The infected cell protein 0 (ICP0) of herpes simplex virus 1, a promiscuous transactivator shown to enhance the expression of genes introduced into cells by infection or transfection, interacts with numerous cellular proteins and has been linked to the disruption of ND10 and degradation of several proteins. ICP0 contains a RING finger domain characteristic of a class of E3 ubiquitin ligases. We report that: (i) in infected cells, ICP0 interacts dynamically with proteasomes and is bound to proteasomes in the presence of the proteasome inhibitor MG132. Also in infected cells, cdc34, a polyubiquitinated E2 ubiquitin-conjugating enzyme, exhibits increased ICP0-dependent dynamic interaction with proteasomes. (ii) In an in vitro substrate-independent ubiquitination system, the RING finger domain encoded by exon 2 of ICP0 binds cdc34, whereas the carboxyl-terminal domain of ICP0 functions as an E3 ligase independent of the RING finger domain. The results indicate that ICP0 can act as a unimolecular E3 ubiquitin ligase and that it promotes ubiquitin-protein ligation and binds the E2 cdc34. It differs from other unimolecular E3 ligases in that the domain containing the RING finger binds E2, whereas the ligase activity maps to a different domain of the protein. The results also suggest that ICP0 shuttles between nucleus and cytoplasm as a function of its dynamic interactions with proteasomes. Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) acts as a promiscuous transactivator of viral and cellular genes (reviewed in ref. 1). ICP0 is critical for viral replication in cells infected at low multiplicity but is not essential in cells infected at high multiplicity (2, 3). In euploid human embryonic lung (HEL) fibroblasts, ICP0 is transported into the cytoplasm between 5 and 7 h after infection. The protein localizes with the promyelocytic leukemia protein, a component of a nuclear structure known as ND10 (4), and causes its disruption (5-7). It also interacts with several proteins such as the BMAL1 transactivator (8), the translation elongation factor 1␦ (9), cyclin D3 (10), and a ubiquitin-specific protease USP7 (11-13).Several lines of investigation have led to the suggestion that ICP0 also interacts with the ubiquitin-proteasomal degradation pathway. The evidence includes the association with USP7 and the functional association with the degradation of sumoylated promyelocytic leukemia protein and other as-yet-unidentified sumoylated proteins (14), the regulatory and catalytic subunits of DNA-dependent protein kinase (15, 16), centromeric proteins C and A (17, 18), and Sp100 (19,20). In addition, this laboratory demonstrated that ICP0 is dynamically associated with proteasomes in untreated cells but remains bound to proteasomes in cells treated with proteasomal inhibitor MG132. Last, the 775-aa ICP0 is translated from a spliced mRNA. The three exons encode 19, 222, and 534 codons, respectively. A RING finger domain characteristic of E3 ubiquitin ligases has been identified in the domain encoded by exon 2. Th...

Herpes simplex virus (HSV) 1 disaggregates the nuclear domain 10 (ND10) nuclear structures and disperses its organizing promyelocytic leukemia protein (PML). An earlier report showed that ectopic overexpression of PML precludes the disaggregation of ND10 but has no effect on viral replication. PML has been reported to mediate the effects of interferon (

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Infected cell protein 0 (ICP0) of herpes simplex virus 1, a multifunctional ring finger protein, enhances the expression of genes introduced into cells by infection or transfection, interacts with numerous cellular and viral proteins, and is associated with the degradation of several cellular proteins. Sequences encoded by exon 2 of ICP0 (residues 20 -241) bind the UbcH3 (cdc34) ubiquitinconjugating enzyme, and its carboxy terminus expresses a ubiquitin ligase activity demonstrable by polyubiquitylation of cdc34 in vitro. We report that: (i) The physical interaction of cdc34 and ICP0 leads to its degradation. Thus, substitution of ICP0 aspartate 199 with alanine attenuates the degradation of cdc34 and its binding to the ICP0 ring finger domain. (ii) Substitution of residue 620 reported to abolish the interaction with a ubiquitin-specific protease has no effect on the function of ubiquitin ligase. (iii) ICP0 contains an additional distinct E3 ligase activity specific for the UbcH5a-and UbcH6 E2-conjugating enzymes mapping to the ring finger domain. This is, to our knowledge, the first identification of a viral protein with at least two physically separated E3 ligase activities with different E2 specificities. The results suggest that each activity may target different proteins.I nfected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is essential for viral replication in cells infected at low multiplicity but is not essential in cells infected at high multiplicity (1-3). The 775-amino acid protein is translated from a spliced mRNA containing three exons encoding 19, 222, and 534 codons, respectively. The interaction of ICP0 with several diverse cellular proteins including the BMAL1 transactivator (4), the translation elongation factor 1␦ (5), cyclin D3 (6), and the ubiquitin (Ub)-specific protease USP7 (7-9), suggests that its phenotype as a promiscuous transactivator reflects the sum of its multiple and diverse functions. Early in infection, ICP0 localizes with the promyelocytic leukemia protein and causes the disruption of ND10 structures (10-13). A zinc-binding RING finger (amino acids 106-149) characteristic of E3 Ub ligase enzymes has been identified in the domain encoded by exon 2 (14).Analyses of ICP0 in the yeast two-hybrid system led to the discovery that ICP0 binds and stabilizes cyclin D3 (6). Mapping studies led to the identification of aspartate 199 as pivotal for both stabilization and binding of cyclin D3. Replacement of aspartate 199 with alanine (D199A) abolishes both binding and stabilization of cyclin D3 mediated by ICP0 and results in attenuated viral growth in quiescent human embryonic lung fibroblasts and reduced neuroinvasiveness (15,16). These studies also revealed that ICP0 stabilizes cyclin D1 in a manner dependent on aspartate 199, even though it does not interact with it in vitro or in the yeast two-hybrid system (15, 16). Because HSV-1 replicates well in both dividing and stationary cells, these observations were pursued at several levels. The transcription of cyclin D3 or D1 ...

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A key early event in the replication of herpes simplex virus 1 (HSV-1) is the localization of infected-cell protein no. 0 (ICP0) in nuclear structures knows as ND10 or promyelocytic leukemia oncogenic domains (PODs). This is followed by dispersal of ND10 constituents such as the promyelocytic leukemia protein (

Our data suggest PPARbeta pharmacological activation as a novel approach to increase cardiac vascularization and cardiac muscle mass.

We reported recently that peroxisome proliferatoractivated receptor β (PPARβ) activation promotes a calcineurin-dependent exercise-like remodelling characterised by increased numbers of oxidative fibres and capillaries. As physical exercise also induces myonuclear accretion, we investigated whether PPARβ activation alters myonuclear density. Transgenic muscle-specific PPARβ over-expression induced 14% increase of myonuclear density. Pharmacological PPARβ activation promoted rapid and massive myonuclear accretion (20% increase after 48 h), which is dependent upon calcineurin/nuclear factor of activated T cells signalling pathway. In vivo bromodeoxyuridine labelling and proliferating cell nuclear antigen immunodetection revealed that PPARβ activation did not promote cell proliferation, suggesting that the PPARβ-promoted myonuclear accretion involves fusion of pre-existing muscle precursor cells to myofibres rather than cell division. Finally, we showed that in skeletal muscle, ageing led to a down-regulation of PPARβ accompanied by decrease of both oxidative fibre number and myonuclear density. PPARβ pharmacological activation counteracts, at least in part, the ageing-driven muscle remodelling.