Permissive human cytomegalovirus infection of a first trimester extravillous cytotrophoblast cell line

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection in the United States and Europe. Despite the significant morbidity associated with prenatal HCMV infection, little is known about how the virus infects the fetus during pregnancy. To date, primary human cytotrophoblasts (CTBs) have been utilized to study placental HCMV infection and replication; however, the minimal mitotic potential of these cells restricts experimentation to a few days, which may be problematic for mechanistic studies of the slow-replicating virus. The aim of this study was to determine whether the human first trimester CTB cell line SGHPL-4 was permissive for HCMV infection and therefore could overcome such limitations. HCMV immediate early (IE) protein expression was detected as early as 3 hours post-infection in SGHPL-4 cells and progressively increased as a function of time. HCMV growth assays revealed the presence of infectious virus in both cell lysates and culture supernatants, indicating that viral replication and the release of progeny virus occurred. Compared to human fibroblasts, viral replication was delayed in CTBs, consistent with previous studies reporting delayed viral kinetics in HCMV-infected primary CTBs. These results indicate that SGHPL-4 cells are fully permissive for the complete HCMV replicative cycle. Our findings suggest that these cells may serve as useful tools for future mechanistic studies of HCMV pathogenesis during early pregnancy.     

Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements

Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.     

A human cytomegalovirus function inhibits replication of herpes simplex virus.

Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 micrograms/ml) for 3 or 24 h, conditions known to result in accumulation of HCMV immediate-early and early mRNA, was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5 degrees C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. However, replication of adenovirus, another DNA virus, was not restricted in these cells under the same conditions. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with [3H]thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection. In addition, superinfection with vesicular stomatitis virus ruled out a role for interferon in restriction of HSV replication in this system.     

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.     

Human Cytomegalovirus Persistently Infects Aortic Endothelial Cells

Endothelial cells (EC) have been implicated as constituting an important cell type in the pathogenesis of human cytomegalovirus (HCMV). Microvascular and macrovascular EC exhibit different biochemical and functional properties depending on the organ of origin. Phenotypic differences between microvascular and macrovascular EC may alter the ability of these cells to support HCMV replication. In this study, we compared the replication of HCMV in primary macrovascular aortic EC (AEC) with that in brain microvascular EC (BMVEC). An examination of IE72, pp65, and gB viral antigen expression in BMVEC and AEC by immunoflourescence revealed similar frequencies of infected cells. Intracellular production of virus was 3 log units greater in BMVEC than in AEC, while equal quantities of extracellular virus were produced in both cell types. HCMV infection of BMVEC resulted in rapid cellular lysis, while the virus was nonlytic and continuously released from HCMV-infected AEC for the life span of the culture. An examination of infected cells by electron microscopy revealed the formation of abundant nucleocapsids in both AEC and BMVEC. However, significant amounts of mature viral particles were only detected in the cytoplasm of BMVEC. These observations indicate that levels of HCMV replication in EC obtained from different organs are distinct and suggest that persistently infected AEC may serve as a reservoir of virus.     

The ESCRT machinery is not required for human cytomegalovirus envelopment

The human cytomegalovirus (HCMV) has been proposed to complete its final envelopment on cytoplasmic membranes prior to its release to the extracellular medium. The nature of these membranes and the mechanisms involved in virus envelopment and release are poorly understood. Here we show by immunogold-labelling and electron microscopy that CD63, a marker of multivesicular bodies (MVBs), is incorporated into the viral envelope, supporting the notion that HCMV uses endocytic membranes for its envelopment. We therefore investigated a possible role for the cellular endosomal sorting complex required for transport (ESCRT) machinery in HCMV envelopment. Depletion of tumour suppressor gene 101 and ALIX/AIP1 with small interfering RNAs (siRNAs) in HCMV-infected cells did not affect virus production. In contrast, siRNAs against the vacuolar protein sorting 4 (VPS4) proteins silenced the expression of VPS4A and VPS4B, inhibited the sorting of epidermal growth factor to lysosomes, the formation of HIV Gag-derived virus-like particles and vesicular stomatitis virus infection, but enhanced the number of HCMV viral particles produced. Treatment of infected cells with protease inhibitors also increased viral production. These studies indicate that, in contrast to some enveloped RNA viruses, HCMV does not require the cellular ESCRT machinery to complete its envelopment.     

Lysis of human cytomegalovirus infected fibroblasts by natural killer cells: demonstration of an interferon-independent component requiring expression of early viral proteins and characterization of effector cells

We investigated the susceptibility of cells infected with human cytomegalovirus (HCMV) to lysis by human natural killer (NK) cells, examining in particular its relationship to sequential viral protein expression, interferon (IFN), and the nature of the effector cells. HCMV-infected fibroblasts were lysed by peripheral blood mononuclear cells from normal seronegative individuals. The effector cells were large granular lymphocytes of Leu-7+, Leu-11+, and to a lesser extent Leu-7- phenotype. Depletion studies suggested they were the same population of NK cells that lyse uninfected fibroblasts, but a subset of NK cells that lyse K562 cells. HCMV-infected cells treated with phosphonoformate and cells infected for 16 hr that only express the nonstructural HCMV immediate early and early proteins and not the late (structural) proteins were susceptible to lysis by IFN-pretreated effector cells, whereas cells expressing immediate early antigens alone were not. This enhanced susceptibility to lysis was associated with increased effector:target binding in target cell binding assays, and was competitively inhibited by uninfected fibroblasts in cold target competition assays. It was independent of IFN release from the infected target cells or effector cells. These results suggest that the increased susceptibility to lysis by NK cells produced by a human herpes virus HCMV i) is manifest when early viral proteins are expressed, ii) is related to enhanced expression of a target structure likely to be present on uninfected fibroblasts, and iii) has a major component that is independent of IFN.     

Mitochondrial epigenetics in bone remodeling during hyperhomocysteinemia

The studies into the pathophysiology of viral miRNAs are still in infancy; the interspecies regulation at the miRNA level fuels the spark of the investigation into the repertoire of virus–host interactions. Reports pertaining to the viral miRNAs role in modulating/evading the host immune response are surging up; we initiated this in silico study to speculate the role of human cytomegalovirus (HCMV)-encoded miRNAs on human antiviral mechanisms such as apoptosis and autophagy. The results indicate that both the above mechanisms were targeted by the HCMV miRNAs, located in the unique long region of the HCMV genome. The proapoptotic genes MOAP1, PHAP, and ERN1 are identified to be the potential targets for the miR-UL70-3p and UL148D, respectively. The ERN1 gene plays a role in the initiation of Endoplasmic reticulum stress-induced apoptosis as well as autophagosome formation. This study shows that HCMV employs its miRNA repertoire for countering the cellular apoptosis and autophagy, particularly the mitochondrial-dependent intrinsic pathway of apoptosis. In addition, the homology studies reveal no HCMV miRNA bears sequence homology with human miRNAs.     

Replication of human cytomegalovirus in human peripheral blood T cells.

The replication of human cytomegalovirus (HCMV) strain AD169 was studied in human peripheral blood granulocytes, monocytes-macrophages, B lymphocytes, and T lymphocytes. Progeny virus was produced in some T-cell cultures stimulated in the allogeneic mixed lymphocyte reaction and was regularly obtained when stimulated T cells were grown in the presence of interleukin 2. Replication of HCMV in these cultures was documented by increases in titer, expression of early and late antigen as assessed by indirect immunofluorescence and Western blot, and viral DNA synthesis as determined by dot-blot assays. Approximately 0.05% of cells in virus-producing cultures formed infectious centers, indicating that only a subset of cells takes part in active virus replication. In double-immunofluorescence experiments this subset was found to consist primarily of the T3+ and T8+ phenotype. By infection of preparatively separated T4+ and T8+ T lymphocytes, however, it could be shown that both T-cell subsets were susceptible to HCMV infection as indicated by increases in titer and by DNA kinetics. We conclude from these data that the T lymphocyte might be a target for HCMV in vitro, which is in accordance with in vivo findings in HCMV-infected patients.     

Inactivating a cellular intrinsic immune defense mediated by Daxx is the mechanism through which the human cytomegalovirus pp71 protein stimulates viral immediate-early gene expression

Human cytomegalovirus (HCMV) masterfully evades adaptive and innate immune responses, allowing infection to be maintained and periodically reactivated for the life of the host. Here we show that cells also possess an intrinsic immune defense against HCMV that is disarmed by the virus. In HCMV-infected cells, the promyelocytic leukemia nuclear body (PML-NB) protein Daxx silences viral immediate-early gene expression through the action of a histone deacetylase. However, this antiviral tactic is efficiently neutralized by the viral pp71 protein, which is incorporated into virions, delivered to cells upon infection, and mediates the proteasomal degradation of Daxx. This work demonstrates the mechanism through which pp71 activates viral immediate-early gene expression in HCMV-infected cells. Furthermore, it provides insight into how a PML-NB protein institutes an intrinsic immune defense against a DNA virus and how HCMV pp71 inactivates this defense.     

Human cytomegalovirus induces drug resistance and alteration of programmed cell death by accumulation of deltaN-p73alpha

Intrauterine transmission of human cytomegalovirus (HCMV) to the fetus following primary infection in early and late pregnancy usually results in severe neurological handicaps and sensorineural hearing loss with typical migrational anomalies, optic atrophy, disturbed myelination, cerebella hypoplasia, microcephaly, hydrocephaly, and lissencephaly. Recently, evidences raised from the phenotype of p73-deficient mice show that an association may exist between the expression of the TP53 homologous gene and HCMV tropism in the brain, suggesting an implication of p73 in viral persistence. In this study, we demonstrated that HCMV-mediated inhibition of apoptosis only occurs in p73-expressing cells. Upon infection, an accumulation of deltaN-p73alpha isoforms was observed in HCMV-infected p73-positive cells. This phenomenon was shown to be responsible for the subsequent acquired resistance to apoptosis of infected cells. Inhibition of apoptosis in p73-positive cells by HCMV may thus contribute both to virus persistency and abnormal nervous cell survival. This finding provides the first molecular basis for HCMV-associated abnormal embryonic development and neurological defects in newborns.     

Human Cytomegalovirus Gene Expression in Long-Term Infected Glioma Stem Cells

The most common adult primary brain tumor, glioblastoma (GBM), is characterized by fifteen months median patient survival and has no clear etiology. We and others have identified the presence of human cytomegalovirus (HCMV) gene products endogenously expressed in GBM tissue and primary cells, with a subset of viral genes being consistently expressed in most samples. Among these viral genes, several have important oncomodulatory properties, regulating tumor stemness, proliferation, immune evasion, invasion and angiogenesis. These findings lead us to hypothesize that a specific HCMV gene signature may be associated with GBM pathogenesis. To investigate this hypothesis, we used glioma cell lines and primary glioma stem-like cells (GSC) infected with clinical and laboratory HCMV strains and measured relative viral gene expression levels along several time points up to 15 weeks post-infection. While HCMV gene expression was detected in several infected glioma lines through week 5 post-infection, only HCMV-infected GSC expressed viral gene products 15 weeks post-infection. Efficiency of infection across time was higher in GSC compared to cell lines. Importantly, HCMV-infected GSC outlived their uninfected counterparts, and this extended survival was paralleled by increased tumorsphere frequency and upregulation of stemness regulators, such as SOX2, p-STAT3, and BMX (a novel HCMV target identified in this study). Interleukin 6 (IL-6) treatment significantly upregulated HCMV gene expression in long-term infected glioma cultures, suggesting that pro-inflammatory signaling in the tumor milieu may further augment HCMV gene expression and subsequent tumor progression driven by viral-induced cellular signaling. Together, our data support a critical role for long-term, low-level HCMV infection in promoting survival, stemness, and proliferation of GSC that could significantly contribute to GBM pathogenesis.     

The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis

Viral subversion of apoptosis regulation plays an important role in the outcome of host/virus interactions. Although human cytomegalovirus (HCMV) encodes several immediate early (IE) antiapoptotic proteins (IE1, IE2, vMIA and vICA), no proapoptotic HCMV protein has yet been identified. Here we show that US28, a functional IE HCMV-encoded chemokine receptor, which may be involved in both viral dissemination and immune evasion, constitutively induces apoptosis in several cell types. In contrast, none of nine human cellular chemokine receptors, belonging to three different subfamilies, induced any significant level of apoptosis. US28-induced cell death involves caspase 10 and caspase 8 activation, but does not depend on the engagement of cell-surface death receptors of the tumour necrosis factor receptor/CD95 family. US28 cell-death induction is prevented by coexpression of C-FLIP, a protein that inhibits Fas-associated death domain protein (FADD)-mediated activation of caspase 10 and caspase 8, and by coexpression of the HCMV antiapoptotic protein IE1. The use of US28 mutants indicated that the DRY sequence of its third transmenbrane domain, required for constitutive G-protein signalling, and the US28 intracellular terminal domain required for constitutive US28 endocytosis, are each partially required for cell-death induction. Thus, in HCMV-infected cells, US28 may function either as a chemokine receptor, a phospholipase C activator, or a proapoptotic factor, depending on expression levels of HCMV and/or cellular antiapoptotic proteins.     

Reduced expression of HLA class II molecules and Iinterleukin-10- and transforming growth factor beta1-independent suppression of T-cell proliferation in human cytomegalovirus-infected macrophage cultures

After a primary infection, human cytomegalovirus (HCMV) establishes lifelong latency in myeloid lineage cells, and the virus has developed several mechanisms to avoid immune recognition and destruction of infected cells. In this study, we show that HCMV utilizes two different strategies to reduce the constitutive expression of HLA-DR, -DP, and -DQ on infected macrophages and that infected macrophages are unable to stimulate a specific CD4+ T-cell response. Downregulation of the HLA class II molecules was observed in 90% of the donor samples and occurred in two phases: at an early (1 day postinfection [dpi]) time point postinfection and at a late (4 dpi) time point postinfection. The early inhibition of HLA class II expression and antigen presentation was not dependent on active virus replication, since UV-inactivated virus induced downregulation of HLA-DR and inhibition of T-cell proliferation at 1 dpi. In contrast, the late effect required virus replication and was dependent on the expression of the HCMV unique short (US) genes US1 to -9 or US11 in 77% of the samples. HCMV-treated macrophages were completely devoid of T-cell stimulation capacity at 1 and 4 dpi. However, while downregulation of HLA class II expression was rather mild, a 66 to 90% reduction in proliferative T-cell response was observed. This discrepancy was due to undefined soluble factors produced in HCMV-infected cell cultures, which did not include interleukin-10 and transforming growth factor beta1. These results suggest that HCMV reduces expression of HLA class II molecules on HCMV-infected macrophages and inhibits T-cell proliferation by different distinct pathways.     

Cytoskeletal disruption during human cytomegalovirus infection of human lung fibroblasts

Human cytomegalovirus (HCMV) infection causes a rapid, progressive disruption of the host cell cytoskeleton that correlates with actin depolymerization. Whole-mount (3D) electron microscopy was used to analyze the cytoskeleton of uninfected and HCMV-infected human lung fibroblast cells. Within 2 min of HCMV infection, localized areas of cytoskeletal disruption were observed. Disruption extended throughout the cytoplasm during the ensuing 45 to 90 min of infection and resulted in generalized cytoskeletal disorganization. Actin depolymerization occurred, as indicated by an increase in DNase I inhibition and alteration in the fluorescence pattern with rhodamine-conjugated phalloidin. Thus, actin appears to be the primary cytoskeletal target involved during HCMV infection. Fractionation of the virus seed inoculum showed that development of DNase I inhibitory activity in infected cells was associated only with the virus-containing fractions. Cytochalasin B treatment at early times of HCMV infection stimulated progeny virus production. This study demonstrates that rapid cytoskeletal disruption occurs during early periods of HCMV infection and indicates that actin depolymerization facilitates viral infectivity.