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.

Figure 1

Antibody to ICP0 pulls down the HC8 proteasome subunit from cells infected with wild-type virus and treated with MG132. HEL fibroblasts infected with 10 pfu of HSV-1(F) per cell were mock-treated or treated for 1 h before harvest at 5, 10, or 18 h after infection, lysed, and reacted with polyclonal rabbit antibody against ICP0-exon 2. The precipitate was solubilized, subjected to electrophoresis in a denaturing gel, and reacted with mouse monoclonal antibody against HC8.

Figure 2

Monoclonal antibody to XAPC7 pulls down cdc34 from lysates of HEL fibroblasts infected with wild-type HSV-1(F) and treated with MG132. Replicate cultures of HEL fibroblasts were infected with 10 pfu or HSV-1(F) or R7914 mutant virus per cell and either left untreated or treated with MG132 for 1 h before harvest at 9 h after infection. The immune precipitates obtained with monoclonal antibody against XAPC7 proteasome subunit were electrophoretically separated in denaturing gels and reacted with polyclonal rabbit serum made against cdc34.

Figure 3

The domain encoded by the carboxyl terminus of exon 3 of ICP0 acts as an E3 ligase, whereas the sequences encoded by exon 2 bind cdc34 ubiquitin-conjugating enzyme. (A) Immunoblots of electrophoretically separated products of substrate-independent in vitro ubiquitination reactions. GST (lanes 1 and 2), GST-exon 2 (lanes 3 and 4), GST-exon 3 (lanes 5 and 6), and no additional protein (lane 7) were added to the substrate-independent in vitro ubiquitination reaction master mix (MM) containing recombinant Uba1 (E1), recombinant cdc34, biotinylated ubiquitin, and ubiquitination buffer in the presence and absence of ATP and an ATP regenerating system, as described in Materials and Methods. The reaction was stopped after 90 min, and the reaction mixture was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. (B) Electrophoretically separated reaction mixtures containing the indicated GST fusion protein in addition to the master mix (lanes 8–13) or the master mix alone (lane 14) in the presence and absence of ATP and an ATP regenerating system were probed with a rabbit polyclonal antibody directed against GST. (C) cdc34 was precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 15–17) or the master mix alone (lane 18) in the presence of ATP and an ATP regenerating system. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. (D) GST or GST fusion proteins were precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 19–21) or the master mix alone (lane 22) in the presence of ATP and an ATP regenerating system by using glutathione Sepharose beads. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with a rabbit polyclonal antibody directed against cdc34. The dots to the right of high molecular weight bands in lanes 6 and 17 identify ubiquitinated proteins; G, GST; 2, GST-exon 2 chimeric protein; 3, GST-carboxyl-terminal domain of exon 3 fusion protein.

Figure 4

ICP0 does not undergo proteasome-dependent degradation. HEL fibroblasts infected with 10 pfu of HSV-1(F) (lanes 2–7) or R7914 (lanes 9–14) per cell were mock-treated or treated with MG132 for 1 h before harvest at indicated times. The lysates were solubilized and subjected to electrophoresis in denaturing gels and reacted with a mouse monoclonal antibody against ICP0.

Figure 5

Model of the shuttling of ICP0 between nucleus and cytoplasm. As discussed, the results of this and preceding studies from this laboratory suggest that ICP0 shuttles between nucleus and cytoplasm depending on the nature of the dynamic association of the protein with proteasomes. In the presence of MG132, ICP0 is sequestered by proteasomes in nuclei either early or late in infection. In the absence of the drug, ICP0 is retained primarily in the nucleus early in infection and in cytoplasm at midpoint and later times after infection.

Figure 6

Models for E3 ubiquitin ligase function. E1 ubiquitin-activating enzymes are shown in dark blue, E2 ubiquitin-conjugating enzymes in green, RING finger subunits and domains in yellow, substrate-binding subunits and domains in magenta, and substrates in gray. (A) Multicomponent E3 complex modeled after SCF (adapted from ref. 40). The substrate is recruited to the complex by a specific substrate-binding domain within a protein that contains an F-box motif. The F-box interacts with SCF components Skp1 and Rbx1. Recruitment of substrate p27 to SCF by the leucine-rich repeat (LRR) of the F-box protein Skp2 is depicted here. Other F-box proteins recruit different substrates (reviewed in ref. 41). SCF contains cullin 1 (CUL1), whereas other cullins are components of other multicomponent E3s. The cullin serves as a scaffold to bind Skp1, the RING finger protein Rbx1, and the E2 cdc34, which also interacts the RING finger. (B) RING finger unimolecular E3 ubiquitin ligase modeled after c-Cbl (adapted from ref. 25). The RING finger interacts with and allosterically activates the E2, UbcH4 (35). A Src-homology 2 (SH2) domain binds phosphotyrosine, such as platelet-derived growth factor receptor β (PDGF-Rβ). (C) Proposed model for ICP0 unimolecular E3 ubiquitin ligase activity. Cdc34 binds the RING finger domain in ICP0-exon 2. Unidentified substrates may bind exon 3, facilitating the transfer of ubiquitin from E2 to the substrate. Also, it is possible that ICP0 E3 activity catalyzes regulatory self-ubiquitination, which does not target ICP0 for degradation.