Adenovirus E4orf3 Targets Transcriptional ... - Journal of Virology

Adenovirus E4orf3 Targets Transcriptional Intermediary Factor 1␥ for Proteasome-Dependent Degradation during Infection Natalie A. Forrester,a Rakesh N. Patel,a Thomas Speiseder,b Peter Groitl,b Garry G. Sedgwick,a,c Neil J. Shimwell,a Robert I. Seed,a,d Pól Ó Catnaigh,a Christopher J. McCabe,a,d Grant S. Stewart,a Thomas Dobner,b Roger J. A. Grand,a Ashley Martin,a and Andrew S. Turnella School of Cancer Sciencesa and School of Experimental Medicine,d College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom; Heinrich Pette Institute for Experimental Virology, Hamburg, Germanyb; and BRIC, University of Copenhagen, Copenhagen, Denmarkc

The ability of adenovirus early region proteins, E1B-55K and E4orf6, to usurp control of cellular ubiquitin ligases and target proteins for proteasome-dependent degradation during infection is well established. Here we show that the E4 gene product, E4orf3 can, independently of E1B-55K and E4orf6, target the transcriptional corepressor transcriptional intermediary factor 1␥ (TIF1␥) for proteasome-mediated degradation during infection. Initial mass spectrometric studies identified TIF1 family members— TIF1␣, TIF1␤, and TIF1␥—as E1B-55K-binding proteins in both transformed and infected cells, but analyses revealed that, akin to TIF1␣, TIF1␥ is reorganized in an E4orf3-dependent manner to promyelocytic leukemia protein-containing nuclear tracks during infection. The use of a number of different adenovirus early region mutants identified the specific and sole requirement for E4orf3 in mediating TIF1␥ degradation. Further analyses revealed that TIF1␥ is targeted for degradation by a number of divergent human adenoviruses, suggesting that the ability of E4orf3 to regulate TIF1␥ expression is evolutionarily conserved. We also determined that E4orf3 does not utilize the Cullin-based ubiquitin ligases, CRL2 and CRL5, or the TIF1␣ ubiquitin ligase in order to promote TIF1␥ degradation. Further studies suggested that TIF1␥ possesses antiviral activity and limits adenovirus early and late gene product expression during infection. Indeed, TIF1␥ knockdown accelerates the adenovirus-mediated degradation of MRE11, while TIF1␥ overexpression delays the adenovirus-mediated degradation of MRE11. Taken together, these studies have identified novel adenovirus targets and have established a new role for the E4orf3 protein during infection.

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uman adenoviruses (Ad) are small, nonenveloped viruses with a linear double-stranded DNA genome and are classified into species A to F according to various criteria (7). Since the observation that Ad12 could induce tumors in newborn rodents, Ad has served as a reliable model for dissecting the molecular basis of the key cellular signaling pathways that underlie the transformation process (28, 33, 69, 70). Studies investigating the roles of the Ad early region proteins in both Ad-transformed and Adinfected cells have led to key advances in the understanding of basic cellular processes and how Ad usurps control of these pathways in order to promote viral replication (8, 33, 67). The Ad early region proteins E1B-55K, E4orf3, and E4orf6 have a complex inter-relationship and serve together to regulate RNA processing, late viral mRNA nuclear export, the shutoff of host-cell protein synthesis, and neutralization of the host cell DNA damage response during infection (4, 29, 57, 61, 67, 73). They can also function synergistically and cooperate with E1A to promote Ad-induced cellular transformation (47–49). It is perhaps not surprising, therefore, that they share many common functions. For instance, E1B-55K interacts directly with p53 to repress transcriptional activity and also promotes p53 sumoylation and targeting to cytoplasmic aggresomes for degradation (41, 46, 53, 76, 77). E4orf6 also interacts directly with p53 to repress p53 transcriptional activity, while E4orf3 inhibits p53 function, by inducing the selective trimethylation of histone H3 K9 at p53 promoters and preventing p53 association with p53-responsive promoter elements (24, 62). Ad E1B-55K and E4orf6 interact directly and cooperate functionally during infection. It has been established that Ad5 E1B55K/E4orf6 recruit subcomplexes of the Cullin 5-containing ubiquitin ligase (CRL5), minimally comprising CUL5, elongins B

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and C, and Rbx1, to promote the ubiquitin- and proteasomedependent degradation of p53 (35, 54). A model has been proposed whereby E1B-55K serves as substrate receptor for p53, while E4orf6 recruits functional CRL5 to the E1B-55K/E4orf6 complex through elongin-interacting BC boxes within its primary sequence (11, 54). Targeting of CRL5 by Ad E1B-55K/E4orf6 has also been shown to be important for viral mRNA export (12, 75). Further studies with Ad5 have indicated that E1B-55K and E4orf6 cooperate to promote the degradation of the MRE11-RAD50NBS1 (MRN) component, MRE11, in order to inhibit ATM and ATR activation and also promote the degradation of DNA ligase IV in order to prevent nonhomologous end joining (5, 18, 64). The E1B-55K binding protein, E1B-AP5 (hnRNPUL1), also plays an important role in regulating ATR during Ad5 and Ad12 infection (9). By targeting ATM, ATR, and DNA ligase IV, E1B-55K and E4orf6 prevent the concatenation of linear double-stranded viral DNA during infection (73). More recent work suggests that E1B-55K/E4orf6 also targets the BLM helicase for degradation in order to inhibit DNA damage repair pathways and integrin ␣3 to possibly inhibit viral reinfection (23, 51). It appears, however, that the relationship between E1B-55K and E4orf6 and the ubiquitin-proteasome pathway is more complicated than previously thought. It has been determined, for in-

Received 18 October 2011 Accepted 22 December 2011 Published ahead of print 28 December 2011 Address correspondence to Andrew S. Turnell, [email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.06583-11

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stance, that Ad12 and Ad40 E1B-55K and E4orf6 utilize exclusively the CUL2-containing CRL2 to promote the degradation of p53 during infection, while Ad16 can utilize either CRL2 or CRL5 (10, 22). Further investigation has revealed that different Ad serotypes have evolved different strategies in order to neutralize the DNA damage response during infection such that the cohort of substrates targeted for degradation varies between viral serotypes (22, 32). In this regard, all Ads studied to date promote the degradation of DNA ligase IV, while they do not necessarily target p53 or MRE11 for degradation; the reasons for these differences await further investigation. Further complexity has been established, such that Ad12 E4orf6 can, independently of Ad12 E1B-55K, utilize CRL2 to promote the degradation of the ATR activator, TOPBP1, and inhibit CHK1 activation during infection (10). In this regard, Ad12 E4orf6 not only recruits CRL2 but also acts as a substrate receptor for TOPBP1 (10). Similarly, Ad5 E1B-55K can function independently of E4orf6 to recruit CRL5 and target the promyelocytic leukemia (PML) nuclear body component, Daxx, for proteasome-dependent degradation (59). Indeed, the SUMO1-conjugated E1B-55K-mediated degradation of Daxx is important for the ability of Ad to promote cellular transformation (60). It has been suggested that E4orf6 shares a number of redundant functions with the E4orf3 protein during infection. Indeed, these proteins have been shown to enhance viral DNA replication, initiate shutoff host-cell protein synthesis, stabilize the nuclear accumulation of late viral mRNAs, and promote late viral protein synthesis, enhancing the production of new virus particles (15, 16, 34, 39). Early work with E4orf3 indicated that it associated with the PML protein and was solely responsible for reorganizing PML-containing nuclear bodies (also called PML oncogenic domains or ND10) into distinctive nuclear track-like structures (19, 25). The importance of PML reorganization during infection was highlighted by observations indicating that overexpression of PML prevented the E4orf3-mediated reorganization of PML bodies and severely delayed adenovirus replication (25). Other work has established that E4orf3 targets the PML II isoform specifically in order to reorganize PML bodies into nuclear tracks and also recruits E1B-55K to these tracks during infection (38, 43). More recent work has determined that the transcriptional intermediary factor 1 (TIF1) family member, TIF1␣, is also recruited in an E4orf3-dependent manner to PML nuclear tracks during infection (78). Although E4orf3s from the divergent Ad serotypes Ad4, Ad5, Ad9, and Ad12 all recruit TIF1␣ to PML tracks, the functional importance of TIF1␣ reorganization during infection remains to be established (78). E4orf3, akin to E1B-55K/E4orf6, also targets DNA damage response and repair pathways during infection in order to prevent viral DNA concatenation and doublestrand break repair (DSBR). Although not conserved among other Ad serotypes, Ad5 E4orf3 sequesters MRN components in PML nuclear tracks prior to their degradation in cytoplasmic aggresomes, and E4orf3, like E4orf6, also interacts with the DNA-PK catalytic subunit, presumably to inhibit DSBR during infection (14, 18, 30, 42, 65). The TRIM/RBCC family of proteins are characterized by an N-terminal tripartite motif that contains a RING finger moiety, one or two zinc-binding motifs named B-boxes, and a coiled-coil domain which is necessary for oligomerization and association with subcellular structures (45, 55). The most prominent TRIM family member is TRIM19, the PML tumor suppressor protein, which is an integral component of PML nuclear bodies and in-

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volved in a number of diverse cellular processes, including cellular senescence, apoptosis, and DNA repair (13). TRIM proteins can, based on homologies, be divided into two main groups, and subdivided into 11 subfamilies (52). The TIF1 subfamily contains four members in mammals ␣ (TRIM24), ␤ (TRIM28), ␥ (TRIM33), and ␦ (TRIM66). TIF1 family members possess an N-terminal tripartite motif, C-terminal plant homeobox (PHD) and bromodomains, and a unique TIF1 signature sequence which is likely to participate in TIF1-dependent transcriptional regulation (72). Here we report that TIF1 family members are evolutionarily conserved targets for Ad early region proteins E1B-55K and E4orf3 during infection. We have determined that E1B-55K associates with TIF1␣, TIF1␤, and TIF1␥ in Ad5- and Ad12transformed and infected cells and that E4orf3 can, independent of E1B-55K, selectively reorganize TIF1␣ and TIF1␥ to PML nuclear bodies during infection. We have also identified a new function for E4orf3, namely, the ability to promote, independent of E1B-55K and E4orf6, the proteasome-mediated degradation of TIF1␥. We show that TIF1␥ possesses antiviral activities and suggest that Ad ablates these activities by targeting TIF1␥ for degradation during infection. Taken together, these results identify TIF1 proteins as major targets for Ad during infection and establish that E4orf3 utilizes the ubiquitin-proteasome pathway in order to promote the degradation of TIF1␥. MATERIALS AND METHODS Cells. Ad12 human embryo retinoblast (HER2) cells, Ad5 human embryo kidney 293 (HEK293) cells, A549 human small cell lung carcinoma cells, and HeLa human cervical carcinoma cells were all grown in HEPESmodified Dulbecco modified Eagle medium (DMEM; Sigma-Aldrich) supplemented with 8% (vol/vol) fetal calf serum (FCS) and 2 mM L-glutamine (Invitrogen). All cells were kept at 37°C in a humidified 5% CO2 atmosphere. A549 and HeLa cells were used interchangeably throughout the present study as reliable cell models for studying Ad infection and viral gene function. Viruses. Human wild-type (wt) Ad5 and wt Ad12 Huie viruses were from the American Type Culture Collection, while wt Ad3, wt Ad7, and wt Ad11 were generously provided by Joe Mymryk. The Ad5 and Ad12 E1B-55K deletion viruses (Ad5 dl1520, Ad12 dl620, and Ad12 hr703) and the E4orf3 (H5pm4150), E4orf6 (H5pm4154), and E4orf3/E4orf6 (H5pm4155) deletion viruses have all been described previously (6, 12, 17, 32, 63). Ad5 and Ad12 viruses were propagated on permissive HEK293 cells and HER3 cells, respectively, and titered by plaque assay on HER911 and HER3 cells, respectively. Plasmids and transfection. Ad5 expression constructs for E1B-55K, HA-E4orf3, and HA-E4orf6 have all been described previously (47–49, 56). FLAG-TIF1␥, Ad12 E1B-55K, HA-E4orf3, and HA-E4orf6 were cloned into pcDNA3 for mammalian expression. Plasmids were transfected into cells using Lipofectamine 2000 according to the manufacturer’s instructions. Antibodies. The monoclonal antibodies against Ad5 E1A (M58), Ad12 E1A (#13), Ad5 E1B-55K (2A6), Ad12 E1B-55K (XPH9), Ad5 E4orf3 (6A11), Ad5 E4orf6 (RSA3), p53 (DO-1), and hemagglutinin (HA) tag were all obtained as supernatant fluid from cultures of the appropriate hybridoma cell lines. The anti-TIF1␥, anti-Ad5 E1B-55K, and anti-Ad12 E1B-55K polyclonal antibodies were raised against respective glutathione S-transferase fusion proteins in conjunction with Eurogentec. Anti-TIF1␣, TIF1␤, p-TIF1␤ S824, and CUL5 antibodies were obtained from Bethyl Laboratories. Anti-RPA32 antibodies were from Calbiochem, anti-MRE11 and anti-NBS1 antibodies were from GeneTex, the CUL2 antibody was from Abcam, and the anti-␤-actin and anti-FLAG antibodies were from Sigma. The TOPBP1 and DNA ligase IV antibodies were

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Role of E4orf3 during Infection

gifts from Iain Morgan and Stephen Jackson, respectively. The anti-Ad5 penton and fiber antibody was from Vivien Mautner. Horseradish peroxidase-conjugated secondary anti-mouse and anti-rabbit antibodies used for Western blotting were from Dako. Secondary anti-mouse and anti-rabbit Alexa 488/546 antibodies used for immunofluorescence were from Molecular Probes. Ad infection. Infection was carried out using subconfluent monolayers of cultured cells in serum-free HEPES-buffered DMEM containing 2 mM L-glutamine. Prior to infection, the cells were washed twice in warmed phosphate-buffered saline (PBS), before addition of the virus at the appropriate infectivity ratio. After 2 h at 37°C, medium containing virus was removed and replaced with HEPES-buffered DMEM supplemented with 8% (vol/vol) FCS and 2 mM L-glutamine. Immunoprecipitation. The cells were washed twice in ice-cold saline, and whole-cell extracts were prepared in immunoprecipitation lysis buffer (50 mM Tris-HCl [pH 7.4], 1% [vol/vol] Nonidet-P40, 0.825 M NaCl). Lysates were clarified by sonication and centrifugation. Immunoprecipitation was carried out overnight at 4°C, typically with 10 ␮g of antibody per 5 mg of cell lysate. Antigen-antibody complexes were isolated using protein G-Sepharose (Sigma-Aldrich). Immunoprecipitates were washed five times in lysis buffer, eluted in sample buffer (9 M urea, 50 mM Tris-HCl [pH 7.4], and 0.15 M ␤-mercaptoethanol–10% sodium dodecyl sulfate [SDS; 2:1, vol/vol] containing 0.1% [wt/vol] bromophenol blue), and then separated by SDS-PAGE. SDS-PAGE and Western blot analysis. Cells were washed twice in ice-cold saline, and whole-cell extracts were prepared in lysis buffer containing 9 M urea, 50 mM Tris-HCl (pH 7.4), and 0.15 M ␤-mercaptoethanol. Samples were sonicated and then cleared by centrifugation. Protein concentrations were subsequently determined by Bradford assay (Bio-Rad). Typically, 50-␮g protein samples and immunoprecipitated samples were separated on 12% (wt/vol) polyacrylamide gels, run in the presence of 0.1 M Tris, 0.1 M Bicine, and 0.1% (wt/vol) SDS. Separated proteins were transferred onto nitrocellulose filters (Pall), followed by incubation for 1 h in TBST (0.1% [vol/vol] Tween 20 in Trisbuffered saline containing 150 mM NaCl and 20 mM Tris-HCl [pH 7.3]) buffer containing 5% (wt/vol) dried milk powder. Nitrocellulose filters were subsequently incubated in TBST-milk with the appropriate primary and secondary antibodies. Antigens were visualized by chemiluminescence. Immunofluorescence. The cells were grown on glass slides and infected with either wt Ad5 or wt Ad12 at 10 PFU/cell. At the appropriate time postinfection, cells were washed with PBS, treated with preextraction buffer (10 mM PIPES [pH 6.8], 20 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.5% Triton X-100) for 5 min, and then fixed in 4% (wt/vol) paraformaldehyde in PBS for 10 min. The cells were then rehydrated in PBS and blocked in blocking buffer (10% [vol/vol] FCS in PBS) for 1 h. After further washing in PBS, the cells were incubated with primary antibodies in 0.1% (vol/vol) FCS in PBS for 2 h. The slides were washed three times in PBS before incubation with secondary antibodies for 1 h. The cells were washed further in PBS and then mounted in Vectashield mounting medium containing DAPI (4=,6=-diamidino-2-phenylindole; Vector Laboratories). Images were obtained using a Zeiss LSM510-Meta laser-scanning confocal microscope and processed with associated software. In instances where images of single cells are presented, these images are representative of the population of cells studied. Transfection of siRNAs. TIF1␣ small interfering RNA (siRNA) oligonucleotides (CGACUGAUUACAUACCGGUtt) and TIF1␥ siRNA oligonucleotides (CCUGCAUCUAGAAAGUGAAdTdT) were purchased from Ambion. CUL2 and CUL5 smart pools were purchased from Dharmacon: the CUL2 sequences were GGAAGUGCAUGGUAAAUUU, CAU CCAAGUUCAUAUACUA, GCAGAAAGACACACCACAA, and UGGU UUACCUCAUAUGAUU, and the CUL5 sequences were GACACGACG UCUUAUAUUA, GCAAAUAGAGUGGCUAAUA, UAAACAAGCUUG CUAGAAU, and CGUCUAAUCUGUUAAAGAA. AllStars Negative Control siRNA was purchased from Qiagen. The cells were plated 24 h

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prior to transfection, so as to be 30% confluent the next day. Oligofectamine reagent (Invitrogen) was used to deliver siRNA duplexes into cells according to the manufacturer’s instructions. The cells were then incubated for 48 h prior to viral infection. Mass spectrometry. Protein bands detected by Coomassie Brilliant Blue G-250 staining were excised and then washed twice with a solution containing 50 mM ammonium bicarbonate and 50% (vol/vol) acetonitrile by agitation for 45 min at 37°C. The protein bands were then incubated under reducing conditions (50 mM dithiothreitol and 50 mM ammonium bicarbonate in 10% [vol/vol] acetonitrile) for 1 h at 56°C prior to 30 min of incubation at room temperature in the dark in an alkylating solution containing 200 mM iodoacetamide, 50 mM ammonium bicarbonate, and 10% (vol/vol) acetonitrile. Protein samples were then washed three times in a solution containing 40 mM ammonium bicarbonate and 10% (vol/vol) acetonitrile and dried in a DNA–mini-vacuum centrifuge for 1 h. The proteins were then digested by rehydration in 12.5 ␮g of modified trypsin (Sigma)/ml. After 1 h of incubation at room temperature, an equal volume of 40 mM ammonium bicarbonate in a 10% (vol/ vol) acetonitrile solution was added to each sample and left to incubate with agitation overnight at 37°C. The resulting peptides were then separated using a Bruker amaZon ion trap mass spectrometer and processed and analyzed by using the ProteinScape central bioinformatic platform (Bruker).

RESULTS

Identification of novel E1B-55K binding partners in Ad12 E1transformed cells. Since known E1B-55K binding proteins function predominantly in fundamental cellular signaling pathways, we undertook an investigation to define more completely the cellular proteins that interact with the less-well-characterized Ad12 E1B-55K protein in Ad12 E1-transformed HER2 cells. In order to identify novel E1B-55K-interacting proteins, we performed immunoprecipitation with an anti-Ad12 E1B-55K antibody, isolated protein bands following SDS-PAGE, and subsequently analyzed tryptic peptides on a Bruker amaZon ion trap mass spectrometer. The data were processed using the Bruker DatAnalysis software to select peaks for a Mascot database search to identify peptides using the Swiss-Prot protein database. In addition to identifying the known E1B-55K-binding partners, E1B-AP5 and MRE11, we also isolated members of the TIF1 family of transcriptional regulators (unpublished data). Of these, TIF1␥ was the most abundant E1B55K interacting protein isolated (unpublished data). E1B-55K associates with TIF1 family members in Adtransformed and Ad-infected cells. To validate these findings, we performed reciprocal coimmunoprecipitation, Western blot analyses. In accordance with our mass spectrometry data, these analyses revealed that the TIF1 family member, TIF1␥ was found associated with E1B-55K in Ad12 HER2 cells (Fig. 1A, panel i). In order to establish whether TIF1␥ is a general target for E1B-55K from different Ad serotypes, we also investigated whether TIF1␥ associated with E1B-55K in Ad5 HEK293 cells. Consistent with the notion that E1B-55K from different Ad serotypes target TIF1␥, these analyses revealed that Ad5 E1B-55K also associated with TIF1␥ in Ad-transformed cells (Fig. 1A, panel ii). Interestingly, the anti-Ad5 E1B-55K polyclonal antibody used for the Western blot identified two E1B-55K-specific bands in Ad5 HEK293 cells; TIF1␥ associated exclusively with the lower-molecular-weight form (Fig. 1A, panel ii). In agreement with the mass spectrometry data, we also determined that TIF1␤ interacted with E1B-55K in both Ad12 HER2 and Ad5 HEK293 cells (Fig. 1B, panels i and ii). In order to establish whether TIF1␥ was also targeted by Ad E1B-55K during infection, we performed coimmunoprecipitation

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FIG 1 Ad E1B-55K associates with TIF1 family members in Ad-transformed and Ad-infected cells. (A) E1B-55K associates with TIF1␥ in Ad12- and Ad5transformed cells. Ad E1B-55K and TIF1␥ were immunoprecipitated from Ad12 HER2 cells (i) and Ad5 HEK 293 cells (ii) and Western blotted for TIF1␥ and E1B-55K, respectively. (B) E1B-55K associates with TIF1␤ in Ad12- and Ad5-transformed cells. Ad E1B-55K was immunoprecipitated from Ad12 HER2 cells (i) and Ad5 HEK 293 cells (ii) and Western blotted for TIF1␤ or E1B-55K. (C) Ad E1B-55K associates with TIF1␥ in Ad12- and Ad5-infected cells. Ad E1B-55K and TIF1␥ were immunoprecipitated from mock-infected and Ad-infected HeLa cells and Western blotted for TIF1␥ (i and ii). TIF1␥ was immunoprecipitated from Ad12- and Ad5-infected HeLa cells and Western blotted for E1B-55K (iii and iv). WCE, whole-cell extract.

experiments from Ad12- and Ad5-infected HeLa cells and assessed the binding of TIF1␥ to E1B-55K by Western blot analysis. In agreement with the binding studies from Ad-transformed cells, TIF1␥ was found to be associated with Ad12 and Ad5 E1B-55K in Ad-infected cells despite an overall reduction in TIF1␥ levels after infection (Fig. 1C, panel i [Ad12] and panel ii [Ad5]). The ability of anti-TIF1␥ antibodies to immunoprecipitate TIF1␥ following infection reflects the limiting amount of TIF1␥ antibody used, and the apparently large pool of TIF1␥ in the cell extracts; overexposure of Western blots indicates that TIF1␥ is expressed in infected cells, albeit at considerably lower levels (data not shown). Consistent with these data, TIF1␥ coimmunoprecipitated E1B-55K from both Ad12- and Ad5-infected HeLa cells (Fig. 1C, panel iii [Ad12] and panel iv [Ad5]). Akin to the binding studies performed in Ad5 HEK293 cells, TIF1␥ also associated with the lower-molecularweight form of E1B-55K in Ad5-infected HeLa cells (Fig. 1C, panel iv). Taken together, these data establish TIF1␥ is a common target for evolutionarily divergent Ad E1B-55K species in both Adinfected and AdE1-transformed cells.

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TIF1␥ is reorganized to PML-containing nuclear tracks in Ad5- and Ad12-infected cells. Since cellular proteins targeted by Ad during infection are often reorganized into discrete intranuclear locations by the particular viral proteins, they associate with, we next used immunofluorescence confocal microscopy to determine the subcellular localization of TIF1␥ early during Ad infection. Since E1B-55K can be found within viral replication centers (VRCs) during infection, we initially assessed whether TIF1␥ also colocalizes to VRCs during infection. To do this, we infected HeLa cells with either Ad5 or Ad12 and fixed the cells at 18 h postinfection. We then costained infected cells with TIF1␥ and the surrogate VRC marker, RPA32. In mock-infected cells TIF1␥ had a pan-nuclear staining distribution (Fig. 2A). Interestingly, TIF1␥ did not appear to associate with VRCs in infected cells, but instead tended to reorganize into extensive nuclear track-like structures in both Ad5- and Ad12-infected cells (Fig. 2A). Since it has previously been established that the TIF1 family member, TIF1␣ is reorganized by E4orf3 to PML-containing nuclear tracks in Adinfected cells (78) we next assessed whether TIF1␥ colocalizes with E4orf3 and PML in Ad-infected cells. Consistent with the notion that TIF1␥ might be recruited along with TIF1␣ to PMLcontaining nuclear tracks, these analyses revealed that TIF1␥ colocalized with PML in both Ad5- and Ad12-infected cells (Fig. 2B). Indeed, further analyses revealed that TIF1␥ colocalized with Ad5 E4orf3 in Ad5-infected cells (Fig. 2B), although the lack of an Ad12 E4orf3 antibody precluded such an analysis in Ad12infected cells. In agreement with published work, we also showed that TIF1␣ was recruited to PML-nuclear tracks following both Ad5 and Ad12 infection (data not shown). To investigate whether TIF1␥ recruitment to PML nuclear tracks during infection is solely dependent upon E4orf3 expression, we transfected A549 cells with either Ad5 HA-E4orf3 or Ad12 HA-E4orf3 and assessed the distribution of TIF1␥ at 6 h posttransfection (Fig. 2C). Consistent with our earlier observations, TIF1␥ had a pan-nuclear distribution in the absence of viral gene expression (Fig. 2C). However, in response to either Ad5 or Ad12 HA-E4orf3 expression, TIF1␥ reorganized to nuclear track-like structures, where it colocalized with HA-E4orf3 (Fig. 2C). These data are consistent with the hypothesis that E4orf3 is solely responsible for TIF1␥ redistribution into PML-containing nuclear tracks. Since TIF1␣ and TIF1␥ are both reorganized to PMLcontaining nuclear tracks during infection, we next investigated whether TIF1␤ is also targeted to these structures. Akin to TIF1␣ and TIF1␥, TIF1␤ displayed a general pan-nuclear staining pattern with a few discrete foci in mock-infected cells (Fig. 2D). In contrast to TIF1␣ and TIF1␥, however, TIF1␤ was not reorganized to PML-containing nuclear structures during infection (Fig. 2D). Since we have shown previously that a proportion of TIF1␤ is phosphorylated after both Ad5 and Ad12 infection (32), we next investigated whether this phospho-TIF1␤ species was recruited to PML-tracks during infection. Thus, we costained mock-infected and Ad-infected cells for PML and phospho-TIF1␤ using a phospho-specific TIF1␤ antibody. As expected, we were unable to detect any phosphorylated TIF1␤ in mock-infected cells (Fig. 2E). Consistent with our previous work, we were able to detect phospho-TIF1␤ in both Ad5- and Ad12-infected cells, but significantly, phospho-TIF1␤ did not colocalize with PML-containing nuclear tracks in these cells (Fig. 2E). Taken together, these results establish that TIF1␥ is reorganized, along with TIF1␣, to E4orf3 and PML-containing nuclear tracks in Ad-infected cells and sug-

Journal of Virology


FIG 2 TIF1␥ is reorganized to PML nuclear tracks during infection. (A) TIF1␥ is not recruited to viral replication centers during infection. HeLa cells were either mock infected or infected with 10 PFU of wt Ad5 or wt Ad12/cell. At 18 h postinfection, the cells were fixed, permeabilized, and costained for TIF1␥ and RPA32. (B) TIF1␥ is recruited to PML nuclear tracks during infection. Ad-infected HeLa cells were fixed and permeabilized 18 h postinfection and costained with either TIF1␥ and PML or TIF1␥ and E4orf3. (C) Recruitment of TIF1␥ to nuclear tracks is dependent upon E4orf3 expression. A549 cells were transfected with Ad5 HA-E4orf3 or Ad12 HA-E4orf3. At 6 h posttransfection, the cells were fixed, permeabilized, and costained for TIF1␥ and E4orf3 (using an anti-HA antibody). (D) TIF1␤ is not reorganized to PML nuclear tracks during infection. Ad-infected HeLa cells were fixed and permeabilized 18 h postinfection and costained with TIF1␤ and PML. (E) Phospho-TIF1␤ is not reorganized to PML nuclear tracks during infection. Ad-infected HeLa cells were fixed and permeabilized at 18 h postinfection and costained with phospho-TIF1␤ and PML. In all instances, colocalization images were recorded using a Zeiss LSM510-Meta confocal microscope. Nuclei were stained with DAPI and are shown in blue.

gest that Ad effects upon TIF1␤ and TIF1␥ are functionally distinct. TIF1␥ is selectively targeted by divergent Ad serotypes during infection. During the course of our studies characterizing the ability of E1B-55K to interact with TIF1␥ in Ad-infected cells, we noted that there was appreciably less TIF1␥ protein in Ad-infected cells than mock-infected cells (Fig. 1C, panels i and ii, cf. lanes 7 and 8). We therefore decided to investigate these findings in more detail. To do this, we initially infected A549 cells with either Ad5 or Ad12 and determined TIF1␥ protein levels at appropriate times postinfection. In accordance with our earlier observations, there was considerably less TIF1␥ protein in A549 cells after both Ad5 and Ad12 infection (Fig. 3A). Significantly, and in agreement with earlier studies (78) TIF1␣ protein levels were not affected by Ad infection (Fig. 3A). In line with observations from a number of laboratories, p53 and the MRN component, MRE11, were both targeted for degradation during infection (Fig. 3A). Since we have recently identified considerable variation in the abilities of different Ad serotypes to target cellular proteins for proteasome-dependent degradation, we next investigated the capabilities of other Ad serotypes to regulate the expression of the TIF1␥ protein (32). In this regard, we chose to investigate the


ability of the group B viruses Ad3, Ad7, and Ad11, which we have shown previously to have a very restrictive capacity to target protein substrates for degradation. Indeed, to date, these viruses have only been shown to target DNA ligase IV for degradation (22, 32). Thus, we infected HeLa cells with the group B viruses Ad3, Ad7, and Ad11, along with Ad5 and Ad12, and determined the effects of infection upon TIF1␥ protein levels. Significantly, Western blot analysis revealed that the protein levels of TIF1␥ were reduced considerably following infection with Ad3, Ad7, and Ad11 (Fig. 3B). In agreement with our previous findings, Ad3, Ad7, and Ad11 were unable, however, to target the MRN component, NBS1, for degradation (Fig. 3B). Given that we also identified TIF1␤ as an E1B-55K binding protein (Fig. 1B), we next investigated whether infection with any of these Ad serotypes would affect TIF1␤ protein levels. Interestingly, infection with Ad3, Ad5, Ad7, Ad11, and Ad12 had no effect upon the levels of the TIF1␤ protein (Fig. 3B). Taken together, these data suggest that the TIF1 family member, TIF1␥, is a preferential and common target for divergent Ad serotypes and that Ad serotypes differentially regulate TIF1␣, TIF1␤, and TIF1␥. TIF1␥ is targeted for proteasome-dependent degradation during Ad infection. Since a number of cellular proteins are tar-

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FIG 4 TIF1␥ is targeted for proteasome-mediated degradation during infection. HeLa cells were infected in serum-free HEPES-buffered DMEM with 10 PFU of wt Ad5 (A) or wt Ad12 (B)/cell for 2 h. After 2 h of incubation in serum-containing HEPES-buffered DMEM, the cells were subsequently incubated in serum-containing HEPES-buffered DMEM containing dimethyl sulfoxide (DMSO) vehicle alone or DMSO containing 1 ␮M proteasome MG132 until they were harvested at appropriate times postinfection (h.p.i.). The cell lysates were then Western blotted for TIF1␥, DNA ligase IV, E1B-55K, and ␤-actin using the appropriate antibodies.

FIG 3 TIF1␥ is targeted for degradation during Ad infection. (A) Ad5 and Ad12 target TIF1␥ for degradation during infection. A549 cells were either mock infected or infected with 10 PFU of wt Ad5 or wt Ad12/cell and then harvested at the appropriate times postinfection. Cell lysates were then Western blotted for TIF1␥, TIF1␣, p53, MRE11, Ad5 E1B-55K, Ad12 E1B-55K, and ␤-actin using the appropriate antibodies. (B) HeLa cells were either mock infected or infected with 10 PFU of wt Ad3, wt Ad5, wt Ad7, wt Ad11, or wt Ad12/cell and then harvested at the appropriate times postinfection (hours postinfection [h.p.i.]). The cell lysates were then Western blotted for TIF1␥, TIF1␤, NBS1, and ␤-actin.

geted for proteasome-dependent degradation during Ad infection, we next decided to examine the role of the proteasome in regulating TIF1␥ protein expression during infection. To do this, we first infected HeLa cells with Ad5 and Ad12 and subsequently incubated infected cells in the absence or presence of the proteasome inhibitor, MG132. In the absence of proteasome inhibitor, Ad5 and Ad12 infection promoted the loss of the TIF1␥ protein (Fig. 4). Significantly, however, treatment of cells with MG132 limited the loss of the TIF1␥ protein following Ad5 and Ad12 infection (Fig. 4). In support of these findings, MG132 had a similar propensity to limit the Ad5- and Ad12-mediated degradation of the known Ad substrate, DNA ligase IV (Fig. 4). Taken together, this series of experiments have revealed that Ad5 and Ad12 target TIF1␥ for proteasome-dependent degradation during infection. Ad does not utilize CRL2 or CRL5 to promote the degradation of TIF1␥. We and others have previously shown that different Ad serotypes can selectively utilize cellular Cullin ring ligases, CRL2 or CRL5 to promote the degradation of protein substrates, such as p53 and TOPBP1 (10, 22, 54). However, our previous studies have also suggested that the Ad-induced degradation of substrates, such as MRE11 and DNA ligase IV, are not dependent exclusively upon CRL2 or CRL5 (32). In light of these findings, we

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wanted to establish whether Ad utilized CRL2 or CRL5 to promote the degradation of TIF1␥. To do this, we initially ablated the expression of the CUL2 or CUL5 proteins in HeLa cells by RNAi, using siRNAs targeted against the CUL2 and CUL5 mRNAs. We then infected these cells with Ad5 or Ad12 and determined the cellular levels of the TIF1␥ protein at appropriate times postinfection (Fig. 5). These experiments revealed that knockdown of CUL2 or CUL5 had no effect on the ability of Ad5 or Ad12 to promote the proteasome-mediated degradation of TIF1␥ (Fig. 5). Consistent with our previous findings, however, CUL5 knockdown did inhibit the ability of Ad5 to promote the degradation of p53 (Fig. 5A), and CUL2 knockdown did inhibit the ability of Ad12 to promote the degradation of TOPBP1 (Fig. 5B). In accordance with our previous findings, the knockdown of CUL2 or CUL5 did not, however, prevent the Ad5- or Ad12-dependent degradation of MRE11 (Fig. 5). These data suggest that Ad5 and Ad12 do not utilize CRL2 or CRL5 in order to promote the degradation of TIF1␥ during infection. Ad does not utilize TIF1␣ to promote the degradation of TIF1␥ during infection. Since TIF1␣ is reorganized along with TIF1␥ to PML-containing nuclear tracks during infection but is not targeted for proteasomal degradation, we reasoned that Ad might utilize TIF1␣ ubiquitin ligase activity in order to promote the degradation of TIF1␥. To investigate this possibility, we first ablated the expression of the TIF1␣ protein in HeLa cells by RNA interference (RNAi), subsequently infected knockdown cells with Ad5 and Ad12, and then monitored TIF1␥ protein levels at appropriate times postinfection (Fig. 6). Although Western blot analyses revealed that TIF1␣ expression in HeLa cells was successfully ablated following treatment with siRNAs specific for TIF1␣ (Fig. 6), both Ad5 and Ad12 retained the ability to target TIF1␥ for degradation (Fig. 6). It appears, therefore, that Ad does not utilize

Journal of Virology


FIG 6 TIF1␣ does not promote the Ad-mediated degradation of TIF1␥ during infection. HeLa cells were treated with nonsilencing siRNAs (siCTRL) or siRNAs against TIF1␣. At 48 h posttransfection, the cells were either mock infected or infected with 10 PFU/cell Ad5 (A) or Ad12 (B). At appropriate times postinfection (h.p.i.), the cells were harvested and Western blotted for TIF1␥, TIF1␣, E1B-55K, and ␤-actin.

FIG 5 CRL2 and CRL5 are not responsible for the Ad-mediated degradation of TIF1␥ during infection. HeLa cells were treated with nonsilencing siRNAs (siCTRL) or siRNAs against CUL2 or CUL5. At 48 h posttransfection, the cells were subsequently infected with either 10 PFU/cell Ad5 (A) or Ad12 (B). At appropriate times postinfection (h.p.i.), the cells were harvested and Western blotted for TIF1␥, p53, TOPBP1, MRE11, CUL5, CUL2, E1B-55K, and ␤-actin.

TIF1␣ in order to promote the degradation of TIF1␥ during infection. TIF1␥ is targeted for degradation in an E4orf3-dependent manner during infection. It is well established that Ad E1B-55K and E4orf6 proteins cooperate to promote the ubiquitin- and proteasome-dependent degradation of numerous cellular proteins during infection. More recent data suggest that E1B-55K and E4orf6 can also function independently to target an, as yet, more limited pool of cellular proteins for degradation. Indeed, the findings presented here suggest that E1B-55K and E4orf3 both have the capacity to interact with TIF1␥ during infection. Given this added complexity, we deemed it important to establish which adenoviral proteins were required to promote the degradation of TIF1␥ during infection. To resolve this, we initially infected HeLa cells with wt Ad5 and a selection of Ad5 mutants that express different complements of E1B-55K, E4orf3, and E4orf6 proteins and then monitored the expression of TIF1␥ at appropriate times postinfection. Interestingly, akin to wt Ad5, E1B-55K (Ad5 dl1520), and E4orf6 (H5pm4154) deletion viruses retained their ability to promote the proteasome-mediated degradation of TIF1␥, suggesting that E1B-55K and E4orf6 do not cooperate in this process (Fig. 7A). Additional Western blots for early region


proteins confirmed the authenticity of the viruses used and that these proteins were expressed to similar levels (Fig. 7A). To delineate further the requirement for early region proteins in the degradation of TIF1␥, we next compared the abilities of wt Ad5, the E4orf3 deletion mutant, H5pm4150, and the E4orf3/ E4orf6 deletion mutant, H5pm4155, to promote the degradation of TIF1␥. As before, we infected A549 cells with these viruses and monitored TIF1␥ protein levels at suitable times after infection. Intriguingly, these experiments indicated that the E4orf3 and E4orf3/E4orf6 deletion viruses lost their capacity to target TIF1␥ for degradation (Fig. 7B). This experiment also reaffirmed the requirement for E4orf6 in the degradation of p53 (Fig. 7B). Again, Western blot analyses for early region proteins confirmed the authenticity and the infectivity ratios of the viruses used (Fig. 7B). Since there are no Ad12 E4 mutants available, we assessed the ability of Ad12 E1B-55K deletion viruses (dl620 and hr703) to promote TIF1␥ degradation. Consistent with the Ad5 studies, Ad12 E1B-55K deletion viruses retained their capacity to target TIF1␥ for degradation (Fig. 7C). As expected, however, these viruses lost their capacity to target MRE11, p53, and DNA ligase IV for degradation (Fig. 7C). Taken together, these data suggest that the viral proteins normally required for Ad-dependent degradation of target substrates, E1B-55K and E4orf6, are not required to promote the degradation of TIF1␥. Rather, these data suggest that degradation of TIF1␥ during infection is dependent upon the expression of the E4orf3 protein. Ad5 and Ad12 E4orf3 are solely responsible for targeting TIF1␥ for degradation. To investigate further the requirement for early region proteins E1B-55K, E4orf3, and E4orf6 in the Adinduced degradation of TIF1␥, we assessed individually the ability of these proteins to target TIF1␥ for degradation. To do this, we transfected HeLa cells with expression plasmids for either Ad5 or

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FIG 7 Role of Ad early region proteins in the targeting of TIF1␥ for degradation. (A) TIF1␥ degradation is not dependent upon Ad5 E1B-55K or E4orf6 expression. HeLa cells were either mock infected, infected with wt Ad5, or infected with E1B-55K (dl1520) or E4orf6 (H5pm4154) deletion viruses at 10 PFU/cell. At appropriate times postinfection (h.p.i.), the cells were harvested and Western blotted for TIF1␥, E1A, E1B-55K, E4orf3, E4orf6, and ␤-actin. (B) TIF1␥ degradation is dependent upon Ad5 E4orf3 expression. A549 cells were either mock infected, infected with wt Ad5, or infected with E4orf3 (H5pm4154) or E4orf3/E4orf6 (H5pm4155) deletion viruses at 10 PFU/cell. At appropriate times postinfection (h.p.i.), the cells were harvested and Western blotted for TIF1␥, p53, E1B-55K, E4orf3, E4orf6, and ␤-actin. (C) TIF1␥ degradation is not dependent upon Ad12 E1B-55K expression. HeLa cells were either mock infected, infected with wt Ad12, or infected with the Ad12 E1B55K deletion viruses dl620 or hr703 at 10 PFU/cell. At appropriate times postinfection (h.p.i.), the cells were harvested and Western blotted for TIF1␥, MRE11, p53, DNA ligase IV, E1A, E1B-55K, and ␤-actin.

Ad12, E1B-55K, HA-E4orf3, and HA-E4orf6. Since E1B-55K is known to associate with both E4orf3 and E4orf6, we also transfected HeLa cells with Ad5 or Ad12, E1B-55K/HA-E4orf3, and E1B-55K/HA-E4orf6. To evaluate the effects of early gene products upon TIF1␥, we harvested cell lysates at 24 h posttransfection and quantified TIF1␥ protein expression by Western blotting. Significantly, and in accordance with the data obtained using mutant viruses, only the expression of Ad5 E4orf3 or Ad12 E4orf3 was able

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to promote the degradation of TIF1␥ (Fig. 8A). Expression of E1B-55K or E4orf6 alone did not affect TIF1␥ protein levels (Fig. 8A). The coexpression of Ad5 or Ad12 E1B-55K with E4orf3 did not affect the ability of E4orf3 to promote TIF1␥ degradation, while coexpression of E1B-55K/E4orf6 did not affect TIF1␥ protein levels (Fig. 8A). These expression studies also revealed that expression of Ad12 E4orf3, but not Ad5 E4orf3, upregulated the expression of the p53 protein, which was more evident when E1B55K was coexpressed with E4orf3 (Fig. 8A). As expected, the expression of Ad5 E1B-55K also upregulated p53 protein levels, while the expression of Ad12 E1B-55K only upregulated p53 modestly (Fig. 8A). As anticipated, the expression of Ad5 or Ad12 E1B-55K and E4orf6 expression plasmids promoted p53 degradation (Fig. 8A). Since CUL2 and CUL5 knockdown did not affect the Admediated degradation of TIF1␥ (Fig. 5), we next investigated whether CUL2 or CUL5 knockdown affected specifically the E4orf3-dependent degradation of TIFI␥. To do this, we first ablated CUL2 and CUL5 expression in HeLa cells by RNAi and then transfected knockdown cells with either pcDNA3, Ad5 HAE4orf3, or Ad12 HA-E4orf3 (Fig. 8B). Consistent with our earlier observations, CUL2 or CUL5 knockdown had no affect on the ability of either Ad5 or Ad12 HA-E4orf3 to promote the degradation of TIF1␥ (Fig. 8B). In order to extend these observations and the TIF1␥-E4orf3 colocalization work presented in Fig. 2C, we next investigated by coimmunoprecipitation whether Ad E4orf3 associates with TIF1␥ in the absence of other Ad proteins in vivo. Therefore, we transfected HeLa cells with FLAG-tagged TIF1␥ and either Ad5 or Ad12 HA-tagged E4orf3 and then immunoprecipitated FLAG-tagged TIF1␥ and assessed E4orf3 binding by Western blotting. Consistent with the TIF1␥-E4orf3 colocalization data, immunoprecipitation of FLAG-tagged TIF1␥ coimmunoprecipitated HA-tagged Ad5 and Ad12 E4orf3 (Fig. 8C). Taken together, these data establish that E4orf3 binds specifically to TIF1␥ and is solely responsible for targeting TIF1␥ for degradation. In this regard, we have determined that E4orf3 does not employ the CRLs utilized by E1B-55K and E4orf6 to promote TIF1␥ degradation. TIF1␥ restricts Ad protein production and the Ad-mediated degradation of MRE11. In order to determine the significance of the Ad-mediated degradation of TIF1␥ during infection, we first investigated the consequences of TIF1␥ knockdown upon Ad protein expression and the Ad-mediated degradation of MRE11. To do this, we treated HeLa cells with either nonsilencing siRNA or TIF1␥ siRNA, infected knockdown cells with either Ad5 or Ad12, and then monitored Ad and MRE11 protein levels by Western blotting (Fig. 9A and B). Initial observations revealed that TIF1␥ expression had been successfully ablated by siRNA and that both Ad5 and Ad12 promoted TIF1␥ degradation in cells treated with nonsilencing siRNA (Fig. 9A and B). Significantly, although Ad5 and Ad12 both promoted the rapid degradation of MRE11 in cells treated with nonsilencing siRNA, the Ad-mediated degradation of MRE11 proceeded more rapidly in cells, where TIF1␥ expression had been ablated by siRNA (Fig. 9A and B). Consistent with these observations, Ad early region proteins were expressed at increased levels and, in some instances, at earlier times during infection in cells where TIF1␥ expression had been knocked down by siRNA (Fig. 9A and B). Late viral proteins were similarly expressed earlier during Ad5 infection of TIF1␥-knockdown cells relative to con-

Journal of Virology


FIG 9 TIF1␥ restricts the expression of Ad proteins and limits the Admediated degradation of MRE11. (A and B) TIF1␥ knockdown accelerates Ad infection. HeLa cells were treated with non-silencing siRNAs (siCTRL) or siRNAs against TIF1␥. At 48 h post-knockdown, the cells were infected with 10 PFU of Ad5 (A) or Ad12 (B)/cell. At appropriate times postinfection (h.p.i.), the cells were harvested and Western blotted for TIF1␥, MRE11, Ad5/Ad12 E1A, Ad5/Ad12 E1B-55K, Ad5 E4orf3, Ad5 E4orf6, Ad5 penton, Ad5 fiber, and ␤-actin. (C and D) TIF1␥ overexpression limits Ad infection. HeLa cells were transfected with either pcDNA3 alone or pcDNA3-FLAG-TIF1␥. At 24 h posttransfection, the cells were infected with 10 PFU/cell Ad5 (A) or Ad12 (B). At appropriate times postinfection (h.p.i.), the cells were harvested and Western blotted for TIF1␥, FLAG-TIF1␥, MRE11, Ad5/Ad12 E1A, Ad5/Ad12 E1B-55K, Ad5 E4orf3, Ad5 E4orf6, Ad5 penton, Ad5 fiber, and ␤-actin. FIG 8 Ad E4orf3 binds to TIF1␥ and promotes its degradation independently of CRLs and E1B-55K and E4orf6. (A) Ad E4orf3 promotes TIF1␥ degradation independently of E1B-55K and E4orf6. HeLa cells were transfected with pcDNA3 vector alone or pcDNA3 constructs for Ad5, Ad12, E1B-55K, HA-E4orf3, and HA-E4orf6. After 24 h, the cells were harvested and Western blotted for TIF1␥, p53, E1B-55K, HA-E4orf6, HA-E4orf3, and ␤-actin. (B) The Ad E4orf3-mediated degradation of TIF1␥ is not dependent upon CRL2 or CRL5. HeLa cells were treated with either control siRNA (siCTRL), CUL2 siRNA (siCUL2), or CUL5 siRNA (siCUL5). After 48 h knockdown, the cells were then transfected with either pcDNA3 vector alone, pcDNA3-Ad5 HA-E4orf3, or pcDNA3-Ad12 HA-E4orf3. At 24 h posttransfection, the cells were harvested and Western blotted for TIF1␥, CUL5, CUL2, HA-E4orf3, and ␤-actin. (C) E4orf3 associates with TIF1␥ in vivo. HeLa cells were transfected with pcDNA3 ⫹ Ad12 HA-E4orf3, pcDNA3 ⫹ Ad5 HA-E4orf3, pcDNA3 ⫹ FLAG-TIF1␥, Ad12 HA-E4orf3 ⫹ FLAG-TIF1␥, and Ad12 HA-E4orf3 ⫹ FLAG-TIF1␥. At 18 h posttransfection, FLAG-TIF1␥ was immunoprecipitated from cells with an anti-FLAG monoclonal antibody and subjected to Western blotting with an anti-HA antibody in order to detect HA-E4orf3. The levels of FLAG-TIF1␥ and HA-E4orf3 in cell lysates were also determined by Western blotting. WCE, whole-cell extract.


trol cells, although the lack of appropriate antibodies precluded such analyses for Ad12 (Fig. 9A and B). These data suggested that TIF1␥ expression might restrict Ad infection. To test this directly, we overexpressed TIF1␥ in HeLa cells and then, following either Ad5 or Ad12 infection, monitored Ad and MRE11 protein levels by Western blotting (Fig. 9C and D). In agreement with the notion that TIF1␥ might restrict Ad infection, the Ad5- and Ad12-mediated degradation of MRE11 was delayed, relative to control cells, in cells overexpressing TIF1␥ (Fig. 9C and D). Consistent with these observations, the expression of Ad5 and Ad12 early region proteins was similarly delayed in cells where TIF1␥ was overexpressed (Fig. 9C and D). Late viral proteins were also expressed later, relative to control cells, in Ad5infected cells where TIF1␥ was overexpressed (Fig. 9C and D). Taken in their entirety, these data are supportive of the proposi-

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tion that TIF1␥ restricts Ad infection and suggests that Ad has evolved to ablate the antiviral function of TIF1␥ by selectively targeting it for degradation during infection. DISCUSSION

Role for Ad E4orf3 in the targeting of TIF1␥ for ubiquitindependent proteolysis. It has long been established that early during infection E4orf3 reorganizes PML nuclear bodies to form distinct nuclear track-like structures that contain the cellular proteins PML, SP-100, the MRN complex, and TIF1␣ (19, 25, 65, 78). Of these, only the MRN component, MRE11, is subsequently targeted to cytoplasmic aggresomes for E1B-55K/E4orf6dependent degradation (2, 30, 44). More recently, it has been determined that E4orf3 can also repress specifically p53-dependent transcription epigenetically by promoting histone H3K9 trimethylation at p53-responsive genes (62). Work presented here identifies a new molecular function for E4orf3, namely, the evolutionarily conserved and selective ability, to promote the proteasome-dependent degradation of TIF1 family member, TIF1␥ (Fig. 3 to 8). Crucial in this respect, we have shown that the E4orf3-dependent degradation of TIF1␥ is wholly independent of E1B-55K and E4orf6 expression (Fig. 7 and 8). Indeed, expression of AdE4orf3 alone was shown to be sufficient to promote the degradation of TIF1␥ (Fig. 8). Immunofluorescence studies revealed that Ad E4orf3 reorganized TIF1␥ to PML tracks during infection (Fig. 2). Consistent with the ability of E4orf3 to reorganize TIF1␥ to nuclear tracks during infection, immunoprecipitation analyses revealed that E4orf3 associated with TIF1␥ in vivo (Fig. 8C). Given that TIF1␥ is not targeted to cytoplasmic aggresomes, we propose that E4orf3 promotes the degradation of TIF1␥ in the PML-containing nuclear tracks formed during infection. Given the established role of CUL2 and CUL5 containing CRLs in promoting E1B-55K-, E4orf6-, and E1B-55K/E4orf6dependent degradation of cellular substrates targeted by Ad, we investigated whether E4orf3 also utilized CRL2 or CRL5 to promote the degradation of TIF1␥. Interestingly, although we could prevent the Ad-dependent and CRL-mediated degradation of p53 and TOPBP1 during infection, we could not inhibit the Adinduced degradation of TIF1␥ (Fig. 5). We also determined that knockdown of CUL2 or CUL5 could not prevent the E4orf3dependent degradation of TIF1␥ (Fig. 8B). These data are consistent with observations suggesting that CUL2 or CUL5 are not required for the Ad-induced degradation of MRE11 or DNA ligase IV (32). Since TIF1␣ is also recruited to PML nuclear tracks during infection, we also investigated whether the TIF1␣ ubiquitin ligase promoted the AdE4orf3-mediated degradation of TIF1␥ (Fig. 6). These experiments revealed, however, that AdE4orf3 does not utilize TIF1␣ to promote TIF1␥ degradation during infection. Taken together, these data suggest that E4orf3 might utilize another cellular ubiquitin ligase activity in order to promote the degradation of TIF1␥. In support of this idea, other viruses have been shown to utilize distinct ubiquitin ligases in order to promote the degradation of specific cellular substrates. For instance, HPV E7 protein utilizes CRL2 to promote the degradation of the retinoblastoma protein, while the HPV E6 protein targets the HECT domain ubiquitin ligase, E6-AP to promote the degradation of p53 (40, 58). It is also possible that E4orf3 association with TIF1␥ activates the intrinsic ubiquitin ligase activity of TIF1␥ to promote TIF1␥ auto-ubiquitylation and degradation.

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Regulation of TIF1 family members TIF1␣, TIF1␤, and TIF1␥ by Ad E1B-55K and E4orf3. Initial mass spectrometric analyses identified TIF1 family members TIF1␣, TIF1␤, and TIF1␥ as Ad12 E1B-55K binding proteins (unpublished data). The ability of Ad5 and Ad12 E1B-55K to bind TIF1␤ and TIF1␥ was corroborated following reciprocal immunoprecipitationWestern blot analyses (Fig. 1). Since E1B-55K can bind to TIF1 family members in Ad5 and Ad12 E1-transformed cells, as well as Ad-infected cells, these data suggest that E1B-55K might be able to regulate the function of these proteins independently of E4orf3 (Fig. 1). Indeed, as E1B-55K associates directly with TIF1␤, whereas E4orf3 is unable to reorganize TIF1␤ to PMLcontaining nuclear tracks during infection, it is possible that E1B-55K and E4orf3 differentially and selectively regulate the functions of TIF1 family members (Fig. 1B and Fig. 2). Undoubtedly, the differential ability of E1B-55K and E4orf3 to regulate TIF1 family members is highlighted by the ability of Ad E4orf3 to promote TIF1␥ degradation independently of E1B-55K expression (Fig. 7). However, since E1B-55K can cooperate functionally with E4orf3 during infection and during the transformation process, it is possible that these proteins also function together to regulate TIF1 family member function. It is interesting that the polyclonal antiserum raised against the Ad5 E1B-55K protein recognizes two specific E1B55K protein species, of which TIF1␥ associates preferentially with the lower-molecular-weight form (Fig. 1). It is tempting to speculate that the higher-molecular-weight species might represent a posttranslationally modified form of Ad5 E1B-55K. Since TIF1␥ does not interact with the higher-molecularweight form of E1B-55K these data suggest that these distinct E1B-55K species perform different roles in transformed and infected cells. Given that TIF1␣, TIF1␤, and TIF1␥ were all initially characterized as transcriptional regulators and that both E1B-55K and E4orf3 can function as transcriptional repressors, it is possible that E1B-55K and E4orf3 target TIF1 family members to coordinately regulate host cell transcription programs during infection or transformation. It is interesting, however, that distinct functions for TIF1 family members have also been established. Recent work has determined that TIF1␣ ubiquitylates p53, targets it for proteasome-dependent degradation, and consequently inhibits p53-dependent apoptosis (1). It is possible, therefore, that at least part of the ability of E1B-55K and E4orf3 to repress p53 transcriptional activity is mediated through their interaction with TIF1␣. ⌱t has also recently been determined that TIF1␤ function is essential for heterochromatic DNA DSBR. It has been established that ionizing radiation induces the ATM-dependent phosphorylation of TIF1␤ and the 53BP1-dependent recruitment of phosphorylated TIF1␤ to ionization-radiation induced foci (50). Since E1B55K functions in concert with E4orf6 during infection to target the MRN component, MRE11, for proteasomal degradation (22, 64), it is possible that E1B-55K also targets TIF1␤ to further disrupt ATM signaling pathways during infection. The work presented here also suggests that E1B-55K and E4orf3 differentially regulate TIF1␥ (Fig. 1, 7, and 8). In addition to its known ability to regulate transcription, it has been determined that TIF1␥ also functions to modulate transforming growth factor ␤ (TGF-␤) signaling (26, 27, 36). It has recently been proposed that TIF1␥ also functions as a tumor suppressor for

Journal of Virology


mouse and human chronic myelomonocytic leukemia and that TIF1␣, TIF1␤, and TIF1␥ suppress murine hepatocellular carcinoma (3, 37). Given these observations, it is possible that E1B-55K and E4orf3 interact with TIF1␥ to modulate TGF-␤ signaling during infection or cellular transformation. Ad counteracts TIF1␥ antiviral activity during infection. It is becoming increasingly apparent that a large number of TRIM proteins exhibit antiviral activities. For instance, the prototypic TRIM protein, PML (TRIM19), can mediate the interferon response to viral infection and impairs virus replication by sequestering viral proteins, inhibiting the synthesis of viral mRNA, and inducing p53-dependent apoptosis (31). Viruses have therefore evolved to counteract such antiviral responses. Indeed, AdE4orf3 is believed to suppress PML function directly by disrupting PML bodies and reorganizing PML into nuclear track-like structures, whereas the herpes simplex virus type 1 ICP0 ubiquitin ligase antagonizes PML function by promoting its ubiquitin-mediated degradation (21). Given these findings, we investigated whether TIF1␥ possesses inherent antiviral activity during Ad infection (Fig. 9). In this regard, we determined that knockdown of TIF1␥ accelerates the Ad-mediated degradation of MRE11 during infection, while overexpression of TIF1␥ delays the Ad-mediated degradation of MRE11 (Fig. 9). In support of these findings, the expression of Ad early region, and late proteins, was evident earlier during infection in TIF1␥-knockdown cells relative to control cells (Fig. 9A and B) and, later, relative to control cells, in cells where TIF1␥ was overexpressed (Fig. 9C and D). Taken together, these data suggest that TIF1␥ limits Ad replication. Since TIF1␥ has not previously been reported to limit viral gene expression and modulate viral responses during infection, the work presented here is the first indication that TIF1␥ actively participates in antiviral responses. In support of the antiviral properties of TRIM proteins, one systematic screen of 55 TRIM proteins identified 20 TRIM proteins which had antiviral activities toward HIV-1, murine leukemia virus (MLV) and avian leukosis virus by affecting viral entry or release (71). In the case of HIV-1, TRIM5␣, TRIM11, and TRIM31 were found to restrict virus entry, TRIM22 and TRIM32 attenuated transcription of the HIV-1 long terminal repeat transcriptional promoter, and TRIM25, TRIM31, and TRIM62 inhibited virus release from cells (66, 68, 71). The transcriptional repressor properties of TIF1␤ have also been implicated in the antiviral response toward MLV and KSHV. TIF1␤ restricts MLV replication in embryonic stem and embryonic carcinoma cells, while KSHV has been found to silence gene expression and maintain a state of latency by exploiting the chromatin remodeling functions of TIF1␤ (20, 74). Furthermore, it has been suggested that the switch from viral latency to lytic replication during KSHV infection is mediated via the viral protein kinase (vPK)-dependent phosphorylation of TIF1␤, which affects the ability of TIF1␤ to condense chromatin on viral promoters (20). Given our observations and the known functions of TIF1␥, it is possible that TIF1␥ limits viral replication by modulating gene expression programs and remodeling chromatin through ubiquitylation. In summary, we have identified TIF1 family members as key targets for Ad early region proteins during infection. We have also determined a novel role for E4orf3 in promoting the proteasomemediated degradation of TIF1␥. We propose that Ad has evolved


specifically to inhibit the antiviral functions of TIF1 proteins in order to promote viral replication. ACKNOWLEDGMENTS We thank Stephen Jackson, Vivien Mautner, Iain Morgan, Joe Mymryk, and Stefano Piccolo for providing some of the reagents used in this study. We also thank Sally Roberts and Elena Odintsova for help with confocal microscopy. This study was supported by a University of Birmingham Medical School Rowbotham Bequest studentship to N.A.F., and a University of Birmingham Medical School studentship to R.N.P. The Heinrich-Pette Institute is supported by the Freie und Hansestadt Hamburg and the Bundesministerium für Gesundheit.

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