Herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 stimulates lytic infection and the reactivation of quiescent viral genomes. These roles of ICP0 require its RING finger E3 ubiquitin ligase domain, which induces the degradation of several cellular proteins, including components of promyelocytic leukemia nuclear bodies and centromeres. ICP0 also interacts very strongly with the cellular ubiquitin-specific protease USP7 (also known as HAUSP). We have shown previously that ICP0 induces its own ubiquitination and degradation in a RING finger-dependent manner, and that its interaction with USP7 regulates this process. In the course of these studies we found and report here that ICP0 also targets USP7 for ubiquitination and proteasomedependent degradation. The reciprocal activities of the two proteins reveal an intriguing situation that poses the question of the balance of the two processes during productive HSV-1 infection. Based on a thorough analysis of the properties of an HSV-1 mutant virus that expresses forms of ICP0 that are unable to bind to USP7, we conclude that USP7-mediated stabilization of ICP0 is dominant over ICP0-induced degradation of USP7 during productive HSV-1 infection. We propose that the biological significance of the ICP0-USP7 interaction may be most pronounced in natural infection situations, in which limited amounts of ICP0 are expressed.An important biological and clinical aspect of herpes simplex virus type 1 (HSV-1) is its ability to establish a latent state in neuronal cell nuclei following an initial epithelial infection (for general reviews, see references 31 and 32). Periodically the latent virus will reactivate and cause an episode of lytic replication at the site of the original epithelial infection. While in most cases this is not serious, in immunocompromised patients the infections can become severe and even life-threatening. Since latent virus is accessible to neither the immune system of the host nor current antiviral treatments, an understanding of the molecular mechanisms that govern the establishment, maintenance, and particularly the reactivation of latent genomes is important for the future development of effective therapies.ICP0 is a member of the immediate-early class of HSV-1 gene products, and it is required for the efficient initiation of viral lytic infection and reactivation of latent virus in animal models. ICP0 also stimulates reactivation of quiescent viral genomes in cultured cells (for reviews, see references 12, 13, 19, and 31). A major biochemical function of ICP0 is its E3 ubiquitin ligase activity, conferred by a RING finger domain near its N terminus (4,20). This activity leads to the proteasome-dependent degradation of several cellular proteins during infection. These include the promyelocytic leukemia protein PML and small ubiquitin-like modifier (SUMO)-modified forms of Sp100 (both of which are major components of nuclear substructures known as ND10 or PML nuclear bodies) (3,7,15,28,29), centromere proteins CENP-C and CENP-A (14, 24), and the ...

Herpes simplex virus type 1 immediate-early regulatory protein ICP0 stimulates lytic infection and reactivation from latency, processes that require the ubiquitin E3 ligase activity mediated by the RING finger domain in the N-terminal portion of the protein. ICP0 stimulates the production of polyubiquitin chains by the ubiquitin-conjugating enzymes UbcH5a and UbcH6 in vitro, and in infected and transfected cells it induces the proteasome-dependent degradation of a number of cellular proteins including PML, the major constituent protein of PML nuclear bodies. However, ICP0 binds strongly to the cellular ubiquitin-specific protease USP7, a member of a family of proteins that cleave polyubiquitin chains and/or ubiquitin precursors. The region of ICP0 that is required for its interaction with USP7 has been mapped, and mutations in this domain reduce the functionality of ICP0. These findings pose the question: why does ICP0 include domains that are associated with the potentially antagonistic functions of ubiquitin conjugation and deconjugation? Here we report that although neither protein affected the intrinsic activities of the other in vitro, USP7 protected ICP0 from autoubiquitination in vitro, and their interaction can greatly increase the stability of ICP0 in vivo. These results demonstrate that RING finger-mediated autoubiquitination of ICP0 is biologically relevant and can be regulated by interaction with USP7. This principle may extend to a number of cellular RING finger E3 ubiquitin ligase proteins that have analogous interactions with ubiquitin-specific cleavage enzymes.Herpes simplex virus type 1 (HSV-1) 1 is a common human pathogen that can establish a lifelong quiescent infection in sensory neurons following primary infection of epithelial cells. Environmental stimuli such as stress and sunlight trigger periodic recurrences of lytic infection, causing cold sores and genital lesions. HSV-1 expresses three broad groups of temporally regulated genes during lytic infection, termed immediate-early (IE), early, and late (for reviews, see Refs. 1 and 2). The IE protein ICP0 has an important role in the mechanisms that govern the switch between lytic and latent infection (reviewed in Refs. 3-5). Although not essential for viral replication, ICP0 increases the probability of the virus entering lytic infection, particularly after low multiplicity infection of human fibroblasts, and in its absence, viral genomes are more likely to become repressed and establish a quiescent infection (5-7). ICP0 stimulates the expression of all three classes of viral genes by as yet uncertain mechanisms that correlate with its ability to induce the degradation of a number of cellular proteins (8 -12). ICP0 includes a zinc-binding RING finger domain in its N-terminal portion, and in its C-terminal third lie a nuclear localization signal and motifs required for self-multimerization and efficient localization at specific nuclear substructures known as ND10 or PML nuclear bodies. Consistent with its ability to induce the degradation of...

The potential for leukocyte-mediated host tissue damage during resolution of inflammatory responses is influenced by the rate at which extravasated apoptotic leukocytes are cleared from inflammatory sites. Regulation of macrophage capacity for clearance of apoptotic granulocytes is likely to be an important factor determining whether inflammation ultimately resolves or progresses to a chronic state. In this study we have investigated the molecular basis for rapid augmentation of macrophage phagocytosis of apoptotic neutrophils, which was observed following macrophage adhesion to fibronectin. We used a combination of monoclonal antibodies, blocking peptides, and recombinant fibronectin fragments to investigate the role of ␤ 1 integrins in mediating the fibronectin effects. Blockade of ␣ 5 ␤ 1 or ␣ 4 ␤ 1 alone did not attenuate fibronectin-augmentation of phagocytosis. In addition, adhesion of macrophages to recombinant fibronectins lacking ␣ 4 ␤ 1 recognition motifs failed to promote phagocytosis of apoptotic neutrophils. Our results would be consistent with a model in which multiple fibronectin receptors, including ␤ 1 integrins, act co-operatively to augment macrophage phagocytic responses. Together, these data suggest that the extracellular matrix environment of macrophages may provide regulatory signals that act indirectly to rapidly alter the potential for removal of apoptotic cells and influence the process of resolution of inflammation. J. Leukoc. Biol. 64: 600-607; 1998.

The ability to destroy a particular protein at a particular time is central to the regulation of many cellular processes. Selective proteolysis in eukaryotic cells is carried out primarily by the ubiquitin-proteasome pathway. Attachment of a ubiquitin polymer to an unwanted protein causes it to be degraded by the proteasome. Several classes of enzyme, known as E1s, E2s and E3s, control the stepwise formation of a ubiquitin-protein conjugate. The specificity of substrate selection lies with the E2s and E3s. Here we describe the cloning of a Drosophila E2 gene, UbcD4, which is only expressed in embryos. Its expression pattern in stage 10-11 embryos suggests a role in germ cell development. UbcD4 can interact with the polyubiquitin-binding subunit of the proteasome.

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