The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity

Abstract

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 1-infected cell protein 0 contains two E3 ubiquitin ligase sites specific for different E2 ubiquitin-conjugating enzymes

“…Substrateindependent in vitro ubiquitylation reactions (17) were performed in 30 l of ubiquitylation buffer (50 mM Tris, pH 7.5͞2.5 mM MgCl 2 ͞0.5 mM DTT) containing 40 ng of recombinant rabbit Ub-activating enzyme E1 (Calbiochem, catalog no. 662070), 2 g of biotinylated Ub (Affiniti Research Products, Mamhead, Exeter, U.K., catalog no.…”

“…In addition to the interaction of USP7 with the carboxy terminus, ICP0 dynamically associates with the 26S proteasome in untreated cells but remains bound to proteasomes in cells treated with the proteasome inhibitor MG132 (17,18). ICP0 similarly promotes the dynamic association of the E2 enzyme cdc34 with the proteasome in a manner dependent on aspartate 199 (17).…”

“…In addition to the interaction of USP7 with the carboxy terminus, ICP0 dynamically associates with the 26S proteasome in untreated cells but remains bound to proteasomes in cells treated with the proteasome inhibitor MG132 (17,18). ICP0 similarly promotes the dynamic association of the E2 enzyme cdc34 with the proteasome in a manner dependent on aspartate 199 (17). Finally, a domain of ICP0 exon 3 (amino acids 543-768) functions as an E3 Ub ligase in conjunction with cdc34 in a substrate-independent in vitro ubiquitylation system, whereas the domain encoded by exon 2 (amino acids 20-241), which contains a RING finger motif, binds cdc34 (17).…”

“…ICP0 similarly promotes the dynamic association of the E2 enzyme cdc34 with the proteasome in a manner dependent on aspartate 199 (17). Finally, a domain of ICP0 exon 3 (amino acids 543-768) functions as an E3 Ub ligase in conjunction with cdc34 in a substrate-independent in vitro ubiquitylation system, whereas the domain encoded by exon 2 (amino acids 20-241), which contains a RING finger motif, binds cdc34 (17). Thus, ICP0 differs from other RING finger E3 ligases in that the domain containing the RING finger binds E2, whereas the ligase activity maps to a different region of the protein.…”

“…We report that ICP0 encodes an additional distinct E3 ligase activity in residues 20-241 containing the RING finger. This site specifically functions in conjunction with a different set of E2 Ub-conjugating enzymes than the E3 activity mapping at the carboxyl-terminal domain of ICP0 (17). Additionally, ICP0 interacts with and promotes the degradation of the E2 enzyme cdc34 in a manner partially dependent on aspartate 199.…”