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• W2002489396 abstract "Article15 August 2000free access Inhibition of S-phase progression by adeno-associated virus Rep78 protein is mediated by hypophosphorylated pRb Philippe Saudan Philippe Saudan Present address: Cytos Biotechnology AG, 8952 Schlieren, Switzerland Search for more papers by this author Jaromir Vlach Jaromir Vlach Present address: Cytos Biotechnology AG, 8952 Schlieren, Switzerland Search for more papers by this author Peter Beard Peter Beard Swiss Institute for Experimental Cancer Research (ISREC), 1066 Epalinges, Switzerland Search for more papers by this author Philippe Saudan Philippe Saudan Present address: Cytos Biotechnology AG, 8952 Schlieren, Switzerland Search for more papers by this author Jaromir Vlach Jaromir Vlach Present address: Cytos Biotechnology AG, 8952 Schlieren, Switzerland Search for more papers by this author Peter Beard Peter Beard Swiss Institute for Experimental Cancer Research (ISREC), 1066 Epalinges, Switzerland Search for more papers by this author Author Information Philippe Saudan2, Jaromir Vlach2 and Peter Beard1 1Swiss Institute for Experimental Cancer Research (ISREC), 1066 Epalinges, Switzerland 2Present address: Cytos Biotechnology AG, 8952 Schlieren, Switzerland The EMBO Journal (2000)19:4351-4361https://doi.org/10.1093/emboj/19.16.4351 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Adeno-associated virus (AAV) has an antiproliferative action on cells. We investigated the effect of the AAV replication proteins (Rep) on the cell division cycle using retroviral vectors. Rep78 and Rep68 inhibited the growth of primary, immortalized and transformed cells, while Rep52 and Rep40 did not. Rep68 induced cell cycle arrest in phases G1 and G2, with elevated CDK inhibitor p21 and reduced cyclin E-, A- and B1-associated kinase activity. Rep78-expressing cells were also impaired in S-phase progression and accumu lated almost exclusively with hypophosphorylated retinoblastoma protein (pRb). The differences between Rep78 and Rep68 were mapped to the C-terminal zinc finger domain of Rep78. Rep78-induced S-phase arrest could be bypassed by adenoviral E1A or papillomaviral E7 proteins but not by E1A or E7 mutants unable to bind pRb. Rb−/− primary mouse embryonic fibroblasts displayed a strongly reduced S-phase arrest when challenged with Rep78, compared with matched Rb+/+ controls. These results suggest that physiological levels of active pRb can interfere with S-phase progression. We propose that the AAV Rep78 protein arrests cells within S-phase by a novel mechanism involving the ectopic accumulation of active pRb. Introduction Adeno-associated virus type 2 (AAV) is a small DNA-containing parvovirus, which infects humans and depends on co-infection with a helper virus in order to produce progeny viruses (Berns and Linden, 1995). Low levels of AAV replication can also occur in cells exposed to genotoxic stress in the absence of helper virus (Yakinoglu et al., 1988). Under conditions that are not permissive for replication, AAV can establish a latent infection by integration of the viral DNA into the cellular genome, preferentially in a site-specific manner (Kotin et al., 1990). The AAV genome consists of two open reading frames, rep and cap, flanked by inverted terminal repeats that contain the replication origins (reviewed in Berns and Linden, 1995). The cap gene encodes three viral structural proteins. Four non-structural proteins are made from the rep gene by alternative promoter utilization and differential splicing. Rep78 and Rep68 are expressed from the p5 promoter and regulate many aspects of the viral life cycle, including DNA replication (Srivastava et al., 1983), transcription (Beaton et al., 1989; Pereira et al., 1997) and site-specific chromosomal insertion (Weitzman et al., 1994). They are multifunctional proteins with activities including specific DNA binding and site- and strand-specific endonuclease, helicase and ATPase activities (Im and Muzyczka, 1990; Wonderling et al., 1995). Rep52 and Rep40, which are expressed from the p19 promoter, do not bind DNA but have been shown to have ATP-dependent helicase activity (Smith and Kotin, 1998) and are involved in packaging single-stranded viral genomes into capsid structures (Chejanovsky and Carter, 1989). Rep68 lacks the 91 C-terminal amino acids of Rep78 and has an additional seven amino acids encoded by exon 2. Rep52 and Rep40 are N-terminally truncated versions of Rep78 and Rep68, respectively. AAV is generally believed to have antiproliferative effects on cells and, since it interferes with tumour formation in animal models, has been proposed to have oncosuppressive activities (reviewed in Rommelaere and Cornelis, 1991; Schlehofer, 1994). However, very little is known about the molecular mechanisms involved. Depending on the cell type used, non-lytic AAV infection at high multiplicity can either induce differentiation (Klein-Bauernschmitt et al., 1992), inhibit cell growth or interfere with the cell cycle (Winocour et al., 1988; Bantel-Schaal, 1990; Hermanns et al., 1997; Kube et al., 1997). These effects do not require de novo Rep synthesis since they were also observed with recombinant or UV-inactivated AAV. Studies with cultured cells revealed that the Rep proteins inhibit transformation by viral and cellular oncogenes (Ostrove et al., 1981; Hermonat, 1989; Khleif et al., 1991), viral and cellular DNA synthesis (Hermonat, 1992; Yang et al., 1994, 1995) and transcription from a variety of promoters (Labow et al., 1987; Hermonat, 1991, 1994; Hörer et al., 1995). Since it is still not clear which components (capsid or the Rep proteins) are responsible for the proposed oncosuppressive activity of AAV, we decided to investigate whether the Rep proteins interfere with the cell division cycle. The cell cycle is driven by a family of protein kinases called the cyclin-dependent kinases (CDKs). Their sequential activation controls the entry and passage through the different phases of the cell cycle: G1, S, G2 and M. In order to be activated, these CDKs must be associated with regulatory subunits, the cyclins, and have their inhibitory phosphates removed by the Cdc25 family of protein phosphatases (reviewed in Morgan, 1997). An additional level of regulation is achieved by two families of small inhibitory proteins the CDK-inhibitors (CKIs). CKIs play a key role in the response of cells to growth-inhibitory signals such as induction of differentiation, p53 activation in response to DNA damage, growth factor deprivation and senescence (reviewed in Sherr and Roberts, 1995; Harper and Elledge, 1996). The Cip/Kip family of CKIs includes p21Cip, p27Kip1 and p57Kip2. These CKIs demonstrate a broad range of specificity and are able to inhibit G1 cyclin–CDK complexes and, to a lower extent, cyclin B–Cdc2 complexes (Xiong et al., 1993; Harper et al., 1995). The second family includes the INK4 proteins p16INK4a, p15INK4b, p18INK4c and p19INK4d, which are specific inhibitors of CDK4 and CDK6 (reviewed in Ruas and Peters, 1998). The decision to complete a round of mitotic division takes place during the G1-phase of the cell cycle at a stage called the restriction point, which precedes the onset of DNA synthesis in the S-phase (Pardee, 1989; Zetterberg et al., 1995). The passage through this point is mainly controlled by the phosphorylation state of the retinoblastoma protein family consisting of pRb, p107 and p130, which are negative growth regulators (reviewed in Grana et al., 1998). In their active hypophosphorylated form, Rb-family proteins associate with transcription factors of the E2F/DP family, repressing their target genes that include regulators of S-phase entry and genes required for DNA replication (for reviews see Weinberg, 1995; Helin, 1998). During the G1–S transition, pRb is inactivated by phosphorylation through sequential activation of CDK4, CDK6 and CDK2. This hyperphosphorylated state is maintained until late mitosis when pRb is dephosphorylated by the PP1 family of phosphatases (reviewed in Mittnacht, 1998; Rubin et al., 1998). Besides the well-documented importance of pRb in the G1–S transition, recent reports based on over-expression of non-phosphorylatable pRb mutants suggest a novel function of active pRb in the inhibition of S-phase progression (Chew et al., 1998; Knudsen et al., 1998). The importance of pRb is further emphasized by the fact that DNA tumour viruses have evolved proteins (e.g. adenovirus E1A, human papillomavirus E7) that can bind and inactivate pRb, allowing progression into S-phase in the absence of mitogenic stimuli (reviewed in Moran, 1993). Results Rep78 and Rep68 have a strong antiproliferative activity Recombinant retroviruses were generated that express the individual Rep proteins (Figure 1A) from the retroviral promoter, together with a puromycin selectable marker (Morgenstern and Land, 1990). Rep68′ and Rep40′ lack the seven amino acids encoded by exon 2 present in Rep68 and Rep40, and therefore correspond to C-terminal truncations of Rep78 and Rep52, respectively. The recombinant retroviruses were named BP78, BP68′, BP52 and BP40′. Figure 1.Rep78 and Rep68′ have a strong antiproliferative activity whereas Rep52 and Rep40′ do not affect cell growth. (A) Schematic representation of the different Rep proteins used in this work. Rep78 is shown at the top with the different functional domains known [modified from Smith et al. (1997)]. The numbers shown on the right refer to amino acid positions in Rep78. (B) NIH 3T3 cells were infected with the indicated recombinant retroviruses, split and seeded in selective medium. Colonies were fixed and stained on dishes 6 days after infection. (C) Outgrowth curves of BGF, BGF78, BGF68′ and BGF52-transduced NIH 3T3 cells. The percentage of GFP-positive cells was monitored at the indicated time point by flow cytometry. (D) Immunoblot analysis of retrovirally expressed Rep proteins, 3 days after infection. Download figure Download PowerPoint NIH 3T3 cells were infected with these recombinant and control (BP, empty vector) retroviruses and seeded at serial dilutions under selective conditions. All uninfected cells had detached within 48 h of selection, at which time the more dense dishes were harvested for biochemical analysis. The lower dilutions were incubated further to assess colony outgrowth. These dishes were fixed and stained after 6 days in selective medium. Rep52 and Rep40′ had no effect on cell growth whereas Rep78 and Rep68′ strongly suppressed colony outgrowth (Figure 1B). Microscopic analysis showed that dishes seeded with BP78- or BP68′-infected cells contained numerous single cells that were enlarged, suggesting that these Rep proteins induce cell cycle arrest as opposed to cell death. Moreover, these results show that the absence of colonies is not due to the down-regulation of the resistance gene by the Rep proteins. Similar results were obtained with primary human lung fibroblasts (HLFs) and the human osteosarcoma cell line U2-OS transduced with pBP78 and 68′ (data not shown). Western blot analysis of infected cells showed that the different Rep proteins are expressed at similar levels (Figure 1D). In BP78 and BP68′, the AAV p19 promoter from which Rep52 and Rep40 are expressed is present and accounts for the low levels of these proteins present in BP78- and BP68′-transduced cells, respectively. To rule out possible effects of the two large Rep proteins on viral titres and expression of the resistance gene, we created a new retroviral vector named BabeGFP (BGF), which instead of the drug resistance, co-expresses the enhanced green fluorescence protein (EGFP). The recombinant versions expressing Rep78, Rep68′ and Rep52 were generated (BGF78, BGF68′ and BGF52) and used to infect NIH 3T3 cells. Infected cells were passed every 2–3 days, and aliquots were fixed and analyzed by flow cytometry for the percentage of infected cells (Figure 1C). Although similar infection rates were observed initially with the four different viruses, the GFP-positive population was rapidly outgrown in BGF78- and BGF68′-infected cells, with kinetics corresponding to a growth arrest, whereas this population remained constant for BGF- and BGF52-infected cells. Living cells were analyzed by fluorescence microscopy. Dishes seeded with BGF78- or BGF68′-transduced cells revealed green cells that were enlarged, with a morphology typical of arrested cells (data not shown) in accordance with the cell cycle block induced by Rep78 and Rep68′. Differential cell cycle arrest mediated by Rep78 and Rep68 Having demonstrated the growth-suppressive activity of Rep78 and Rep68′ in this system, we wanted to know whether these cells are blocked in specific phases of the cell cycle. Asynchronously growing NIH 3T3 cells were infected with BP, BP78 and BP68′, and 4 days after infection subconfluent cells were pulse-labelled with bromodeoxyuridine (BrdU) for 45 min and fixed for flow cytometry. Nuclei were prepared, stained with propidium iodide and fluorescein isothiocyanate (FITC)-coupled anti-BrdU antibody, and analyzed by bivariate flow cytometry. We found that Rep78 and Rep68′ have different effects on cell cycle distribution (Figure 2). Rep68′ led to a strong reduction of S-phase accompanied by accumulation of cells at the G2–M boundary, while the percentage of cells in G1 was not significantly changed. This pattern shows that the cells were blocked both in G1 and G2. In contrast, cells expressing Rep78 not only arrested in G1 and G2, but also in S-phase, as documented by the appearance of cells with S-phase DNA content that did not incorporate BrdU. Similar results were obtained with primary mouse embryonic fibroblasts (MEFs) and primary human lung fibroblasts (HLFs) (Figure 2) suggesting that the arrest observed here reflects a general action of the two Rep proteins. Figure 2.Rep78 and Rep68′ arrest the cells at different phases of the cell division cycle. (A) Cell cycle distribution of Rep78- and Rep68′-expressing cells compared with cells transduced with empty vector as measured by BrdU incorporation and DNA-content flow cytometry on nuclei. BrdU incorporation is monitored with an FITC-anti-BrdU antibody (vertical axis) and DNA content by propidium iodide staining (horizontal axis). The different cell types are indicated at the top, and the different retroviruses at the right of the panels. (B) Quantitation of the different phases of the cell cycle in transduced NIH 3T3 cells and HLFs. The S-phase is differentiated into cells that synthesize DNA (S+) as measured by BrdU incorporation, and cells with S-phase DNA content that do not synthesize DNA (S−). BP-infected cells are shown as black, BP78 as white and BP68′ as grey bars. Error bars represent the standard deviations of four independent experiments. Download figure Download PowerPoint Analysis of cell cycle proteins To characterize the nature of the cell cycle arrest caused by the Rep proteins, we examined the expression of several cell cycle regulatory proteins by immunoblot analysis. NIH 3T3 cells were transduced with empty vector or viruses expressing either Rep78 or Rep68′, and the cells were collected for analysis 3–4 days after infection (Figure 3A). Both Rep proteins were expressed at similar levels in the transduced cell population. Neither Rep78 nor Rep68′ had a significant effect on the protein levels of CDK4, 6 and 2, the G1 cyclins D1 and E, and the CKI p27. In contrast, expression of Rep78 and Rep68′ led to the induction of the CKI p21. Figure 3.(A) Immunoblot analysis of cellular proteins in transduced NIH 3T3 cells. Cells were infected with BP, BP78 and BP68′ as indicated at the top. At the time of analysis (3–4 days post-infection) all cells were subconfluent. The cellular proteins visualized in each panel are indicated to the right. (B) Cyclin E, cyclin A or cyclin B1 as indicated were immunoprecipitated from NIH 3T3 cell lysates. Histone H1 kinase activities were measured in the immunoprecipitates and normalized to the activities in control cells (BP, 100%). Error bars represent the standard deviation of at least three independent experiments. Download figure Download PowerPoint Expression of Rep78 and Rep68′ had opposite effects on the levels of the S-phase cyclin (A) and the G2 cyclin (B1). Both cyclins were strongly down-regulated in BP78-transduced cells but present at higher levels in BP68′-transduced cells. The two Rep proteins also differentially affected Rb-family proteins. The phosphorylation status of these proteins can be monitored by SDS–PAGE, in which the hyperphosphorylated forms have slower migration. Rep68′-expressing cells accumulated with a mixture of hyper- and hypo-phosphorylated pRb and slightly increased levels of p130, whilst the levels and phosphorylation status of p107 remained unchanged. However, BP78-transduced cells arrested with increased levels of p130, decreased levels of p107 and essentially only the hypophosphorylated form of pRb. To gain further insight into the nature of the cell cycle arrest induced by the Rep proteins, we measured kinase activities associated with the cyclins E, A and B1 in a histone H1 phosphorylation assay (Figure 3B). Rep78- and Rep68′-expressing cells had a 10-fold reduced cyclin B1-associated kinase activity whereas cyclin E-associated kinase activity was reduced by a factor of two. Cyclin A-associated kinase activity was strongly reduced in BP78, and reduced ∼2-fold in BP68′-transduced cells. In summary, Rep68-expressing cells are arrested in G1 and G2–M. The increase in the fraction of hypophosphorylated pRb and the slight decrease in cyclin E activity appears consistent with the G1 arrest. We presume that the elevated levels of the cyclins A and B1 can be attributed to the increased population in G2, since we observed similar effect on these cyclins in NIH 3T3 cells chemically blocked in G2 with etoposide (data not shown). In addition, Rep78 interferes with S-phase progression. Although Rep78-expressing cells are arrested throughout the cell cycle, they accumulate almost exclusively with the hypophosphorylated form of pRb. The reduced activity of cyclin A- and B1-associated kinases can be attributed to the markedly reduced levels of the corresponding cyclins. A zinc finger motif in the C-terminus of Rep78 is responsible for the differential response of cells to Rep78 and Rep68′ We wanted to explore further the difference between Rep78 and Rep68′. As mentioned earlier, Rep68′ represents a C-terminal truncation of Rep78. A closer look at the C-terminus absent in Rep68′ reveals a zinc finger motif composed of three CXXC and three CXXH repeats (Figure 4C). This motif was shown to be capable of binding zinc in vitro. Individual mutation of either of these two repeated motifs, (CXXC)3 to (RXXR)3, and (CXXH)3 to (SXXH)3, abolished zinc binding suggesting that both repeats are necessary to form a functional zinc finger (Hörer et al., 1995). To test whether this motif is responsible for the functional differences observed here between Rep78 and Rep68′, recombinant retroviruses were generated that express the (RXXR)3 mutant version of Rep78 (BP78CXXC), and analyzed as before in NIH 3T3 cells. Cells transduced with 78CXXC behaved like cells transduced with Rep68′ (Figure 4A and B). They arrested in G1 and G2, accumulated with increased levels of p21 and cyclins A and B1, and displayed hypo- and hyper-phosphorylated pRb. Similar results were obtained with the (SXXH)3 mutant (data not shown). In conclusion, a functional zinc finger motif is required for the S-phase arrest observed with Rep78 and for the quantitative accumulation of pRb in its hypophosphorylated form, as well as for the down-regulation of cyclins A and B1. Figure 4.The zinc finger motif in the C-terminus of Rep78 is responsible for the S-phase arrest. NIH 3T3 cells were transduced with BP, BP78, BP68′ and BP78CXXC. (A) Cell cycle distribution of the different samples, analyzed as described in Figure 2. (B) Immunoblot analysis of cellular proteins as described in Figure 3. (C) Sequence of the zinc finger motif in the C-terminus of Rep78 in comparison with the mutant constructs 78CXXC and 78CXXH (Hörer et al., 1995). Download figure Download PowerPoint Rapid appearance of hypophosphorylated pRb upon Rep78 expression Given the profound effect of Rep78 on the pRb phosphorylation pattern, we determined the kinetics of pRb accumulation in its hypophosphorylated form. NIH 3T3 cells were transduced with BGF or BGF78, and the phosphorylation status of pRb was examined in cells harvested at different time points between 24 and 48 h later (Figure 5). The percentage of infected cells was 80–90% as determined by flow cytometry (data not shown). Maximal Rep78 protein levels were observed 28 h after infection and remained constant thereafter. At this time point, the pRb phosphorylation pattern was still unchanged as compared with BGF-infected cells. However, 4 h later already half of the Rb protein was found in its hypophosphorylated form, and after an additional 4 h most of the Rb protein was hypophosphorylated. In contrast, cyclin A started to decline only 8 h after the onset of Rep expression. These results are in accordance with a central role played by pRb in response to Rep78, and suggest that the disappearance of cyclin A is subsequent to, rather than a cause of, pRb dephosphorylation. Figure 5.Rapid appearance of hypophosphorylated pRb upon Rep78 expression. NIH 3T3 cells were infected with pBGF (−) and pBGF78 (+) for 12 h, split and seeded at different dilutions. Whole-cell lysates of samples harvested at the indicated time points were analyzed by immunoblotting for Rep and cyclin A protein levels and pRb phosphorylation status. Download figure Download PowerPoint Bypass of the Rep78-mediated arrest by the adenovirus E1A protein We wanted to elucidate further the possible role of pRb in the S-phase arrest mediated by Rep78. The fact that in contrast to Rep68′, Rep78 arrests cells throughout the cell cycle with almost exclusively hypophosphorylated pRb prompted us to hypothesize that pRb could be responsible for the observed S-phase arrest. Two recent reports show that over-expression of active pRb can interfere with S-phase progression (Chew et al., 1998; Knudsen et al., 1998). This effect could be attenuated by over-expression of the adenovirus E1A protein, which functionally inactivates pRb by direct binding. Thus, we reasoned that if the Rep78-mediated S-phase arrest is due to active pRb, the adenovirus E1A protein should overcome this block. To test this hypothesis we used serial retroviral infections as described previously (Vlach et al., 1996). NIH 3T3 cells were first infected (INF.1) with pBH (control vector) or pBH–E1A, expanded under hygromycin selection, superinfected (INF.2) with BP or BP78, and seeded in puromycin selective medium. Cells were harvested 4 days after infection, and analyzed by bivariate flow cytometry (Figure 6A). E1A partially rescued Rep78-mediated cell cycle arrest. In comparison with control cells arrested with BP78, the number of cells expressing both E1A and Rep78 arrested in S and G2 was reduced concomitant with an increased population of cells actively synthesizing DNA. Figure 6.Bypass of the Rep78-mediated cell cycle arrest by the Ad-E1A protein. (A) Cell cycle distribution of infected cells prepared and analyzed by flow cytometry as described in Figure 2. NIH 3T3 cells were infected first (INF.1) with a retrovirus expressing E1A or with the empty control vector (pBH), followed by BP retroviruses with or without Rep78 (INF.2). (B) pRb binding but not p300 binding of E1A is required to rescue Rep78 mediated S-phase arrest. Quantitation of the different phases of the cell cycle serially-infected NIH 3T3 cells. NIH 3T3 cells were infected first with BH retroviruses expressing E1A, E1AR2G, E1A124/135A and E1AΔ26–35 or with the empty control vector (BH) followed by a second infection with BP or BP78. The different cell populations infected are indicated above the graphs. BP-infected cells are shown as black bars and BP78 as white bars. The S-phase is differentiated into cells that synthesize DNA (S+) as measured by BrdU incorporation and cells with S-phase DNA content that do not synthesize DNA (S−). Error bars represent the standard deviations of three independent experiments. (C) pRb inactivation by E1A is required to restore cyclin A, B1 and cdc2 protein levels in cells expressing Rep78. Cell populations expressing E1A, E1A124/135A or infected with control virus (BH) were infected with BP (−) or BP78 (+) as indicated at the top. Immunoblot analysis was performed as described in Figure 3. The antibodies used are indicated to the right of the individual panels. (D) Cells expressing both E1A and Rep78 die. Cells infected with BH, BHE1A and BHE1AΔ26–35 retroviruses were superinfected with BP and BP78 viruses as described above. After complete selection (48 h post-splitting) resistant cells were counted at 12 h intervals. Averages and the standard deviation of triplicates are shown. Download figure Download PowerPoint Two functional domains of E1A are required to bypass Rep78 mediated arrest E1A is a multifunctional protein. To identify which functional domains are required to bypass Rep78-mediated cell cycle arrest, we analyzed a panel of different E1A mutants. These were E1A124/135A, which is unable to bind and inactivate pRb; E1AR2G, which has lost the ability to bind to p300/CBP but can still functionally inactivate pRb (reviewed Bayley and Mymryk, 1994); and an N-terminal deletion (E1AΔ26–35), which still functionally inactivates pRb but has lost the ability to bind p400, a cellular protein with as yet unknown functions (Barbeau et al., 1994). Cell populations expressing wild-type (wt) E1A, E1A124/135A, E1AR2G, E1AΔ26–35 and control cells (BH) were generated by retroviral transduction as described above, and superinfected with BP and BP78. Four days after infection, cells were harvested and analyzed by flow cytometry (Figure 6B). The mutant that cannot bind pRb (E1A124/135A) failed to rescue the Rep78-mediated arrest. Similar results were obtained with HPV16 E7 protein. Wild-type E7 partially restored a normal cell cycle distribution, whereas the pRb non-binding mutant E7C24G had no effect on the Rep78-imposed cell cycle arrest (data not shown). E1AR2G, which has lost the ability to bind p300/CBP, showed a similar rescue to the one observed with wt E1A. Interestingly, E1AΔ26-35 rescued the Rep78-mediated S and G2 phase arrest, as documented by the reduced populations blocked in S and G2 compared with control cells. However, this mutant did not rescue the Rep78-mediated G1 arrest, since these cells accumulated in G1. In conclusion, the pocket-binding domain and an N-terminal domain are required to bypass Rep78-mediated cell cycle arrest. Whereas the N-terminal domain residues 26–35 are essential to rescue the G1 arrest, this domain is dispensable for the bypass of the S and G2 arrest. These results provide further evidence for the implication of pRb in the S-phase arrest, and in addition reveal that Rep78 also targets a pRb-independent function required for the G1–S transition. We have shown above that Rep78 leads to the down-regulation of several cell cycle regulatory proteins including the E2F target genes, cyclin A and Cdc2, as well as the mitotic cyclin B1. We wondered whether the reduction of these protein levels was a consequence of the activation of pRb or whether this reflected a pRb-independent activity of Rep78. In order to test this, we analyzed the levels of these proteins in control cells (BH) and cells expressing E1A or E1A124/135A superinfected with BP and BP78 (Figure 6C). Cells expressing the E1A mutant deficient in pRb binding (E1A124/135A) displayed similar patterns to control cells when challenged with Rep78. In contrast, the levels of cyclin A and Cdc2 were restored in cells expressing both E1A and Rep78. Interestingly, the levels of cyclin B1, which is not an E2F target gene, were also restored in cells expressing E1A. However, recently J.Lukas et al. (1999) provided a link between pRb phosphorylation status in S-phase and cyclin B1 stability (see Discussion). In conclusion, these results suggest that the reduced levels of cyclins A and B1, and of Cdc2 are a consequence of pRb activation rather than a pRb-independent function of Rep78. Cells expressing both E1A and Rep78 die Although E1A partially rescued a Rep78-mediated cell cycle arrest, E1A-expressing cells transduced with BP78 fail to form colonies in a colony outgrowth assay (data not shown). Microscopic analysis revealed that there were very few cells recovered expressing both E1A and Rep78, suggesting that these cells were dying. To test this further we compared the growth properties of these cells with control and E1AΔ26–35-expressing cells that are both arrested by Rep78. The different cell populations were transduced with BP or BP78, selected in puromycin and, 48 h post splitting, were counted at 12 h intervals (Figure 6D). The number of control and E1AΔ26–35-expressing cells transduced with BP78 remained constant over this time period, reflecting cell cycle arrest. However, cells expressing the E1A protein decreased in number, suggesting cell death. The cell populations infected with BP showed exponential growth. Taken together with the observations from the flow cytometry analysis above, these results show that E1A-expressing cells that fail to arrest upon Rep78 expression, die. Reduced S-phase" @default.
• W2002489396 created "2016-06-24" @default.
• W2002489396 creator A5002928235 @default.
• W2002489396 creator A5030616724 @default.
• W2002489396 creator A5067856155 @default.
• W2002489396 date "2000-08-15" @default.
• W2002489396 modified "2023-09-25" @default.
• W2002489396 title "Inhibition of S-phase progression by adeno-associated virus Rep78 protein is mediated by hypophosphorylated pRb" @default.
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