Fig 1.
pUL78 is a late protein that is partially localized to juxtanuclear structures in fibroblasts and copurifies with virions. (A) FLAG-tagged pUL78 accumulates during the late phase of TB40/E-mCherry-inUL78-3xF infection, and its accumulation is dependent on viral DNA replication. Fibroblasts were infected (0.5 TCID50/cell), and whole-cell lysates were immunoblotted for the FLAG epitope tag to detect pUL78-3xF. A separate culture was treated with PAA after adsorption and collected at 72 hpi. To ensure equal loading, lysates were assayed for α-tubulin (α-tub). (B) Expression of FLAG-tagged pUL78 after infection with a different HCMV clinical isolate. Fibroblasts were infected (0.5 TCID50/cell) with either TB40/E-mCherry-inUL78-3xF (T) or BFX-GFP-inUL78-3xF (F). Lysates were collected at 96 hpi and immunoblotted for the FLAG epitope. α-Tubulin is shown as a loading control. M, mock. (C) Immunofluorescence detection of pUL78-3xF following TB40/E-mCherry-inUL78-3xF infection. Fibroblasts were infected (0.5 TCID50/cell) with TB40/E-mCherry-inUL78-3xF and then fixed and permeabilized after various time intervals. mCherry (red) is shown as a marker of infection. pUL78-3xF expression was assayed with an antibody directed to the FLAG epitope (green), and nuclei were stained with Hoechst dye (blue). (D) pUL78 copurifies with virions. TB40/Ewt-mCherry (TB40/Ewt) or TB40/E-mCherry-inUL78-3xF (inUL78-3xF) virions were partially purified by centrifugation through a 20% sorbitol cushion. The presence of pUL78-3xF was assessed by immunoblot assay with an antibody to the FLAG epitope. The tegument protein pUL83 is shown in the lower panels as a loading control.