In early studies, herpes simplex virus 1 (HSV-1) proteins were identified on the basis of two criteria. The first consisted of characterization of proteins contained in virions purified from cells whose proteins were labeled prior to infection. These proteins designated by the prefix VP were numbered in the order of decreasing apparent molecular weight or, conversely, ascending electrophoretic mobility in denaturing gels (147). The second criterion identified putative viral proteins accumulating in infected cells but absent from uninfected cells. These proteins, designated infected-cell proteins (ICPs), were also numbered in order of decreasing apparent molecular weight (73). One protein, however, while clearly apparent only in infected cells, varied with respect to electrophoretic mobility depending on the composition of the denaturing gel (74). This anomalously migrating protein was designated ICP0 (74). In subsequent studies, viral proteins were designated either by their known primary function (e.g., DNA polymerase, etc.) or the position of the gene along the unique long (U L ) or short (U S ) components of the viral genome (105, 107). The original ICP designation, however, was retained primarily for five proteins recognized in early studies as being the products of ␣ or immediate-early genes expressed after infection in the absence of de novo viral protein synthesis (74, 75). The five proteins, ICP0, ICP4, ICP22, ICP27, and ICP47, have been extensively studied, and for the most part, there is at least a semblance of concordance between the phenotype of cells infected with the mutant lacking the gene, the behavior of the protein in transduced cells, and the molecular functions expressed by the protein (reviewed in reference 132). ICP4 is an essential positive and negative regulator of gene expression (reviewed in reference 132). The protein blocks gene expression by binding to high-affinity DNA consensus sites located at transcription initiation sites of at least two genes. The mechanism of gene activation is less understood, although ICP4's affinity for transcription factors and binding to highly degenerate or nonconsensus sites are suggestive of how it might act. ICP27 is also a multifunctional protein whose phenotype can be largely explained by its ability to block RNA splicing but not transport of unspliced RNA early in infection and by its activity as a chaperone of newly made viral mRNA across the nuclear membranes at late times after infection (reviewed in reference 137).Available data indicate that the carboxyl-terminal half of ICP22 enables full expression of a subset of late (␥ 2 ) viral genes by causing cdc2 cyclin-dependent kinase activity to survive the degradation of its physiologic partners cyclins A and B, by an aberrant partnership with the U L 42 DNA polymerase processivity factor (2,5,6,19,141). Optimal transcription of late genes requires binding and posttranslational modification of topoisomerase II␣ by the two proteins (7). The sole known mission of ICP47 is to bind to and preclude TAP1...

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...

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 ...

Infected cell protein 0 (ICP0) of herpes simplex virus-1 is a multifunctional protein translated from a spliced mRNA containing three exons encoding 19, 222, and 534 codons (reviewed in ref. 1). Substitution of aspartate-199 in ICP0 with alanine (D199A) abolishes the ability of ICP0 to interact with and stabilize cyclin D3, attenuates viral growth in quiescent primary fibroblasts, and impairs neuroinvasiveness (2-4). In addition, ICP0 also stabilizes cyclin D1 even though a physical interaction between ICP0 and cyclin D1 has not been detected (3, 4).Multiple lines of evidence indicate that ICP0 functionally and physically interacts with the ubiquitin (Ub)-proteasomal degradation pathway (reviewed in ref. 5). ICP0 interacts with the Ub-specific protease (USP)7 (6-8) and dynamically associates with the 26S proteasome and remains bound to proteasomes in the presence of the proteasome inhibitor MG132 (9, 10). Furthermore, a zinc-binding really interesting new gene (RING) finger motif (amino acids 106-149) characteristic of a class of ubiquitin ligase (E3) enzymes is encoded by ICP0 exon 2 (5, 11).Two different regions of ICP0 have been shown to possess E3 Ub ligase activity in conjunction with different E2 Ubconjugating enzymes in substrate-independent in vitro ubiquitylation reactions (9, 12, 13). The one reported first and designated herpesvirus Ub ligase 1 (HUL-1) was initially mapped to the C terminus of exon 3 (amino acids 543-768) and functions in conjunction with the E2 enzyme cdc34 (UbcH3) but not with UbcH5a, UbcH6, or UbcH7 (9, 12). As the RING finger domain is not required for HUL-1 E3 function and no homology has been detected between the HUL-1 domain and other known classes of E3 enzymes, it appears to represent a novel class of E3 enzymes (12). Cdc34 was shown to interact with the domain encoded by ICP0 exon 2 (amino acids 20-241), and this interaction was attenuated but not ablated by the D199A mutation (9, 12). The second E3 site designated here as HUL-2 was mapped to exon 2 containing the RING finger and in in vitro ubiquitylation reactions functionally interacts with UbcH6 and UbcH5a but not with UbcH7 or cdc34 (9, 12, 13). These results led to the unprecedented conclusion that ICP0 encodes two physically separate E3 ligase domains that belong to different classes of E3 enzymes and have different E2 specificities (12).In vivo, ICP0 promotes the proteasome-dependent degradation of multiple cellular proteins in a RING finger-dependent manner, indicating they are substrates of the HUL-2 RING finger E3 ligase (reviewed in refs. 1 and 11). In addition, aspartate-199 is required for ICP0 to efficiently promote the interaction of ubiquitylated cdc34 with proteasomes in the presence of MG132 and the proteasome-dependent degradation of cdc34 in infected cells (9, 12).On the basis of these observations, we proposed a model where the HUL-1 E3 ligase promotes the ubiquitylation of the E3 cdc34 whereas the HUL-2 E3 ligase promotes degradation of other cellular proteins (12). Thus HUL-2 may target proteins whose...

The fusion (F) protein of the paramyxovirus SV5 promotes both virus-cell and cell-cell fusion. Recently, the atomic structure at 1.4 A of an extremely thermostable six-helix bundle core complex consisting of two heptad repeat regions of the F protein has been described (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S Jardetsky, Mol. Cell 3, 309-319, 1999). To analyze the conformations of the F protein at various stages of the membrane fusion process and to understand further the role of formation of the six-helix bundle core complex in promotion of membrane fusion, antibodies to peptides corresponding to regions of the F protein were obtained. Major changes in F protein antibody recognition were found after cleavage of the precursor protein F(0) to the fusogenically active disulfide-linked heterodimer, F(1) + F(2), and antibodies directed against the heptad repeat regions recognized only the uncleaved form. A monoclonal antibody directed against the F protein showed increased recognition at the cell surface of the cleaved form of the F protein as compared to uncleaved F protein, again indicating changes in conformation between the uncleaved and cleaved forms of the F protein. Anti-peptide antibodies specific for the heptad repeat regions were unable to precipitate a synthetic protein that consisted of the heptad repeat regions separated only by a small spacer, suggesting that the antibodies are unable to recognize their target regions when the heptad repeats are present in the six-helix bundle core complex. Taken together, these data indicate that the six-helix bundle core complex is not present in the precursor molecule F(0) and that significant conformational changes occur subsequent to cleavage of the F protein.