Cecilia Söderberg-Nauclér

Reactivation of Latent Human Cytomegalovirus in CD14+ Monocytes Is Differentiation Dependent

ABSTRACT We have previously demonstrated reactivation of latent human cytomegalovirus (HCMV) in myeloid lineage cells obtained from healthy donors. Virus was obtained from allogenically stimulated monocyte-derived macrophages (Allo-MDM), but not from macrophages differentiated by mitogenic stimulation (ConA-MDM). In the present study, the cellular and cytokine components essential for HCMV replication and reactivation were examined in Allo-MDM. The importance of both CD4+ and CD8+ T cells in the generation of HCMV-permissive Allo-MDM was demonstrated by negative selection or blocking experiments using antibodies directed against both HLA class I and HLA class II molecules. Interestingly, contact of monocytes with CD4 or CD8 T cells was not essential for reactivation of HCMV, since virus was observed in macrophages derived from CD14+monocytes stimulated by supernatants produced by allogeneic stimulation of peripheral blood mononuclear cells. Examination of the cytokines produced in Allo-MDM and ConA-MDM cultures indicated a significant difference in the kinetics of production and quantity of these factors. Further examination of the cytokines essential for the generation of HCMV-permissive Allo-MDM identified gamma interferon (IFN-γ) but not interleukin-1 or -2, tumor necrosis factor alpha, or granulocyte-macrophage colony-stimulating factor as critical components in the generation of these macrophages. In addition, although IFN-γ was crucial for reactivation of latent HCMV, addition of IFN-γ to unstimulated macrophage cultures was insufficient to reactivate virus. Thus, this study characterizes two distinct monocyte-derived cell types which can be distinguished by their ability to reactivate and support HCMV replication and identifies the critical importance of IFN-γ in the reactivation of HCMV.

Effects of Human Cytomegalovirus Infection on Ligands for the Activating NKG2D Receptor of NK Cells: Up-Regulation of UL16-Binding Protein (ULBP)1 and ULBP2 Is Counteracted by the Viral UL16 Protein

Human CMV (HCMV) interferes with NK cell functions at various levels. The HCMV glycoprotein UL16 binds some of the ligands recognized by the NK-activating receptor NKG2D, namely UL16-binding proteins (ULBP) 1 and 2 and MHC class I-related chain B, possibly representing another mechanism of viral immune escape. This study addressed the expression and function of these proteins in infected cells. HCMV induced the expression of all three ULBPs, which were predominantly localized in the endoplasmic reticulum of infected fibroblasts together with UL16. However, while at a lower viral dose ULBP1 and 2 surface expression was completely inhibited compared to ULBP3, at a higher viral dose cell surface expression of ULBP1 and ULBP2 was delayed. The induction of ULBPs correlated with an increased dependency on NKG2D for recognition; however, the overall NK sensitivity did not change (suggesting that additional viral mechanisms interfere with NKG2D-independent pathways for recognition). Infection with a UL16 deletion mutant virus resulted in a different pattern compared to the wild type: all three ULBP molecules were induced with similar kinetics at the cell surface, accompanied by a pronounced, entirely NKG2D-dependent increase in NK sensitivity. Together our findings show that upon infection with HCMV, the host cell responds by expression of ULBPs and increased susceptibility to the NKG2D-mediated component of NK cell recognition, but UL16 limits these effects by interfering with the surface expression of ULBP1 and ULBP2.

HCMV-Encoded Chemokine Receptor US28 Mediates Proliferative Signaling Through the IL-6–STAT3 Axis

A viral G protein–coupled receptor may initiate a positive feedback loop to promote tumor proliferation and vascularization. A Viral Pathway to Tumor Development Human cytomegalovirus (HCMV), a widespread human herpesvirus that persists in a latent form, is associated with pathological processes in immunocompromised hosts and has been implicated in the development of several forms of cancer, including glioblastoma. HCMV encodes a G protein–coupled receptor, US28, that resembles a chemokine receptor and constitutively activates signaling pathways associated with cell proliferation. Slinger et al. expressed US28 in cultured cells to explore the mechanisms through which it could promote tumor development. They found that US28 stimulated the production and secretion of both vascular endothelial growth factor (VEGF) and the cytokine interleukin-6 (IL-6) and defined a signaling pathway whereby US28 increased cell proliferation through IL-6–dependent activation of the JAK1-STAT3 axis. IL-6 is itself a target of STAT3, leading the authors to propose that US28-dependent production and secretion of IL-6 and consequent autocrine and paracrine STAT3 activation lead to establishment of a positive feedback loop that promotes proliferation of both infected and neighboring cells. Analyses of human glioblastoma tissue revealed US28 and activated STAT3 in cells lining blood vessels, suggesting that US28 may play a role in tumor vascularization. US28 is a viral G protein (heterotrimeric guanosine triphosphate–binding protein)–coupled receptor encoded by the human cytomegalovirus (HCMV). In addition to binding and internalizing chemokines, US28 constitutively activates signaling pathways linked to cell proliferation. Here, we show increased concentrations of vascular endothelial growth factor and interleukin-6 (IL-6) in supernatants of US28-expressing NIH 3T3 cells. Increased IL-6 was associated with increased activation of the signal transducer and activator of transcription 3 (STAT3) through upstream activation of the Janus-activated kinase JAK1. We used conditioned growth medium, IL-6–neutralizing antibodies, an inhibitor of the IL-6 receptor, and short hairpin RNA targeting IL-6 to show that US28 activates the IL-6–JAK1–STAT3 signaling axis through activation of the transcription factor nuclear factor κB and the consequent production of IL-6. Treatment of cells with a specific inhibitor of STAT3 inhibited US28-dependent [3H]thymidine incorporation and foci formation, suggesting a key role for STAT3 in the US28-mediated proliferative phenotype. US28 also elicited STAT3 activation and IL-6 secretion in HCMV-infected cells. Analyses of tumor specimens from glioblastoma patients demonstrated colocalization of US28 and phosphorylated STAT3 in the vascular niche of these tumors. Moreover, increased phospho-STAT3 abundance correlated with poor patient outcome. We propose that US28 induces proliferation in HCMV-infected tumors by establishing a positive feedback loop through activation of the IL-6–STAT3 signaling axis.

HCMV microinfections in inflammatory diseases and cancer

Human cytomegalovirus (HCMV) is a wide-spread human virus that was mainly known to cause disease in immunocompromised patients. A new entity of infection can be diagnosed with high sensitive techniques; HCMV microinfections that often exhibit an altered pattern of IE protein expression. We have recently discovered that HCMV microinfections are very common in patients with inflammatory diseases and certain cancers. The discovery of active HCMV infections in tissue specimens from patients with inflammatory diseases raises the question of whether the infection is an epiphenomenon or whether the virus plays a causative role in disease development. After a primary infection, which is generally asymptomatic in immunocompetent individuals, HCMV establishes latency and persists in its host. In infected cells, the virus can produce over 250 proteins, but only about 50-60 are believed to be essential for viral replication. Thus, the vast majority of these viral proteins enable the virus to co-exist with its host. Such proteins act through highly sophisticated mechanisms to control different cellular and immunological functions in order to facilitate viral production and to avoid detection and elimination of the virus by the immune system. These proteins may also contribute to the development of common inflammation-related diseases.

Survival in Patients with Glioblastoma Receiving Valganciclovir

A retrospective analysis of selected patients with glioblastoma who received treatment for cytomegalovirus with their anticancer treatment showed surprisingly good 2-year survival. A randomized trial is needed.

Envelopment of Human Cytomegalovirus Occurs by Budding into Golgi-Derived Vacuole Compartments Positive for gB, Rab 3, Trans-Golgi Network 46, and Mannosidase II

ABSTRACT Although considerable progress has been made towards characterizing virus assembly processes, assignment of the site of tegumentation and envelopment for human cytomegalovirus (HCMV) is still not clear. In this study, we examined the envelopment of HCMV particles in human lung fibroblasts (HF) HL 411 and HL 19, human umbilical vein endothelial cells, human pulmonary arterial endothelial cells, and arterial smooth muscle cells at different time points after infection by electron microscopy (EM), immunohistochemistry, and confocal microscopy analysis. Double-immunofluorescence labeling experiments demonstrated colocalization of the HCMV glycoprotein B (gB) with the Golgi resident enzyme mannosidase II, the Golgi marker TGN (trans-Golgi network) 46, and the secretory vacuole marker Rab 3 in all cell types investigated. Final envelopment of tegumented capsids was observed at 5 days postinfection by EM, when tegumented capsids budded into subcellular compartments located in the cytoplasm, in close proximity to the Golgi apparatus. Immunogold labeling and EM analysis confirmed staining of the budding compartment with HCMV gB, Rab 3, and mannosidase II in HL 411 cells. However, the markers Rab 1, Rab 2, Rab 7, Lamp 1 (late endosomes and lysosomes), and Lamp 2 (lysosomes) neither showed specific staining of the budding compartment in the immunogold labeling experiments nor colocalized with gB in the immunofluorescent colocalization experiments in any cell type studied. Together, these results suggest that the final envelopment of HCMV particles takes place mainly into a Golgi-derived secretory vacuole destined for the plasma membrane, which may release new infectious virus particles by fusion with the plasma membrane.

Detection of human cytomegalovirus in medulloblastomas reveals a potential therapeutic target

Medulloblastomas are the most common malignant brain tumors in children. They express high levels of COX-2 and produce PGE2, which stimulates tumor cell proliferation. Human cytomegalovirus (HCMV) is prevalent in the human population and encodes proteins that provide immune evasion strategies and promote oncogenic transformation and oncomodulation. In particular, HCMV induces COX-2 expression; STAT3 phosphorylation; production of PGE2, vascular endothelial growth factor, and IL-6; and tumor formation in vivo. Here, we show that a large proportion of primary medulloblastomas and medulloblastoma cell lines are infected with HCMV and that COX-2 expression, along with PGE2 levels, in tumors is directly modulated by the virus. Our analysis indicated that both HCMV immediate-early proteins and late proteins are expressed in the majority of primary medulloblastomas. Remarkably, all of the human medulloblastoma cell lines that we analyzed contained HCMV DNA and RNA and expressed HCMV proteins at various levels in vitro. When engrafted into immunocompromised mice, human medulloblastoma cells induced expression of HCMV proteins. HCMV and COX-2 expression correlated in primary tumors, cell lines, and medulloblastoma xenografts. The antiviral drug valganciclovir and the specific COX-2 inhibitor celecoxib prevented HCMV replication in vitro and inhibited PGE2 production and reduced medulloblastoma tumor cell growth both in vitro and in vivo. Ganciclovir did not affect the growth of HCMV-negative tumor cell lines. These findings imply an important role for HCMV in medulloblastoma and suggest HCMV as a novel therapeutic target for this tumor.

Human Cytomegalovirus Inhibits Neuronal Differentiation and Induces Apoptosis in Human Neural Precursor Cells

ABSTRACT Human cytomegalovirus (HCMV) is the most common cause of congenital infections in developed countries, with an incidence varying between 0.5 and 2.2% and consequences varying from asymptomatic infection to lethal conditions for the fetus. Infants that are asymptomatic at birth may still develop neurological sequelae, such as hearing loss and mental retardation, at a later age. Infection of neural stem and precursor cells by HCMV and consequent disruption of the proliferation, differentiation, and/or migration of these cells may be the primary mechanism underlying the development of brain abnormalities. In the present investigation, we demonstrate that human neural precursor cells (NPCs) are permissive for HCMV infection, by both the laboratory strain Towne and the clinical isolate TB40, resulting in 55% and 72% inhibition of induced differentiation of human NPCs into neurons, respectively, when infection occurred at the onset of differentiation. This repression of neuronal differentiation required active viral replication and involved the expression of late HCMV gene products. This capacity of HCMV to prevent neuronal differentiation declined within 24 h after initiation of differentiation. Furthermore, the rate of cell proliferation in infected cultures was attenuated. Surprisingly, HCMV-infected cells exhibited an elevated frequency of apoptosis at 7 days following the onset of differentiation, at which time approximately 50% of the cells were apoptotic at a multiplicity of infection of 10. These findings indicate that HCMV has the capacity to reduce the ability of human NPCs to differentiate into neurons, which may offer one explanation for the abnormalities in brain development associated with congenital HCMV infection.

Activation of Telomerase by Human Cytomegalovirus

BACKGROUND The mechanism by which human cytomegalovirus (HCMV) stimulates oncogenesis is unclear. Because cellular immortalization and transformation require telomerase activation by expression of the telomerase reverse transcriptase (hTERT) gene, we examined the role of HCMV in telomerase activation. METHODS Normal human diploid fibroblasts (HDFs) and human malignant glioma (MG) cell lines were infected with HCMV or transfected with expression vectors encoding HCMV immediate early (IE) antigen 72 or 86. hTERT expression and promoter activity and telomerase activity were evaluated using reverse transcription-polymerase chain reaction, a luciferase reporter assay, and a telomeric repeat amplification protocol, respectively. hTERT promoter occupancy by the transcription factor Sp1, IE antigens, and histone deacetylases (HDACs) was assessed by chromatin immunoprecipitation. hTERT and IE protein expression in human primary glioblastoma multiforme (GBM) was determined immunohistochemically. All statistical tests were two-sided. RESULTS In telomerase and hTERT-negative HDFs, HCMV infection induced constitutive hTERT expression and telomerase activation. The hTERT promoter activity in HDFs and MG cell lines was statistically significantly enhanced by HCMV in a dose-dependent manner (mean luciferase activity [arbitrary units] in control HDFs and in HDFs infected with HCMV at multiplicities of infection [MOIs] of 0.1 = 6 and 521, respectively, difference = 515, 95% CI = 178 to 850; mean activity at MOI of 1 and 10 = 8828 and 59,923, respectively; P < .001 comparing control with HCMV-infected cells at all MOIs). Ectopic expression of HCMV IE-72 protein also stimulated hTERT promoter activity in HDFs. HCMV-mediated transactivation of the hTERT gene was dependent on the presence of Sp1-binding sites in the hTERT promoter and was accompanied by increases in Sp1 binding, acetylation of histone H3, and a reduction in HDAC binding at the core promoter. In specimens of GBM, HCMV IE and hTERT proteins were colocalized in malignant cells and their levels paralleled each other. CONCLUSIONS HCMV activates telomerase in both HDFs and malignant cells. These findings begin to reveal a novel mechanism by which HCMV infection may be linked to or modulate oncogenesis through telomerase activation.

Human Cytomegalovirus Latency and Reactivation – A Delicate Balance between the Virus and Its Host’s Immune System

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that still causes severe morbidity and mortality in immunocompromised individuals. During its evolution, the virus has developed sophisticated methods to evade immune recognition and to establish life-long persistence in its host. Today, we know that the virus establishes latency in myeloid lineage cells and that the virus is dependent on immune activation mechanisms to reactivate it from latency to produce a new viral progeny. During this process, a number of viral proteins are produced that interfere with different immune recognition pathways. The current knowledge of the delicate balance between the virus‘ continuous existence and its host’s immune system will be summarized in this chapter.