Transforming Potential of the Adenovirus Type 5 E4orf3 Protein

Plasmids used in this study.

pAd5 XhoI-C (30) contains the leftmost 15.5% of the Ad5 genome, including the E1A and E1B genes with their endogenous promoters. Plasmid pAd5 dl338XhoI-C is a derivative of pAd5XhoI-C that contains a deletion in the E1B-55kDa coding regions (30). Plasmids pCMV-E4orf3 and pSVHA-E4orf3 express wild-type and influenza virus hemagglutinin (HA) epitope-tagged Ad5 E4orf3 under the control of the cytomegalovirus (CMV) immediate-early promoter and the SV40 promoter/enhancer, respectively. These expression plasmids were constructed by PCR amplification of the E4orf3 coding sequence from pXbaC (16) with primers E4orf3fw (5′-CAGGGATCCGTCATGATTCGCTGCTTGAGGC-3′) and E4orf3rev (5′-CGCGGGATCCGTCGACTTATTCCAAAAGATTATCC-3′) containing BamHI restriction sites. To generate pCMV-E4orf3, the PCR product was cloned into the BamHI site of pcDNA3 (InVitrogen). For pSVHA-E4orf3, the same PCR fragment was first inserted into the BamHI site of pAS2 (19). From this plasmid (pAS-E4orf3), an EcoRI fragment containing the HA epitope-tagged E4orf3 coding sequence was isolated and cloned into plasmid vector pSVK3 (Pharmacia) to generate pSVHA-E4orf3. The sequence of each plasmid was confirmed by DNA sequencing. Plasmids pCMV-E4orf6 and pCMV-E1A express the Ad5 E4orf6 and Ad5 E1A proteins, respectively, from the CMV immediate-early promoter and have been described previously (8, 36). To generate pCMV-PML, an EcoRI fragment corresponding to the human full-length PML cDNA was isolated from pSG5-PML and inserted into the EcoRI site of pcDNA3 (InVitrogen). pSG5-PML was kindly provided by Anne Dejean.

Transformation assays and cell lines.

Sprague-Dawley rats were randomly bred at the university’s animal facilities under standardized conditions. Primary cultures of BRK cells were prepared from 6- to 7-day-old rats as described previously (37) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). For transformation assays, subconfluent cells were transfected 2 days postplating by the calcium phosphate procedure (13) with salmon sperm carrier (Boehringer) and plasmid DNA as described elsewhere (37). To avoid effects of promoter competition, the total amount of CMV or SV40 promoter was held constant by inclusion of empty pcDNA3 or pSVK3 vector plasmid. Three to four weeks after transfection, cultures were stained with crystal violet (1% in 25% methanol) and dense foci of morphologically transformed cells were counted. Representative plates were scanned and cropped by using Adobe Photoshop. Alternatively, foci were pooled or cloned and subsequently expanded into permanent cell lines.

The transformed BRK cell lines AB7, AB16, ABS1, and ABS6 have been described previously (38). AB7 and AB16 cells express the Ad5 E1 region; ABS1 and ABS6 cells express different levels of the Ad5 E4orf6 protein in addition to the Ad5 E1A and E1B genes. ABS16, ABT29, ABST4, and ABST5 are G418-selected cell lines of polyclonal origin derived from transfections of primary BRK cells with pAd5 XhoI-C and pCMV-E4orf6 (ABS16), pCMV-E4orf3 (ABT29), or both E4 plasmids (ABST4 and ABST5). Cell lines ABT9 and ABT10 were derived from transfections with pAd5 XhoI-C plus pSVHA-E4orf3 and originated from single transformed BRK cell foci. ABT27 cells were established from foci obtained from transfection of pSVHA-E4orf3, pcDNA3, and pAd5 dl338XhoI-C. All BRK cell lines were maintained in DMEM with 10% FCS.

Growth studies were performed exactly as described previously (38). Cell morphology was examined and photographed on Scotch Chrome 640T films with a camera-mounted Olympus AX70 microscope using phase contrast.

Antibodies.

Unless otherwise noted, all mouse monoclonal antibodies (MAbs) used in this study were obtained from supernatants of hybridoma cell cultures grown in RPMI supplemented with 10% FCS. RSA3 recognizes the amino terminus of the Ad5 E4orf6 protein (32), 2A6 (44) is specific for E1B-55kDa, M73 (17) is directed against E1A proteins and PAb421 (18) is specific for p53 from different species. The anti-PML MAb 5E10 was generously provided by Luitzen de Jong, and the p53-specific rabbit polyclonal antibody CM-1 was kindly provided by David Lane. Anti-HA mouse MAb 12CA5 and anti-E1B-19kDa rat MAb 1G11 were obtained from Boehringer and Oncogene Research, respectively. The mouse MAb AC-15 is specific for β-actin and was collected from ascites fluid (Sigma).

The anti-Ad5 E4orf3 rat MAb 6A11 was raised against a glutathione S-transferase E4orf3 fusion protein produced in Escherichia coli and will be described elsewhere.

Immunoprecipitation and immunoblotting.

For analysis of proteins by immunoprecipitation and immunoblotting assays, cells were lysed on ice in radioimmunoprecipitation assay buffer (50 mM Tris-chloride [pH 8.0], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 1% Nonidet P-40 [NP-40], 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 0.3 μM aprotinin, 1 μM leupeptin, 1 μM pepstatin) or NP-40 lysis buffer (50 mM Tris-chloride [pH 8.0], 150 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 0.3 μM aprotinin, 1 μM leupeptin, 1 μM pepstatin). After normalization for protein concentration, whole-cell extracts were subjected to Western blotting or immunoprecipitation. For immunoprecipitations, protein A- or protein G-Sepharose (Sigma) was incubated with 100 μl of the appropriate hybridoma supernatant. Immune complexes were resuspended in SDS-sample buffer, separated on SDS–10 to 15% polyacrylamide gels, and blotted onto nitrocellulose membranes (Schleicher & Schüll). The filters were blocked in phosphate-buffered saline (PBS) containing 5% nonfat dry milk for at least 1 h and then overnight in PBS containing the appropriate antibodies. The proteins were visualized by a secondary antibody linked to horseradish peroxidase (Amersham) followed by enhanced chemiluminescence (NOWA; Energene). Autoradiograms were scanned and cropped by using Adobe Photoshop, and figures were prepared by using Macromedia FreeHand software on an Apple Macintosh computer.

Indirect immunofluorescence and confocal laser scan microscopy.

For indirect immunofluorescence studies, cells were grown on coverslips to subconfluent densities. They were fixed with methanol for 20 min at −20°C and reacted with undiluted hybridoma supernatants and appropriate fluorescein isothiocyanate (FITC)- or Cy3-conjugated secondary antibodies (Dianova) at a concentration of 7.5 μg/ml as described elsewhere (38). Samples were analyzed and photographed on Scotch Chrome 640T films with a camera-mounted Olympus AX70 microscope. For double-label studies, a TCS-NT confocal laser scanning microscope (Leica) was used. Excitation wavelengths of 488 nm (FITC) and 568 nm (Cy3) were selected from an argon-krypton laser. Each fluorochrome was independently selected; pseudocolor images of both signals were generated and superimposed.

Tumorigenicity in nude mice.

Analyses of tumor growth in NMRI(nu/nu) mice were exactly as described previously (38). Briefly, transformed cells were harvested by scraping into PBS. Nude mice were injected subcutaneously with 10⁶ cells in serum-free DMEM, and tumor growth was recorded weekly with an electronic caliper.

The Ad5 E4orf3 protein can cooperate with E1A to stably transform primary BRK cells.

To explore the possibility that E4orf3 has transforming potential, we transfected primary BRK cells with plasmids expressing the E1A gene (pCMV-E1A) in combination with E4orf3 (pCMV-E4orf3), E4orf6 (pCMV-E4orf6), and E4orf3 plus E4orf6 (Fig. 1A). The E4orf3 protein alone was unable to induce the formation of transformed colonies, while transfection of BRK cells with DNA encoding E1A alone resulted in the appearance of a few, mostly abortive foci (Fig. 1B). In contrast, coexpression of E1A with E4orf3 resulted in a two- to threefold increase in the number of dense foci, which usually could be established into permanent cell lines. Thus, E4orf3, like E4orf6, can cooperate with E1A to stably transform primary rat cells, although on average, E4orf6 appeared to promote E1A-induced focus formation more efficiently than E4orf3. No further increase in the transformation frequency was obtained when increasing amounts of pCMV-E4orf3 were included in the transformation mixture or when both E4 plasmids were simultaneously introduced with E1A into BRK cells (Fig. 1A). Curiously, protein analyses revealed that all established cell lines derived from these experiments did not express the viral gene products (data not shown). Consistent with our previous work on transformed cells generated by E1A and E4orf6 (37), subsequent PCR analyses confirmed that all of these cell lines did not contain the viral E4orf3 gene (data not shown). This result strongly suggests that expression of E1A with E4orf3 or E4orf6 is sufficient for transformation but is not required to maintain the transformed cell phenotype.

E4orf3 and E4orf6 can synergistically promote focus formation in cooperation with E1A and E1B.

Expression of E4orf6 also enhances transformation by E1A and E1B proteins (33, 37). More importantly, these foci develop much more rapidly, contain more cells, and differ significantly from the E1A/E1B-transformed ones (33, 38). Similar to E4orf6, cotransfection of pCMV-E4orf3 with a plasmid expressing the E1A and E1B gene products (pAd5 XhoI-C) substantially increased the frequency of focus formation over transfection with pAd5 XhoI-C alone (Fig. 2A). Identical results were obtained when an epitope-tagged E4orf3 protein expressed from the SV40 promoter (pSVHA-E4orf3) was used in these assays (data not shown). Many of these foci grew rapidly and to a high density, closely resembling the transformed colonies produced by E1A/E1B and E4orf6 (Fig. 2B). The total number of transformed cells was further greatly increased when both E4 plasmids were simultaneously cotransfected with pAd5 XhoI-C, suggesting that both E4 proteins can synergistically promote focus formation in the presence of the E1B gene products. However, like E4orf6 (33, 37), the E4orf3 protein did not cooperate with E1A and E1B-19kDa (pAd5 dl338XhoI-C) in the same transformation assays (Fig. 2A). Rather, the E4orf3 protein reduced the number of foci produced by E1A and E1B-19kDa. We also noticed that as in the experiments using pCMV-E1A (Fig. 1A), a two- to fivefold increase of pCMV-E4orf3 resulted in a reduction of transformed foci (Fig. 2A). Interestingly, this negative effect was reproducibly not observed in cultures transfected with pAd5 XhoI-C and increasing amounts of pCMV-E4orf6 (data not shown). The decrease in focus-forming activity suggests that high levels of E4orf3 may cause cytotoxic effects, but the reason for the apparent difference between both E4 proteins in these assays is unclear.

Together with the results presented above, these experiments demonstrate that E4orf3 can cooperate with E1A and E1A/E1B proteins to transform primary rat cells, and they show that there is cooperation between E4orf3 and E4orf6 in the presence of E1B proteins.

The E4orf3 protein does not antagonize E1A-induced metabolic stabilization of p53 in transformed rat cells.

Adenovirus E1-transformed cells contain high levels of p53 due to the metabolic stabilization of this protein induced by E1A (31). The stability of p53 is further increased in the presence of the E1B gene products, although this effect may not require the binding of p53 to the large E1B protein (52). Recent studies demonstrated that the E4orf6 protein antagonizes this process. Expression of E4orf6 in E1-transformed rat cells induces a dramatic decrease of p53 steady-state levels (33, 37, 38), most likely by reducing the half-life of the tumor suppressor protein (33). Therefore, it was of interest to determine whether E4orf3 expression has similar effects on p53 levels. Following selection with G418, several different pools of foci from cultures transfected with pAd5 XhoI-C and pCMV-E4orf3 (ABT cells) and pAd5 XhoI-C with pCMV-E4orf6 plus pCMV-E4orf3 (ABST cells) were generated. These cells were compared with respect to expression of viral and cellular proteins to the previously described AB cells (expressing E1A and E1B) and ABS cells (expressing E1A, E1B, and E4orf6 proteins) (38). The levels of E1A and E1B proteins varied among the transformed cell lines (Fig. 3A). Significantly, ABS and ABST cells contained substantially lower levels of E1B-55kDa compared with AB and ABT cells. The molecular basis for this effect is unknown but may be related to the expression of the E4orf6 protein in these cell lines. In ABT and ABST cells, expression of E4orf3 was easily detectable; ABT9 cells (Fig. 3B, lane 7) contained significantly lower levels of the adenovirus protein compared with ABT10, ABT29, and both ABST cell lines (Fig. 3B, lanes 8 to 11). As expected, the p53 steady-state levels were considerably decreased in all ABS cells (Fig. 3A, lanes 4 to 6) relative to AB cells, where the p53 protein accumulated to high levels in the absence of the E4orf6 gene product (Fig. 3B, lanes 2 and 3). In contrast, no reduction of p53 was observed in the E4orf3-expressing ABT cell lines (Fig. 3B, lanes 7 to 9), which contained p53 protein in amounts comparable to those in both AB cells. Thus, the E4orf3 protein, unlike E4orf6, does not induce a decrease of p53 steady-state levels in E1A/E1B-transformed rat cells.

Interestingly, ABST5 cells exhibited higher levels of the p53 protein than ABS and ABST4 cells (Fig. 3B, lanes 4 to 6 and 10). These cell lines expressed comparable levels of both E1A and E1B proteins, excluding the possibility that different levels of these viral gene products contributed to this effect. Instead, this difference may be linked to the slightly lower E4orf6 levels in ABST5 cells, since we found that the reduction of p53 inversely correlates with E4orf6 expression (38). From these results, we conclude that E4orf3 does not interfere with the E4orf6-induced destabilization of p53 in transformed rat cells.

E4orf3 colocalizes with the PML nuclear structure in transformed rat cells.

Adenovirus infection causes a drastic redistribution of PODs (6, 10). Analyses of adenovirus mutants and transient transfections demonstrated that the E4orf3 gene product alone is sufficient to induce this reorganization and that the viral protein colocalizes with the PML protein in these structures. We therefore tested whether expression of the E4orf3 protein affects the integrity of the POD structure in transformed rat cells. By conventional fluorescence microscopy, all three ABT cell lines, but not AB7 cells, incubated with the anti-E4orf3 MAb 6A11 exhibited a punctate and nuclear fluorescence characteristic of the POD (Fig. 4), which was also apparent in ABST cells (data not shown). Double-labeling experiments and confocal laser scanning microscopy confirmed that essentially all of the E4orf3 protein localized with PML in the same intranuclear structures (Fig. 5). It appeared, however, that not all of the PML-containing bodies were associated with E4orf3, most likely due to an excess of PML or PODs. Remarkably, in some structures where PML colocalized with the E4orf3 protein (Fig. 5c), the PML punctate bodies were changed into slightly elongated, less tightly stained structures, indicating that E4orf3 can induce a relocalization of the PML protein in transformed rat cells, although these alterations were less pronounced than those induced by E4orf3 in adenovirus-infected and transiently transfected human cells (6, 10).

The E4orf3 protein can bind to E1B-55kDa in transformed rat cells.

The transforming potential of E4orf6 correlates with the ability of the adenovirus protein to bind to and modulate the function and stability of p53 (37). In addition, the results from the colocalization studies suggested that E4orf3 might physically interact with the PML protein. To address these questions, we analyzed whether E4orf3 can bind to p53 and/or PML in ABT and ABST cells. Using combined immunoprecipitation-immunoblotting assays, we failed to coprecipitate E4orf3 with antibodies directed against p53 (PAb421) or PML (5E10) (data not shown). Instead, we found that a substantial amount of E4orf3 present in ABT9 and ABT10 cells coprecipitated with E1B-55kDa (Fig. 6B, lanes 2 and 3), whereas no E4orf3 was detected in precipitates from ABT27 cells, which do not express the large E1B protein (Fig. 6A, lane 5). The reduced amount of coprecipitated E4orf3 in ABT9 cells is most likely due to the lower expression levels of the E4 protein in these cells (Fig. 6A, lane 2). The significance of these observations is not known, but they may hint at the possibility that the redundant functions of E4orf3 and E4orf6 are linked, at least in part, to complex formation with E1B-55kDa.

The E4orf3 protein does not interfere with PML-mediated suppression of E1A/E1B-induced focus formation.

The observation that the POD structure is consistently disrupted in lymphocytes from patients with APL, together with the demonstration that viral oncoproteins are found associated with these subnuclear domains, has led to the suggestion that PODs represent a target in oncogenic processes (6, 9). In addition, accumulating evidence indicates that the POD-associated protein PML is a growth suppressor (34) that can inhibit oncogenic transformation (29), possibly by modulating cell cycle progression (35) and programmed cell death (3). These findings prompted us to investigate the effect of PML expression on E1A/E1B-mediated transformation of primary BRK cells (Fig. 7). Cotransfection of a plasmid encoding human wild-type PML (pCMV-PML) with pAd5 XhoI-C reduced the number of foci by over 60% compared with pAd5 XhoI-C alone. Hence, this result demonstrates for the first time that PML can efficiently suppress Ad5 E1A/E1B-mediated focus formation of primary rat cells. In light of the fact that E4orf3 colocalizes with the PML protein in transformed rat cells (Fig. 5), we also examined whether simultaneous expression of E4orf3 overcomes the inhibitory effect of PML. While E4orf3 alone promoted focus formation in combination with E1A and E1B, no increase in the number of foci was observed when E4orf3 was expressed in combination with PML and E1A plus E1B proteins. Thus, E4orf3 does not interfere with PML-mediated suppression of E1A/E1B-induced focus formation. These data, together with the results from coimmunoprecipitation assays, indicate that E4orf3 enhances focus formation by mechanisms that are independent from interactions with PML.

E4orf3 expression in transformed cells induces morphological alterations and enhanced growth rates.

In our previous studies, we have demonstrated that transformed BRK cells stably expressing E4orf6 display additional properties commonly associated with a high grade of oncogenic transformation, including morphological alterations, markedly enhanced growth rates, and growth to higher saturation densities (38). To reveal the effect of E4orf3 expression on the transformed cell phenotype, we compared the morphology and growth rates of E4orf3 expressing cells with those of AB and ABS cells. Subtle differences existed in the morphology of the cell lines depending on whether they expressed the E4orf3, E4orf6, or both E4 proteins. Representative samples of these morphological differences are presented in Fig. 8. ABT cells differed from AB cells in that they were smaller, although these E4orf3-dependent alterations were less pronounced compared with those induced by E4orf6 in ABS1 cells. The E4orf3-expressing cell lines, and in particular ABT29 cells (Fig. 8e), tended to grow in colonies, which resembled the morphology of ABS1 cells (Fig. 8f), while ABST cells displayed an intermediate phenotype which seemed to reflect the contributions of both E4 proteins. Expression of the E4orf3 protein also affected the growth rates of these cells. Compared with AB7 cells, ABT10 and ABT29 cells expressing high levels of E4orf3 started to divide at higher growth rates at 4 days after plating and reached 2.6- and 1.8-times-higher saturation densities, respectively when this experiment was ended at 12 days after plating (Fig. 9). This positive effect was greatly enhanced when E4orf6 was coexpressed with E4orf3 in ABST cells. Clearly, ABST4 cells had the capacity to grow to a higher density than ABS1 cells, indicating a combinatorial promoting effect of both E4 proteins. Shortly after they reached confluency, these cells, like ABS1 and ABST5 cells, rapidly acidified the media and detached from the tissue culture plates as a result of cell death. The difference in growth rates between ABST4 and ABST5 cells might directly reflect the different levels of p53 which inversely correlate with E4orf6 expression levels in these cell lines (Fig. 3B). In support of this view, ABS1 cells expressing low levels of p53 divided at significantly higher rates than ABST5 cells containing higher amounts of p53. In sum, these findings demonstrated that the E4orf3 protein is capable of producing morphological alterations associated with the transformed cell phenotype, allowing the cells to grow more rapidly and to higher densities.

E4orf3 expression increases the tumorigenicity of transformed BRK cells in nude mice.

Because E4orf3 appeared to affect the growth properties of E1-transformed BRK cells in vitro, these cells were tested together with AB7 and ABS1 cell lines for tumorigenicity in nude mice (Table 1). During a 56-day observation period, all of the animals receiving 10⁶ ABS1, ABST4, or ABST5 cells developed visually apparent, rapidly growing tumors as early as 7 days after injection whereas no tumors were induced by AB7 cells during the observation period. In contrast, four of six and three of six animals given the same number of ABT10 and ABT29 cells, respectively, produced tumors 7 days after injection. Two solid tumors were also observed with ABT9 cells but only after an extended delay of 41 days. Consistent with our data from the in vitro experiments, tumors induced by ABST4 cells grew more rapidly than those produced by ABS1 or ABST5 cells and reached sizes of approximately 120 mm² at 28 days after injection (Table 1). Significantly smaller tumors were obtained with ABT cells after the same incubation period. Apparently, the ability of ABST4 and ABS1 cells to induce rapid tumor growth in nude mice correlated with lower p53 steady-state levels, and was dependent on the expression of the E4orf6 protein. Taken together, these in vivo studies demonstrated that E4orf3 expression increases the tumorigenicity of E1-transformed BRK cells.