Advances in the treatment of cytomegalovirus.

BACKGROUND: Human cytomegalovirus (HCMV) is a threat to immunologically weak patients. HCMV cannot yet be eliminated with a vaccine, despite recent advances. SOURCES OF DATA: Sources of data are recently published research papers and reviews about HCMV treatments. AREAS OF AGREEMENT: Current antivirals target the UL54 DNA polymerase and are limited by nephrotoxicity and viral resistance. Promisingly, letermovir targets the HCMV terminase complex and has been recently approved by the FDA and EMA. AREAS OF CONTROVERSY: Should we screen newborns for HCMV, and use antivirals to treat sensorineural hearing loss after congenital HCMV infection? GROWING POINTS: Growing points are developing drugs against latently infected cells. In addition to small molecule inhibitors, a chemokine-based fusion toxin protein, F49A-FTP, has shown promise in killing both lytically and latently infected cells. AREAS TIMELY FOR DEVELOPING RESEARCH: We need to understand what immune responses are required to control HCMV, and how best to raise these immune responses with a vaccine.

Targeting the latent cytomegalovirus reservoir with an antiviral fusion toxin protein.

Reactivation of human cytomegalovirus (HCMV) in transplant recipients can cause life-threatening disease. Consequently, for transplant recipients, killing latently infected cells could have far-reaching clinical benefits. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, and one viral gene expressed by latently infected myeloid cells is US28. This viral gene encodes a cell surface G protein-coupled receptor (GPCR) that binds chemokines, triggering its endocytosis. We show that the expression of US28 on the surface of latently infected cells allows monocytes and their progenitor CD34+ cells to be targeted and killed by F49A-FTP, a highly specific fusion toxin protein that binds this viral GPCR. As expected, this specific targeting of latently infected cells by F49A-FTP also robustly reduces virus reactivation in vitro. Consequently, such specific fusion toxin proteins could form the basis of a therapeutic strategy for eliminating latently infected cells before haematopoietic stem cell transplantation.

Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells.

Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens.

Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries.

UNLABELLED: Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication. IMPORTANCE: Human cytomegalovirus (HCMV), a betaherpesvirus, is a leading cause of morbidity and mortality during congenital infection and among immunosuppressed individuals. HCMV infection significantly changes cellular metabolism. Akin to tumor cells, in HCMV-infected cells, glycolysis is increased and glucose carbon is shifted from the tricarboxylic acid cycle to fatty acid biosynthesis. However, unlike in tumor cells, HCMV induces mitochondrial biogenesis even under aerobic glycolysis. Here, we have affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We find that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication.

An in vitro model for the regulation of human cytomegalovirus latency and reactivation in dendritic cells by chromatin remodelling.

Human cytomegalovirus (HCMV) is a frequent cause of major disease following primary infection or reactivation from latency in immunocompromised patients. Infection of non-permissive mononuclear cells is used for analyses of HCMV latency in vitro. Using this approach, it is shown here that repression of lytic gene expression following experimental infection of CD34+ cells, a site of HCMV latency in vivo, correlates with recruitment of repressive chromatin around the major immediate-early promoter (MIEP). Furthermore, long-term culture of CD34+ cells results in carriage of viral genomes in which the MIEP remains associated with transcriptionally repressive chromatin. Finally, specific differentiation of long-term cultures of infected CD34+ cells to mature dendritic cells results in acetylation of histones bound to the MIEP, concomitant loss of heterochromatin protein 1 and the reactivation of HCMV. These data are consistent with ex vivo analyses of latency and may provide a model for further analyses of the mechanisms involved during latency and reactivation.

Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers.

Human cytomegalovirus (HCMV) persists as a subclinical, lifelong infection in the normal human host, but reactivation from latency in immunocompromised subjects results in serious disease. Latency and reactivation are defining characteristics of the herpesviruses and are key to understanding their biology; however, the precise cellular sites in which HCMV is carried and the mechanisms regulating its latency and reactivation during natural infection remain poorly understood. Here we present evidence, based entirely on direct analysis of material isolated from healthy virus carriers, to show that myeloid dendritic cell (DC) progenitors are sites of HCMV latency and that their ex vivo differentiation to a mature DC phenotype is linked with reactivation of infectious virus resulting from differentiation-dependent chromatin remodeling of the viral major immediate-early promoter. Thus, myeloid DC progenitors are a site of HCMV latency during natural persistence, and there is a critical linkage between their differentiation to DC and transcriptional reactivation of latent virus, which is likely to play an important role in the pathogenesis of HCMV infection.

Human cytomegalovirus infection inhibits tumor necrosis factor alpha (TNF-alpha) signaling by targeting the 55-kilodalton TNF-alpha receptor.

Infection with human cytomegalovirus (HCMV) results in complex interactions between viral and cellular factors which perturb many cellular functions. HCMV is known to target the cell cycle, cellular transcription, and immunoregulation, and it is believed that this optimizes the cellular environment for viral DNA replication during productive infection or during carriage in the latently infected host. Here, we show that HCMV infection also prevents external signaling to the cell by disrupting the function of TNFRI, the 55-kDa receptor for tumor necrosis factor alpha (TNF-alpha), one of the receptors for a potent cytokine involved in eliciting a wide spectrum of cellular responses, including antiviral responses. HCMV infection of fully permissive differentiated monocytic cell lines and U373 cells resulted in a reduction in cell surface expression of TNFRI. The reduction appeared to be due to relocalization of TNFRI from the cell surface and was reflected in the elimination of TNF-alpha-induced Jun kinase activity. Analysis of specific phases of infection suggested that viral early gene products were responsible for this relocalization. However, a mutant HCMV in which all viral gene products known to be involved in down-regulation of major histocompatibility complex (MHC) class I were deleted still resulted in relocalization of TNFRI. Consequently, TNFRI relocalization by HCMV appears to be mediated by a novel viral early function not involved in down-regulation of cell surface MHC class I expression. We suggest that upon infection, HCMV isolates the cell from host-mediated signals, forcing the cell to respond only to virus-specific signals which optimize the cell for virus production and effect proviral responses from bystander cells.

Human cytomegalovirus infection inhibits epidermal growth factor (EGF) signalling by targeting EGF receptors.

Infection with human cytomegalovirus (HCMV) is known to involve complex interactions between viral and cellular factors resulting in perturbation of a number of cellular functions. Specifically, HCMV infection targets control of the cell cycle, cellular transcription and immunoregulation, presumably to optimize the cellular environment for virus persistence and productive infection. Here, we show that HCMV infection also prevents external signalling to the cell by disrupting the function of epidermal growth factor receptor (EGFR). Infection with HCMV resulted in a decrease in cell-surface expression of EGFR. This decrease was correlated with a concomitant decrease in steady-state levels of EGFR protein. Consistent with this, HCMV inhibited EGF-mediated receptor autophosphorylation. Infection with a mutant HCMV deleted of all viral gene products known to be involved in down-regulation of MHC Class I receptors still resulted in this down-regulation, implying that EGFR down-regulation by HCMV is mediated by a novel virus function. We suggest that a primary goal of HCMV is to 'isolate' the infected cell from host-mediated signals so that the cell responds solely to an array of virus-specific signals which optimize the cell for virus production.

The human cytomegalovirus 86-kilodalton major immediate-early protein interacts physically and functionally with histone acetyltransferase P/CAF.

The major immediate-early proteins of human cytomegalovirus (HCMV) play a pivotal role in controlling viral and cellular gene expression during productive infection. As well as negatively autoregulating its own promoter, the HCMV 86-kDa major immediate early protein (IE86) activates viral early gene expression and is known to be a promiscuous transcriptional regulator of cellular genes. IE86 appears to act as a multimodal transcription factor. It is able to bind directly to target promoters to activate transcription but is also able to bridge between upstream binding factors such as CREB/ATF and the basal transcription complex as well as interacting directly with general transcription factors such as TATA-binding protein and TFIIB. We now show that IE86 is also able to interact directly with histone acetyltransferases during infection. At least one of these factors is the histone acetyltransferase CBP-associated factor (P/CAF). Furthermore, we show that this interaction results in synergistic transactivation by IE86 of IE86-responsive promoters. Recruitment of such chromatin-remodeling factors to target promoters by IE86 may help explain the ability of this viral protein to act as a promiscuous transactivator of cellular genes.

Disruption of PML-associated nuclear bodies mediated by the human cytomegalovirus major immediate early gene product.

The PML gene product is associated with a defined nuclear structure (10-20 per cell) known variously as PML-bodies, ND10, PODs or Kr bodies. Certain conditions are known to compromise the integrity of PML-bodies; these include environmental stress (e.g. heat shock), a chromosomal translocation-associated acute promyelocytic leukaemia, and infection with certain viruses [including human cytomegalovirus (HCMV), herpes simplex virus type 1 and adenovirus]. Expression of the HCMV major immediate early (IE) protein (IE1(491aa)) is by itself sufficient to cause disruption of PML-bodies, resulting in the dispersal of the PML antigen uniformly throughout the nucleus. In uninfected cells undergoing mitosis PML is excluded from chromatin. However, both IE1(491aa) and PML were observed to associate with mitotic chromosomes in cells infected with HCMV or transfected with the IE1 gene. A series of in-frame IE1 deletion mutants was used in DNA transfection experiments to identify two large sequence elements (aa 132-274 and the C-terminal aa 347-491) not required for dispersal of the PML antigen. However, a putative leucine-zipper domain (aa 105-139), a putative zinc-finger domain (aa 267-286) and exon 2 and 3 coding sequences (aa 6-85) were required. The association of the IE1 gene product with chromatin required an acidic domain near the C terminus (aa 421-486). The interaction of IE1(491aa) with chromatin was therefore not required for the disruption of PML-bodies. Exon 2 (aa 1-24) was shown to encode a nuclear localization signal.

The human cytomegalovirus IE1-72 protein interacts with the cellular p107 protein and relieves p107-mediated transcriptional repression of an E2F-responsive promoter.

The Rb-related p107 protein has been implicated as an important control element in proper cell cycle progression. The p107 protein is thought to restrict cellular proliferation in part through its interaction with the E2F family of transcription factors and is, therefore, a specific target for regulation by several DNA viruses. Here, we demonstrate that p107 protein levels are induced in a biphasic manner in human fibroblasts during productive infection by the human cytomegalovirus (HCMV). Expression patterns of p107 protein levels during HCMV infection of human embryonic lung cells (HELs) demonstrate a sustained induction from early to late times of infection. We also demonstrate that the HCMV immediate-early protein IE1-72 complexes in vivo with the p107 protein and that this interaction can be reconstituted in an in vitro system by using reticulocyte-translated protein. Our data demonstrate that the interaction between p107 and the IE1-72 protein occurs at times of infection that temporally match the second tier of p107 protein induction and the phosphorylation pattern of the IE1-72 protein. Furthermore, we show here that the ability of p107 to transcriptionally repress E2F-responsive promoters can be overcome by expression of the IE1-72 protein. This effect appears to be specific, since the IE1-72 protein is not capable of relieving Rb-mediated repression of an E2F-responsive promoter. Finally, our data demonstrate that HCMV infection can induce cellular proliferation in quiescent cells and that IE1-72 expression alone can, to a degree, drive a similar progression through the cell cycle. These data suggest that IE1-72-mediated transactivation of E2F-responsive promoters through alleviation of p107 transcriptional repression may play a key role in the cell cycle progression stimulated by HCMV infection.

What is the future of third molar removal? A serious presentation for not performing the removal of third molars.

There are three causes for third molar removal: pain, anterior crowding and the 'time-bomb' theory, which are used to justify the removal of otherwise asymptomatic third molars. Arguments are presented to show that these may not be as true as we think and perhaps we need to reconsider our whole approach to third molar removal. Carrying out a procedure which has a significant risk of morbidity without good reason is unacceptable.

CCAAT box-dependent activation of the TATA-less human DNA polymerase alpha promoter by the human cytomegalovirus 72-kilodalton major immediate-early protein.

Human cytomegalovirus (HCMV) immediate-early (IE) proteins are known potent transregulators of viral and cellular gene expression upon HCMV infection. HCMV is known to activate a number of cellular genes intimately associated with the cell cycle and DNA replication by mechanisms involving the viral major IE 86-kDa protein (IE2). We have recently shown that IE2 mediates this activation in a TATA-dependent manner and interacts directly with the TATA-binding protein. However, a number of TATA-less cellular promoters, e.g., DNA polymerase alpha and dihydrofolate reductase, are also activated by HCMV infection. Consequently, we have asked how HCMV mediates this activation. We show that, consistent with its known TATA dependency, IE2 does not activate the DNA polymerase alpha promoter. In contrast, this promoter is strongly activated by the major IE 72-kDa protein (IE1). Whilst deletion of ATF or E2F sites within the DNA polymerase alpha promoter had little effect on IE1-mediated activation, removal of the CCAAT box appeared to abolish high levels of activation by IE1. Consistent with this observation, we also find that IE1 interacts directly with the CCAAT box binding factor CTF1 in vitro and massively augments CTF1-mediated activation of the DNA polymerase alpha promoter in transient transfection assays.

The transcription factor YY1 binds to negative regulatory elements in the human cytomegalovirus major immediate early enhancer/promoter and mediates repression in non-permissive cells.

We have previously shown that repression of human cytomegalovirus (HCMV) major immediate early (IE) gene expression in non-permissive human teratocarcinoma (T2) cells is associated with a number of nuclear factors which bind to the imperfect dyad symmetry located in the modulator region upstream of the major IE enhancer as well as to the 21 bp repeat elements within the enhancer. Differentiation of T2 cells with retinoic acid (RA) results in a decrease in binding of some of these nuclear factors to these sites and deletion of these specific binding sites from major IE promoter/reporter constructs results in increased IE promoter activity in normally non-permissive cells. In this study, we demonstrate that the transcription factor YY1, which can negatively regulate the adeno-associated virus P5 promoter, directly binds to both the imperfect dyad symmetry and the 21 bp repeat elements in the HCMV major IE promoter/regulatory region and mediates repression of HCMV IE gene expression. This strongly suggests that YY1 plays an important role in regulating HCMV expression in non-permissive cells.

Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow.

Peripheral blood monocytes (PBM) are one site of persistence of human cytomegalovirus (HCMV) in healthy carriers. However, because PBM circulate only briefly before entering the tissues and are difficult to infect with HCMV, it has been suggested that they may acquire HCMV during development in the bone marrow. Consistent with this, we show evidence that bone marrow progenitors from healthy HCMV carriers contain endogenous HCMV DNA as detected by PCR. We also show that bone marrow precursors are readily infected by clinical isolates of HCMV in vitro but that no viral gene expression occurs until these cells become differentiated. In contrast, incubation of these cells at any developmental stage with the laboratory strain AD169 resulted in few cells expressing viral immediate-early genes, and this correlated with a lack of entry of AD169 virus. These observations are consistent with bone marrow progenitors acting as a reservoir for HCMV and transmitting the viral genome to PBM, in the absence of lytic-gene expression, until they leave the circulation and undergo tissue-specific differentiation to macrophages.

In vitro expression of calcitonin gene-related peptide in human endothelial cells transfected with plasmid and retroviral vectors.

Introduction of cDNA encoding human alpha-calcitonin gene-related peptide (CGRP), a potent vasodilator, into cultured human umbilical vein endothelial cells (HUVEC) was undertaken by using plasmids and retroviral vectors. In order to improve expression, modification of context coding sequence for the initiation of CGRP translation and deletion of nontranslated regions of CGRP cDNA in the transfection vectors were tested. Stable transfer of neo in the HUVEC was achieved with both plasmid and retroviral vectors. Integration rates obtained by using retrovirus (approximately 1%), where higher than those achieved with plasmid-mediated transfection (< 1/1000). CGRP expressed in the transfected HUVEC was secreted into culture medium when a leading sequence was included in the expression vectors. CGRP was detected by enzyme-linked immunosorbent assay (ELISA) in the supernatants of both transiently transfected and stably transfected/infected HUVEC. Higher levels of expression were achieved by using plasmid (giving a maximum CGRP concentration of 6.5 +/- 0.5 pM in the supernatant) than retroviruses. Lipofectin-mediated transfer of CGRP cDNA also resulted in transient expression of CGRP in the HUVEC.

Inhibition of human cytomegalovirus major immediate early gene expression by antisense RNA expression vectors.

We have used antisense oligonucleotides and expression vectors to inhibit human cytomegalovirus (HCMV) major immediate early (IE) gene expression. We find that oligonucleotides complementary to the HCMV 72K IE protein (IE1) coding region do inhibit HCMV infection, but this is non-specific. However, the use of certain antisense expression vectors, which express short oligonucleotides complementary to IE1, specifically inhibits IE1 expression at the protein level after introduction of IE expression vectors into cells or after HCMV infection.

Polymorphonuclear cells are not sites of persistence of human cytomegalovirus in healthy individuals.

Polymorphonuclear leukocytes (PMNL) have been shown to harbour human cytomegalovirus (HCMV) in viraemic patients, but to date PMNL of asymptomatic healthy subjects have not been examined directly to determine whether this is a normal site of HCMV persistence. Using the polymerase chain reaction (PCR), paired DNA samples prepared from adherent peripheral blood mononuclear cells (PBMC), which are known to be a site of persistence of HCMV, and PMNL of 10 healthy adults were analysed. All of seven individuals who were HCMV seropositive, and one of three who were seronegative gave a reproducible signal for HCMV DNA in their adherent PBMC, whereas none of the paired PMNL DNA samples gave a positive result. The remaining two seronegative subjects showed no HCMV DNA in either the PBMC or PMNL samples. In every case where PCR for HCMV was negative, PCR amplification of a control human gene was used to show there was no inability to amplify the DNA. We conclude that within the leukocyte population of normal asymptomatic HCMV carriers, PMNL do not appear to harbour persistent HCMV whereas adherent PBMC in the same subjects are a site of persistence.

The 72K IE1 and 80K IE2 proteins of human cytomegalovirus independently trans-activate the c-fos, c-myc and hsp70 promoters via basal promoter elements.

Growth-regulating cellular genes or genes encoding proteins involved in cell cycle control are likely to be major targets of viral gene products in the establishment of a cellular state favourable for a permissive infection. We have examined whether infection of permissive fibroblasts with human cytomegalovirus (HCMV) results in trans-regulation of such cellular genes. Here we have shown that the proto-oncogenes c-fos and c-myc are specifically induced during immediate early (IE) and early times of HCMV infection, as has recently been shown for the heat shock protein 70 gene (hsp70). Deletion analyses and transfection assays of all three promoters showed that previously defined control sequences upstream of the constitutive promoters and downstream of the mRNA cap site are not required for this up-regulation by HCMV, such that the minimal inducible promoters of c-fos, c-myc and the hsp70 gene contained only 50 to 60 bp upstream of the transcription start site. Cotransfection assays with vectors expressing HCMV major IE cDNAs showed that the 72K IE1 and 80K IE2 proteins are involved in the up-regulation of these promoters. IE1 and IE2 products independently were able to up-regulate the minimal constitutive promoters of the constructs tested here, but trans-activation by IE1 and IE2 together was synergistic. In the case of the hsp70 promoter, promoter constructs containing a variety of different TATA elements could be activated by the 72K IE1 and 80K IE2 proteins.