesis and disease result from viral spread to multiple organ sites following primary HCMV infection, a process which appears to be a critical step in the viral persistence strategy, as it allows for the establishment of lifelong persistence within the host, as well as for viral shedding and spread to additional hosts (1,35,36). Monocytes are the primary blood-borne targets for HCMV infection and are thought to be centrally involved in the hematogenous dissemination of the virus to target organ systems (37-41). We propose that HCMV reprograms the biology of infected monocytes, creating the ideal cell type to serve as "Trojan horses" to carry HCMV to target host organ sites and then to