Daxx-mediated accumulation of human cytomegalovirus tegument protein pp71 at ND10 facilitates initiation of viral infection at these nuclear domains

Abstract

Human cytomegalovirus (HCMV) starts immediate-early transcription at nuclear domains 10 (ND10), forming a highly dynamic immediate transcript environment at this nuclear site. The reason for this spatial correlation remains enigmatic, and the mechanism for induction of transcription at ND10 is unknown. We investigated whether tegument-based transactivators are involved in the specific intranuclear location of HCMV. Here, we demonstrate that the HCMV transactivator tegument protein pp71 accumulates at ND10 before the production of immediate-early proteins. Intracellular trafficking of pp71 is facilitated through binding to a coiled-coil region of Daxx. The C-terminal domain of Daxx then interacts with SUMO-modified PML, resulting in the deposition of pp71 at ND10. In Daxx-deficient cells, pp71 does not accumulate at ND10, proving in vivo the necessity of Daxx for pp71 deposition. Also, HCMV forms immediate transcript environments at sites other than ND10 in Daxx-deficient cells, and so does the HCMV pp71 knockout mutant UL82(-/-) in normal cells. This result strongly suggests that pp71 and Daxx are essential for HCMV transcription at ND10. Lack of Daxx had the effect of reducing the infection rate. We conclude that the tegument transactivator pp71 facilitates viral genome deposition and transcription at ND10, possibly priming HCMV for more efficient productive infection.

FIG. 1.

Localization of pp71 during HCMV infection. Human primary WI38 fibroblasts were infected at 0.1 MOI with HCMV (a to h and m to t) or with UV-inactivated HCMV (i to l) for 1.5 h (a to d) or 3 h (e to t) and triple stained for pp71 with monoclonal antibody CMV355 (b, f, j, and r), for IE2 with polyclonal rabbit antibody (c, g, k, o, and s), and for ND10 with human antibody 1745 reacting with the ND10-associated proteins Sp100 and PML (d, h, l, p, and t). Merged images demonstrate colocalization of pp71 and ND10 at 1.5 h postinfection, before IE proteins are visible (a), at 3 h postinfection, when only some cells start producing IE proteins (e), and upon infection by UV-inactivated virus (i). Another HCMV tegument protein, pp65, does not accumulate at ND10 (m to p). Details of pp71 accumulation are presented at the enlarged sections (q to t). IE2 accumulated juxtaposed to pp71/ND10 accumulations (q, arrow).

FIG. 2.

pp71 accumulation at ND10 is Daxx dependent and required for the formation of ITE juxtaposed to ND10. MPEF-T (a, d, and i), PML^−/− T (b and e), or Daxx^−/− T (c and f) mouse cells or WI38 human fibroblasts (g and h) were infected with HCMV (0.1 MOI) for 3 h (a to c) or 5 h (d to g) or with HCMV UL82^−/− (0.1 MOI) for 5 h (h and i), fixed, and immunostained with the indicated antibody. While pp71 in MPEF-T infected cells accumulated at ND10 (a), pp71 does not colocalize with this structure in Daxx^−/− cells (c) or with Daxx at condensed chromatin in PML^−/− T cells (b). Upon infection of MPEF-T cells, the IE2 protein marker of ITE localizes juxtaposed to ND10 (d), but no such correlation between ND10 and IE2 is observed in Daxx^−/− T cells (f). In PML^−/− T cells (e), Daxx is transiently relocated upon infection from condensed chromatin (lower cell) to accumulations juxtaposed to IE2 (left and upper cell), and finally is distributed homogeneously in the nucleoplasm (right cell). IE2 accumulates juxtaposed to ND10 upon HCMV infection of WI38 (g) but not upon HCMV UL82^−/− infection of WI38 (h) or MPEF-T (i) cells.

FIG. 3.

Daxx interacts with pp71 in immunoprecipitation experiments. COS-7 cells were cotransfected with pcDNApp71FLAG or pHP1FLAG and pETDaxx plasmids encoding Daxx deletion mutants or wild-type Daxx fused with GFP-NLS or pET encoding GFP-NLS. Cell extracts were incubated with ProBond resin or protein G (ProtG)-Sepharose as indicated and probed for Daxx and deletion mutants of this protein with anti-GFP polyclonal antibody (a). An unbound fraction is presented at panel b, and 15% of input used for immunoprecipitation is presented in panel c. The same cell extracts were used in lanes 5 and 9 (a and b). Arrows indicate wild-type Daxx or mutants fused to GFP-NLS or GFP-NLS; asterisks mark nonspecific bands. Wild-type Daxx and mutants in which amino acids 1 to 290, 1 to 407, and 1 to 595 were deleted are precipitated under these conditions (lanes 5 and 2 to 4 of panel a), but mutants in which amino acids 1 to 142 and 624 to 740 were deleted are not. Note that wild-type Daxx is not precipitated in the absence of ProBond resin or pp71-Flag (lanes 8 and 9, panel a). GFP-NLS is also not precipitated (lane 7, panel a), confirming the specificity of the Daxx-pp71 interaction. Sizes are shown at the left in kilodaltons.