CTCF Binding to the First Intron of the Major Immediate Early (MIE) Gene of Human Cytomegalovirus (HCMV) Negatively Regulates MIE Gene Expression and HCMV Replication

Abstract

Human cytomegalovirus (HCMV) gene expression during infection is highly regulated, with sequential expression of immediate-early (IE), early (E), and late (L) gene transcripts. To explore the potential role of chromatin regulatory factors that may regulate HCMV gene expression and DNA replication, we investigated the interaction of HCMV with the cellular chromatin-organizing factor CTCF. Here, we show that HCMV-infected cells produce higher levels of CTCF mRNA and protein at early stages of infection. We also show that CTCF depletion by short hairpin RNA results in an increase in major IE (MIE) and E gene expression and an about 50-fold increase in HCMV particle production. We identified a DNA sequence (TTAACGGTGGAGGGCAG TGT) IMPORTANCEOur findings that CTCF binds to intron A of the cytomegalovirus (CMV) major immediate-early (MIE) gene and functions to repress MIE gene expression and viral replication are highly significant. For the first time, a chromatin-organizing factor, CTCF, has been found to facilitate human CMV gene expression, which affects viral replication. We also identified a CTCF-binding motif in the first intron (also called intron A) that directly binds to CTCF and is required for CTCF to repress MIE gene expression. Finally, we show that the CTCF-binding motif is conserved in CMV because a similar DNA sequence was found in murine CMV (MCMV) that is required for CTCF to bind to MCMV MIE gene to repress MCMV MIE gene expression. Human cytomegalovirus (HCMV) is a human betaherpesvirus that infects a large percentage of the human population and causes serious disease in immunocompromised individuals, especially in the setting of HIV-AIDS (1-3). CMV infection in permissive host cells consists of the following sequence of viral events (2): viral entry, immediate-early (IE) and early (E) gene expression, DNA replication, late gene expression, and finally, viral packaging and release. Major IE (MIE) gene expression is one of the earliest events during CMV infection. The MIE gene is the most abundantly expressed viral gene at the IE stage and gives rise to several nuclear phosphoproteins that are critical for the regulation of viral and cellular gene expression and viral DNA replication (4-14). Hence, the HCMV MIE gene has been the focus of much study.HCMV MIE gene expression is under the control of the MIE promoter (MIEP). MIEP activity is regulated predominantly by a long upstream DNA sequence referred to as the MIE enhancer. The MIE enhancer contains an array of cis-acting sites that bind cellular transcription factors such as NF-B, CREB/ATF, NF1, SP-1, AP1, RAR-RXR, ISG, YY1, Gfi-1, ETS, serum response factor, Elk-1, and CAAT/enhancer-binding protein (15). Many of these binding sites (BSs) are repetitive and have complex roles in regulating CMV IE gene transcription. Several transcriptional suppressor proteins can also regulate MIE gene expression, including members of the histone deacetylase family (16, 17), histone chaperone and chromatin remodeling factors (DAXX and ATRX) (18), and anti...

CTCF interacts with the lytic HSV-1 genome to promote viral transcription

“…In KSHV, CTCF and cohesin initially facilitate KSHV gene transcription but inhibit lytic gene transcription at a later time, suggesting their complex roles in KSHV lytic infection16. In HCMV, CTCF binds to the first intron of major IE gene and functions as repressor of major IE gene expression and HCMV particle production17. This diversity partly reflects the multiple activities of CTCF and partially reflects the highly evolved viral strategies viruses use to take advantage of host factors.…”

“…CTCF has been reported to interact with or regulate several DNA viruses including Herpes Simplex Virus (HSV)-1, Epstein–Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), Human cytomegalovirus (HCMV) and adenovirus13141516171819. CTCF interacts with the latent HSV-1 genome and may control its latency by protecting an area of active chromatin around the LAT promoter from silencing202122.…”

“…CTCF interacts with the latent HSV-1 genome and may control its latency by protecting an area of active chromatin around the LAT promoter from silencing202122. In EBV and KSHV, CTCF is reported to play a role in regulating viral gene expression during latent infection and maintaining epigenetic states, including chromatin loop structures, while in HCMV, CTCF binding sites directly regulate CMV gene transcription1314172324.…”

Positive Expression of Human Cytomegalovirus Phosphoprotein 65 in Atherosclerosis

“…Therefore, ISH may be not sensitive enough for HCMV detection in these tissues. Indeed, a recent study showed that, for enterovirus detection, RT-PCR was the most sensitive technique, followed by LC/MRM/MS/MS and IHC, while ISH turned to be the least sensitive method [22]. Secondly, the patients analyzed in this study indeed had HCMV infection, but involvement of the peripheral artery tissue is inconsistent and the assessed samples were inadequate to detect HCMV presence by ISH.…”

Negative Glucocorticoid Response-Like Element from the First Intron of the Chicken Growth Hormone Gene Represses Gene Expression in the Rat Pituitary Tumor Cell Line

“…Introns, particularly the first intron, have large positive or negative effects on the regulation of gene expression in mammalian and fish cells [1,2]. In physiologically relevant cell lines, reporter gene assays on 107 genes in humans revealed that most regulatory elements were in the proximal regulatory promoter regions of the genes, which were located in the 5′-flanking and the intron 1 sequences [3].…”