Human Cytomegalovirus Gene Expression Is Silenced by Daxx-Mediated Intrinsic Immune Defense in Model Latent Infections Established In Vitro

Abstract

In addition to productive lytic infections, herpesviruses such as human cytomegalovirus (HCMV) establish a reservoir of latently infected cells that permit lifelong colonization of the host. When latency is established, the viral immediate-early (IE) genes that initiate the lytic replication cycle are not expressed. HCMV IE gene expression at the start of a lytic infection is facilitated by the viral pp71 protein, which is delivered to cells by infectious viral particles. pp71 neutralizes the Daxx-mediated cellular intrinsic immune defense that silences IE gene expression by generating a repressive chromatin structure on the viral major IE promoter (MIEP). In naturally latently infected cells and in cells latently infected in vitro, the MIEP also adopts a similar silenced chromatin structure. Here we analyze the role of Daxx in quiescent HCMV infections in vitro that mimic some, but not all, of the characteristics of natural latency. We show that in these "latent-like" infections, the Daxx-mediated defense that represses viral gene expression is not disabled because pp71 and Daxx localize to different cellular compartments. We demonstrate that Daxx is required to establish quiescent HCMV infections in vitro because in cells that would normally foster the establishment of these latent-like infections, the loss of Daxx causes the lytic replication cycle to be initiated. Importantly, the lytic cycle is inefficiently completed, which results in an abortive infection. Our work demonstrates that, in certain cell types, HCMV must silence its own gene expression to establish quiescence and prevent abortive infection and that the virus usurps a Daxx-mediated cellular intrinsic immune defense mechanism to do so. This identifies Daxx as one of the likely multiple viral and cellular determinants in the pathway of HCMV quiescence in vitro, and perhaps in natural latent infections as well.

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2–CDK-dependent sensor of the host cell cycle and differentiation state

“…In differentiated cells, pp71 translocates to the nucleus after virus entry and associates with nuclear domain 10 (ND10)-structures, where it prevents histone deacetylase (HDAC)-mediated IE gene silencing by neutralizing the ATRX-Daxx repressor complex (3,4). In contrast, in undifferentiated cells, like CD34+ hematopoietic progenitors or embryonic cell lines, pp71 remains cytoplasmic and consequently a quiescent or latent mode of infection is favored (5,6).…”

Promyelocytic Leukemia-Nuclear Body Proteins: Herpesvirus Enemies, Accomplices, or Both?

“…A similar chromatin structure is also observed on viral genomes when HCMV infects NT2 and THP-1 cells in vitro, two model systems that are used to study quiescent HCMV infections [86][87][88]. Infections in NT2 and THP-1 cells are referred to as quiescent (or latent like) because treatments that can reactivate expression from the silenced viral genomes in these cells (if they exist) have not yet been identified [89][90][91][92]. Additionally, in lytically-infected fibroblasts prior to pp71-mediated degradation of Daxx, HCMV genomes adopt a chromatin structure indistinguishable from that observed in CD34 + and NT2 cells [45].…”

“…The mechanism by which the repressive chromatin structure is established on latent viral genomes in CD34 + cells has yet to be analyzed, but it is now evident that PML-NB proteins and HDACs contribute to this process at the start of lytic and quiescent HCMV infections. Chemical HDAC inhibitors permit IE gene expression in NT2 and THP-1 cells [87,90,92,93], indicating that HDACs play a role in transcriptional repression during HCMV quiescence. A study from our laboratory found that the knockdown of Daxx prior to HCMV infection also permits IE gene expression in NT2 and THP-1 cells [92].…”

“…Chemical HDAC inhibitors permit IE gene expression in NT2 and THP-1 cells [87,90,92,93], indicating that HDACs play a role in transcriptional repression during HCMV quiescence. A study from our laboratory found that the knockdown of Daxx prior to HCMV infection also permits IE gene expression in NT2 and THP-1 cells [92]. Artificial knockdown of Daxx is not required for HCMV IE gene expression upon lytic infection of fibroblasts (as it is in quiescent infections of NT2 and THP-1 cells) because tegument-delivered pp71 migrates to the nucleus, binds to Daxx and induces its degradation in these cells [2,4].…”

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells

“…Also, it is unclear if hDaxx has a major role in repression of HCMV gene transcription during latency. In the embryonic carcinoma cell line NT2D1, which can recapitulate the differentiation-dependent regulation of IE gene expression observed during latency, repression of IE gene expression has been reported in the presence (Saffert & Kalejta, 2007) and absence (Groves & Sinclair, 2007) of hDaxx in undifferentiated NT2D1 cells. This may be due to differences in the virus strains used in each study.…”