The Herpes Simplex Virus Type 1 (HSV-1) Regulatory Protein ICP0 Interacts with and Ubiquitinates p53
2003; Elsevier BV; Volume: 278; Issue: 38 Linguagem: Inglês
10.1074/jbc.m300776200
ISSN1083-351X
AutoresChris Boutell, Roger D. Everett,
Tópico(s)Virus-based gene therapy research
ResumoHerpes simplex virus type 1 regulatory protein ICP0 contains a zinc-binding RING finger and has been shown to induce the proteasome-dependent degradation of a number of cellular proteins in a RING finger-dependent manner during infection. This domain of ICP0 is also required to induce the formation of unanchored polyubiquitin chains in vitro in the presence of ubiquitin-conjugating enzymes UbcH5a and UbcH6. These data indicate that ICP0 has the potential to act as a RING finger ubiquitin ubiquitin-protein isopeptide ligase (E3) and to induce the degradation of certain cellular proteins through ubiquitination and proteasome-mediated degradation. Here we demonstrate that ICP0 is a genuine RING finger ubiquitin E3 ligase that can interact with and mediate the ubiquitination of the major oncoprotein p53 both in vitro and in vivo. Ubiquitination of p53 requires ICP0 to have an intact RING finger domain and occurs independently of its ability to bind to the ubiquitin-specific protease USP7. Herpes simplex virus type 1 regulatory protein ICP0 contains a zinc-binding RING finger and has been shown to induce the proteasome-dependent degradation of a number of cellular proteins in a RING finger-dependent manner during infection. This domain of ICP0 is also required to induce the formation of unanchored polyubiquitin chains in vitro in the presence of ubiquitin-conjugating enzymes UbcH5a and UbcH6. These data indicate that ICP0 has the potential to act as a RING finger ubiquitin ubiquitin-protein isopeptide ligase (E3) and to induce the degradation of certain cellular proteins through ubiquitination and proteasome-mediated degradation. Here we demonstrate that ICP0 is a genuine RING finger ubiquitin E3 ligase that can interact with and mediate the ubiquitination of the major oncoprotein p53 both in vitro and in vivo. Ubiquitination of p53 requires ICP0 to have an intact RING finger domain and occurs independently of its ability to bind to the ubiquitin-specific protease USP7. Herpes simplex virus type 1 (HSV-1) 1The abbreviations used are: HSV-1, Herpes simplex virus type 1; GST, glutathione S-transferase; FACS, fluorescence-activated cell sorter; IE, immediate early; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; HCMV, human cytomegalovirus; PML, promyelocytic leukemia; CENP, centromere-associated proteins; EYFP, enhanced yellow fluorescent protein. is a common human pathogen that is capable of establishing a quiescent state of infection within sensory neurons following primary lytic infection of epithelial cells. HSV-1 gene expression during lytic infection occurs in a regulated temporal cascade with three groups of genes: immediate early (IE), early (E), and late (L) (reviewed in Ref. 1Roizman R. Knipe D.M. Fields B.N. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Knipe D.M. Fields Virology. 4th Ed. Vol. 2. Lippincott Williams & Wilkins, Philadelphia, PA2001: 2399-2459Google Scholar). Although the majority of IE genes have been associated with the regulation of viral gene expression, only the IE protein ICP0 (also known as Vmw110) is capable of trans-activating all three classes of viral genes (reviewed in Ref. 2Everett R.D. Bioessays. 2000; 22: 761-770Crossref PubMed Scopus (258) Google Scholar). Virus mutants that do not express ICP0 are severely impaired in their ability to replicate in limited passage human fibroblasts at low doses of input virus and are more likely to establish a quiescent infection. ICP0 is sufficient to reactivate quiescent viral genomes in both cultured cells and mouse models (2Everett R.D. Bioessays. 2000; 22: 761-770Crossref PubMed Scopus (258) Google Scholar, 3Preston C.M. J. Gen. Virol. 2000; 81: 1-19Crossref PubMed Scopus (133) Google Scholar, 4Halford W.P. Schaffer P.A. J. Virol. 2001; 75: 3240-3249Crossref PubMed Scopus (138) Google Scholar, 5Halford W.P. Kemp C.D. Isler J.A. Davido D.J. Schaffer P.A. J. Virol. 2001; 75: 6143-6153Crossref PubMed Scopus (99) Google Scholar). ICP0 has been reported to interact with various cellular proteins, including cyclin D3 (6Kawaguchi Y. Van Sant C. Roizman B. J. Virol. 1997; 71: 7328-7336Crossref PubMed Google Scholar), elongation factor EF-1δ (7Kawaguchi Y. Bruni R. Roizman B. J. Virol. 1997; 71: 1019-1024Crossref PubMed Google Scholar), and the transcription factor BMAL1 (8Kawaguchi Y. Tanaka M. Yokoymama A. Matsuda G. Kato K. Kagawa H. Hirai K. Roizman B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1877-1882PubMed Google Scholar), and it forms a strong and specific interaction with ubiquitin-specific protease USP7 (also known as HAUSP) (9Everett R.D. Meredith M. Orr A. Cross A. Kathoria M. Parkinson J. EMBO J. 1997; 16: 1519-1530Crossref PubMed Scopus (318) Google Scholar). Although the exact mechanism by which ICP0 functions has yet to be determined, one crucial feature is its zinc-binding RING finger domain. Recently, it has been demonstrated that the RING finger domain of ICP0 has ubiquitin E3 ligase activity (10Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar, 11Hagglund R. Van Sant C. Lopez P. Roizman B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 631-636Crossref PubMed Scopus (92) Google Scholar). E3 ligases provide the substrate specificity to mediate the transfer of ubiquitin from ubiquitin-conjugating enzymes (E2s) to substrate proteins targeted for ubiquitin-dependent proteasome-mediated degradation (reviewed in Ref. 12Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2944) Google Scholar). ICP0 has also been shown to induce the accumulation of co-localizing conjugated ubiquitin in vivo (13Everett R.D. J. Virol. 2000; 74: 9994-10005Crossref PubMed Scopus (90) Google Scholar) and to induce the proteasome-mediated degradation of certain cellular proteins, including the major ND10 (PML nuclear body) constituent proteins PML and Sp100 (14Everett R.D. Freemont P. Saitoh H. Dasso M. Orr A. Kathoria M. Parkinson J. J. Virol. 1998; 72: 6581-6591Crossref PubMed Google Scholar, 15Chelbi-Alix M.K. de The H. Oncogene. 1999; 18: 935-941Crossref PubMed Scopus (280) Google Scholar, 16Muller S. Dejean A. J. Virol. 1999; 73: 5137-5143Crossref PubMed Google Scholar, 17Parkinson J. Everett R.D. J. Virol. 2000; 74: 10006-10017Crossref PubMed Scopus (149) Google Scholar), the centromeric proteins CENP-A and CENP-C (18Lomonte P. Sullivan K.F. Everett R.D. J. Biol. Chem. 2001; 276: 5829-5835Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 19Everett R.D. Earnshaw W.C. Findlay J. Lomonte P. EMBO J. 1999; 18: 1526-1538Crossref PubMed Scopus (213) Google Scholar), and the catalytic subunit of DNA-dependent protein kinase (20Parkinson J. Lees-Miller S.P. Everett R.D. J. Virol. 1999; 73: 650-657Crossref PubMed Google Scholar). The degradation of all these proteins requires that ICP0 has an intact RING finger domain and provides strong evidence that ICP0 acts as an E3 ligase in vivo. However, to date, no specific substrate has been shown to be directly ubiquitinated by full-length ICP0. A number of DNA viruses have been shown to affect the stability of the major oncoprotein p53. The E6 protein of human papillomaviruses 16 and 18, in association with E6-AP, and the adenovirus E1B-55K/E4-orf6 complex, have both been shown to induce the degradation of p53 through ubiquitination (21Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1993) Google Scholar, 22Querido E. Blanchette P. Yan Q. Kamura T. Morrison M. Boivin D. Kaelin W.G. Conaway R.C. Conaway J.W. Branton P.E. Genes Dev. 2001; 15: 3104-3117Crossref PubMed Scopus (401) Google Scholar, 23Harada J.N. Shevchenko A. Pallas D.C. Berk A.J. J. Virol. 2002; 76: 9194-9206Crossref PubMed Scopus (183) Google Scholar). Conversely, the simian virus 40 (SV40) large T antigen and the adenovirus E1B protein have been shown to inhibit specific p53 transcriptional functions through its sequestration (24Lowe S.W. Ruley H.E. Genes Dev. 1993; 7: 535-545Crossref PubMed Scopus (613) Google Scholar, 25Pipas J.M. Levine A.J. Semin. Cancer Biol. 2001; 11: 23-30Crossref PubMed Scopus (170) Google Scholar). There is limited information on the fate of p53 during HSV-1 infection. A number of cellular proteins, including p53, have been shown to be recruited into HSV-1 DNA replication compartments (26Wilcock D. Lane D.P. Nature. 1991; 349: 429-431Crossref PubMed Scopus (202) Google Scholar), although the significance of this recruitment remains unclear. The available evidence suggests that the overall p53 levels are not greatly affected in the HSV-1 infection systems so far examined. Analysis of HSV-1 mutants restricted in their ability to express IE proteins has shown that cell cycle arrest occurs independently of p53 (27Hobbs 2nd, W.E. DeLuca N.A. J. Virol. 1999; 73: 8245-8255Crossref PubMed Google Scholar) and that the ICP0-induced mitotic block is a direct result of its ability to induce the degradation of CENP-A and CENP-C (19Everett R.D. Earnshaw W.C. Findlay J. Lomonte P. EMBO J. 1999; 18: 1526-1538Crossref PubMed Scopus (213) Google Scholar, 18Lomonte P. Sullivan K.F. Everett R.D. J. Biol. Chem. 2001; 276: 5829-5835Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Cellular levels of p53 are typically maintained at a low level where p53 turnover is tightly regulated by ubiquitination and proteasome-mediated degradation. Mdm2, the RING finger ubiquitin E3 ligase responsible for p53 ubiquitination, binds to the N terminus of p53, and in conjunction with UbcH5, mediates the ubiquitination of C-terminal lysine residues, resulting in its eventual degradation by the 26 S proteasome (28Fuchs S.Y. Adler V. Buschmann T. Wu X. Ronai Z. Oncogene. 1998; 17: 2543-2547Crossref PubMed Scopus (212) Google Scholar, 29Rodriguez M.S. Desterro J.M. Lain S. Lane D.P. Hay R.T. Mol. Cell Biol. 2000; 20: 8458-8467Crossref PubMed Scopus (309) Google Scholar, 30Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1604) Google Scholar). Stabilization of p53 has been shown to occur by several mechanisms including phosphorylation of p53 and ADP ribosylation factor-mediated sequestration of mdm2. These mechanisms result in the inhibition of mdm2 interacting with and therefore ubiquitinating p53 (reviewed in Ref. 31Woods D.B. Vousden K.H. Exp. Cell Res. 2001; 264: 56-66Crossref PubMed Scopus (292) Google Scholar). Recently, Li et al. (32Li M. Chen D. Shiloh A. Luo J. Nikolaev A.Y. Qin J. Gu W. Nature. 2002; 416: 648-653Crossref PubMed Scopus (802) Google Scholar) demonstrated an alternative mechanism by which p53 could be stabilized. They showed that USP7 could bind directly to p53 and that the ubiquitin protease activity of USP7 was sufficient to deubiquitinate p53 targeted for degradation by mdm2 (32Li M. Chen D. Shiloh A. Luo J. Nikolaev A.Y. Qin J. Gu W. Nature. 2002; 416: 648-653Crossref PubMed Scopus (802) Google Scholar). The authors proposed that this deubiquitination and consequent stabilization of p53 by USP7 provided an additional mechanism by which cells could regulate their p53 transcriptional response. Due to the strong and specific interaction formed between ICP0 and USP7 and the ability of ICP0 to act as a RING finger ubiquitin E3 ligase, we wanted to determine whether ICP0 had any effect on p53. Our results show that ICP0 can recruit p53 in vivo independently of other viral proteins and during viral infection and that it can interact directly with p53 in vitro, independent of its USP7-binding domain. This interaction, in conjunction with the E3 ligase activity, of ICP0 is sufficient to allow ICP0 to mediate the ubiquitination of p53 both in vitro and in vivo in a RING finger-dependent manner. We also demonstrate that U2OS cells infected with an HSV-1 mutant that fails to express functional ICP0 are more susceptible to UV-induced apoptosis then those infected with wild type HSV-1. These results demonstrate that ICP0 is a genuine ubiquitin E3 ligase and suggest that one of its functions is to influence p53-mediated pathways. Plasmids—pCDNA-p53 and pCDNA-mdm2 were kind gifts from Ron Hay (University of St. Andrews). pGex2T-p53 was a kind gift from Nick La Thangne (Glasgow University). Plasmids expressing the E2-conjugating enzymes UbcH5a, UbcH6, and cdc34 were gifts from Seth Sadis (Millennium Pharmaceuticals); the open reading frames were amplified from their respective pC-CMVFLAG backbone vectors by PCR and cloned into the pET28a vector (Novagen) as NdeI/NotI fragments allowing the insertion of sequences encoding a polyhistidine tag. pET3aUbcH7 and pTriexUbcH10 were kind gifts from Phil Robinson (University of Leeds). Plasmids pCI-110, pCI-FXE, pCI-M1, and pGex241 have been described previously (19Everett R.D. Earnshaw W.C. Findlay J. Lomonte P. EMBO J. 1999; 18: 1526-1538Crossref PubMed Scopus (213) Google Scholar, 10Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar). pCW7 (expressing polyhistidine-tagged ubiquitin) was a kind gift from Ron Kopito (University of Standford). Construction of Baculoviruses—Viruses Ac.ICP0His, Ac.FXEHis, Ac.110, Ac.FXE, and Ac.E52X have been described previously (10Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar, 33Meredith M. Orr A. Elliott M. Everett R. Virology. 1995; 209: 174-187Crossref PubMed Scopus (69) Google Scholar). Baculoviruses Ac.CMV.EYFP and Ac.CMV.EYFP-ICP0 were constructed to express the relevant proteins in mammalian cells as follows. Plasmid pEYFP-ICP0 contains the NcoI-HpaI genomic fragment containing the ICP0 coding region inserted into the SmaI site of pEYFP-C1 (Clontech) to express an enhanced yellow fluorescent protein (EYFP)-ICP0 fusion protein. The BglII-NdeI fragment of pCIneo that contains the 5′ part of the HCMV IE promoter region, was linked to the NdeI-HindIII fragment of pEYFP-ICP0 (containing the 3′ part of the HCMV promoter and the EYFP-ICP0 fusion fragment) and inserted into vector pFastBacHTa (Invitrogen) between its BamHI and HindIII sites. The resultant plasmid was used to generate recombinant baculovirus Ac.C-MV.EYFP-ICP0 by following the Bac-toBac protocol (Invitrogen). Similarly, the NdeI-XhoI EYFP coding region of pEYFP-C1 was inserted with the BglII-NdeI fragment of pCI-neo into the BamHI-SalI sites of pFastBacHTa, and the resultant plasmid was used to generate Ac.CMV.EYFP. Cells and Co-Transfection Assays—U2OS and mouse p53: mdm2(–/–) cells were grown in Dulbecco's modified Eagles' medium supplemented with 10% fetal calf serum. Cells were seeded into 35-mm dishes at a cell density of 1.5 × 105 cells/dish and transfected using LipofectAMINE Plus reagent (Invitrogen) with 10 ng of pCDNA-p53 plasmid, 50 ng of pCW7, and 100 ng of pCDNA-mdm2, pCI-110, pCI-FXE, or pCI-M1 according to the manufacturer's instructions. Promoter competition was balanced by including appropriate amounts of pCI-neo vector, and empty pUC-9 was used to bring the total DNA amounts to 500 ng. At 16 h after transfection, the cells were treated with MG132 (final concentration 10 μm) and incubated for an additional 8 h. Cell monolayers were subsequently washed twice in ice-cold phosphate-buffered saline before being harvested in either 1× SDS-PAGE boiling mix buffer containing 3 m urea and 25 mm dithiothreitol or 1 ml of guanidine HCl buffer A (phosphate-buffered saline plus 6 m guanidine HCl, 0.1% Nonidet P-40, 10 mm β-mercaptoethanol, and 5% glycerol), pH 8.0. His-tagged ubiquitinated proteins were isolated by nickel affinity chromatography using 35 μl of equilibrated nickel-nitrilotriacetic acid beads (Qiagen) per sample as described in Ref. 34Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (847) Google Scholar. Samples were resolved by 10% SDS-PAGE and Western blotted, and membranes were probed with either anti-p53 (Oncogene Ab-6) or anti-ICP0 (11060) monoclonal antibodies. Immunofluorescence and Confocal Microscopy—Aliquots of 1 × 105 U2OS cells were seeded onto 12-mm coverslips in 24-well dishes and infected with 50 plaque-forming units/cell of either Ac.CMV.EYFP or Ac.CMV.EYFP-ICP0. The cells were stained for immunofluorescence 16 h after infection using an anti-p53 monoclonal antibody (Oncogene Ab-6, 1500) and a secondary Cy5-conjugated goat anti-rabbit IgG antibody (Amersham Biosciences, 1500) and examined by confocal microscopy as described previously (10Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar). Human fetal foreskin fibroblast cells (HFFF-2; European Collection of Cell Cultures) were grown in Dulbecco's modified Eagles' medium supplemented with 10% fetal calf serum, seeded onto coverslips at 1 × 105 cells/well, and then infected with HSV-1 strain 17+ at a multiplicity of 10 plaque-forming units/cell or with HSV-1 virus FXE (35Everett R.D. J. Gen. Virol. 1989; 70: 1185-1202Crossref PubMed Scopus (145) Google Scholar) that expresses the FXE RING finger mutant ICP0 protein, at a multiplicity of 0.1 plaque-forming units/cell. The strain 17+ and FXE-infected cells were fixed at 2 or 24 h after infection respectively and then stained for ICP0 (monoclonal antibody 11060) and p53 (rabbit FL-393, Santa Cruz Biotechnology). Fluorescein isothiocyanateand Cy5-labeled secondary antibodies were used to prevent channel overlap. Expression and Purification of Proteins—Human ubiquitin-activating enzyme (E1), full-length ICP0, RING finger deletion mutant FXE, and GST-241 were purified as described (10Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar). Ubiquitin was purchased from Sigma. Clones expressing recombinant E2-conjugating enzymes and pGex2T-p53 were transformed into BL21 (DE3) pLysS bacteria, and single colonies were used to inoculate 100 ml YTB broth. Cultures were grown to mid-log phase at 37 °C before being induced with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside for 4 h at 25 °C. Bacterial pellets (equivalent to 10 ml of bacterial culture) were resuspended in 10 ml of the appropriate buffer, lysed by probe sonication, and clarified by centrifugation at 13,000 rpm for 10 min. UbcH5a, UbcH6, UbcH10, and cdc34 were purified by nickel affinity chromatography using 150 μl of equilibrated nickel-nitrilotriacetic acid beads (Qiagen) in buffer A (50 mm Hepes, pH 7.2, 150 mm NaCl, 10% glycerol, 0.1% Nonidet P-40, and 10 mm β-mercaptoethanol). Supernatants were tumbled at 4 °C for 1 h before being washed with 3 × 1 ml of buffer A plus 20 mm imidazole (pH 7.5). Recombinant protein was eluted from the beads in 300 μl of buffer A plus 250 mm imidazole (pH 7.5) and dialyzed against 50 mm Tris, pH 7.5, 50 mm NaCl, 2.5 mm β-mercaptoethanol, aliquoted, and stored at –70 °C. UbcH7 was purified by a combination of cation and anion exchange in buffer B (50 mm Tris (pH 7.5), 25 mm NaCl, 1 mm EDTA, 2 mm benzamidine, 5 mm β-mercaptoethanol). The soluble supernatant fraction was bound to 1 ml of Q-Sepharose (Sigma) for 1 h at 4 °C, and the flow-through was collected and bound to 1 ml of SP-Sepharose (Sigma) for 2 h at 4 °C. The column was extensively washed in buffer B before proteins were eluted using an NaCl gradient (0–0.6 m NaCl) in 50 mm Tris (pH 7.5) and 5 mm β-mercaptoethanol. Fractions containing UbcH7 were identified by SDS-PAGE and Coomassie Brilliant Blue staining. GST-p53 was purified in buffer C (100 mm Tris-HCl (pH 8.0), 150 mm NaCl, 5% glycerol, 0.1% Nonidet P-40, 5 mm β-mercaptoethanol) using 150 μl of equilibrated GST beads (50% w/v) for 1 h at 4 °C. The beads were subsequently washed three times with 1 ml of buffer C. GST-p53 was eluted from the beads in 200 μl of buffer C plus 50 mm reduced glutathione (pH 7.5) and dialyzed against buffer C to remove glutathione. GST-p53 Pull-down Assays—Frozen SF21 cell pellets (equivalent to 4 × 106 cells) infected previously for 72 h at a multiplicity of infection of 2 with recombinant baculoviruses expressing ICP0, E52X, FXE, or mock-infected were resuspended in 500 μl of buffer C plus protease inhibitors (Roche Applied Science). Cells were lysed by gentle bath sonication, and the extracts were clarified by ultracentrifugation at 30,000 rpm for 25 min at 4 °C. Extracts were precleared using 50 μl (50% w/v) of equilibrated GST beads in buffer C end-over-end for 45 min at 4 °C. 20 μl of GST beads prebound to either GST or GST-p53 were mixed with precleared supernatants for 2 h at 4 °C. The beads were washed five times with 200 μl in ice-cold buffer C. Soluble protein complexes were eluted from the beads in 30 μl of ice-cold buffer C containing 50 mm reduced glutathione (pH 7.5). The samples and 1100 input material were denatured by adding 10 μl of 1× SDS-PAGE boiling mix (as described above) and resolved by 7.5% SDS-PAGE and analyzed by Western blotting with an anti-ICP0 monoclonal antibody 11060. In Vitro Transcription/Translation and Ubiquitination Assays—In vitro transcription/translation was performed using the rabbit reticulocyte lysate system (Promega) in the presence of [35S]methionine following the manufacturer's instructions. In vitro ubiquitination assays were performed either in the presence of 0.5 μl of [35S]methionine-labeled substrate or in the presence of 50 ng of purified substrate protein. Reactions were carried out in buffer D (50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 5 mm ATP) containing an ATP regenerating system (10 mm creatine phosphate, 3.5 units/ml creatine kinase, 0.6 units/ml inorganic pyrophosphatase) containing 20 ng of E1, 50 ng of E2, 5 μg of ubiquitin, and 50 ng of purified full-length ICP0, RING finger mutant (FXE), or GST-241. Reactions were carried out in a final volume of 10 μl for 3 h at 28 °C and terminated by the addition of 5 μlof3× SDS-PAGE boil mix buffer containing 9 m urea and 100 mm dithiothreitol. Samples were resolved either by 7.5% SDS-PAGE or by 4–12% NuPAGE gels (Invitrogen). Gels were either Western blotted and probed with antiubiquitin (Santa Cruz Biotechnology P4D1) or with anti-p53 (Oncogene Ab-6) monoclonal antibodies or stained with Coomassie Brilliant Blue, destained, dried, and analyzed by phosphorimaging. FACS Analysis—Aliquots of 4 × 105 U2OS cells were seeded onto 35-mm dishes and were either mock-infected or infected with five plaque-forming units/cell with wild type HSV-1 syn17+ or dl1403 (ΔICP0) (35Everett R.D. J. Gen. Virol. 1989; 70: 1185-1202Crossref PubMed Scopus (145) Google Scholar), an HSV-1 mutant that fails to express ICP0. Following absorption, the cells were overlaid with medium containing 10 μm acycloguanosine to prevent the initiation of viral DNA replication. At 3 h after infection, the medium was removed, and then the cells were washed in phosphate-buffered saline and subjected to a UV dose of 50 J/m2 (UV cross-linker, Stratagene). The cells were subsequently incubated in normal medium again containing acycloguanosine, and then detached, and adherent cells were harvested 24 h after UV irradiation using cell dissociation buffer (Sigma). The cells were pelleted by centrifugation and washed twice with 1 ml in phosphate-buffered saline. Cell pellets were either stained directly for apoptotic markers using annexin V-PE apoptosis detection kit (BD PharMingen) following the manufacturer's instructions or fixed and permeabilized using FIX & PERM cell permeabilization kit (Caltag Laboratories) and stained for the presence of ICP4 using the monoclonal antibody 58 S (36Showalter S.D. Zweig M. Hampar B. Infect. Immun. 1981; 34: 684-692Crossref PubMed Google Scholar) and anti-mouse fluorescein isothiocyanate (Sigma) following the manufacturer's instructions. Cells were subsequently analyzed by flow cytometry (FACS Calibur, BD Biosciences). ICP0 Ubiquitinates p53 in Vitro—We have reported previously that full-length ICP0 has ubiquitin E3 ligase activity in vitro and generates unanchored polyubiquitin chains in the presence of E2-conjugating enzymes UbcH5a and UbcH6 (10Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar). This activity requires ICP0 to have an intact RING finger domain. To test whether p53 could be ubiquitinated by ICP0, radiolabeled p53 was incubated in the presence or absence of a number of purified ubiquitin-conjugating enzymes (Fig. 1). Ubiquitinated p53 was readily detected in reactions containing ICP0 and a full complement of ubiquitin conjugation enzymes; ubiquitin, E1, and an E2, either UbcH5a or UbcH6. No significant p53 ubiquitination could be detected if either ICP0 or ubiquitin were omitted from the reaction mixture. The RING finger deletion mutant of ICP0, termed FXE (deletion of amino acids 106–146), was inactive in this assay. To determine whether the RING finger domain of ICP0 was sufficient to induce the ubiquitination of p53, reactions were carried out either in the presence of full-length ICP0 or in the presence of an N-terminal fragment of ICP0 containing the RING finger (amino acids 1–241) that has efficient E3 ligase activity in vitro (10Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar). Ubiquitinated p53 was detected in complete reaction mixtures containing full-length ICP0 but not in those containing the RING finger fragment (Fig. 2A), despite the fact that this fragment is able to produce unanchored polyubiquitin chains with activity comparable with that of full-length ICP0 (Fig. 2B). This indicates that ubiquitination of p53 by ICP0 occurs in a substrate-specific manner and that ICP0 residues downstream of amino acid 241 are required for this activity. To confirm that ICP0 acted directly on p53 and not through additional proteins found within the rabbit reticulolysate used for the production of the p53 substrate, GST-tagged p53 was purified from bacteria and was found to be ubiquitinated only in the presence of a full complement of ubiquitin-conjugating enzymes and ICP0 (Fig. 3). Therefore, ICP0 acts directly on p53 independently of other cellular proteins apart from those associated with the basic ubiquitination machinery. ICP0 has been reported previously by others to contain an additional E3 ligase domain within the C-terminal region of the protein (amino acid residues 616–680) (11Hagglund R. Van Sant C. Lopez P. Roizman B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 631-636Crossref PubMed Scopus (92) Google Scholar, 37Hagglund R. Roizman B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7889-7894Crossref PubMed Scopus (48) Google Scholar). The authors reported that this domain of ICP0 stimulated the production of autoubiquitinated forms of the E2-conjugating enzyme cdc34 (UbcH3). However, in our original E2 screening assay using full-length ICP0, we could not detect the formation of any polyubiquitinated proteins in the presence of cdc34 (10Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar). To test whether ICP0 stimulated the activity of cdc34 in a substrate-dependent manner, p53 ubiquitination reactions were performed utilizing a variety of different E2-conjugating enzymes. Ubiquitination of p53 was only detected in the presence of UbcH5a and UbcH6 and not with UbcH7, UbcH10, or cdc34 (Fig. 4), despite all the E2 preparations being able to form thiol-ester intermediates with ubiquitin (data not shown). ICP0 Interacts with p53 Independent of Its USP7-binding Domain—ICP0 forms a strong and specific interaction with USP7, resulting in increased amounts of USP7 co-localizing with ICP0 in ND10 domains during the early stages of infection (9Everett R.D. Meredith M. Orr A. Cross A. Kathoria M. Parkinson J. EMBO J. 1997; 16: 1519-1530Crossref PubMed Scopus (318) Google Scholar). This interaction has been mapped to a region containing two lysine residues within the C-terminal region of ICP0 (33Meredith M. Orr A. Elliott M. Everett R. Virology. 1995; 209: 174-187Crossref PubMed Scopus (69) Google Scholar). To test whether the USP7-binding domain is required for ICP0 to interact with p53, GST-p53 pull-down assays were performed using soluble extracts containing ICP0 or ICP0 mutant proteins (Fig. 5). GST-p53, but not GST, formed soluble complexes with full-length ICP0, the RING finger deletion mutant FXE (deletion of amino acids 106–146), and the USP7 negative binding mutant E52X (deletion of amino acids 594–775) (Fig. 5B). These data indicate that p53 binds to ICP0 via sequences that do not include its RING finger or USP7-binding domains. As p53 binds to E52X but is not ubiquitinated by the N-terminal RING finger domain of ICP0 (Fig. 2), the p53 interaction domain appears to be located between amino acids 241 and 594 of ICP0. Consistent with this interpretation, we found that the E52X protein ubiquitinated p53 with activity comparable with that of full-length ICP0 (data not shown), demonstrating that ICP0 does not require its USP7-binding domain, nor an interaction with USP7, to bind to and ubiquitinate p53. ICP0 Ubiquitinates and Recruits p53 in Vivo—To determine whether ICP0 could ubiquitinate p53 in vivo, p53(–/–): mdm2(–/–) mouse cells were transfected with plasmids expressing p53, polyhistidine-tagged ubiquitin, mdm2, wild type ICP0, or various ICP0 mutants. The cells were treated with the proteasome inhibitor MG132 at 16 h after transfection and incubated for a further 8 h before analysis (Fig. 6). Ubiquitinated p53 in whole cell extracts could readily be detected in the presence of p53 and mdm2 (Fig. 6A) but not in the presence of any of the ICP0 proteins tested (Fig. 6B). As p53 undergoes numerous post-translational modifications, including phosphorylation, sumoylation, and acetylation, we investigated whether a minor subpopulation of p53 molecules could be ubiquitin
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