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

Abstract

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 hDaxxmediated 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 (HCMV)3 is an extremely widespread, opportunistic pathogen of considerable clinical significance. Primary infection of healthy, immunocompetent individuals with HCMV is normally asymptomatic. However, as is the case with all herpesviruses, after the initial control of primary infection by the immune system, HCMV establishes lifelong latency in the host. During latency and throughout the lifetime of the infected individual, the herpesviruses can reactivate. Although generally asymptomatic, such reactivation can result in severe disease. As such, the disease burden of herpesviruses can be life-long and particularly severe when associated with immunosuppression, such as in transplant patients and in human immunodeficiency virus-infected patients who have developed immunodeficiency (1, 2).After infection with herpes simplex virus-1, the viral genomes appear to co-localize with cellular nuclear structures known as nuclear domain 10 (ND10) and give rise to replication centers in close proximity to these sites (3, 4). ND10 are discrete interchromosomal accumulations of a number of proteins, many of which are transcriptional repressors. Intriguingly, many of the human herpesviruses encode an immediate early protein, which functions to destroy or redistribute components of ND10 (5-17), and a number of studies have attempted to address the role of ND10 during infection. One of the first observations was that ICP0 (int...

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

“…Supernatants from all HCMV-infected cells were harvested 5 days after infection and titrated on fresh HFFs by IE1 fluorescence, combining 2 previously described approaches. 55,56 The percentage of the IE1-positive cells was statistically significantly reduced in the fresh HFFs infected with supernatants of the RhoB-depleted cells, compared to the ones infected with the supernatants of the control non-induced TRIPZshRhoB fibroblasts ( Fig. 7A and B).…”

“…Moreover, in order to compare the viral yield in different experimental conditions, supernatants from HCMV-infected cells were harvested when indicated and titrated on fresh HFFs by IE1 fluorescence, combining 2 previously described approaches. 55,56 Briefly, equal volumes of each supernatant were used to infect fresh HFFs grown on coverslips, and after 24 hours incubation, they were stained for IE1. Cell nuclei were counterstained using DAPI.…”

Nuclear Domain 10 Components Promyelocytic Leukemia Protein and hDaxx Independently Contribute to an Intrinsic Antiviral Defense against Human Cytomegalovirus Infection

“…At the same time, the ND10 constituent hDaxx was identified as a repressor of HCMV IE gene expression and replication (8,45,46,50). hDaxx silences IE gene expression through the action of a histone deacetylase (HDAC), thereby inducing a transcriptionally inactive chromatin state around the major IE enhancer promoter (MIEP) of HCMV (46,50). Taken together, these findings revealed the ND10 proteins PML and hDaxx as cellular restriction factors that are able to induce silencing of HCMV gene expression, thus controlling viral replication.…”

“…In the case of human cytomegalovirus (HCMV), a member of the ␤-subgroup of herpesviruses, only lately two cellular proteins, promyelocytic leukemia protein (PML) and hDaxx, have been identified as restriction factors that are involved in mediating intrinsic immunity against HCMV infection (8,45,46,48,50). Interestingly, both proteins, PML and hDaxx, are components of a cellular subnuclear structure known as nuclear domain 10 (ND10) or PML nuclear bodies.…”

“…Similarly, plaque formation and IE gene expression of an ICP0-deficient herpes simplex virus type 1 was also shown to be enhanced in PML-negative fibroblasts (15), further supporting the notion of PML and ND10 as mediators of an intrinsic immune defense against herpesvirus infections in general. At the same time, the ND10 constituent hDaxx was identified as a repressor of HCMV IE gene expression and replication (8,45,46,50). hDaxx silences IE gene expression through the action of a histone deacetylase (HDAC), thereby inducing a transcriptionally inactive chromatin state around the major IE enhancer promoter (MIEP) of HCMV (46,50).…”