Identification of pUL79 interacting proteins.
(A) Schematic diagram for creating pADflagUL79, the recombinant HCMV BAC clone used to produce virus ADflagUL79. A cassette that contained a 3×FLAG tag followed by the FRT-bracketed GalK/kanamycin dual selection marker was amplified by PCR and recombined into the wildtype HCMV BAC clone (pADwt) at the 5′ terminus of the UL79 coding sequence. The selection marker was then removed by Flp/FRT recombination. The final clone, pADflagUL79, carried the UL79 coding sequence tagged at its 5′ terminus with 3×FLAG. (B) Single step viral growth analysis. HFF cells were infected with HCMV recombinant virus ADflagUL79 (derived from pADflagUL79) or ADwt (derived from pADwt) at an MOI of 3. Infected culture supernatants were collected at indicated days post infection and virus titers were determined by TCID50 assay. The mean virus titers were derived from two independent experiments and two technical replicates. Standard deviations are presented. The detection limit is indicated by the dashed line. (C) Viral protein expression profile. HFFs were infected as described in (B), and harvested at indicated times post infection. Accumulations of host and viral proteins were determined by immunoblot analysis. FLAG-tagged pUL79 was detected by an anti-FLAG antibody. Actin was used as a loading control. Representative results from three independent experiments are shown. (D) Polyacrylamide gel electrophoresis to resolve pUL79 protein complexes. HFFs were infected as described in (B), and at 72 hpi, cell lysates were prepared for immunoprecipitation using an anti-FLAG antibody. Immunoprecipitated proteins were resolved on a gradient polyacrylamide gel and silver stained. Protein bands containing RNAP II subunits identified by mass spectrometry are indicated. Molecular size markers (in kilodaltons) are shown.
