Functional Interaction between the pp71 Protein of Human Cytomegalovirus and the PML-Interacting Protein Human Daxx

Abstract

The tegument protein pp71 (UL82) of human cytomegalovirus (HCMV) has previously been shown to transactivate the major immediate-early enhancer-promoter of HCMV. Furthermore, this protein is able to enhance the infectivity of viral DNA and to accelerate the infection cycle, suggesting an important regulatory function during viral replication. To gain insight into the underlying mechanisms that are used by pp71 to exert these pleiotropic effects, we sought for cellular factors interacting with pp71 in a yeast two-hybrid screen. Here, we report the isolation of the human Daxx (hDaxx) protein as a specific interaction partner of HCMV pp71. hDaxx, which was initially described as an adapter protein involved in apoptosis regulation, has recently been identified as a nuclear protein that interacts and colocalizes with PML in the nuclear domain ND10. In order to assess whether pp71 can also be detected in ND10 structures, a vector expressing pp71 in fusion with the green fluorescent protein was used for transfection of human fibroblasts. This revealed a colocalization of pp71 with the ND10 proteins PML and Sp100. In addition, cotransfection of a hDaxx expression vector resulted in an enhanced recruitment of pp71 to ND10. Targeting of pp71 to nuclear dots could also be observed in infected human fibroblasts in the absence of de novo viral protein synthesis. Moreover, cotransfection experiments revealed that pp71-mediated transactivation of the major immediate-early enhancer-promoter was synergistically enhanced in the presence of hDaxx. These results suggest an important role of hDaxx for pp71 protein function.Human cytomegalovirus (HCMV), a member of the betasubgroup of herpesviruses, is characterized by its narrow host range and prolonged replicative cycle in cell culture, as well as in the infected human host. Generally, HCMV causes asymptomatic infections in immunocompetent individuals; severe disease, however, can result from infection in immunocompromised patients and after intrauterine infection (4).Similar to other herpesviruses, the HCMV open reading frames are expressed in a temporally regulated cascade consisting of three sequential phases, termed immediate early (IE), early (E) and late (L) (19,27,44,47,69,70). IE gene expression results in the synthesis of viral regulatory factors, in particular the major IE proteins IE1-p72 and IE2-p86, which act as strong transactivators of viral early promoters and are therefore required for efficient productive infection (44, 51, 53, 65). Expression from the major IE gene locus is driven by a very strong, complex regulatory element known as the major IE enhancer-promoter (MIEP), which contains several binding sites for known eucaryotic transcription factors (for a review, see reference 48). These in turn modulate gene expression not only in various cell types but also in both differentiated and undifferentiated cells (6,12,23,43,48,50,60,63). In addition to cellular transcription factors, activation of IE transcription is controlled by structural protein components of th...

Noncytotoxic Inhibition of Cytomegalovirus Replication through NK Cell Protease Granzyme M-Mediated Cleavage of Viral Phosphoprotein 71

“…Upon infection, tegument pp71 translocates to the nucleus and facilitates the degradation of nuclear Daxx, thereby relieving suppression of the MIEP and promoting viral replication (41). In agreement with other studies (13,34,38), we observed a ∼5-fold induction of the MIEP by pp71 in fibroblasts (Fig. 6).…”

Nuclear domain 10 of the viral aspect

“…It is supported by the facts that SUMO-1 molecules aggregate in ND10 and ND10 might be the hot sites for SUMOylation, that the acetylation and phosphorylation of p53 at PML bodies enhance the activity of p53 [16,81,82] , and that the 19S and 20S proteasome subunits localize at some PML bodies [83] .…”

“…The first ND10 protein investigated for its role in HCMV gene expression and viral replication was Daxx. In that study, Daxx was found to interact functionally with HCMV tegument protein pp71 [16] . The Stamminger group [99] also investigated PML to see whether PML could have any effects on viral gene expression or on viral replication.…”

“…Once inside the nucleus, viral genomes distribute randomly, but it appears that only those at ND10 replicate and transcribe predominantly [10][11][12][13] , suggesting specifically that the environment at ND10 is particularly advantageous for the virus. However, the ND10 proteins [such as PML, Speckled Protein of 100 kDa (SP100), and Daxx] are interferon-upregulated and have repressive effects on viral replication [14][15][16][17][18][19][20][21][22][23][24][25] . Moreover, most DNA viruses encode an immediate-early protein that induces the dispersion of ND10 [10,[26][27][28][29] , and in the absence of these viral proteins, replication is severely retarded [13,29,30] .…”

Promyelocytic Leukemia-Nuclear Body Proteins: Herpesvirus Enemies, Accomplices, or Both?

“…Additional evidence demonstrated that pp71 induced the degradation of Daxx [2,4] through an unusual proteasome dependent, ubiquitin independent manner [38], but did not disrupt PML-NBs [2]. However, pp71-mediated Daxx degradation induces the expression of the viral IE1 protein [2,[39][40][41][42], and IE1 subsequently disrupts PML-NBs by altering the SUMOylation state of PML [27,43,44] and perhaps other proteins as well. As stated above, the fact that these herpesviral proteins degraded and disrupted PML-NBs implied, but did not demonstrate, that PML-NB proteins antagonize herpesviral infections.…”