Association of the viral core nuclear egress complex (NEC) constituent pUL53 with intranuclear human cytomegalovirus (HCMV) capsids and capsids budding at the inner nuclear membrane (INM). (a,b) HCMV-infected primary human foreskin fibroblasts (HFFs) were harvested at 3 dpi and subjected to immunogold staining of viral pUL53. Samples were analysed by transmission electron microscopy (TEM), 35,970-fold magnification. NE, nuclear envelope; open arrowheads, intranuclear HCMV capsids; filled arrowheads, HCMV capsids budding at nuclear membranes; (c) Co-immunoprecipitation (CoIP) analysis with HCMV-infected whole cell lysates (WCL). HCMV- or uninfected (mock)-HFFs were lysed at 4 dpi followed by immunoprecipitation with monoclonal antibody (mAb)-UL50.01 coupled to protein A sepharose (lanes 3–4) or empty beads as a CoIP negative control (lanes 1–2). CoIP samples (lanes 1–4) and expression control samples (input, representing one-twentieth of CoIP samples; lanes 5–6) were subjected to Western blot analysis using protein-specific antibodies. Ig-HC, cross-reactive band for immunoglobulin heavy chain; MCP, major capsid protein.