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Adeno-associated Virus Protects the Retinoblastoma Family of Proteins from Adenoviral-induced Functional Inactivation¹

Myeloma and Transplantation Research Center [R. B. B., J. F., N. R., N. C. M.], and Central Arkansas Veteran’s Healthcare System [M. A. S., J. Y. W., N. C. M.], University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, and Dana Farber Cancer Institute, Boston Massachusetts 02115 [M. A. S., N. C. M.]

	ABSTRACT

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Adeno-associated virus type 2 (AAV) is known to inhibit virally mediated oncogenic transformation. One of the early events of adenovirus (Ad) infection is the functional inactivation of cell cycle regulatory retinoblastoma (RB) family of proteins, which consists of retinoblastoma protein (pRB), p107, and p130. In an effort to understand the molecular basis of anti-oncogenic properties of AAV, we studied the effects of AAV expression on these proteins in cells infected with Ad. Western blot analysis showed that AAV interferes with the adenoviral-induced degradation and hyperphosphorylation of the pRB family of proteins in normal human fibroblasts as well as in HeLa and 293 cell lines. RNase protection assay showed enhanced expression of pocket protein gene by AAV expression. We also demonstrate that Rep proteins, the major AAV regulatory proteins, bind to E1A, the immediate early gene of Ad responsible for hyperphosphorylation and dissociation of pRB-E2F complex. This binding of AAV Rep proteins to E1A leads to decreased association between E1A and pRB leading to protection of pocket proteins from degradation, decreased expression of S phase genes and inhibition of cell cycle progression. These results suggest that the antiproliferative activity of AAV against Ad is mediated, at least in part, by effects of AAV Rep proteins on the Rb family of proteins.

	INTRODUCTION

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AAV,³ a nonpathogenic 4.7-kb single-stranded DNA virus belongs to the Parvoviridae family (1) . AAV latently infects the cell and integrates preferentially into human chromosome 19 at q13.3-qter (2) , and requires special cellular conditions, provided by Ad for its productive replication (1 , 3) . Members of Parvoviridae, particularly AAV, show growth inhibition and tumor suppression in a variety of cells (3, 4) . Suppression of tumor formation induced by various DNA tumor viruses (5, 6, 7) , particularly Ad, is well documented (8, 9, 10, 11) .

Although the exact molecular basis of tumor-suppressive properties of AAV has not been elucidated, Rep, its regulatory gene has been characterized to confer these properties. AAV Rep gene is transcribed from two promoters and encodes four overlapping multifunctional proteins, Rep78, -68, -52, and -40 (12, 13) . The two large Rep proteins, Rep78 and Rep68 have been characterized as interfering with cell proliferation (5 , 14 , 15) .

Cell cycle progression from G₁ to S phase is tightly controlled by the p53 and pRB family of "pocket proteins," which comprises pRB, p107, and p130 (16 , 17) . Ad encodes proteins that inactivate the function of these cell cycle regulatory proteins, presumably to facilitate a productive viral infection in an otherwise quiescent cell (18) . Recently, we demonstrated that the tumor suppressor gene p53 is protected from adenoviral-mediated degradation by AAV Rep proteins (19) . We also observed the inhibition of the activity of E2F-1, a major cell cycle regulatory protein that preferentially binds to pRB. Because the RB gene product is one of the early genes targeted for adenoviral-mediated inactivation, we investigated whether this process is also inhibited by AAV as part of its anti-oncogenic activity. In this report, we show that the adenoviral-mediated inactivation of pRB family of proteins is protected by AAV Rep proteins leading to the inhibition of cell proliferation.

	MATERIALS AND METHODS

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Plasmids.
pE2-luciferase was provided by Dr. Ed Harlow (Harvard University, Boston, MA). pCMV Rep78 was constructed as described previously (20) .

Viruses and Cell Lines.
Cell lines 293 and HeLa and normal human fibroblasts were obtained from American Type Culture Collection (Manassas, VA) and cultured in DMEM, supplemented with 10% fetal bovine serum, 1% penicillin G-streptomycin, 2 mM L-glutamine (Life Technologies, Inc., Grand Island, N.Y) at 37°C under a humidified atmosphere containing 5% CO₂. AAV type 2 was prepared from HeLa cells that were infected with Ad-2 and AAV-2 and was purified as described previously (19) . These experiments were carried out with 10 multiplicities of infection of Ad-2 and/or AAV-2 on semiconfluent fibroblasts HeLa or 293 cells, and the cells were harvested at the described time intervals.

Lipofection and Luciferase Assay.
Plasmid pE2 luciferase was transfected into human cells using the Effectene transfection kit (Qiagen Inc., Valencia, CA), as recommended by the manufacturer. One day before transfection, 10⁵ cells were plated per well of a six-well tissue-culture plate. DNA (0.5 µg per transfection) was sequentially mixed with enhancer and with Effectene reagent and layered on monolayer cells. After 2–4 h of incubation, the medium was replaced with fresh regular-growth medium and cells were further subjected to viral infections with Ad and/or AAV as described earlier. The cells were lysed inside the wells by using 200 µl of lysis buffer and were harvested. The luciferase assay was performed using Promega’s luciferase assay detection system (Promega Corporation, Madison, WI).

RNase Protection Assay.
RNase protection assay was performed as described previously (20) . Briefly, a human cell cycle regulator multiprobe template set (PharMingen, San Diego, CA), hTS-1 (containing pRB, p107, and p130, and GAPDH cDNA sequences) was used to synthesize [³²P]UTP-labeled antisense RNA probe. Template DNA molecules were digested with RNase-free DNase, and the probe was purified by phenol-chloroform extractions and ethanol precipitation. Purified probe (10⁵ cpm) was mixed with 10 µg of total RNA from 293 cells in hybridization buffer. Hybridization was carried out for 16 h at 56°C. Free-probe and single-stranded RNA molecules were digested with a mixture of RNase A and T1. The "RNase-protected" molecules were purified and resolved on a denaturing polyacrylamide gel and dried. Autoradiographic signal was scanned on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and signal intensity of each band was quantitated by "ImageQuant" software, (Molecular Dynamics).

Affinity Chromatography with Rep78.
His-Rep78 protein was expressed as described earlier (20) . Protein was adsorbed to nickel-nitrilo-triacetic acid spin columns (Qiagen, Santa Clarita, CA) according to the instructions given by the manufacturer. Approximately 100 µg of adenoviral-infected 293 cell lysate was chromatographed on the Rep78 affinity column by incubating at 4°C for 30 min. His-Rep78 was eluted with 250 mM imidazole and subjected to 8% SDS-PAGE, and the eluted proteins were transferred to nitrocellulose blot and probed with pRB and E1A antibodies.

Electrophoresis and Western Blotting.
Approximately 100 µg of protein were suspended in Laemmli’s sample buffer [0.1 M Tris-HCl buffer (pH 6.8), containing 1% SDS, 0.05% ß-mercaptoethanol, 10% glycerol, and 0.001% bromphenol blue], boiled for 2 min, and applied on 8% glycerol gradient SDS-acrylamide along with a M_r 10,000 protein ladder (Life Technologies, Inc.), electrophoresed for 4 h at 120 V. Gels were electroblotted onto nitrocellulose paper (Trans-Blot, 0.2-µm transfer membrane; Bio-Rad Laboratories, Hercules, CA) at 40 V for 3 h in a Tris-glycine buffer system, as described previously (19 , 20) . Incubation with various antibodies, AAV Rep78 (American Research Products; San Jose, CA), E1A, pRB, p107, p130 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or ß-actin (Sigma Chemicals Co, St. Louis, MO) was performed for 2 h in PBS-Tween 20 (PBST) containing 1% BSA with constant rocking. Blots were washed with PBST and incubated in either antirabbit or antimouse horseradish peroxidase (HRPO) conjugates for 2 h in PBST containing 3% nonfat dry milk. After washing, specific proteins were detected using an enhanced chemiluminescence, according to the instructions provided in the manual (Amersham Life Sciences Inc., Arlington Heights, IL). For immunoprecipitation experiments, cell culture plates (100 mm) after various treatments, were washed and incubated with lysis buffer for 30 min with protease inhibitor mixture (Boehringer Mannheim, Indianapolis, IN). A cell pellet was freeze-thawed three times and incubated on ice at 4°C with constant shaking for 30 min. Cell debris were removed by refrigerated centrifugation, supernatants were collected, and protein content was estimated using Micro BCA kit (Pierce, Rockford, IL). Immunoprecipitations were conducted as described previously (19) . Protein bands were detected using an enhanced chemiluminescence reagent (Amersham Life Sciences Inc., Arlington Heights, IL) and quantitated by a laser densitometer (Molecular Dynamics, Sunnyvale CA).

Cell Cycle Analysis.
Approximately 5 x 10⁶ viral-infected and control cells were trypsinized and washed with cold PBS. Cells were resuspended in 100 µl of cold PBS, and 480 µl of 100% ethanol was added drop-wise to a final concentration of 70% ethanol; the resulting suspension was left overnight at 4°C. The next day the samples were spun down to remove the ethanol, and the cells were resuspended in 1 ml of cold PBS. Later the cells were stained with propidium iodide for FACScan. The number of cells in S phase was determined using ModFit software.

	RESULTS

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Protection of Adenoviral-mediated Changes in pRB Family of Proteins by AAV.
The pRB family of proteins were targeted by Ad to induce cell proliferation. Because AAV is known to reverse the transforming ability of Ad, we evaluated the effect of AAV infection on Ad-induced changes in pRB family proteins. After infection of normal human fibroblasts with Ad, or AAV, or both, cell lysates were prepared, resolved on gradient SDS polyacrylamide gels and were probed with antibodies against p130, pRB, and p107. Corresponding protein bands were detected by chemiluminescence and quantitated by a laser densitometer. As seen in Fig. 1 , coinfection with AAV prevented adenoviral-mediated degradation of p130 in these cells. In the case of pRB, AAV coinfection inhibited adenoviral-mediated phosphorylation and the accumulation of underphosphorylated pRB. It is important to note here that the pRB family of proteins undergoes functional inactivation with phosphorylation (21) . Interestingly, AAV infection alone was also observed to decrease the hyperphosphorylated form of pRB, however, with an increase in total pRB protein. Although the change in p107 after Ad-infection was less marked, AAV coinfection also protected against adenoviral-induced changes. The blots were also probed with antibodies against ß-actin and AAV Rep proteins, confirming equal loading and the expression of all 4 Rep proteins: Rep78, -68, -52, and -40.

View larger version (64K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Transcriptional transactivation and functional protection of pRB family of proteins by AAV. A, viral infections were carried out on 60–80% confluent normal human fibroblast cells as mentioned in "Materials and Methods." Cell extracts were prepared at optimal AAV Rep protein expression and probed with indicated antibodies after Western blotting. Protein bands were visualized by enhanced chemiluminescence and quantitated by a laser densitometer. B, HeLa cells were infected with viruses and the cell extracts prepared and probed as above with indicated antibodies. C, 293 cells were infected with viruses and the cell extracts prepared and probed as above with indicated antibodies.

Enhanced Transcription of pRB Family of Proteins by AAV.
To evaluate the possibility that AAV may also affect expression of p130, p107, and pRB at transcription level, we performed a RNase protection assay in Ad-infected normal human fibroblasts with and without coinfection with AAV. As seen in Fig. 2 , adenoviral infection, by itself, markedly decreased the transcription of both p130 (by 4.1-fold) and pRB (by 2.75-fold). However, coinfection with AAV not only overcame the adenoviral-induced decrease but substantially increased the transcription above the basal level. The level of p107 was undetectable in control, AAV-infected, or Ad-infected cells, however, there was a substantial increase after coinfection of Ad-infected cells with AAV. Surprisingly, AAV infection alone was also able to increase expression of p130 and pRB. These results indicated that AAV superinfection reverses the adenoviral-mediated inhibition of pocket protein transcription.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. AAV-mediated transcriptional increase of pRB family genes. Autoradiograph of duplex-protected p130, pRB, and p107 (top panel) and GAPDH (bottom panel) transcripts after digestion with RNase A and T1. RNA, isolated from human diploid fibroblasts after viral infection, was subjected to RNase protection assay using RiboQuant Multi-Probe RNase Protection Assay System (PharMingen). Autoradiographic signal was scanned on a phosphorimager, and signal intensity of each band was quantitated. Numbers at bottom, steady-state levels of p130, pRB, and p107 mRNA levels normalized to GAPDH.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Interaction of AAV Rep 78 with adeno-viral E1A inhibits its affinity to pRB. A, cells were infected with viruses as indicated and total cell lysates were prepared. Immunoprecipitations were carried out with E1A polyclonal antibodies. Precipitates were resolved on 8–12% SDS-polyacrylamide gels, transferred to nitrocellulose blots, and probed with AAV Rep monoclonal antibodies. B, E1A binding to Rep78 in vitro. His-Rep78 fusion protein was conjugated to nickel-nitrilo-triacetic acid column. Adenoviral-treated cell extracts were incubated in the column, and the affinity column was eluted with 250 mM imidazole. The eluents were electrophoresed, transferred, and probed with both pRB and E1A monoclonal antibodies. C, inhibition of pRB-E1A interaction with AAV expression. Total cell lysates were prepared after indicated viral infections. Immunoprecipitations were carried out with pRB polyclonal antibodies. Precipitates were resolved on 8–12% SDS-polyacrylamide gels, transferred to nitrocellulose blots and probed with both pRB and E1A monoclonal antibodies.

Next, we evaluated the effect of AAV Rep78 on the pRB binding to E1A. We immunoprecipitated adenoviral-infected cells, with and without AAV coinfection, with pRB antibody and probed the extracts for E1A. In the presence of AAV, substantially decreased coimmunoprecipitation of E1A with pRB was observed, which suggested decreased affinity of E1A to pRB in the presence of AAV Rep proteins. (Fig. 3C , Lane 2).

Effect of Change in pRB Family of Proteins after AAV Rep Expression.
The pRB family of proteins complex with, and sequester, the E2F family of transcription factors, thereby preventing transcription of critical genes that are essential for cell cycle progression (16 , 22) . It is well known that E1A affects this interaction, releasing E2F-1, which in turn sets the stage for cell cycle progression. Cells that express E1A have little or none of the pRB-E2F complex. Earlier experiments indicated that AAV Rep proteins protects the pRB family of proteins from E1A-mediated functional inactivation by decreasing their interaction. This stabilization of the pRB-E2F-1 complex by AAV expression in adenoviral-infected cells should lead to a decrease in E2F-1-mediated expression of cell cycle-specific genes. A plasmid construct with four E2F-1 binding sites, along with a luciferase open reading frame (E2-luc), was used to evaluate the functional consequences of change in free E2F-1 after AAV infection of adenoviral-treated cells. Adenoviral-treated cells, with or without AAV infection, were transiently transfected with E2-luc plasmid. Here, the measured luciferase activity would be directly proportional to the E2F-1 transcriptional activity. As expected, AAV decreased the adenoviral-induced luciferase activity, which indicated reduced levels of free E2F-1 (Fig. 4A) . To evaluate the effect of reduced E2F-1 activity on AAV expression, we analyzed the cell cycle status of these cells after Ad infection or AAV infection, or both. As seen in Fig. 4B , AAV coinfection of adenoviral-treated cells decreased the number of cells in S phase by 25%.

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Rep78 inhibits E2F-1-responsive E2 promoter activity. A, 0.5 µg pE2 luciferase plasmids were transfected into normal human fibroblasts by Effectene as mentioned in "Materials and Methods." After overnight incubation, cells were further subjected to adenoviral/AAV infections. Cell lysates were prepared, and luciferase activity was measured in light units. Data presented are means of three experiments and are statistically significant with regard to decrease in AAV+Ad compared with Ad alone (P < 0.001). B, reduction in the percentage of S phase cells with AAV. Approximately 5 x 106 cells were subjected to fluorescence-activated cell-sorting analysis after propidium iodide staining and S-phase cells were measured using ModFit software. Data presented are the means of three experiments.

	DISCUSSION

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Previously, we observed that the major regulatory protein of AAV, Rep78 interacts with p53 and protects it from adenoviral-mediated degradation (19) . Here, we show that AAV protects the pRB family of proteins from adenoviral-mediated functional inactivation and degradation. The specificity of our observation is confirmed using purified AAV virus with all contaminants removed, and by transfection of a plasmid containing the Rep gene alone. Underphosphorylated forms of the pRB family of proteins bind to, and negatively regulate, the E2F family of cellular transcription factors. E1A interaction with pRB contributes to its phosphorylation and the release of E2F-1, which leads to a transcriptional activation of S phase genes and to G₁-to-S-phase transition (18 , 23 , 24) . AAV Rep proteins not only provide protection from functional inactivation of pocket proteins by phosphorylation but also protect it from degradation by Ad.

Degradation of pRB after an adenoviral infection is facilitated by its binding with E1A. Our data indicate a specific binding between E1a and AAV Rep78, Rep68, and Rep52 proteins. Because Rep40 is expressed at a very low level, binding of E1a to Rep40 was not detected. However, we can’t rule out the possibility that E1a interacts preferentially with Rep52. Our results suggest that Rep binding to E1A reduces E1A binding to pRB, which decreases its degradation. At this point, it is unclear whether Rep also inhibits the effect of E1A on other kinases or whether it achieves this by directly preventing its binding to pRB. The observed effect on all of the three pocket proteins, pRB, p107, and p130, raises an important question regarding the possibility of a central control-protein responsible for their function and degradation. We also observed a substantial transcription activation of pocket proteins in response to AAV infection in the presence or in the absence of adenoviral infection (Fig. 2) . We postulate that Rep must either act as a transcription factor or may indirectly affect suppression of the transcription leading to higher expression.

The functional consequence of protection of pRB activity by AAV Rep in adenoviral-infected cells reflects the trans-inhibition of S phase genes followed by a reduced number of cells in S phase. Using luciferase vector construct with multiple E2F-1 binding sites, we confirmed significant reduction in the E2F activity on AAV treatment of adenoviral-infected cells.

AAV requires special cellular conditions or adenoviral helper function for its productive infection (1 , 3) . We observed that in fibroblasts, AAV infection alone prevents pRB phosphorylation, despite its inability to substantially express any regulatory genes (Fig. 1) . This confirmed earlier reports (24) . Although AAV does not express Rep proteins without the helper functions provided by Ad, it is possible that the encapsulated Rep protein in the virus may enter the cell and, thus, mimic the expression of Rep proteins (25) . Recent observations from our laboratory indicate that Rep proteins also exert their influence on E2F-1 to interfere with cell cycle progression besides their direct effects on E1A (20) . The data presented here elucidate the possible molecular mechanisms by which AAV negates the tumorigenic properties of Ad.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by grants from the National Cancer Institute (CA71092) and the Veterans Administration Merit Award (to N. C. M.) N. C. M. is a Leukemia Society Scholar in Clinical Research.

² To whom requests for reprints should be addressed, at Dana Farber Cancer Institute, 44 Binney Street, M557, Boston MA 02115. Phone: (617) 632-2144; Fax: (617) 632-2140; E-mail: Nikhil_munshi{at}dfci.harvard.edu

³ The abbreviations used are: AAV, adeno-associated virus type 2; RB, retinoblastoma; Ad, adenovirus; GAPDH, glyceryldehyde phosphate dehydrogenase.

Received 10/24/01. Accepted 3/18/02.

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