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...