Regulation of host and viral promoters during human cytomegalovirus latency via US28 and CTCF

Viral latency is an active process during which the host cell environment is optimized for latent carriage and reactivation. This requires control of both viral and host gene promoters and enhancers often at the level of chromatin, and several viruses co-opt the chromatin organiser CTCF to control gene expression during latency. While CTCF has a role in the latencies of alpha- and gamma-herpesviruses, it was not known whether CTCF played a role in the latency of the beta-herpesvirus human cytomegalovirus (HCMV). Here, we show that HCMV latency is associated with increased CTCF expression and CTCF binding to the viral major lytic promoter, the major immediate early promoter (MIEP). This increase in CTCF binding is dependent on the virally encoded G protein coupled receptor, US28, and contributes to suppression of MIEP-driven transcription, a hallmark of latency. Furthermore, we show that latency-associated upregulation of CTCF represses expression of the neutrophil chemoattractants S100A8 and S100A9 which we have previously shown are downregulated during HCMV latency. As with downregulation of the MIEP, CTCF binding to the enhancer region of S100A8/A9 drives their suppression, again in a US28-dependent manner. Taken together, we identify CTCF upregulation as an important mechanism for optimizing latent carriage of HCMV at both the levels of viral and cellular gene expression.

Human cytomegalovirus major immediate early transcripts arise predominantly from the canonical major immediate early promoter in reactivating progenitor-derived dendritic cells

Human cytomegalovirus latency and reactivation is a major source of morbidity in immune-suppressed patient populations. Lifelong latent infections are established in CD34+progenitor cells in the bone marrow, which are hallmarked by a lack of major lytic gene expression, genome replication and virus production. A number of studies have shown that inhibition of the major immediate early promoter (MIEP) – the promoter that regulates immediate early (IE) gene expression – is important for the establishment of latency and that, by extension, reactivation requires reversal of this repression of the MIEP. The identification of novel promoters (termed ip1 and ip2) downstream of the MIEP that can drive IE gene expression has led to speculation over the precise role of the MIEP in reactivation. In this study we show that IE transcripts arise from both the MIEP and ip2 promoter in the THP1 cell macrophage cell line and also CD14+monocytes stimulated with phorbol ester. In contrast, we show that in in vitro generated dendritic cells or macrophages that support HCMV reactivation IE transcripts arise predominantly from the MIEP and not the intronic promoters. Furthermore, inhibition of histone modifying enzyme activity confirms the view that the MIEP is predominantly regulated by the activity of cellular chromatin. Finally, we observe that ip2-derived IE transcription is cycloheximide-sensitive in reactivating DCs, behaviour consistent with an early gene designation. Taken together, these data argue that MIEP activity is still important for HCMV reactivation but ip2 activity could play cell-type-specific roles in reactivation.

Human cytomegalovirus G protein-coupled receptor US28 promotes latency by attenuating c-fos

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that undergoes latency in cells of the hematopoietic compartment, although the mechanisms underlying establishment and maintenance of latency remain elusive. We previously reported that the HCMV-encoded G protein-coupled receptor (GPCR) homologUS28is required for successful latent infection. We now show that US28 protein (pUS28) providedin transcomplements the US28Δ lytic phenotype in myeloid cells, suggesting that sustained US28 expression is necessary for long-term latency. Furthermore, expression of pUS28 at the time of infection represses transcription from the major immediate early promoter (MIEP) within 24 h. However, this repression is only maintained in the presence of continual pUS28 expression providedin trans. Our data also reveal that pUS28-mediated signaling attenuates both expression and phosphorylation of cellular fos (c-fos), an AP-1 transcription factor subunit, to repress MIEP-driven transcription. AP-1 binds to the MIEP and promotes lytic replication, and in line with this we find that US28Δ infection results in an increase in AP-1 binding to the MIEP, compared with WT latent infection. Pharmacological inhibition of c-fos represses the MIEP during US28Δ infection to levels similar to those we observe during WT latent infection. Together, our data reveal that US28 is required for both establishment and long-term maintenance of HCMV latency, which is modulated, at least in part, by repressing functional AP-1 binding to the MIEP.

Alternative Promoters Drive Human Cytomegalovirus Reactivation from Latency

ABSTRACTReactivation from latency requires reinitiation of viral gene expression and culminates in the production of infectious progeny. The major immediate early promoter (MIEP) of human cytomegalovirus (HCMV) drives the expression of crucial lytic cycle transactivators but is silenced during latency in hematopoietic progenitor cells (HPCs). Because the MIEP is poorly active in HPCs, it is unclear how viral transactivators are expressed during reactivation. We demonstrate that transcripts originating from alternative promoters within the canonical major immediate early locus are abundantly expressed upon reactivation, whereas MIEP-derived transcripts remain undetectable. Further, we show that these promoters are necessary for efficient reactivation in primary CD34+ HPCs. Our findings change the paradigm for HCMV reactivation by demonstrating that promoter switching governs reactivation from viral latency in a context-specific manner.

Chromatin-Remodeling Factor SPOC1 Acts as a Cellular Restriction Factor against Human Cytomegalovirus by Repressing the Major Immediate Early Promoter

ABSTRACTThe cellular protein SPOC1 (survival time-associated PHD [plant homeodomain] finger protein in ovarian cancer 1) acts as a regulator of chromatin structure and the DNA damage response. It binds H3K4me2/3-containing chromatin and promotes DNA condensation by recruiting corepressors such as KAP-1 and H3K9 methyltransferases. Previous studies identified SPOC1 as a restriction factor against human adenovirus (HAdV) infection that is antagonized by E1B-55K/E4-orf6-dependent proteasomal degradation. Here, we demonstrate that, in contrast to HAdV-infected cells, SPOC1 is transiently upregulated during the early phase of human cytomegalovirus (HCMV) replication. We show that the expression of immediate early protein 1 (IE1) is sufficient and necessary to induce SPOC1. Additionally, we discovered that during later stages of infection, SPOC1 is downregulated in a glycogen synthase kinase 3β (GSK-3β)-dependent manner. We provide evidence that SPOC1 overexpression severely impairs HCMV replication by repressing the initiation of viral immediate early (IE) gene expression. Consistently, we observed that SPOC1-depleted primary human fibroblasts displayed an augmented initiation of viral IE gene expression. This occurs in a multiplicity of infection (MOI)-dependent manner, a defining hallmark of intrinsic immunity. Interestingly, repression requires the presence of high SPOC1 levels at the start of infection, while later upregulation had no negative impact, suggesting distinct temporal roles of SPOC1 during the HCMV replicative cycle. Mechanistically, we observed a highly specific association of SPOC1 with the major immediate early promoter (MIEP), strongly suggesting that SPOC1 inhibits HCMV replication by MIEP binding and the subsequent recruitment of heterochromatin-building factors. Thus, our data add SPOC1 as a novel factor to the endowment of a host cell to restrict cytomegalovirus infections.IMPORTANCEAccumulating evidence indicates that during millennia of coevolution, host cells have developed a sophisticated compilation of cellular factors to restrict cytomegalovirus infections. Defining this equipment is important to understand cellular barriers against viral infection and to develop strategies to utilize these factors for antiviral approaches. So far, constituents of PML nuclear bodies and interferon gamma-inducible protein 16 (IFI16) were known to mediate intrinsic immunity against HCMV. In this study, we identify the chromatin modulator SPOC1 as a novel restriction factor against HCMV. We show that preexisting high SPOC1 protein levels mediate a silencing of HCMV gene expression via a specific association with an important viralcis-regulatory element, the major immediate early promoter. Since SPOC1 expression varies between cell types, this factor may play an important role in tissue-specific defense against HCMV.