Intracellular localization and expression of the human cytomegalovirus matrix phosphoprotein pp71 (ppUL82): evidence for its translocation into the nucleus

Hensel, Gabriele; Meyer, Hemmo; Buchmann, Inga; Pommerehne, Diamanto; Schmolke, S.; Plachter, Bodo; Radsak, K.; Kern, Horst F.

doi:10.1099/0022-1317-77-12-3087

Cited by 62 publications

(76 citation statements)

References 21 publications

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“…This protein is discussed as the HCMV functional analogue of the HSV-1 VP16 transactivator since pp71 is able to activate IE gene expression in the infected cell via stimulation of the HCMV MIEP, as well as additional viral and cellular promoters (15,17,34,42,74). Consistent with these findings, it was demonstrated that pp71 is imported into the nucleus immediately after infection (30). Furthermore, pp71 has been shown to enhance the infectivity of viral DNA and to accelerate the viral infectious cycle (9).…”

supporting

confidence: 66%

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

Hofmann

Sindre²,

Stamminger

2002

J Virol

153

183

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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...

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supporting

confidence: 66%

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

Hofmann

Sindre²,

Stamminger

2002

J Virol

153

183

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“…The protein pp71 localizes to nuclear domain 10 (ND10) intranuclear structures at early times after infection with HCMV and also when introduced into cells by transfection of pp71-expressing plasmids (17,19,23,37). The cell factor Daxx was identified as an interaction partner of pp71 in yeast twohybrid analyses, and this finding was confirmed by immunoprecipitation studies with intact cells (19,23).…”

supporting

confidence: 56%

“…This effect could not simply be accounted for by the increased quantities of IE proteins, suggesting that pp71 exerts a more general effect on the transcription of the viral genome. The importance of pp71 for HCMV replication was underscored by the finding that a mutant with a deletion of the UL82 sequences exhibited impaired IE gene expression and a consequent defect in the initiation of productive infection (5).The protein pp71 localizes to nuclear domain 10 (ND10) intranuclear structures at early times after infection with HCMV and also when introduced into cells by transfection of pp71-expressing plasmids (17,19,23,37). The cell factor Daxx was identified as an interaction partner of pp71 in yeast twohybrid analyses, and this finding was confirmed by immunoprecipitation studies with intact cells (19,23).…”

mentioning

confidence: 99%

Human Cytomegalovirus Tegument Protein pp71 Directs Long-Term Gene Expression from Quiescent Herpes Simplex Virus Genomes

Preston

Nicholl

2005

J Virol

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The human cytomegalovirus tegument protein pp71 is important for transactivation of immediate-early (IE) gene expression and for the efficient initiation of virus replication. We have analyzed the properties of pp71 by assaying its effects on gene expression from the genome of in1312, a herpes simplex virus type 1 (HSV-1) mutant devoid of functional VP16, ICP0, and ICP4. Upon infection of human fibroblasts, in1312-derived viruses are repressed and retained in a quiescent state, but the presence of pp71 prevented the quiescent state from being attained. Reporter gene cassettes cloned into the in1312 genome, in addition to the endogenous IE promoters, remained active for at least 12 days postinfection, and infected cells were viable and morphologically normal. Cells expressing pp71 remained responsive to the HSV-1 transactivating factors VP16 and ICP4 and to trichostatin A. The C-terminal 61 amino acids, but not the LACSD motif, were required for pp71 activity. In addition to preventing attainment of quiescence, pp71 was able to disrupt the quiescent state of in1312 derivatives and promote the resumption of viral gene expression after a lag of approximately 3 days. The results extend the functional analysis of pp71 and suggest a degree of similarity with the HSV-1 IE protein ICP0. The ability to provoke slow reactivation of quiescent genomes, in conjunction with cell survival, represents a novel property for a viral structural protein.Many herpesviruses utilize virion proteins as transactivators of immediate-early (IE) gene expression to ensure the rapid initiation of productive replication. In the case of herpes simplex virus type 1 (HSV-1), the well-studied tegument protein VP16 activates IE transcription through specific TAATGA RAT elements that are present in all IE promoters (6, 43). For human cytomegalovirus (HCMV), the 559-amino-acid tegument phosphoprotein pp71, encoded by the gene UL82 (9, 42, 51), fulfills an equivalent role, although the mode of action of this protein is not understood in detail.The transactivating properties of pp71 were first recognized in cotransfection assays. Expression from cytomegalovirus IE promoters on plasmid templates was stimulated by the presence of a plasmid that expressed pp71, and the effect was mediated by sequence elements that bind the cellular transcription factors ATF or AP-1 (33). Dependence on ATF recognition sites was also observed in cotransfection studies that investigated the response of the HCMV US11 promoter in the presence of pp71, although in this case the IE protein IE86 was also present (7). The promoter for intercellular adhesion molecule 1 was activated by pp71, in conjunction with IE86, via Sp1 recognition sites (32). Limited stimulation of endogenous intercellular adhesion molecule 1 expression by pp71 alone was also reported in these studies. When HCMV virion DNA was delivered to cells by transfection, the addition of plasmids that expressed pp71 increased the numbers of progeny plaques and the rate of progress of infection (3). This effect could no...

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“…Whether NK cells recognize HCMV-infected cells in the earliest phases of infection remains unknown. In this context, however, it is of importance to mention that the pp71 gene UL82 is expressed with early-late kinetics (49). Therefore, it cannot be excluded that GrM-mediated cleavage of pp71 may nevertheless affect HCMV replication 1) by acting in an IE-independent manner at later time points of infection and/or 2) by modulating the infectiousness of newly formed HCMV virion particles that are packed with dysfunctional GrM-cleaved pp71.…”

Section: Discussionmentioning

confidence: 99%

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

Domselaar¹,

Philippen²,

Quadir³

et al. 2010

The Journal of Immunology

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Granzyme M (GrM) is highly expressed in cytotoxic granules of NK cells, which provide the first line of defense against viral pathogens. GrM knockout mice show increased susceptibility toward murine CMV infection. Although GrM is a potent inducer of cell death, the mechanism by which GrM eliminates viruses remains elusive. In this paper, we show that purified human GrM in combination with the perforin-analog streptolysin O (SLO) strongly inhibited human CMV (HCMV) replication in fibroblasts in the absence of host cell death. In a proteomic approach, GrM was highly specific toward the HCMV proteome and most efficiently cleaved phosphoprotein 71 (pp71), an HCMV tegument protein that is critical for viral replication. Cleavage of pp71 occurred when viral lysates were incubated with purified GrM, when intact cells expressing recombinant pp71 were challenged with living cytotoxic effector cells, and when HCMV-infected fibroblasts were incubated with SLO and purified GrM. GrM directly cleaved pp71 after Leu439, which coincided with aberrant cellular localization of both pp71 cleavage fragments as determined by confocal immunofluorescence. In a luciferase reporter assay, cleavage of pp71 after Leu439 by GrM completely abolished the ability of pp71 to transactivate the HCMV major immediate-early promoter, which is indispensable for effective HCMV replication. Finally, GrM decreased immediate-early 1 protein expression in HCMV-infected fibroblasts. These results indicate that the NK cell protease GrM mediates cell death-independent antiviral activity by direct cleavage of a viral substrate.

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Intracellular localization and expression of the human cytomegalovirus matrix phosphoprotein pp71 (ppUL82): evidence for its translocation into the nucleus

Cited by 62 publications

References 21 publications

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

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

Human Cytomegalovirus Tegument Protein pp71 Directs Long-Term Gene Expression from Quiescent Herpes Simplex Virus Genomes

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

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