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. 2021 May;102(5):001609.
doi: 10.1099/jgv.0.001609.

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

Elizabeth G Elder  1   2 , Benjamin A Krishna  1 , Emma Poole  1 , Marianne Perera  1 , John Sinclair  1
Affiliations

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

Elizabeth G Elder et al. J Gen Virol. 2021 May.

Abstract

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.

Keywords: CTCF; chromatin; cytomegalovirus; latency; viral gene expression; virus-host interactions.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
CTCF binds the MIEP during HCMV latency. (a) Schematic of the MIE locus, indicating the positions of the predicted and experimentally verified CTCF binding sites. Exons and introns are denoted by the letters E and i, respectively. (b) Primary CD14+ monocytes were treated with UV-inactivated virus, or infected with HCMV (strain Titan, MOI 3). At six dpi, cells were fixed with formaldehyde and subjected to chromatin extraction and analysis by ChIP using a CTCF antibody or isotype control. Fold enrichment of MIEP sequences is shown with respect to isotype control. Statistical analysis by two-tailed t-test, * indicates P <0.05. (c) Primary CD14+ monocytes were infected with HCMV strain TB40/Egfp at MOI 3. At three dpi, GFP positive cells (latent) were separated from GFP negative cells (bystander) by FACS. CTCF protein level was then analysed by Western blot, using actin as a loading control.
Fig. 2.
Fig. 2.
CTCF binds and represses the MIEP in a US28 dependent manner. (a) THP-1 cells were transfected with control or CTCF overexpression vector and after 2 days cell lysates were analysed for CTCF overexpression by Western blot using actin as a loading control (b) THP-1 cells were transfected with the control or CTCF overexpression vectors as (a) along with an MIEP luciferase vector and transfection control renilla vector. After 2 days, luciferase activity was measured and is quantified as relative light units (RLU). (c) THP-1 cells transfected with CTCF overexpression or control vector were then infected with HCMV Titan WT. At four dpi, total RNA was harvested and analysed for IE gene expression. (d) THP-1 cells were transduced with control lentivirus or CTCF shRNA lentivirus. CTCF knockdown was confirmed by RT-qPCR for CTCF mRNA. (e) Cells from (d) were infected as per (c) and analysed for IE gene expression. (f) THP-1 cells transfected with control or CTCF overexpression vector as (a) or transduced with control or CTCF shRNA lentivirus as (d) were infected with HCMV Titan WT. After 16 h, cells were pelleted, washed with citrate buffer to remove externally bound virions, and analysed for HCMV genome levels by qPCR. Fold change in HCMV genome levels is presented with respect to the relevant control vectors. (g) THP-1 cells transduced with Empty vector (EV), US28-WT, US28-R129A, and CTCF-targeting-shRNA were lysed and CTCF protein was analysed by Western blot, using GAPDH as a loading control. (h) Primary CD14+ monocytes were left uninfected, infected with Titan WT (WT), or Titan ΔUS28 (Δ). At six dpi, cells were fixed with formaldehyde and subject to chromatin extraction and analysis by ChIP using a CTCF antibody or isotype control. Following qPCR for the MIEP region, PCR products from input, anti-CTCF antibody, and isotype control precipitations were separated on a 1.5 % agarose gel. Molecular mass markers (MM) are indicated in numbers of base pairs. The results of statistical analysis by two-tailed t-test are indicated, **, P <0.01; *, P <0.05, or P values are given numerically.
Fig. 3.
Fig. 3.
US28 mediates S100A8/A9 downregulation via CTCF. (a) Enrichment of CTCF on the S100A8/A9 enhancer in control and US28-expressing THP-1 cells was detected by ChIP analysis using a CTCF or isotype control antibody and is expressed as % input. Statistical analysis by two-tailed t-test, *** indicates P <0.001. (b) RT-qPCR analysis of US28 expression in transduced cell lines, using GAPDH as housekeeping control. Results are shown for reactions with reverse transcriptase (+RT) and without reverse transcriptase (-RT) to control for gDNA contamination. (c) Western blot analysis of CTCF expression in control or US28-expressing THP-1 cell lines, using actin as a loading control. (d) S100A8/A9 enhancer luciferase assay in cells from B. (e) S100A8/A9 ELISA in supernatants from cells in B. (f) CTCF expression was detected by RT-qPCR in monocytes left uninfected (UI) or treated with UV-inactivated HCMV (UV), or infected with HCMV Titan WT (WT), or Titan ΔUS28 (ΔUS28) for 6 days. (g) CTCF expression was detected by RT-qPCR in control and US28-expressing THP-1 cells (h) IFI16 protein expression in cells from B. Statistical analysis for C, D by one-way ANOVA followed by Tukey’s multiple comparison test, *** indicates P <0.001, *****P <0.0001, ns=not significant.
Fig. 4.
Fig. 4.
S100A8/A9 downregulation is associated with CTCF binding to an enhancer region. (a) Schematic showing the organisation of the S100A8, S100A9, and S100A12 gene region and enhancer region, which contains a CTCF binding site (black triangle). Genomic coordinates of the enhancer are given. (b) Enrichment of CTCF on the S100A8/A9 enhancer in sorted latently infected monocytes was detected by ChIP analysis of latently infected, or uninfected monocytes at six dpi using a CTCF or isotype control antibody. Statistical analysis by two-tailed t-test, * indicates P <0.05. (c) S100A8/A9 heterodimer concentration was measured in the supernatants from uninfected monocytes, monocytes infected with Titan WT or with Titan ΔUS28 at six dpi. Statistical analysis by one-way ANOVA followed by Tukey’s multiple comparison test, * indicates P <0.05, **P <0.01.
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