Human Cytomegalovirus Clinical Strain-Specific microRNA miR-UL148D Targets the Human Chemokine RANTES during Infection

The human cytomegalovirus (HCMV) clinical strain Toledo and the attenuated strain AD169 exhibit a striking difference in pathogenic potential and cell tropism. The virulent Toledo genome contains a 15-kb segment, which is present in all virulent strains but is absent from the AD169 genome. The pathogenic differences between the 2 strains are thought to be associated with this additional genome segment. Cytokines induced during viral infection play major roles in the regulation of the cellular interactions involving cells of the immune and inflammatory systems and consequently determine the pathogenic outcome of infection. The chemokine RANTES (Regulated on activation, normal T-cell expressed and secreted) attracts immune cells during inflammation and the immune response, indicating a role for RANTES in viral pathogenesis. Here, we show that RANTES was downregulated in human foreskin fibroblast (HFF) cells at a later stage after infection with the Toledo strain but not after infection with the AD169 strain. miR-UL148D, the only miRNA predicted from the UL/b'' sequences of the Toledo genome, targeted the 3′-untranslated region of RANTES and induced degradation of RANTES mRNA during infection. While wild-type Toledo inhibited expression of RANTES in HFF cells, Toledo mutant virus in which miR-UL148D is specifically abrogated did not repress RANTES expression. Furthermore, miR-UL148D-mediated downregulation of RANTES was inhibited by treatment with a miR-UL148D-specific inhibitor designed to bind to the miR-UL148D sequence via an antisense mechanism, supporting the potential value of antisense agents as therapeutic tools directed against HCMV. Our findings identify a viral microRNA as a novel negative regulator of the chemokine RANTES and provide clues for understanding the pathogenesis of the clinical strains of HCMV.     

Human cytomegalovirus inhibits differentiation of monocytes into dendritic cells with the consequence of depressed immunological functions

Human cytomegalovirus (HCMV) infections in immunocompromised patients are associated with impaired immunological functions. Blood monocytes, which can differentiate into dendritic cells upon cytokine stimulation, play a central role in adequate immune reactivity and are believed to carry latent HCMV. We demonstrate here that HCMV infection of monocytes results in a block in the cytokine-induced differentiation of monocytes into functionally active CD1a-positive dendritic cells, which exhibited severely depressed immunological functions in vitro. The HCMV-infected cells exhibited a significantly reduced ability to endocytose fluorescein isothiocyanate-labeled dextran particles as well as a more than 90% reduced ability to migrate in response to the chemoattractant factors RANTES, MIP-1alpha, and MIP-3beta. Interestingly, HCMV-infected cells expressed high levels of the costimulatory molecule CD86, in contrast to the low levels of expression that was observed on uninfected monocytes and uninfected immature dendritic cells. Furthermore, HCMV-infected CD1a-negative cells were unable to induce a T-cell response. Thus, these observations suggest that HCMV infection of monocytes in vitro blocks cytokine-induced dendritic cell differentiation, and since dendritic cells play a central role in initiating immune responses, these findings suggest a powerful tactic to avoid immune recognition and to blunt the immune response at early phases of infection.     

Human cytomegalovirus subverts the functions of monocytes, impairing chemokine-mediated migration and leukocyte recruitment

Despite their role in innate and adaptive immunity, during human cytomegalovirus (HCMV) infection, monocytes are considered to be an important target of infection, a site of latency, and vehicles for virus dissemination. Since chemokine receptors play crucial roles in monocyte activation and trafficking, we investigated the effects of HCMV on their expression and function. By using endotheliotropic strains of HCMV, we obtained high rates (roughly 50%) of in vitro-infected monocytes but only restricted viral gene expression. At 24 h after infection, while the chemokine receptors CX3CR and CCR7 were unaffected, CCR1, CCR2, CCR5, and CXCR4 were downmodulated on the cell surface and retained intracellularly. Structural components of the viral particles, but not viral gene expression or soluble factors released from infected cells, accounted for the changed localization of the receptor molecules and for the block of chemokine-driven migration. HCMV-infected monocytes indeed became unresponsive to inflammatory and homeostatic chemokines, although the basal cell motility and responsiveness to N-formyl-Met-Leu-Phe were unaffected or slightly increased. The production of inflammatory mediators responsible for the recruitment of other immune cells was also hampered by HCMV. Whereas endothelial and fibroblast cells infected by HCMV efficiently recruited leukocytes, infected monocytes were unable to recruit lymphocytes, monocytes, and neutrophils. Our data further highlight the complex level of interference exerted by HCMV on the host immune system.     

Coxiella burnetii stimulates production of RANTES and MCP-1 by mononuclear cells: modulation by adhesion to endothelial cells and its implication in Q fever

Q fever is an infectious disease caused by Coxiella burnetii, which may become chronic when cytokine network and cell-mediated immune responses are altered. Chemokines, such as Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES, CCL5) and Monocyte Chemoattractant Protein-1 (MCP-1, CCL2), are specialized in the trafficing of peripheral blood mononuclear cells (PBMC), and are associated with T cell polarization that is essential for intracellular survival of C. burnetii. The present study investigated whether or not the infection status (no infection and acute or chronic infection with C. burnetii) of donors, affected the production of the two chemokines by PBMC with or without stimulation with virulent and avirulent C. burnetii. Our findings indicate that in vitro exposure to virulent or avirulent C. burnetii stimulated the production of RANTES and MCP-1 in PBMC obtained from healthy adults. The co-cultivation of endothelial cells and human PBMC resulted in an increased production of MCP-1 and the up-regulation of RANTES, which were contact-dependent. Unstimulated PBMC from patients with acute or chronic Q fever overproduced MCP-1. Interestingly, the addition of C. burnetii resulted in an increased production of RANTES and MCP-1 by PBMC obtained from patients with chronic Q fever, and the co-cultivation of PBMC with endothelial cells amplified increased production of chemokines. Circulating levels of RANTES and MCP-1 were also increased in chronic Q fever. We suggest that the overproduction of RANTES and MCP-1 secondary to the contact of PBMC with endothelium may perpetuate exaggerated inflammatory responses leading to inappropriate PBMC trafficking and to the pathogenesis of Q fever.     

Cytomegalovirus (CMV)

Human cytomegalovirus (HCMV) is a ubiquitous beta human herpesvirus type 5. Compared to other human herpesviruses, HCMV is the largest, with a genome of approximately 235 kb containing approximately 250 ORFs with the potential to encode proteins. Usually, HCMV asymptomatically infects the host during childhood, and establishes life-long latency. The infection is life-threatening for infants and immunocompromised individuals, because of direct cytopathicity by viral replication, causing systemic organ injuries. Intrauterine infection occasionally causes microcephaly, sensorineural hearing loss and mental retardation. HCMV genome contains a number of accessory genes. Most of them are engaged in immune evasion or inhibition of cell death, possibly, resulting in a symbiosis between virus and host. CD34-positive myeloid progenitor cells are considered as a site of latency. However, the molecular mechanisms by which HCMV establishes and maintains latency and reactivates remain poorly understood. Recently in Japan, the decline of maternal HCMV seropositivity may increase the risk of intrauterine infection. It needs to immediately establish the protection against transplacental HCMV infection, such as a new type of neutralizing antibody or vaccine, which effectively interferes viral entry specific to endothelial and epithelial cells. Furthermore, HCMV infection might be considered as the most important factor for driving immune senescence in the elderly.     

Human cytomegalovirus (HCMV) infection of endothelial cells promotes naive monocyte extravasation and transfer of productive virus to enhance hematogenous dissemination of HCMV

Human cytomegalovirus (HCMV) pathogenesis is dependent on the hematogenous spread of the virus to host tissue. While data suggest that infected monocytes are required for viral dissemination from the blood to the host organs, infected endothelial cells are also thought to contribute to this key step in viral pathogenesis. We show here that HCMV infection of endothelial cells increased the recruitment and transendothelial migration of monocytes. Infection of endothelial cells promoted the increased surface expression of cell adhesion molecules (intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and platelet endothelial cell adhesion molecule 1), which were necessary for the recruitment of na?ve monocytes to the apical surface of the endothelium and for the migration of these monocytes through the endothelial cell layer. As a mechanism to account for the increased monocyte migration, we showed that HCMV infection of endothelial cells increased the permeability of the endothelium. The cellular changes contributing to the increased permeability and increased na?ve monocyte transendothelial migration include the disruption of actin stress fiber formation and the decreased expression of lateral junction proteins (occludin and vascular endothelial cadherin). Finally, we showed that the migrating monocytes were productively infected with the virus, documenting that the virus was transferred to the migrating monocyte during passage through the lateral junctions. Together, our results provide evidence for an active role of the infected endothelium in HCMV dissemination and pathogenesis.     

MicroRNA-217 Promotes Angiogenesis of Human Cytomegalovirus-Infected Endothelial Cells through Downregulation of SIRT1 and FOXO3A

Human cytomegalovirus(HCMV) infection has been shown to contribute to vascular disease through the induction of angiogenesis. However, the role of microRNA in angiogenesis induced by HCMV infection remains unclear. The present study was thus designed to explore the potential effect of miR-1217 on angiogenesis and to disclose the underlying mechanism in endothelial cells. We found that HCMV infection of endothelial cells(ECs) enhanced expression of miR-217 and reduced SIRT1 and FOXO3A protein level in 24 hours post infection(hpi). Transfection of miR-217 inhibitor not only depressed cellular migration and tube formation induced by HCMV infection, but also enhanced SIRT1 and FOXO3A protein expression. Additionally, luciferase assay confirmed that miR-217 directly targeted FOXO3A mRNA 3`UTR. Furthermore, pretreatment with resveratrol depressed motility and tube formation of HCMV-infected ECs, which could be reversed by SIRT1 siRNA. Similarly, delivery of FOXO3A overexpression lentivirus suppressed proliferative rate, migration and tube formation of HCMV-infected ECs, which reversed by transfection of FOXO3A siRNA. In summary, HCMV infection of endothelial cells induces angiogenesis by both of miR-217/SIRT1 and miR-217/FOXO3A axis.     

MicroRNA Editing Facilitates Immune Elimination of HCMV Infected Cells

The human cytomegalovirus (HCMV) is extremely prevalent in the human population. Infection by HCMV is life threatening in immune compromised individuals and in immune competent individuals it can cause severe birth defects, developmental retardation and is even associated with tumor development. While numerous mechanisms were developed by HCMV to interfere with immune cell activity, much less is known about cellular mechanisms that operate in response to HCMV infection. Here we demonstrate that in response to HCMV infection, the expression of the short form of the RNA editing enzyme ADAR1 (ADAR1-p110) is induced. We identified the specific promoter region responsible for this induction and we show that ADAR1-p110 can edit miR-376a. Accordingly, we demonstrate that the levels of the edited-miR-376a (miR-376a(e)) increase during HCMV infection. Importantly, we show that miR-376a(e) downregulates the immune modulating molecule HLA-E and that this consequently renders HCMV infected cells susceptible to elimination by NK cells.     

Intracellular Signaling by the Chemokine Receptor US28 during Human Cytomegalovirus Infection

In patients with impaired cell-mediated immune responses (e.g., lung transplant recipients and AIDS patients), cytomegalovirus (CMV) infection causes severe disease such as pneumonitis. However, although immunocompetency in the host can protect from CMV disease, the virus persists by evading the host immune defenses. A model of CMV infection of the endothelium has been developed in which inflammatory stimuli, such as the CC chemokine RANTES, bind to the endothelial cell surface, stimulating calcium flux during late times of CMV infection. At 96 h postinfection, CMV-infected cells express mRNA of the CMV-encoded CC chemokine receptor US28 but do not express mRNA of other CC chemokine receptors that bind RANTES (CCR1, CCR4, CCR5). Cloning and stable expression of the receptor CMV US28 in human kidney epithelial cells (293 cells) with and without the heterotrimeric G protein α₁₆ indicated that CMV US28 couples to both Gα_i and Gα₁₆ proteins to activate calcium flux in response to the chemokines RANTES and MCP-3. Furthermore, cells that coexpress US28 and Gα₁₆ responded to RANTES stimulation with activation of extracellular signal-regulated kinase, which could be attributed, in part, to specific Gα₁₆ coupling. Thus, through expression of the CC chemokine receptor US28, CMV may utilize resident G proteins of the infected cell to manipulate cellular responses stimulated by chemokines.     

cGAS Senses Human Cytomegalovirus and Induces Type I Interferon Responses in Human Monocyte-Derived Cells

Human cytomegalovirus (HCMV) infections of healthy individuals are mostly unnoticed and result in viral latency. However, HCMV can also cause devastating disease, e.g., upon reactivation in immunocompromised patients. Yet, little is known about human immune cell sensing of DNA-encoded HCMV. Recent studies indicated that during viral infection the cyclic GMP/AMP synthase (cGAS) senses cytosolic DNA and catalyzes formation of the cyclic di-nucleotide cGAMP, which triggers stimulator of interferon genes (STING) and thus induces antiviral type I interferon (IFN-I) responses. We found that plasmacytoid dendritic cells (pDC) as well as monocyte-derived DC and macrophages constitutively expressed cGAS and STING. HCMV infection further induced cGAS, whereas STING expression was only moderately affected. Although pDC expressed particularly high levels of cGAS, and the cGAS/STING axis was functional down-stream of STING, as indicated by IFN-I induction upon synthetic cGAMP treatment, pDC were not susceptible to HCMV infection and mounted IFN-I responses in a TLR9-dependent manner. Conversely, HCMV infected monocyte-derived cells synthesized abundant cGAMP levels that preceded IFN-I production and that correlated with the extent of infection. CRISPR/Cas9- or siRNA-mediated cGAS ablation in monocytic THP-1 cells and primary monocyte-derived cells, respectively, impeded induction of IFN-I responses following HCMV infection. Thus, cGAS is a key sensor of HCMV for IFN-I induction in primary human monocyte-derived DC and macrophages.     

HCMV infection attenuates hydrogen peroxide induced endothelial apoptosis-- involvement of ERK pathway

Human cytomegalovirus (HCMV) exerts anti-apoptotic effect during early stage of infection, which provides HCMV time for propagation. We investigated pathways mediating the resistance to H(2)O(2)-induced cell death - a self-defense mechanism to remove infected cells. We found that human aortic endothelial cells (HAECs) infected with VHL/E strain of HCMV during first 3 days were resistant to H(2)O(2) (0-2 mM) induced apoptosis. This anti-apoptotic effect may be mediated by the upregulation of Bcl-2, an anti-apoptotic protein through the activation pro-survival pathway extracellular signal regulated kinase (ERK). Through this mechanism, HCMV is able to propagate and causes endothelial dysfunction, hence vascular disease.     

Angiotensin II-induced mononuclear leukocyte interactions with arteriolar and venular endothelium are mediated by the release of different CC chemokines

Angiotensin II (Ang-II) is associated with atherogenesis and arterial subendothelial mononuclear leukocyte infiltration. We have demonstrated that Ang-II causes the initial attachment of mononuclear cells to the arteriolar endothelium. We now report on the contribution of CC chemokines to this response. Intraperitoneal administration of 1 nM Ang-II induced MCP-1, RANTES, and MIP-1alpha generation, maximal at 4 h, followed by mononuclear leukocyte recruitment at 8 and 24 h. Using intravital microscopy within the rat mesenteric microcirculation 4 h after exposure to 1 nM Ang-II, arteriolar mononuclear cell adhesion was 80-90% inhibited by pretreatment with Met-RANTES, a CCR1 and CCR5 antagonist, or an anti-MCP-1 antiserum, without affecting the increased endothelial expression of P-selectin and VCAM-1. Conversely, leukocyte interactions with the venular endothelium, although inhibited by Met-RANTES, were little affected by the anti-MCP-1. Using rat whole blood in vitro, Ang-II (100 nM) induced the expression of monocyte CD11b that was inhibited by Met-RANTES but not by anti-MCP-1. Stimulation of human endothelial cells (human umbilical arterial endothelial cells and HUVECs) with 1-1000 nM Ang-II, predominantly acting at its AT(1) receptor, induced the release of MCP-1 within 1 h, RANTES within 4 h, and MCP-3 within 24 h. Eotaxin-3, a natural CCR2 antagonist, was released within 1 h and may delay mononuclear cell responses to MCP-1. Therefore, Ang-II-induced mononuclear leukocyte recruitment at arterioles and venules is mediated by the production of different CC chemokines. Thus, Ang-II may be a key molecule in the initial attachment of mononuclear cells to the arterial endothelium in cardiovascular disease states where this event is a characteristic feature.     

Mu-opioid induction of monocyte chemoattractant protein-1, RANTES, and IFN-gamma-inducible protein-10 expression in human peripheral blood mononuclear cells

Strong evidence for the direct modulation of the immune system by opioids is well documented. Mu-opioids have been shown to alter the release of cytokines important for both host defense and the inflammatory response. Proinflammatory chemokines monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-gamma-inducible protein-10 (IP-10) play crucial roles in cell-mediated immune responses, proinflammatory reactions, and viral infections. In this report, we show that [D-Ala(2),N:-Me-Phe(4),Gly-ol(5)]enkephalin (DAMGO), a mu-opioid-selective agonist, augments the expression in human PBMCs of MCP-1, RANTES, and IP-10 at both the mRNA and protein levels. Because of the proposed relationship between opioid abuse and HIV-1 infection, we also examined the impact of DAMGO on chemokine expression in HIV-infected cells. Our results show that DAMGO administration induces a significant increase in RANTES and IP-10 expression, while MCP-1 protein levels remain unaffected in PBMCs infected with the HIV-1 strain. In contrast, we show a dichotomous effect of DAMGO treatment on IP-10 protein levels expressed by T- and M-tropic HIV-infected PBMCs. The differential modulation of chemokine expression in T- and M-tropic HIV-1-infected PBMCs by opioids supports a detrimental role for opioids during HIV-1 infection. Modulation of chemokine expression may enhance trafficking of potential noninfected target cells to the site of active infection, thus directly contributing to HIV-1 replication and disease progression to AIDS.     

A cyclooxygenase-2 homologue encoded by rhesus cytomegalovirus is a determinant for endothelial cell tropism

Cyclooxygenase-2 (COX-2) is a cellular enzyme in the eicosanoid synthetic pathway that mediates the synthesis of prostaglandins from arachidonic acid. The eicosanoids function as critical regulators of a number of cellular processes, including the acute and chronic inflammatory response, hemostasis, and the innate immune response. Human cytomegalovirus (HCMV), which does not encode a viral COX-2 isoform, has been shown to induce cellular COX-2 expression. Importantly, although the precise role of COX-2 in CMV replication is unknown, COX-2 induction was shown to be critical for normal HCMV replication. In an earlier study, we identified an open reading frame (Rh10) within the rhesus cytomegalovirus (RhCMV) genome that encoded a putative protein (designated vCOX-2) with high homology to cellular COX-2. In the current study, we show that vCOX-2 is expressed with early-gene kinetics during RhCMV infection, resulting in production of a 70-kDa protein. Consistent with the expression of a viral COX-2 isoform, cellular COX-2 expression was not induced during RhCMV infection. Finally, analysis of growth of recombinant RhCMV with vCOX-2 deleted identified vCOX-2 as a critical determinant for replication in endothelial cells.     

Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification

Platelets, in addition to exerting hemostatic activity, contribute to immunity and inflammation. The recent report that platelets express CD40 led us to hypothesize that CD40 ligand (CD40L)-positive T cells could bind to platelets, cause their activation, and trigger granular RANTES release, creating a T cell recruitment feedback loop. Platelets were cocultured with resting or activated autologous T cells and their activation was assessed by P-selectin expression. RANTES binding to endothelial cells was assessed by confocal microscopy, and its biological activity was demonstrated by a T cell adhesion assay. CD40L-positive T cells induced platelet activation through a contact-mediated, CD40-dependent pathway resulting in RANTES release, which bound to endothelial cells and mediated T cell recruitment. Soluble CD40L induced the same events via p38, but not extracellular signal-regulated kinase, phosphorylation. These results show the existence of a novel platelet-dependent pathway of immune response amplification which brings these nonimmune cells close to the level of pathogenic relevance traditionally attributed to classical immune cells.     

Hantavirus infection induces the expression of RANTES and IP-10 without causing increased permeability in human lung microvascular endothelial cells

Sin Nombre virus (SNV) and Hantaan virus (HTN) infect endothelial cells and are associated with different patterns of increased vascular permeability during human disease. It is thought that such patterns of increased vascular permeability are a consequence of endothelial activation and subsequent dysfunction mediated by differential immune responses to hantavirus infection. In this study, the ability of hantavirus to directly induce activation of human lung microvascular endothelial cells (HMVEC-Ls) was examined. No virus-specific modulation in the constitutive or cytokine-induced expression of cellular adhesion molecules (CD40, CD54, CD61, CD62E, CD62P, CD106, and major histocompatibility complex classes I and II) or in cytokines and chemokines (eotaxin, tumor necrosis factor alpha, interleukin 1beta [IL-1beta], IL-6, IL-8, MCP-1, MIP-1alpha, and MIP-1beta) was detected at either the protein or message level in hantavirus-infected HMVEC-Ls. Furthermore, no virus-specific enhancement of paracellular or transcellular permeability or changes in the organization and distribution of endothelial intercellular junctional proteins was observed. However, infection with either HTN or SNV resulted in detectable levels of the chemokines RANTES and IP-10 (the 10-kDa interferon-inducible protein) in HMVEC-Ls within 72 h and was associated with nuclear translocation of interferon regulatory factor 3 (IRF-3) and IRF-7. Gamma interferon (IFN-gamma)-induced expression of RANTES and IP-10 could also be detected in uninfected HMVEC-Ls and was associated with nuclear translocation of IRF-1 and IRF-3. Treatment of hantavirus-infected HMVEC-Ls with IFN-gamma for 24 h resulted in a synergistic enhancement in the expression of both RANTES and IP-10 and was associated with nuclear translocation of IRF-1, IRF-3, IRF-7, and NF-kappaB p65. These results reveal a possible mechanism by which hantavirus infection and a TH1 immune response can cooperate to synergistically enhance chemokine expression by HMVEC-Ls and trigger immune-mediated increases in vascular permeability.     

Factors affecting human cytomegalovirus gene expression in human monocyte cell lines

To understand the mechanisms for establishing and reactivating monocytes and macrophages from latency by human cytomegalovirus (HCMV), human monocyte cell lines were infected and HCMV gene expression was investigated. Indirect immunofluorescence assay (IFA) with monoclonal antibody to HCMV major immediate early (MIE) IE1 or IE2 proteins revealed that HCMV MIE genes were expressed at low levels in relatively more differentiated THP-1 cells with TPA treatment after virus infection (posttreatment). Less differentiated cells such as U937 or HL60 did not support MIE gene expression even after TPA treatment. If THP-1 cells were pretreated before virus infection with TPA and became differentiated at the time of HCMV infection, MIE gene expression increased by 5-6 fold. Therefore, the relative degree of monocyte cell differentiation appears to be an important factor for regulating HCMV gene expression. Further IFA studies using monoclonal antibodies specific for IE1 or IE2 proteins indicate that the sequence and general pattern of IE1 and IE2 gene expression in THP-1 cells treated with TPA were similar to those in permissive human fibroblast cells with some delay in time. Formation of the replication compartment detected with monoclonal antibody to HCMV polymerase accessory protein UL44 in THP-1 cells suggests a fully productive replication process of HCMV in these cells. Monocytes are known to be induced to differentiate by hydrocortisone (HC), tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. HC, which is known to stimulate HCMV replication in permissive human fibroblast (HF) cells, enhanced HCMV gene expression by 2-3 fold in TPA-pre or posttreated THP-1 cells, but TNF-alpha or IFN-gamma had little effect. Nitric oxide (NO) is released by immune cells in the defense against foreign stimuli and was shown to inhibit HCMV gene expression in HF cells. Increasing NO by nitroprusside significantly reduced HCMV gene expression in THP-1 cells. Therefore, it appears that the expression of HCMV immediate early genes in THP-1 cells treated with TPA closely resembles those in permissive HF cells.     

Upregulation of CD40 expression on endothelial cells infected with human cytomegalovirus

CD40 has been identified as an important molecule for a number of processes, such as immune responses, inflammation, and the activation of endothelia. We investigated CD40 in endothelial cells (EC) following infection with an endotheliotropic strain of human cytomegalovirus (HCMV). Between 8 and 72 h postinfection, we observed a significant increase in CD40 levels on the surface of infected EC, as measured by fluorescence-activated cell sorting analysis. As a consequence of CD40 upregulation, increased levels of E-selectin were found on infected EC after stimulation with CD154-expressing T cells. Enhanced expression of CD40 was specific for EC, since infection of fibroblasts did not result in the upregulation of CD40. The addition of neutralizing antibodies as well as UV inactivation of virus completely prevented the upregulation of CD40 on EC. Also, laboratory-adapted HCMV strain AD169 was not able to induce CD40 on EC. De novo protein synthesis was necessary for the increased surface expression. At early times (4 to 24 h) postinfection, this change was not accompanied by increased levels of CD40 protein or mRNA. At late times (48 to 96 h) postinfection, increased amounts of CD40 protein and mRNA were detected. Immunohistochemical analysis of infected tissues demonstrated elevated levels of CD40 on HCMV-infected EC in vivo. Thus, infection of EC by HCMV may result in the activation of endothelia and in the augmentation of inflammatory processes.