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. 2017 May 26;91(12):e02384-16. doi: 10.1128/JVI.02384-16

FIG 2.

FIG 2

Reduced amounts of the terminase subunits in cells transfected with HCMV BAC genomes in which either UL51, UL56, UL89, or UL52 is disrupted. (A, left) RPE-1 cells were adenofected with the indicated BACs or were mock transfected. On day 4 posttransfection, cells were harvested, whole-cell lysates were prepared, and immunoblot analysis was performed with the antibodies indicated. The HCMV late protein pUL52 and the early protein pUL44 served as controls for both transfection efficiency and viral gene expression, and GAPDH was used as a loading control. The asterisks denote unspecific reactivity with a cellular protein. (Right) Immunoblot signals of three independent experiments (means ± standard deviations [SD] [error bars]) were quantified with ImageJ software using membranes exposed for a few seconds only. For normalization, UL51, UL56, and UL89 protein levels of cells transfected with the parental BAC pHG-UL51-SF/HA were set at 100%. (B) RPE-1 cells were adenofected with the BAC genome pHG-ΔUL52 carrying a deletion within the UL52 ORF or with the parental BAC pHG and analyzed by immunoblotting as in panel A with the antibodies indicated (HCMV major capsid [late] protein [MCP]). Quantification of signals was done as described above for panel A. (C) RPE-1 cells adenofected with the indicated HCMV BACs were used for preparation of total RNA on day 4 posttransfection. UL51, UL56, UL89, and UL52 transcript levels were determined by quantitative RT-PCR, relative RNA levels were calculated using UL52 as the internal control and normalized to the values for cells transfected with the parental BAC pHG-UL51-SF/HA. Data are representative of two independent experiments. (D and E) Interactions between the terminase subunits in RPE-1 cells adenofected with the indicated BACs. pUL51-SF/HA was pulled down using Strep-Tactin Sepharose (D), or pUL56 and pUL89 were immunoprecipitated (IP) with specific antibodies from cell lysates prepared on day 4 posttransfection (E). Eluted proteins were analyzed by immunoblotting using antibodies directed against the HA tag (for pUL51), pUL56, or pUL89. IgG heavy chains (HC) and light chains (LC) served as controls.