Matthew B. Reeves

Human Daxx-mediated Repression of Human Cytomegalovirus Gene Expression Correlates with a Repressive Chromatin Structure around the Major Immediate Early Promoter

Upon herpesvirus infection, viral DNA becomes associated with nuclear structures known as nuclear domain 10 (ND10). The role of ND10 during herpesvirus infection has long been contentious; data arguing for a role for ND10 in repression of infection have been countered by other data showing little effect of ND10 on virus infection. Here we show that knockdown of human Daxx (hDaxx) expression, an important component of ND10, prior to infection with human cytomegalovirus resulted in increased levels of viral immediate early RNA and protein expression and that this correlated with an increased association of the major immediate early promoter with markers of transcriptionally active chromatin. Conversely, we also show that stable overexpression of hDaxx renders cells refractory to cytomegalovirus immediate early gene expression. Intriguingly, this hDaxx-mediated repression appears to be restricted to cells stably overexpressing hDaxx and is not recapitulated in transient transfection assays. Finally, hDaxx-mediated repression of cytomegalovirus major immediate early gene expression was overcome by infecting at higher virus titers, suggesting that an incoming viral structural protein or viral DNA is responsible for overcoming the repression of viral gene expression in hDaxx superexpressing cells. These data suggest that hDaxx in ND10 functions at the site of cytomegalovirus genome deposition to repress transcription of incoming viral genomes and that this repression is mediated by a direct and immediate effect of hDaxx on chromatin modification around the viral major immediate early promoter.

Human cytomegalovirus encodes an MHC class I-like molecule (UL142) that functions to inhibit NK cell lysis.

Clinical and low passage strains of human CMV (HCMV) encode an additional MHC class I-related molecule UL142, in addition to the previously described UL18. The UL142 open reading frame is encoded within the ULb′ region which is missing from a number of common high passage laboratory strains. Cells expressing UL142 following transfection, and fibroblasts infected with a recombinant adenovirus-expressing UL142, were used to screen both polyclonal NK cells and NK cell clones, in a completely autologous system. Analysis of 100 NK cell clones derived from five donors, revealed 23 clones that were inhibited by fibroblasts expressing UL142 alone. Small-interfering RNA-mediated knockdown of UL142 mRNA expression in HCMV-infected cells resulted in increased sensitivity to lysis. From these data we conclude that UL142 is a novel HCMV-encoded MHC class I-related molecule which inhibits NK cell killing in a clonally dependent manner.

The pathogenesis of human cytomegalovirus

Human cytomegalovirus (HCMV) is a recognized cause of disease in the fetus, the allograft recipient and AIDS patients. More recently, it has been recognized as a pathogen for those admitted to intensive care units, for the elderly and for the general population. The epidemiology and molecular and cellular pathology of this virus are summarized to provide an overarching model of pathogenesis, able to account for these varying clinical presentations. In brief, HCMV has the potential to spread in the bloodstream to all organs, but only produces overt disease if the viral load increases to high levels. This is normally prevented by a robust immune response, so that the infected individual usually remains asymptomatic. However, this benefit comes at the cost of committing more and more immunological resources to controlling HCMV with time, so that the overall function of the immune system is impaired. Fortunately, recent progress in developing novel antiviral drugs and vaccines suggests the possibility that the diverse effects of HCMV may soon become controllable at the individual and population level, respectively. Copyright

Analysis of latent viral gene expression in natural and experimental latency models of human cytomegalovirus and its correlation with histone modifications at a latent promoter

Human cytomegalovirus (HCMV) is an opportunistic human pathogen that establishes a lifelong latent infection, which can reactivate periodically. If unchecked by a robust immune response, this reactivation can result in severe disease in immunocompromised patients. Reactivation of latent virus in myeloid progenitor cells is concomitant with cellular differentiation through regulation of the major immediate-early promoter (MIEP) by chromatin remodelling. In this study, we analysed the expression of the latent gene transcript UL81-82as (LUNA). LUNA is expressed in latently infected CD34(+) cells and its expression decreases as CD34(+) cells differentiate to immature dendritic cells. Upon maturation (and HCMV reactivation), a second wave of transcription occurs, consistent with expression during lytic infection. Furthermore, we show that the LUNA promoter is associated with acetylated histones during HCMV latency in experimentally and naturally infected CD34(+) cells, thus suggesting that latent gene promoters are, like the MIEP, regulated by post-translational modifications of their associated histone proteins.

Distribution of the voltage‐dependent calcium channel α1G subunit mRNA and protein throughout the mature rat brain

The molecular identity of a gene which encodes the pore‐forming subunit (α1G) of a member of the family of low‐voltage‐activated, T‐type, voltage‐dependent calcium channels has been described recently. Although northern mRNA analyses have shown α1G to be expressed predominantly in the brain, the detailed cellular distribution of this protein in the central nervous system (CNS) has not yet been reported. The current study describes the preparation of a subunit specific α1G riboprobe and antiserum which have been used in parallel in situ mRNA hybridization and immunohistochemical studies to localize α1G in the mature rat brain. Both α1G mRNA and protein were widely distributed throughout the brain, but variations were observed in the relative level of expression in discrete nuclei. Immunoreactivity for α1G was typically localized in both the soma and dendrites of many neurons. Whilst α1G protein and mRNA expression were often observed in cells known to exhibit T‐type current activity, some was also noted in regions, e.g. cerebellar granule cells, in which T‐type activity has not been described. These observations may reflect differences between the subcellular distribution of channels that can be identified by immunohistochemical methods compared with electrophysiological techniques.

Autorepression of the Human Cytomegalovirus Major Immediate-Early Promoter/Enhancer at Late Times of Infection Is Mediated by the Recruitment of Chromatin Remodeling Enzymes by IE86

ABSTRACT The human cytomegalovirus major immediate-early protein IE86 is pivotal for coordinated regulation of viral gene expression throughout infection. A relatively promiscuous transactivator of viral early and late gene transcription, IE86 also acts during infection to negatively regulate its own promoter via direct binding to a 14-bp palindromic IE86-binding site, the cis repression sequence (crs), located between the major immediate-early promoter (MIEP) TATA box and the start of transcription. Although such autoregulation does not involve changes in the binding of basal transcription factors to the MIEP in vitro, it does appear to involve selective inhibition of RNA polymerase II recruitment. However, how this occurs is unclear. We show that autorepression by IE86 at late times of infection correlates with changes in chromatin structure around the MIEP during the course of infection and that this is likely to result from physical and functional interactions between IE86 and chromatin remodeling enzymes normally associated with transcriptional repression of cellular promoters. Firstly, we show that IE86-mediated autorepression is inhibited by histone deacetylase inhibitors. We also show that IE86 interacts, in vitro and in vivo, with the histone deacetylase HDAC1 and histone methyltransferases G9a and Suvar(3-9)H1 and that coexpression of these chromatin remodeling enzymes with IE86 increases autorepression of the MIEP. Finally, we show that mutation of the crs in the context of the virus abrogates the transcriptionally repressive chromatin phenotype normally found around the MIEP at late times of infection, suggesting that negative autoregulation by IE86 results, at least in part, from IE86-mediated changes in chromatin structure of the viral MIEP.

Human cytomegalovirus activation of ERK and myeloid cell leukemia-1 protein correlates with survival of latently infected cells

The ability of human CMV (HCMV) to enter and establish a latent infection in myeloid cells is crucial for survival and transmission in the human population. Initial pathogen binding and entry triggers a number of antiviral responses, including the activation of proapoptotic cell death pathways, which must be countered during latency establishment. However, mechanisms responsible for a prosurvival state in myeloid cells upon latent HCMV infection remain completely undefined. We hypothesized that the cellular antiapoptotic machinery must be initially activated by HCMV to promote early survival events upon entry. Here we show that HCMV transiently protects nonpermissive myeloid cells from chemical and virus entry induced cell death by up-regulating a key myeloid cell survival gene, myeloid cell leukemia (MCL)-1 protein. The induction of MCL-1 expression was independent of viral gene expression but dependent on activation of the ERK–MAPK pathway by viral glycoprotein B. Inhibition of ERK-MAPK signaling, inhibition of HCMV fusion, antibody-mediated neutralization of glycoprotein B signaling or expression of a shRNA against MCL-1 all correlated with increased cell death in response to virus infection or chemical stimulation. Finally we show that activation of ERK–MAPK signaling impacts on long-term latency and reactivation in hematopoietic cells. Thus, HCMV primes myeloid cells for from the initial virus-cell encounter. Given the importance of ERK and MCL-1 for myeloid cell survival, the successful establishment of HCMV latency in myeloid progenitors begins at the point of virus entry.

Chromatin-mediated regulation of cytomegalovirus gene expression

Following primary infection, whether Human cytomegalovirus (HCMV) enters either the latent or lytic lifecycle is dependent on the phenotype of the cell type infected. Multiple cell types are permissive for lytic infection with HCMV whereas, in contrast, well characterized sites of latency are restricted to a very specific population of CD34+ cells resident in the bone marrow and the immature myeloid cells they give rise to. It is becoming increasingly clear that one of the mechanisms that promote HCMV latency involves the recruitment of histone proteins to the major immediate early promoter (MIEP) which are subject to post-translational modifications that promote a transcriptionally inactive state. Integral to this, is the role of cellular transcriptional repressors that interact with histone modifying enzymes that promote and maintain this repressed state during latency. Crucially, the chromatin associated with the MIEP is dynamically regulated-myeloid cell differentiation triggers the acetylation of histones bound to the MIEP which is concomitant with the reactivation of IE gene expression and re-entry into lytic infection. Interestingly, this dynamic regulation of the MIEP by chromatin structure in latency extends not only into lytic infection but also for the regulation of multiple viral promoters in all phases of infection. HCMV lytic infection is characterised by a timely and co-ordinated pattern of gene expression that now has been shown to correlate with active post-translational modification of the histones associated with early and late promoters. These effects are mediated by the major IE products (IE72 and IE86) which physically and functionally interact with histone modifying enzymes resulting in the efficient activation of viral gene expression. Thus chromatin appears to play an important role in gene regulation in all phases of infection. Furthermore, these studies are highly suggestive that an intrinsic cellular anti-viral response to incoming viral genomes is to promote chromatinisation into a transcriptionally repressed state which the virus must overcome to establish a lytic infection. What is becoming evident is that chromatin structure is becoming as increasingly important for the regulation of viral gene expression as it is for cellular gene expression and thus understanding the mechanisms employed by HCMV to modulate chromatin function could have broader implications on our understanding of the control of gene expression in general.

Circulating dendritic cells isolated from healthy seropositive donors are sites of human cytomegalovirus reactivation in vivo.

ABSTRACT Primary infection with human cytomegalovirus (HCMV) is generally asymptomatic in healthy individuals and results in a lifelong infection of the host. In contrast, in immunosuppressed transplant recipients and late-stage AIDS patients, HCMV infection and reactivation can result in severe disease or death. In vivo, latency is established in bone marrow CD34+ progenitor cells with reactivation linked with their differentiation to macrophages and dendritic cells (DCs). However, previous analyses have relied on ex vivo differentiation of myeloid progenitor cells to DCs in culture. Here, we now report on the isolation and analysis of circulating blood myeloid DCs, resulting from natural differentiation in vivo, from healthy HCMV-seropositive carriers. We show that these in vivo-differentiated circulating DCs are fully permissive for HCMV and exhibit a phenotype similar to that of monocyte-derived DCs routinely used for in vitro studies of HCMV. Importantly, we also show that these DCs from healthy HCMV-seropositive donors carry HCMV genomes and, significantly, are typically positive for viral immediate-early (IE) gene expression, in contrast to circulating monocytes, which carry genomes with an absence of IE expression. Finally, we show that HCMV reactivation from these circulating DCs is enhanced by inflammatory stimuli. Overall, these data argue that the differentiation in vivo of myeloid progenitors to circulating DCs promotes the reactivation of HCMV lytic gene expression in healthy individuals, thereby providing valuable confirmation of studies performed using in vitro generation of DCs from myeloid precursors to study HCMV reactivation.

Human cytomegalovirus manipulation of latently infected cells.

Primary infection with human cytomegalovirus (HCMV) results in the establishment of a lifelong infection of the host which is aided by the ability of HCMV to undergo a latent infection. One site of HCMV latency in vivo is in haematopoietic progenitor cells, resident in the bone marrow, with genome carriage and reactivation being restricted to the cells of the myeloid lineage. Until recently, HCMV latency has been considered to be relatively quiescent with the virus being maintained essentially as a “silent partner” until conditions are met that trigger reactivation. However, advances in techniques to study global changes in gene expression have begun to show that HCMV latency is a highly active process which involves expression of specific latency-associated viral gene products which orchestrate major changes in the latently infected cell. These changes are argued to help maintain latent infection and to modulate the cellular environment to the benefit of latent virus. In this review, we will discuss these new findings and how they impact not only on our understanding of the biology of HCMV latency but also how they could provide tantalising glimpses into mechanisms that could become targets for the clearance of latent HCMV.