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Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus
Published on: October 7, 2011
Functional interaction between the pp71 protein of human cytomegalovirus and the PML-interacting protein human Daxx
Heike Hofmann1, Hilde Sindre, Thomas Stamminger
1Institut für Klinische und Molekulare Virologie der Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
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.
Insights
Human cytomegalovirus (HCMV) tegument protein pp71 interacts with human Daxx (hDaxx). This interaction enhances pp71
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Human cytomegalovirus (HCMV) tegument protein pp71 (UL82) regulates viral replication by transactivating the viral major immediate-early enhancer-promoter and enhancing viral DNA infectivity.
- Cellular factors interacting with pp71 are crucial for understanding its regulatory functions during HCMV infection.
- Human Daxx (hDaxx) is a nuclear protein known to interact with PML within nuclear domain 10 (ND10) structures.
Purpose of the Study:
- To identify cellular proteins that interact with HCMV pp71.
- To investigate the role of these interactions in pp71's function during HCMV replication.
- To determine if pp71 localizes to ND10 structures and if hDaxx influences this localization.
Main Methods:
- Yeast two-hybrid screening was employed to identify pp71 interacting partners.
- Expression vectors for pp71-GFP fusion protein and hDaxx were used for transfection of human fibroblasts.
- Immunofluorescence microscopy was utilized to assess protein colocalization with ND10 components (PML, Sp100).
- Co-transfection experiments were performed to evaluate the effect of hDaxx on pp71-mediated promoter transactivation.
Main Results:
- The human Daxx (hDaxx) protein was identified as a specific interaction partner of HCMV pp71.
- pp71 was found to colocalize with ND10 proteins PML and Sp100 in transfected and infected human fibroblasts.
- Co-expression of hDaxx enhanced the recruitment of pp71 to ND10 structures.
- hDaxx synergistically enhanced pp71-mediated transactivation of the HCMV major immediate-early enhancer-promoter.
Conclusions:
- hDaxx specifically interacts with the HCMV tegument protein pp71.
- pp71 localizes to ND10 nuclear structures, and this localization is promoted by hDaxx.
- hDaxx plays a significant role in potentiating pp71's function in viral gene regulation, suggesting a critical role in the HCMV replication cycle.
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