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. 2021 May 27;102(5):001609. doi: 10.1099/jgv.0.001609

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.