Interaction of Adeno-associated Virus Rep78 with p53: Implications inGrowth Inhibition 1
Ramesh B. Batchu,2 Masood A. Shammas,2 Jing Yi Wang, and Nikhil C. Munshi 3
Central Arkansas Veterans Health Care System and Myeloma and Transplantation Research Center, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Abstract
Adeno-associated virus (AAV) is a nonpathogenic, single-strandedDNA virus belonging to the parvoviridae family. Onco-suppressive prop-erties of AAV against adenovirus, a DNA tumor virus, have been welldocumented. Rep78, a major regulatory protein of AAV, is believed to beresponsible for its antioncogenic properties. Most DNA tumor virusesdisturb the cell cycle pathways by essentially abrogating the functions ofp53. Here we present evidence that AAV acts as an antiproliferative agentagainst adenovirus by protecting the adenoviral-mediated degradation ofp53 as confirmed by both Western blot analysis and immunoprecipitationanalysis with anti-p53 antibody. Coimmunoprecipitation experiments re-vealed that the AAV Rep78 is physically bound to p53in vivo. Further-more, the binding of purified p53 to the AAV Rep78 affinity columnconfirms their interaction. These results document for the first time thatthe antiproliferative effects of AAV against adenovirus are mediated, atleast in part, by the interaction of AAV Rep78 with p53.
Introduction
AAV, 4 a 4.7-kb single-stranded DNA virus, belongs to the familyof parvoviridae and can infect various species, including humans (1).However, it is not known to be associated with any disease. In fact, ithas been shown to inhibit the transforming potential of various agents(2). AAV latently infects the cell and integrates into a specific site,AAVS1, on chromosome 19 (3). It generally requires coinfection withhelper viruses for its productive replication (1).
Several reports have demonstrated that tumors generated by ade-novirus in chicks (4) and in hamsters (5, 6) were inhibited in thepresence of AAV. Coinfection of AAV with adenovirus leads to thegeneration of a reduced number of plaques when compared withinfection by adenovirus alone (7, 8). It has been observed that in thepresence of AAV, the transforming potential of adenovirus in variouscell lines is significantly reduced (7, 9, 10). These tumor-suppressiveand antiproliferative properties have been mapped to the left half ofthe AAV genome, which codes for the multifunctional regulatoryprotein Rep78 (11, 12).
Although the tumor-suppressive and antiproliferative properties ofAAV Rep78 have been well documented, nothing is known about themolecular mechanisms behind this phenomenon. To induce cell pro-liferation, the adenovirus first has to remove the cell cycle block. Animportant cell cycle checkpoint in mammalian cells that acts in the G1
phase is mediated by the p53 tumor suppressor gene (13). Adenoviral
E1B, an early gene product, has been shown to form a complex withp53 (14). This interaction of adenoviral E1B with p53 induces ubiq-uitin-mediated degradation of p53 (15). Because AAV inhibits the cellproliferation potentials of adenovirus, this work was undertaken tostudy whether AAV interferes with the adenoviral-mediated abroga-tion of p53 functions. As these antioncogenic properties of AAV areconfined to its regulatory protein, AAV Rep78, we have investigatedwhether it interacts with p53, potentially protecting it from adenovi-ral-mediated degradation. Here we document that p53 levels arestabilized by AAV in adenovirus-infected cells. Furthermore, wedocument the interaction of AAV Rep78 with p53 that may beresponsible for the observed protection of p53.
Materials and Methods
Plasmids, Viruses, and Cell Lines.The HeLa and 293 cell lines andfibroblasts cells were procured from the American Type Culture Collection(Manassas, VA) and maintained at 37°C under a humidified atmospherecontaining 5% CO2 in air in DMEM supplemented with 10% fetal bovineserum, 1% penicillin G-streptomycin, and 2 mM L-glutamine (Life Technolo-gies, Inc., Grand Island, NY). AAV-2 was prepared from HeLa cells infectedwith adenovirus-2 and AAV-2. At 24 h after plating (63 106 cells; twenty100-mm dishes), semiconfluent HeLa cultures were inoculated with 10 mul-tiplicity of infection units of both AAV-2 and adenovirus-2 in DMEM withoutserum or antibiotics for 1 h at 37°C. Infectious medium was replaced withDMEM supplemented with 5% FCS, 1% penicillin G-streptomycin, and 2 mM
L-glutamine (Life Technologies, Inc.). Cultures showing adenovirus-inducedcytopathic effects observed after approximately 48 h were washed withDMEM without serum or antibiotics, and the cells were harvested in 10 ml ofDMEM and subjected to three freeze/thaw cycles. Cells were spun at 10,000rpm at 4°C for 20 min, and the supernatant was adjusted to 5 mM manganesechloride and incubated at 37°C for 30 min with 5,000 units of DNase I andRNase (0.2 mg/ml). This preparation was spun again at 10,000 rpm at 4°C for20 min, and the supernatant was incubated at 56°C for 1 h to inactivateadenovirus. The absence of adenovirus was confirmed by inoculating HeLacells with this preparation and probing the total cell extracts for adenoviralproteins. This preparation was adjusted to 5% glycerol, and the aliquots werestored in liquid nitrogen. All of the experiments were carried out with 10multiplicity of infection units of adenovirus-2 and/or AAV-2 on semiconfluentHeLa or 293 cells, and the cells were harvested at the desired time intervals.
Production of the His-Rep78 Protein. AAV Rep78 was PCR-amplifiedusing linearized plasmid pSub201 (16). Primers designed to amplify Rep78were as follows: (a) primer 1, 59-CCGGGGTTTTACGAGATTGT-39; and (b)primer 2, 59-TTGTTCAAAGATGCAGTCAT-39.
Both primers were designed to haveBglII/BamHI overhangs. The PCRproduct was gel-purified using the Qiaex II gel extraction kit (Qiagen,Chatsworth, CA). The resulting open reading frame of Rep78 was cloned intopQE16 (QIAexpressionist; Qiagen) vector as follows. First, the pQE16 vectorwas digested withBamHI andBglII, and the resulting linearized plasmid,which has a 6 His tag at the COOH-terminal end, was gel-purified. PCR-amplified Rep78 was ligated to linearized pQE16 overnight at 16°C. Cloneswere selected after bacterial transformation and analyzed for protein expres-sion. Briefly, small overnight cultures were diluted 1:50 and grown until theabsorbance (A600) reached 0.7. Isopropyl-b-D-thiogalactoside was added to afinal concentration of 1 mM, and the incubation was continued for 4 h after theinduction. Purification of His-Rep78 was carried out under native conditions,
Received 3/29/99; accepted 6/15/99.The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by Grant DHP 153 from the American Cancer Society, GrantHL-55695 from the USPHS National Heart Blood and Lung Institute, and National CancerInstitute Grant CA71092 and VA merit award (to N. C. M.). N. C. M. is a LeukemiaSociety Scholar.
2 R. B. B. and M. A. S. contributed equally to this work.3 To whom requests for reprints should be addressed, at University of Arkansas for
Medical Sciences, 4301 West Markham, Slot 776, Little Rock, AR 72205. Phone: (501)686-8250; Fax: (501) 686-6442; E-mail: [email protected].
essentially following the protocol provided by Qiagen. The purity of theprotein was confirmed by 8% SDS-PAGE.
Affinity Chromatography. The His-Rep78 protein was expressed as de-scribed previously, and protein was adsorbed to Ni-NTA spin columns (Qia-gen) according to the instructions given in the manual. p53 protein (Santa CruzBiotechnology, Santa Cruz, CA) was chromatographed on the Rep78 affinitycolumn by incubating at 4°C for 30 min. His-Rep78 was then eluted with 250mM imidazole and subjected to 8% SDS electrophoresis. Some blots were alsotransferred to nitrocellulose membrane as mentioned earlier for probing withantibodies.
Immunoprecipitation. After various treatments, 100-mm cell cultureplates were washed three times with PBS and incubated with 13 lysis buffer[50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 0.5% NP40, 1 mMEDTA, and 1 mM EGTA] for 30 min with a protease inhibitor mixture fromBoehringer Mannheim. The cell pellet was subjected to three freeze/thawcycles and left on ice at 4°C with constant shaking for 30 min. Cell debris wasremoved by refrigerated centrifugation for 5 min at 12,000 rpm. Supernatantswere collected, and the protein content was estimated using the Micro BCA kit(Pierce, Rockford, IL). Protein contents of all of the samples were normalizedto 10 mg/ml with lysis buffer, aliquoted, and stored at270°C. Immunopre-cipitations were conducted essentially as described previously (17).
Electrophoresis and Western Blotting. After different virus treatments,aliquots of total protein extracts (100mg) from cells were suspended inLaemmli’s sample buffer [0.1M Tris-Cl (pH 6.8) containing 1% SDS, 0.05%b-mercaptoethanol, 10% glycerol, and 0.001% bromphenol blue]; boiled for 2min; applied on either 8%, 12%, or 8–12% (18) glycerol gradient SDS-acrylamide along with aMr 10,000 protein ladder from Life Technologies,Inc.; and electrophoresed for 16 h using the Bio-Rad PROTEIN II system at 60V. Gels were electroblotted overnight onto nitrocellulose paper (Trans-Blottransfer membrane, 0.2mm; Bio-Rad; Hercules, CA) at 40 V for 3 h in aTris-glycine buffer system. The transfer was confirmed by Ponceu S stainingof the blot. Nonspecific sites on the blots were blocked with PBST. Incubationwith various antibodies [AAV Rep78 (American Research Products), p53(Santa Cruz Biotechnology), orb-actin (Sigma, St. Loius, MO)] was per-
formed for 2 h inPBST containing 1% BSA with constant rocking. Blots werewashed three times with PBST and incubated in either antirabbit or antimousehorseradish peroxidase conjugates for 2 h in PBST. After washing, specificproteins were detected using ECL according to the instructions provided in themanual (Amersham Life Sciences, Inc., Arlington Heights, IL).
Results
Protection of Adenoviral-mediated Degradation of p53 byAAV. E1B-mediated active degradation of p53 protein in adenovirus-infected cells is well known (19). After adenovirus infection, normallevels of p53 transcript are observed. However, ubiquitin-mediatedprotein degradation leads to lower levels of the p53 protein, which isessential for adenoviral propagation and cell proliferation (15, 19).Because AAV inhibits cell proliferation initiated by the adenovirus,we examined the effect of AAV expression on p53 levels with regardto adenovirus infection. Total cell lysates were prepared at varioustime points after viral infections and probed for p53 by Western blotanalysis. As seen in Fig. 1, no appreciable change in the protein levelsis observed before 48 h in HeLa cells. However, there is a significantreduction in p53 levels at 48 and 72 h with adenovirus infection.Coinfection of AAV with adenovirus protects against adenoviral-mediated degradation of p53 (Fig. 1A). Probing the same blot withb-actin confirmed equal protein loading in each lane. As seen in Fig.1B, the quantitation of p53 levels in comparison with theb-actinlevels shows a significant block in the adenoviral-mediated degrada-tion of the p53 by AAV. In an effort to correlate the inhibition of p53degradation and AAV expression, we probed the cell lysates for AAVRep protein expression at various time points. As seen in Fig. 1C, Repprotein expression starts only at 30 h after AAV infection and reachesa plateau after 48 h, correlating with the observed inhibition ofadenoviral-mediated p53 degradation by AAV at 48 h. The effect ofadenovirus infection was also examined in 293 cells because, unlikeHeLa cells, these cells do not contain HPV genes. Decreased levels ofp53 after adenovirus infection, apparent after 24 h in this cell line,were also partially blocked by AAV (Fig. 2A). In the 293 cell line, weobserve a significant expression of AAV Rep proteins by 24 h, whichis consistent with the observed inhibition at that time point (Fig. 2B).
In Vitro Interaction of p53 and His-Rep78.Next, we evaluatedwhether protein-protein interactions are involved in the stabilizationof p53 protein as it has been reported that Rep78, is responsible foronco-suppressive properties of AAV (11, 12). To investigate theinteraction between AAV Rep78 and p53, we produced the Rep78affinity column as described in “Materials and Methods” and incu-
Fig. 1. A, Western blotting of viral-infected HeLa cell extracts with p53 andb-actinantibodies. Viral infections were carried out on 80% confluent HeLa cells as described in“Materials and Methods,” and the cell lysates were prepared at the indicated timeintervals. Proteins were visualized by ECL with Amersham ECL detection reagents.B,quantification of relative p53 expression in HeLa cells after various viral treatments. Bothp53 and actin protein bands on the X-ray films developed by the ECL assay werequantitated using Gel Doc 1000 (Bio-Rad). p53 band intensities were normalized withactin bands of the same lane. The intensity of the p53 bands relative to the actin bands wasplotted against various time intervals.C, total cell lysates at various time points after viralinfections were applied onto 8% SDS-polyacrylamide gels, transferred to a blot, anddeveloped with anti-Rep as well asb-actin monoclonal antibodies.
Fig. 2. A, detection of p53 protein levels in 293 cells after various viral infections.AAV and adenoviral infections were carried out in the 293 cell line. Protein bands werevisualized by using ECL reagents.B, the total cell extracts of 293 cells 24 and 36 h afterviral infections were electrophoresed, transferred to a blot, and probed for the expressionof Rep proteins.
bated it with p53. After several washings, eluted proteins were elec-trophoresed, transferred onto nitrocellulose blots, and probed with thep53 antibody. As seen in Fig. 3A, p53 was eluted along with Rep78,indicating a physical interaction between both proteinsin vitro. Thesame blot was also reprobed with both AAV Rep78 and p53 antibod-ies together. Fig. 3B shows that the p53 is eluted with Rep78,confirming the identity and the interaction of the proteins.
AAV Rep78 Forms a Complex with p53 in Vivo. Althoughprevious experiments have shown the interaction of p53 and Rep78invitro, we wanted to confirm whether the two proteins interactin vivo.Cells were lysed 36 h after infection with the viruses, and one set wasimmunoprecipitated with Rep78 antibody, and the other set wasimmunoprecipitated with p53 antibody. These extracts were subjectedto electrophoresis and Western blotting and probed for p53. Asexpected, when Rep78 precipitates were probed with p53 antibodies,we observed the presence of p53 in the cells infected with bothadenovirus and AAV (Fig. 4A). AAV is a helper-dependent virus, andthe expression of AAV proteins occurs only with adenovirus super-infection in humans. Therefore, Rep78 expression is not expected ineither mock-transfected cells, cells infected with adenovirus alone, orcells infected with AAV alone, as observed in this figure. When these
extracts were immunoprecipitated with p53 and probed with p53antibody (Fig. 4B), adenovirus-infected cells did not show the pres-ence of p53, confirming the degradation of p53 after adenovirusinfection. In the presence of AAV superinfection, the p53 band is seenjust below the IgG heavy chain, which is aroundMr 55,000. Toconfirm the specificity of thesein vivo interactions, we probed the p53immunoprecipitations with Rep78 antibodies after electrophoresis andWestern blot transfer. As seen in Fig. 5 AAV Rep78 and Rep52proteins coimmunoprecipitated with p53. AAV produces four regula-tory proteins, Rep78, Rep68, Rep52, and Rep40. All of them areproduced by differential splicing of a transcript from a single openreading frame. The monoclonal antibody we used for probing AAVRep78 recognizes all four regulatory proteins; however, the amountsof all four proteins vary to a significant extent in both HeLa (Fig. 1C)and 293 cells (Fig. 2B). Rep78 and Rep52 are overexpressed com-pared to the other two regulatory proteins, Rep68 and Rep40, thusthese two proteins were seen on the blot.
Discussion
Adenovirus depends on host cell mechanisms to replicate the viralgenome. In consequence, it encodes early gene products capable ofactivating host cell cycle progression and proliferative processes (14).The transition from G1 to S phase is an important regulatory point incell cycle progression. p53, a well-characterized tumor suppressorprotein, is antithetical to growth by blocking the cell in the G1 phaseof the cell cycle (13). Adenovirus large E1B protein, an early geneproduct, has been shown to form a complex with p53 (14). Thisinteraction not only inactivates p53 but targets it for ubiquitination,leading to its active degradation (15). These reports indicate that theinactivation and degradation of p53 is essential for adenoviral-medi-ated cell proliferation.
AAV has been shown to possess a tumor suppressor propertyagainst adenovirus in various systems (4–7). This property has beenmapped to the left half of the AAV genome, which codes for amultifunctional regulatory protein, Rep78 (11, 12). When the adeno-virus-infected HeLa cells were challenged with AAV, p53 degrada-tion by adenovirus was substantially inhibited (Fig. 1,A andB). Wechose the time points to observe p53 protein levels by exploring thetiming of AAV expression after the initial infection (Fig. 1C). AAVexpression starts 36 h after the viral infection, and we observed theinhibition of adenoviral-mediated p53 degradation at 48 h. Similarly,
Fig. 3. In vitro interaction of Rep78 and p53. AAV Rep78 was produced as a fusionprotein with 6 His amino acids at the COOH-terminal end, as described in “Materials andMethods,” and the protein was conjugated to Ni-NTA columns that have a very highaffinity for the histidine tag.Lanes 1–3show increasing concentrations (20, 40, and 60 ngof Rep78) incubated with the affinity matrix.Lanes 4and5 were incubated with 10 and20 ng of p53, which was produced as a glutathioneS-transferase-fusion protein.Lane 6was incubated with 20 ng of p53 alone in the affinity matrix. The affinity columns wereeluted with 250 mM imidazole. The elutents were electrophoresed, transferred, and probedwith p53 antibody. InB, the blot used inA was stripped off the antibodies and reprobedwith both AAV Rep78 antibody and p53 antibody.
Fig. 4. Detection of p53 by Western blotting in Rep78 and p53 immunoprecipitationsof cell lysates. The 80% confluent 293 cells were treated with the viruses as indicated, andthe total cell lysates were prepared after 36 h.A shows the probing of Rep78 immuno-precipitates with p53 monoclonal antibodies. Because the immunoprecipitations withmouse monoclonal antibodies were finally subjected to a reaction with antimouse horse-radish peroxidase conjugate in an effort to detect p53 in the precipitates, we observed astrong immunoglobulin heavy and light chain signals. InB, immunoprecipitations wereconducted with p53 antibodies, transferred to Western blot, and probed with p53. In bothcases, the blots were developed with ECL reagents.
Fig. 5. Detection of Rep proteins in p53 immunoprecipitates. 293 cells were infectedwith the viruses as indicated, and the total cell lysates were prepared after 36 h.Immunoprecipitation was carried out with p53 monoclonal antibodies, and the precipitateswere transferred to nitrocellulose filters and probed with Rep78 antibodies.
in 293 cells, adenoviral-mediated degradation of p53 protein wassubstantially reduced in the presence of AAV at 24 and 36 h, when293 cells express the maximum amounts of AAV Rep proteins (Fig.2B). The reduction in p53 after adenoviral infection is less marked in293 cells, possibly due to the presence of adenoviral early genes inthis cell line. The AAV-mediated p53 protection in 293 cells is alsoless marked compared to that in the HeLa cells. This may be due toobservation at earlier time points, the presence of adenoviral earlygenes in this cell line, or a high background of p53 expression incontrol cells. Interestingly, the AAV-mediated protection of p53 be-comes more obvious at 36 h than at 24 h.
Whereas these data indicate that AAV rescues p53 from adenovi-rus-mediated degradation, they do not provide a possible mechanism.To address this question, we have investigated the role of Rep78 inexerting growth-inhibitory effects on primary human cells (20) andthe transforming potentials of various DNA tumor viruses (2). Withthis background, we checked whether AAV Rep78 has any bindingaffinity with p53 that may be responsible for its protection fromadenovirus-mediated degradation. The binding of p53 with the Rep78affinity column in a dose-dependent manner confirmed itsin vitrointeraction with AAV Rep78 (Fig. 3A). Neither glutathioneS-trans-ferase moiety nor p53 moiety of the fusion protein has any affinitytoward the Ni-NTA affinity column (data not shown), and furthercoelution of Rep78 with p53 was confirmed by probing the same blotwith both antibodies (Fig. 3B). These protein-protein interactionswere confirmedin vivoby immunoprecipitating 293 cells with Rep78,followed by p53 probing. We observed a strong p53 signal in coim-munoprecipitates of Rep78 when it is expressed in the cells. Also theabsence of p53 signal in either control, with AAV or with adenovirusinfection, shows that this interaction is specific for Rep78 (Fig. 4A).It is important to observe that Rep78 is expressed only when adeno-virus is coinfected with AAV, because AAV expression is dependenton the adenovirus helper functions in humans (1). In view of theonco-suppressive nature of Rep78, its interaction with p53 bothinvitro and in vivo leading to elevated amounts of p53 suggests apossible stabilization of p53 from ubiquitin-mediated degradation byadenovirus. By protecting p53 from ubiquitin-mediated degradation,the function of p53 as a cell cycle blocking agent is restored. Protein-protein interactions leading to protection from ubiquitin-mediateddegradation are not uncommon. For example, the binding of retino-blastoma, another onco-suppressor protein, to E2F-1, which is aubiquitous cell cycle-related transcription factor, confers protectionagainst ubiquitin-mediated degradation (21).
Interestingly, of the four spliced versions of AAV Rep78, wedetected only Rep78 and Rep52 in coimmunoprecipitations with p53(Fig. 5A). It is possible that the lower expressions of Rep68 andRep40, compared to the other two Rep proteins (Figs. 1C and 2B),resulted in the much lower coprecipitations and the inability to detectthem on the blot. At present, we are trying to determine how theinteraction between AAV Rep proteins and p53 protects it fromadenoviral-mediated degradation.
This report suggests for the first time the possible molecular mech-anism behind the onco-suppressive nature of AAV, which has beenwidely reported over the years. Insights into the molecular mecha-nisms underlying the onco-suppressive nature of AAV not only con-tribute to our basic understanding about oncogenesis but may alsosuggest a possible use of AAV Rep78 as an antiproliferative agent. Itis worthwhile to note that AAV infects humans without any knownpathological consequences (1), and the sero-epidmeological data
clearly suggest a negative correlation between the occurrence ofcervical cancer and the presence of AAV (22, 23). In summary, wepresent evidence that the onco-suppressive properties of AAV againstadenovirus are mediated, at least in part, by the protection of p53degradation, presumably by the interaction with AAV Rep78.
Acknowledgments
We thank Jeana Cromer for editorial assistance.
References
1. Berns, K. I., and Bohenzky, R. A. Adeno-associated viruses: an update. Adv. VirusRes.,32: 243–306, 1987.
2. Schlehofer, J. R. The tumor suppressive properties of adeno-associated viruses.Mutat. Res.,305: 303–313, 1994.
3. Samulski, R. J., Zhu, X., Xiao, X., Brook, J. D., Housman, D. E., Epstein, N., andHunter, L. A. Targeted integration of adeno-associated virus (AAV) into humanchromosome 19 (erratum appears in EMBO J.,11: 228, 1992). EMBO J.,10:3941–3950, 1991.
4. Pronovost, A. D., Yates, V. J., and Fry, D. E. Inhibition and enhancement of avianadenovirus plaque production by heavy and light avian adenovirus-associated viralparticles. Am. J. Vet. Res.,40: 549–551, 1979.
5. Mayor, H. D., Houlditch, G. S., and Mumford, D. M. Influence of adeno-associatedsatellite virus on adenovirus-induced tumours in hamsters. Nat. New Biol.,241:44–46, 1973.
6. de la Maza, L. M., and Carter, B. J. Inhibition of adenovirus oncogenicity in hamstersby adeno-associated virus DNA. J. Natl. Cancer Inst.,67: 1323–1326, 1981.
7. Casto, B. C., and Goodheart, C. R. Inhibition of adenovirus transformationin vitro byAAV-1. Proc. Soc. Exp. Biol. Med.,140: 72–78, 1972.
8. Mayor, H. D., Jordan, L. E., and Gorman, C. Hamster embryo cells transformed byherpes simplex virus: reactions with adeno-associated satellite virus (AAV) and itsadenovirus helpers. Cancer Biochem. Biophys.,2: 25–31, 1977.
9. Gilden, R. V., Kern, J., Beddow, T. G., and Huebner, R. J. Oncogenicity of mixturesof adeno-associated virus and adenovirus type 12. Nature (Lond.),220: 1139, 1968.
10. Ostrove, J. M., Duckworth, D. H., and Berns, K. I. Inhibition of adenovirus-transformed cell oncogenicity by adeno-associated virus. Virology,113: 521–533,1981.
11. Yang, Q., Kadam, A., and Trempe, J. P. Mutational analysis of the adeno-associatedvirus rep gene. J. Virol.,66: 6058–6069, 1992.
12. Heilbronn, R., Burkle, A., Stephan, S., and zur Hausen, H. The adeno-associated virusrep gene suppresses herpes simplex virus-induced DNA amplification. J. Virol.,64:3012–3018, 1990.
13. Kastan, M. B., Zhan, Q., el-Deiry, W. S., Carrier, F., Jacks, T., Walsh, W. V.,Plunkett, B. S., Vogelstein, B., and Fornace, A. J., Jr. A mammalian cell cyclecheckpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia.Cell, 71: 587–597, 1992.
14. Sarnow, P., Ho, Y. S., Williams, J., and Levine, A. J. Adenovirus E1b–58kd tumorantigen and SV40 large tumor antigen are physically associated with the same 54 kdcellular protein in transformed cells. Cell,28: 387–394, 1982.
15. Ciechanover, A., Shkedy, D., Oren, M., and Bercovich, B. Degradation of the tumorsuppressor protein p53 by the ubiquitin-mediated proteolytic system requires a novelspecies of ubiquitin-carrier protein, E2. J. Biol. Chem.,269: 9582–9589, 1994.
16. Samulski, R. J., Chang, L. S., and Shenk, T. A recombinant plasmid from which aninfectious adeno-associated virus genome can be excisedin vitro and its use to studyviral replication. J. Virol.,61: 3096–3101, 1987.
17. Hermonat, P. L., Santin, A. D., and Batchu, R. B. The adeno-associated virus Rep78major regulatory/transformation suppressor protein binds cellular Sp1in vitro andevidence of a biological effect. Cancer Res.,56: 5299–5304, 1996.
18. Tyagi, R. K., Babu, B. R., and Datta, K. Simultaneous determination of native andsubunit molecular weights of proteins by pore limit electrophoresis and restricted useof sodium dodecyl sulfate. Electrophoresis,14: 826–828, 1993.
19. Maheswaran, S., Englert, C., Lee, S. B., Ezzel, R. M., Settleman, J., and Haber, D. A.E1B 55K sequesters WT1 along with p53 within a cytoplasmic body in adenovirus-transformed kidney cells. Oncogene,16: 2041–2050, 1998.
20. Kube, D. M., Ponnazhagan, S., and Srivastava, A. Encapsidation of adeno-associatedvirus type 2 Rep proteins in wild-type and recombinant progeny virions: Rep-mediated growth inhibition of primary human cells. J. Virol.,71: 7361–7371, 1997.
21. Hofmann, F., Martelli, F., Livingston, D. M., and Wang, Z. The retinoblastoma geneproduct protects E2F-1 from degradation by the ubiquitin-proteasome pathway.Genes Dev.,10: 2949–2959, 1996.
22. Georg-Fries, B., Biederlack, S., Wolf, J., and zur Hausen, H. Analysis of proteins,helper dependence, and seroepidemiology of a new human parvovirus. Virology,134:64–71, 1984.
23. Sprecher-Goldberger, S., Thiry, L., Lefebvre, N., Dekegel, D., and Halleux, F. D.Complement-fixation antibodies to adenovirus-associated viruses, cytomegalovirusesand herpes simplex viruses in patients with tumors and in control individuals. Am. J.Epidemiol.,94: 351–358, 1971.
1999;59:3592-3595. Cancer Res Ramesh B. Batchu, Masood A. Shammas, Jing Yi Wang, et al. Implications in Growth InhibitionInteraction of Adeno-associated Virus Rep78 with p53:
![Data Workflows in Stata and Python · Data Workflows in Stata and Python ... auto.foreign.value_counts() In [21]: ... [22]: foreign Domestic Foreign rep78 1 2 0 2 8 0 3 27 3](https://static.documents.pub/doc/80x56/5b080b0f7f8b9a520e8c065d/data-workflows-in-stata-and-python-workflows-in-stata-and-python-auto-in.jpg)
