Daxx is degraded early during HCMV infection. (A) HFs were uninfected (−) or infected (+) with HCMV at an MOI of 3. Lysates were harvested at the indicated hours postinfection (hpi) and analyzed by Western blotting. Tubulin (Tub) was analyzed as an internal loading control. (B) HFs were either mock infected (M) or infected with HSV-1 or HCMV at an MOI of 3. Lysates harvested at 4 hpi were analyzed by Western blotting. (C) HFs were treated as in panel A. pp28 is an HCMV protein that serves as a marker for the late stages of viral infection.

Daxx is degraded by a tegument protein in a proteasome-dependent manner. (A) HFs infected with HCMV at an MOI of 3 were treated with DMSO (D), lactacystin (L), or E64. Lysates were harvested 6 h postinfection (6 hpi) and analyzed by Western blotting. Tub, tubulin. (B) HFs were mock infected, infected with HCMV (V), or infected with HCMV particles exposed to UV light (UV-V) at an MOI of 3. Lysates were harvested at 6 hpi and analyzed by Western blotting. (C) Untreated HFs or HFs preincubated with cycloheximide (C) were mock infected or infected with HCMV at an MOI of 3. Lysates were harvested at 6 hpi and analyzed by Western blotting. (D) HFs were mock infected, infected with HCMV, or infected with HCMV treated with heparin (hep) at an MOI of 0.1. Lystates were harvested at 6 hpi and analyzed by Western blotting. (E) HFs were mock infected or infected with purified HCMV particles (PV) at an MOI of 3. Lysates were harvested at 6 hpi and analyzed by Western blotting.

Low-multiplicity infection is sufficient to deliver tegument proteins and induce the degradation of Daxx. (A) HFs were mock infected (M) or infected with HCMV at the indicated MOIs. Lysates were harvested 6 h postinfection (hpi) and analyzed by Western blotting. Tub, tubulin. (B) HFs grown on coverslips were infected with HCMV at an MOI of 0.5, 0.05, or 0.005. Cells were fixed, and delivery of pp71 to the nucleus was determined by indirect immunofluorescence. The nuclei were counterstained with Hoechst. (C) The percentage of nuclei positive for either pp71 or IE1 was compared from HCMV-infected HFs at an MOI of 0.5, 0.05, or 0.005. (D) HFs were infected at an MOI of 0.5 with gradient-purified HCMV for 4 h in the presence of cycloheximide and analyzed by indirect immunofluorescence. Fixed cells were stained for pp71 and either Daxx or PML, and nuclei were counterstained with Hoechst. Two separate panels are shown for the pp71-Daxx costaining, and a single panel is shown for the pp71-PML costaining.

pp71 is necessary for Daxx degradation in HCMV-infected cells. (A) HFs were mock infected (M), infected with HCMV (V), or infected with the pp71-null HCMV virus (Δ71) at an MOI of 1. Lysates were harvested at 6 h postinfection (hpi) and analyzed by Western blotting. Tub, tubulin. (B) HFs were mock infected or infected with UV-inactivated wild-type HCMV (UV-V), UV-inactivated pp71-null HCMV (UV-Δ71), wild-type HCMV, or an IE1-null HCMV (CR208) at an MOI of 1. Lysates were harvested at 6 hpi and analyzed by Western blotting.

pp71 induces the proteasome-dependent degradation of Daxx in the absence of all other HCMV proteins. (A) HFs mock transduced (M) or transduced with rAD71 (lanes 71) at 10,000 particles per cell (ppc) were treated with lactacystin (L), DMSO (D), or E64 at 6 h posttransduction (hpt). Lysates were harvested at 18 hpt and analyzed by Western blotting. (B) HFs were mock transduced or transduced with recombinant adenoviruses that express pp71 (lane 71), pp65 (lane 65), or E2F-1 at 30,000 ppc. Lysates were harvested at 24 hpt and analyzed by Western blotting. (C) HFs were mock transduced or transduced with a recombinant adenovirus that expresses pp71 (lane 71) at 3,000 ppc, a mutant pp71 which fails to bind Daxx (Did 2-3) at 30,000 ppc, or a pp71 mutant that fails to degrade Rb and p130 (C219G) at 3,000 ppc. Lysates were harvested 24 hpt. (D) Lysates from HFs either mock transduced or transduced with rAD71 at 30,000 ppc were harvested at the indicated times (hpt) and analyzed by Western blotting. (E) HFs were transduced with rAD71 at decreasing ppc (from left to right: 30,000, 10,000, 3,000, 1,000, 300, 100, and 0 ppc), and lysates harvested at 24 hpt were analyzed by Western blotting.

Daxx silences the HCMV MIEP. (A) Mock-infected HFs (M) or HFs infected with HCMV at the indicated MOIs were treated with DMSO (D) or lactacystin (L). Lysates were harvested 6 h postinfection (hpi) and analyzed by Western blotting. Tub, tubulin. (B) HFs transfected with siRNA directed at either Daxx or Skp-1 and subsequently infected with HCMV were treated with either DMSO or lactacystin. Lysates were harvested 6 hpi and analyzed by Western blotting. Lysates from HCMV-infected (MOI of 0.1) and mock-infected cells not treated with siRNA were also analyzed.

Daxx represses HCMV immediate-early gene expression through the action of an HDAC. HFs pretreated with trichostatin A (T) were mock infected (M) or infected with HCMV (V) at an MOI of 0.05 and then treated with lactacystin (L). Lysates were harvested 6 h postinfection and analyzed by Western blotting. Tub, tubulin.

Model for gene expression from the viral MIEP at the onset of lytic viral replication in HCMV-infected cells. (A) In HFs infected with the pp71-null virus or with wild-type virus in the presence of lactacystin, Daxx represses viral transcription through its interactions with an HDAC and an unknown transcription factor bound to the promoter. (B) During infection with HCMV, tegument-delivered pp71 enters the nucleus, degrades Daxx, and thus activates viral immediate-early gene expression.