Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection

Abstract

Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP-E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP-E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells.

Introduction

HAdv E1A has been widely used as a prototypical viral oncogene for dissecting the molecular mechanisms of cell proliferation and cell transformation. The E1A gene codes for two major proteins that are expressed from two alternatively spliced mRNA species namely the 13S and 12S. The 13S mRNA codes for a 289 amino acids protein (L-E1A) and the 12S mRNA codes for a 243 amino acids protein (S-E1A). The 13S product differs from the 12S product by the presence of a unique 46 amino acids internal sequence. Both E1A protein products can immortalize primary cells and can transform them in co-operation with other viral and cellular oncogenes (Graham et al., 1974; Houweling et al., 1980; Ruley, 1983; Zerler et al., 1986). The transforming activities of E1A have been linked to the interaction with various cellular protein complexes (reviewed in references, (Chinnadurai, 2011; Pelka et al., 2008). Through interaction with the cellular protein complexes, E1A deregulates the cell cycle and induces cell transformation. These cellular protein complexes include the histone acetyl transferases, p300/CBP, the TRRAP/p400/GCN5 multi protein chromatin remodeling complex and the retinoblastoma (Rb) tumor suppressor family proteins (reviewed in (Chinnadurai, 2011)). The interaction of E1A with Rb results in the activation of E2F family of transcription factors (Chellappan et al., 1992) which activate the S-phase genes resulting in cell cycle progression. The E1A gene also reprograms host cell gene expression to block cell differentiation (reviewed in (Berk, 2005; Frisch and Mymryk, 2002; Gallimore and Turnell, 2001).

In addition to the transforming function, the E1A proteins also possess a paradoxical transformation suppression function that is encoded within the C-terminal region (reviewed by (Chinnadurai, 2011; Yousef et al., 2012)). The E1A C-terminal mutants induced high frequency transformation of BRK cells in co-operation with the activated Ras oncogene (Boyd et al., 1993; Douglas et al., 1991; Fischer and Quinlan, 1998; Schaeper et al., 1995; Subramanian et al., 1989)). In addition, the transformed cells expressing the C-terminal mutants were highly tumorigenic in athymic mice and syngeneic rats whereas the wild-type E1A-transformed cells were less tumorigenic in athymic mice and non-tumorigenic in syngeneic rats. Moreover, the E1A mutant transformed cells were highly metastatic when injected into athymic mice. At present the mechanisms by which the C-terminus of E1A suppresses the cell transformation, tumorigenesis and tumor metastasis are not fully understood. Our laboratory identified and cloned the first cellular protein, C-terminal binding protein 1 (CtBP1) that interacts with the C-terminus of E1A through a conserved motif, PLDLS (Boyd et al., 1993; Schaeper et al., 1995). E1A also interacts with a highly homologous protein, CtBP2 (Zhao et al., 2006). In addition to CtBP1/2 (collectively designated as CtBP), the C-terminal region of E1A interacts with two other protein complexes, DYRK1A/1B/HAN11 (designated here as DYRK1/HAN11) and FOXK1/K2 through distinct conserved domains (Komorek et al., 2010; Zhang et al., 2001). E1A mutants that are individually defective in interaction with DYRK1A/1B/HAN11 and FOXK1/K2 complexes exhibit hyper transforming activities (Komorek et al., 2010). To understand the importance of interaction of CtBP with E1A C-terminus in cell transformation and virus replication, we mutated E1A C-terminus within the CtBP-binding motif and characterized the effect of interaction of E1A with CtBP in cell transformation and virus replication. We found that the interaction of E1A with CtBP reduced immortalization and Ras co-operative transformation in primary rodent epithelial (BRK) cells while enhancing virus replication in human cells.

MATERIALS AND METHODS

Cells

Baby rat kidney (BRK) cells were prepared from 2-day old neonatal rats (Subramanian et al., 1989) and grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10 % fetal bovine serum. Normal human bronchial epithelial cells (HBEpC) were purchased from Cambrex, USA and grown in bronchial epithelial cell growth medium (Lonza Walkersville, Inc.). HeLa, A549, U2OS and 293 cells were grown in DMEM containing 10 % fetal bovine serum.

Plasmids and Viruses

The plasmids p12S, p12S-dl1132, p12S-dl1133, p12S-177-9, pBABEpuro, pBABE-12S, pBABE-dl1132 and pBABE-dl1133 were described previously (Komorek et al., 2010). Plasmids pBABE-177-9, p12S-DL→AS and pBABE12S-DL→AS were constructed by oligo nucleotide directed mutagenesis. Plasmids CtBP1shRNA2/pL6 and CtBP2shRNA1/pL7 were generous gifts from Dr. Dana Madison (Madison and Lundblad, 2010).

For the construction of HAdv5-DL→AS mutant virus, the plasmid, pE1A (nucleotide 1 to 1770 containing genomic E1A of HAdv5 in pUC19) was mutated to change residues D281 and L282 to A and S using an oligo nucleotide directed mutagenesis kit from Stratagene. The resulting plasmid pE1A-DL→AS, was co-transfected with the HAdv5 genomic plasmid pBHGE3 (Bett et al., 1994) onto human 293 cells and the cells were maintained in DMEM containing 2% fetal bovine serum until clear cytopathic effects were seen. The cells were freeze thawed three times and the supernatant was plaque assayed on 293 cells. Plaques were screened for the mutation by restriction analysis and confirmed by DNA sequencing. The mutant virus (HAdv5-DL→AS) was purified by CsCl banding and titrated on 293 cells. In a similar way the p12S-177-9 mutation (Subramanian et al., 1989) was introduced into the E1A plasmid and the virus (HAdv5-177-9 (Δ224-284 aa in 13S product)) was constructed as described above. The genomic version of mutants HAdv5-dl1132 and HAdv5-dl1133 have been described previously (Jelsma et al., 1989; Mymryk and Bayley, 1993). The plasmids/retrovirus vectors and HAdv5 viral mutants harboring different E1A mutations and their defect in interaction with cellular proteins are listed in Table 1.

Table 1. Plasmids, retroviral vectors and viral mutants used in the study.

Plasmids that express E1A12S in pUC19 were used in transformation studies. pLPC-based vectors that express E1A12S were used for transient E1A protein expression. pBABE(E1A12S)-based retroviral vectors were used in immortalization studies. HAdv5 based adenovirus mutants (genomic E1A) were used in viral replication studies. The coordinates of E1A mutations are based on the larger 289-amino acid protein (L-E1A).

Plasmid/viral mutant	E1A background	aa mutated	Interaction with cellular proteins
			FOXK	DYRK1	CtBP
Plasmids(pUC18/pBABE/pLPC)
E1A(12S)	12S (243R)	-	+	+	+
E1A(12S) dl1132	12S (243R)	Δ224–238	−	+	+
E1A(12S)dl1133	12S (243R)	Δ239–254	+	−	+
E1A(12S)DL→AS	12S (243R)	D281L282→AS	+	+	−
E1A(12S)177-9	12S (243R)	Δ224–284	−	−	−
Adenoviruses
HAdv5dl309	Genomic(12S+13S)	-	+	+	+
HAdv5dl1132	Genomic(12S+13S)	Δ224–238	−	+	+
HAdv5dl1133	Genomic(12S+13S)	Δ239–254	+	−	+
HAdv5DL→AS	Genomic(12S+13S)	D281L282→AS	+	+	−
HAdv5177-9	Genomic(12S+13S)	Δ224–284	−	−	−

Immortalization

Retrovirus vectors (pBABE) expressing E1A12S or E1A12S mutants or empty vector were pseudotyped in Phoenix E retrovirus packaging cell line. Forty eight hr after transfection, the supernatant containing retroviruses were used to infect BRK cells in six-well plates. Twenty four hr after infection, cells were selected with 0.25 μg/mL of puromycin for 2 weeks. Every fourth day, the medium was replaced with fresh medium containing puromycin. At the end of 10 days, colonies were stained with crystal violet, photographed and counted. In the second set of experiments, 24 puromycin selected colonies were randomly picked from retrovirus-transduced cells and plated in 24-well plates with 1 mL of growth medium. The growth medium was replaced with fresh medium every fourth day and the cells were stained with crystal violet after four weeks. The total number of wells with proliferated cells were counted and considered as immortalized.

Ras co-operative transformation and tumorigenesis

Primary BRK cells were plated in six-well plates with 2.0 mL of DMEM containing 10 % fetal bovine serum one day prior to transfection. Each well was transfected with 1.0 μg of E1A12S or mutants, 1.0 μg of pUC18-Ras and 1.0 μg of pcDNA3 (Invitrogen), using jetPEI (Polyplus) transfection reagent according to the manufacturer’s specification. Twenty four hr post transfection, cells were selected with 50.0 μg/mL of G418 for 10 days and stained with crystal violet, the transformed colonies were counted and plotted. To establish cell lines, another batch of transfected BRK cells were selected with G418, the colonies from each transfectant were pooled and grown as a pooled cell line. The established cell lines were tested for their tumorigenesity in athymic mice and two-day-old neonatal rats. Each athymic mice were subcutaneously injected with 1 X 10⁶ cells in 100.0 μL of DMEM without serum and the tumor volumes were measured every fifth day for 20 days. For neonatal rats, 1 X 10⁶ cells in 50.0 μL of DMEM without serum were injected subcutaneously. At the end of 3 weeks period, all the rats were euthanized and the number of tumor bearing rats were counted.

Establishment of CtBP knockdown cell lines

The human 293T cell line was transfected with lentiviral vectors expressing shRNAs against CtBP1 (CtBP1shRNA2/pL6) or CtBP2 (CtBP2shRNA/pL7) along with lentivirus packaging plasmids. After 48 hr of transfection, the supernatant containing lentivirus was collected and used for infecting the human U2OS cell line. The infected cells were selected with 6 μg/mL of blasticidin for CtBP1 knockdown or 300 μg/mL of hygromycin for CtBP2 knockdown. The selected colonies were pooled, tested for the expression of CtBP1 and 2 and established as cell lines.

Virus growth

The human cells (A549 and HBEpC) were infected with 5 PFU/cell of HAdv5-dl309 or genomic versions of different E1A mutant viruses (DL→AS, dl1132, dl1133 or 177-9). After 1 hr incubation at 37°C, cells were washed 3 times with 2.5 mL of DMEM containing 2% fetal bovine serum and replaced with 2.5 mL of DMEM containing 2% fetal bovine serum. At the indicated times after infection, cells were frozen and the virus contents were titrated on 293 cells. In another set of experiments, the virus infected cells were lysed in SDS-sample buffer and subjected to PAGE and western blot analyses of adenoviral early and late proteins.

Immunoprecipitation and western blot analysis

Transiently transfected HeLa cells (24 hr after of transfection) or established BRK cells were lysed and cleared by centrifugation. The clear cell lysates were incubated with anti-E1A mAb (M58; BD Biosciences) overnight and the bound protein complexes were pulled with protein A agarose beads. The immunoprecipitated protein complexes and cell lysates were resolved by SDS-PAGE and analyzed by western blot using antibodies against indicated proteins (E1A, CtBP1, CtBP2, FOXK1 and DYRK1A). For western blot analysis of viral structural proteins, human cells in 35 mm dishes were infected with 5 PFU/cell of wt HAdv5 (dl309) or mutants. At the indicated times after infection, cells were lysed in 300 μL of SDS-sample buffer and western blots were carried out using antibodies specific to viral early and late proteins.

RESULTS

The effect of CtBP interaction with E1A on oncogenic transformation was previously examined using a mutant (dl1135) that contains a deletion of sequences coding for 14 residues encompassing the CtBP binding motif (Boyd et al., 1993). The discovery of additional protein interactions and the mapping of their interaction regions within E1A (Komorek et al., 2010) indicated that dl1135 is defective in interaction with both the DYRK1/HAN11 and CtBP1/2 complexes. To investigate the role of CtBP in E1A-mediated cell transformation and in virus replication, we constructed an E1A mutant that results in two amino acid substitution within the CtBP-binding motif, PLDLS (PLDLS→PLASS). The interaction of cellular proteins with the newly engineered mutant (E1A12S-DL→AS) was determined by immunoprecipitation/western-blot analysis of E1A proteins from HeLa cells transfected with different E1A12S plasmids expressing wt S-E1A or mutant proteins (Fig. 1A) or BRK cells transformed with E1A-12S and the Ras oncogene (Fig. 1B). As expected, the mutant DL→AS made comparable level of protein as wt S-E1A and did not bind to CtBP1 and CtBP2 (Fig. 1A and 1B). However, it interacted as well as wt S-E1A with FOXK1 and DYRK1A, suggesting that the newly engineered E1A mutant is deficient in interaction only with CtBP.

FIG. 1. Interaction of cellular proteins with E1A C-terminal region.

A. HeLa cells were transfected with E1A12S wt and mutant DL→AS in pLPC vectors and 24 hr after transfection cells were lysed and immunoprecipitated with E1A specific antibody M58. The proteins were separated by SDS-PAGE (4–12%) and analyzed by western-blotting. B. Western-blot analysis of E1A immunoprecipitated from BRK cells transformed with E1A and Ras. The blots were probed with antibodies specific to CtBP1, CtBP2, FOXK1, DYRK1A and E1A (M58). Background protein bands that migrate slower than CtBP1/CtBP2 bands appear to correspond to IgH bands.

Interaction with CtBP suppresses the immortalization activity of E1A

The E1A gene has been shown to immortalize primary rodent epithelial cells and efficiently transform them in cooperation with the Ras oncogene (Graham et al., 1974; Houweling et al., 1980; Ruley, 1983; Zerler et al., 1986). Previous studies have shown that different mutations within the C-terminal region of E1A resulted in a hyper transformation phenotype and also in an overall defect in immortalization (Douglas et al., 1991; Quinlan and Douglas, 1992; Subramanian et al., 1991; Subramanian et al., 2007). To determine the specific effect of CtBP on the immortalization function of E1A, we transduced retrovirus (pBABE) vectors that express E1A12S wt or DL→AS mutant or mutants that are specifically defective in FOXK1/K2 interaction (12S-dl1132)) or defective in DYRK1/HAN11 binding (12S-dl1133) into primary BRK cells and subjected them to dominant selection with puromycin. The resulting colonies were quantified (Fig. 2A). As expected, there was no colony in cells transduced with pBABE vector while transduction of E1A-12S wt resulted in significant colony formation. Interestingly, BRK cells transduced with 12S-DL→AS or 12S-dl1133 contained more colonies than cells transduced with E1A12S wt. Cells transduced with 12S-dl1132 formed only about one half of number of colonies induced by E1A12S wt. These results suggest that the interaction of CtBP as well as DYRK1/HAN11 may negatively regulate the immortalization activity of E1A while the interaction of FOXK1/K2 transcription factors may enhance the immortalization function. Since the colony formation assay may not exclude the possibility of abortive colonies, we also tested the ability of isolated colonies to establish as immortalized cell lines (Fig. 2B). Among the mutants tested for colony establishment, 35% of colonies induced by 12S-DL→AS established as cell lines while less than 10% of 12S wt-induced colonies established. Mutant dl1133 induced colonies established less than 12S-wt colonies did. A higher percent of colonies (~17 %) from cells transduced with dl1132 established compared to cells transduced with E1A12S wt. To ascertain the cells transduced with different E1A retrovirus vectors express E1A proteins, two pooled cell lines in each category were analyzed by western-blot analysis (Fig. 2C). The colony establishment assay clearly showed that the 12S-DL→AS mutant immortalized better than wt 12S as well as other C-terminal mutants. Taken together these results suggest that the interaction of CtBP with E1A C-terminus suppresses the immortalization function of E1A.

FIG. 2. Effect of E1A C-terminal mutants on immortalization of BRK cells.

BRK cells were plated in six-well plates and infected with retrovirus vectors expressing E1A12S wt, E1A12S-DL→AS, E1A12S-dl1132, E1A12S-dl1133 or the empty vector. Next day, cells were subjected to selection with 0.25 μg/mL of puromycin with media change every fourth day. On tenth day, cells were stained with crystal violet and the colonies were quantified (from three experiments) (A). The colonies selected with puromycin were also isolated (24 colonies each time) and plated in 24-well plates and propagated for four weeks and the percentage of established colonies were calculated (B). The expression of E1A proteins in pooled immortalized cell lines (two different lines in each category designated A and B) were determined by western blot analysis (C). The statistical differences were determined using one-way ANOVA followed by Newman-Keuls Multiple Comparison Test using GraphPad Prism software. P-value was determined by comparing 12S with DL→AS, dl1132 and dl1133 mutants. Significance was accepted at a value of * p<0.05, ** p<0.01 and *** p<.001.

Interaction of CtBP suppresses Ras co-operative transformation activity of E1A

Different E1A C-terminal mutants that are deficient in interaction with one or more of the cellular protein complexes have previously been shown to be hyper-transforming in Ras cooperative transformation assays (Boyd et al., 1993; Douglas et al., 1991; Schaeper et al., 1995; Subramanian et al., 1989). However, the effect of E1A mutants that are specifically deficient in interaction with CtBP is not known (a previously examined mutant dl1135 is defective in interaction with both CtBP1/2 and the DYRK1/HAN11 complexes). Here, we examined the effect of E1A12S-DL→AS mutant on Ras co-operative transformation of BRK cells (Fig. 3). Cotransfection of 12S-DL→AS with the Ras oncogene resulted in about twice as many transformed foci as that of Ras and 12S wt. Other mutants that are individually deficient in interaction in FOXK1/K2 and DYRK1/HAN11 also resulted in enhanced cooperative transformation while 12S-177-9 that is deficient in interaction with all three protein complexes induced more transformed foci (that were also qualitatively more aggressive) compared to other mutants. These results suggest that the interaction of E1A C-terminus with CtBP also suppresses the frequency of Ras co-operative transformation.

FIG. 3. Ras cooperative transformation by E1A C-terminal mutants.

BRK cells in 6-well plates were transfected with E1A12S wt or mutants along with the activated Ras oncogene and pcDNA3 vector. The cells were selected with 50 μg/mL of G418 for 10 days, stained with crystal violet and the colonies were counted. The average values from three independent experiments are plotted (A). Pooled transformed cell lines were established from transformed colonies that were selected for 10 days with 50 μg/mL of G418, and were trypsinized and passaged three times. 1 X 10⁶ cells in 100 μL of growth medium without serum were injected subcutaneously into 4 to 6 week-old female athymic mice (n = 4) and the tumor volumes were measured periodically. The averages of three experiments are shown (B). The expression of E1A in transformed cell lines (1x10⁶ cell/cell line) was analyzed by western-blotting using M58 Ab. The statistical significances were determined as in Fig. 2.

We have previously reported that BRK cells transformed from cells transfected with E1A12S wt and Ras were less tumorigenic in athymic mice than cells transformed with E1A12S C-terminal truncation mutants and Ras. In syngeneic rats, the transformed cells expressing E1A C-terminal truncation mutants were highly tumorigenic while cells transformed with E1A12S wt and Ras were non-tumorigenic (Subramanian et. al., 1989). To evaluate the tumorigenic potentials of transformed cells that express E1A12S-DL→AS and Ras, we established pooled cell lines and transplanted them in athymic mice as well as in new born rats. The tumor volumes in athymic mice were measured at five day-intervals (Fig. 3B) and the tumor incidence in syngeneic rats was noted after three weeks (Table 2). All mice injected with various cell lines formed tumors of varying sizes. Among the different cell lines, the cell line expressing E1A12S-dl1133 (defective in interaction with DYRK1/HAN11) formed the largest tumors followed by cells that express E1A12S-177-9 (defective in binding with all three known C-terminus interacting protein complexes). Cells expressing E1A12S-DL→AS formed similar size tumors as cells expressing E1A12S wt whereas cells expressing E1A12S-dl1132 formed tumors of size in between cells expressing E1A12S wt and cells expressing E1A12S-177-9. Since E1A12S wt and Ras transformed cell lines did not induce tumor in new born rats as reported (Subramanian et al., 1989), we made only qualitative determination of tumor formation in rats (Table 2). As expected, there was no tumor in rats injected with E1A12S wt and Ras transformed cells. Again transformed cells that express E1A12S-dl1133 cells (88%) or mutant E1A12S-117-9 (76%) formed maximum percentage of tumors. Cells expressing E1A12S-dl1132 formed tumors only 50% of the total rats injected. Only 10% of the rats injected with the cells expressing E1A12S-DL→AS formed tumors. These tumors were also smaller in size compared to those induced by cells that express other E1A mutants. Taken together, these results suggest that the mutant E1A12S-DL→AS clearly showed enhanced immortalization and Ras cooperative transformation, they were not as tumorigenic as cells that express E1A12S-dl1133 or E1A12S-117-9.

Table 2. Tumorigenesis of transformed BRK cell lines in syngeneic rats.

Pooled transformed cell lines were established as in Fig. 3B and 1 X 10⁶ cells in 50 μL of growth medium without serum were injected subcutaneously into 3-day old neonatal Fisher rats. The animals were monitored for three weeks period for the formation of tumors. The experiment was repeated three times with one liter of new born rats (n = 5–9) for each cell lines in independent experiments.

Cell line	Tumors/pups	Percentage
12S + Ras	0/20	0.0
12S-DL→AS + Ras	(3)/30	10.0
12S-dl1132 + Ras	10/20	50.0
12S-dl1133 + Ras	15/17	88.2
12S-177-9 + Ras	22/29	75.9

CtBP interaction with E1A is needed for efficient virus replication

Having determined the effect of CtBP interaction on the transforming activities of E1A, we then determined the role of E1A-CtBP interaction on viral replication. For this purpose, we constructed an HAdv5 recombinant virus that expresses genomic E1A harboring the DL→AS mutation and compared its replicative potential with other E1A C-terminal mutants. Primary human bronchial epithelial cells (HBEpC) and the human lung cancer cell line, A549 were infected with different viruses and viral replication was determined by performing single step growth curves. In HBEpC, replication of the C-terminal truncation mutant HAdv5-177-9 was reduced about 5-fold compared to HAdv5-dl309 (wt). Mutant HAdv5-dl1132 replicated to comparable levels as HAdv5-dl309 whereas mutant HAdv5-dl1133 replicated at reduced levels (Fig. 4A). The mutant HAdv5-DL→AS replicated about 3-fold lower than wt. Since the mutants HAdv5-DL→AS and HAdv5-177-9 exhibited defects in viral replication, we analyzed the expression of viral early and late genes in infected cells by western-blot analysis (Fig. 4B). The C-terminal mutants, except HAdv5-177-9 expressed E1A proteins at levels comparable to that of HAdv5-dl309 between 12 to 48 hr post-infection. Mutant HAdv5-177-9 made reduced levels of E1A at 24 hr post-infection and was not readily detectable at late times of infection. The levels of other early (E1B-19K, E2-DNA binding protein, E3-19K glycoprotein and E4-ORF4) and late (fiber and capsid) proteins were lower in cells infected with HAdv5-DL→AS than in wt (dl309)-infected cells. Mutants HAdv5-dl1132 and HAdv5-dl1133 were not significantly defective in the expression of viral early and late genes. These results suggest that interaction of CtBP with E1A C-terminal region may be required for optimal activation of viral early and late genes.

FIG. 4. Effect of E1A C-terminal mutations on HAdv5 replication.

A. Replication in HBEpC. Cells (1 X 10⁶) were infected with 5 PFU/cell of HAdv5-dl309 (wt) or mutants and the infected cells were frozen at every 12, 24, 36 and 48 hr post infection. The cells were thawed, sonicated, cleared and titrated on 293 cells. The experiment was done in triplicate and the average virus yields were plotted. B. Effect on expression of viral early and late genes. In separate experiments, the infected cells were lysed in SDS-sample buffer and analyzed by western blotting for viral early and late proteins. C, D. Experiments similar to A and B were repeated with human A549 cells.

In similar experiments with A549 cells, the virus replication in general was about to 20 to 30-fold higher than in primary HBEpC. In A549 cells also mutant HAdv5- DL→AS replicated less efficiently than HAdv5-dl309. However, the replication defect of HAdv5-DL→AS was less pronounced in A549 cells than in HBEpC cells. The overall patterns of expression of viral early and late proteins were also comparable to that observed in primary cells. The mutant HAdv5-DL→AS expressed lower levels of various early and late genes (Fig. 4D). Taken together, these results suggest that CtBP interaction with E1A C-terminus is required for efficient expression of viral genes and for virus replication.

CtBPs knockdown enhances virus replication

To further confirm our results on the effect of CtBPs on virus replication, we determined the levels of virus replication in human U2OS cells that were stably depleted for CtBP1 or CtBP2. Pooled cell lines transduced with lentiviral vectors that stably express shRNA targets for CtBP1 or CtBP2 were established (Fig. 5A). The infection of HAdv5-dl309 in cells depleted for CtBP2 resulted in large plaques compared to the plaques on the parental U2OS cells or CtBP1-depleted cells (Fig. 5B). The plaques produced by the mutant HAdv5-DL→AS were relatively larger in CtBP2-depleted cells than those in the parental U2OS cells or in CtBP1-depleted cells. However, they were smaller than the plaques produced by dl309 in CtBP2-depleted cells. We also tested virus replication in CtBP-knockdown cells by infecting these cells with 5 pfu/cell of HAdv5-dl309 or HAdv5-DL→AS. At 36 hr of post infection, the virus yield was quantified by titrating on 293 cells (Fig. 5C). These results indicated that dl309 replicated better in CtBP2-depleted cells than in U2OS cells or CtBP1-depleted cells. Infection with the mutant DL→AS resulted in about 10-fold reduction in virus yield in U2OS cells and in CtBP1- or CtBP2-depleted cells compared to HAdv5-dl309. The levels of expression of viral early and late proteins (Fig. 5D) paralleled the levels of virus replication. Taken together, the results presented in figure 5 suggests that knockdown of CtBP2 in U2OS cells enhances the virus replication and the inability of E1A to interact with CtBP (i.e., mutant DL→AS) drastically retards virus replication.

FIG. 5. Effect of CtBPs knockdown on virus growth.

A. Western blot analysis of CtBP expression in U2OS and CtBP knockdown (indicated by ↓ arrow) cell lines. B. Plaque morphology of HAdv5-dl309 and HAdv5-DL→AS mutant in U2OS cell lines. Ten days after infection, the cells were stained with crystal violet and photographed. C. Growth of HAdv5-dl309 and HAdv5-DL→AS mutant. U2OS cell lines were infected with 5 pfu/cell of HAdv5-dl309 or HAdv5-DL→AS mutant. Thirty six hr post infection, the virus contents were titrated on 293 cells. D. Expression of viral early and late proteins. Cells were infected with wt and mutant viruses and the expression early and late proteins were analyzed at the indicated time points by western blots.

DISCUSSION

HAdv E1A proteins have been extensively studied as model viral transforming proteins and transcriptional regulators. Most studies have concentrated on the N-terminal half of E1A proteins (reviewed by (Berk, 2005; Chinnadurai, 2011; Pelka et al., 2008)). At present, there is only a limited understanding of the functions encoded by the C-terminal region of E1A which targets three different cellular protein complexes. Here, we have investigated the functional significance of E1A interaction with one of the protein complexes, CtBP1/2 by the use of a specific E1A mutant (DL→AS) that obliterates the core CtBP binding motif PLDLS (Schaeper et al., 1995).

Our current studies have shown that the DL→AS mutant of E1A12S immortalized primary BRK cells better than E1A12S wt, suggesting that the interaction of CtBP with the C-terminus suppresses the immortalization function of E1A. Although it is well known that E1A immortalizes primary rodent cells, immortalization of primary epithelial cells (e.g., BRK) by HAdv5 E1A is generally inefficient (while inducing efficient Ras-cooperative transformation). Our current results suggest that interaction of CtBP with the E1A C-terminus may contribute to the inefficient immortalization by wt E1A. Previous gene expression profiling studies have identified that CtBP suppresses the expression of several pro-apoptotic genes (Grooteclaes et al., 2003). It is possible that E1A may relieve CtBP-mediated repression of pro-apoptotic genes in BRK cells leading to reduced immortalization. Consistent with this notion, we have observed enhanced apoptotic response in BRK cells transduced with wt E1A-12S compared to cells transduced with E1A 12S-DL→AS mutant (results not shown).

Our results also indicated that the DL→AS mutant enhanced Ras-cooperative transformation, suggesting that CtBP interaction with E1A suppresses the transforming activity of E1A as well. It is possible that the cellular pathways deregulated by E1A-CtBP interaction that contribute to the suppression of immortalization may also suppress Ras-cooperative transformation. In addition to proliferation and apoptotic pathways deregulated as a result of E1A-CtBP interaction, deregulation of cell differentiation may also contribute the effect on cell transformation. CtBP has been reported to repress several epithelial genes that were relieved as a result of interaction with E1A (Grooteclaes et al., 2003; Grooteclaes and Frisch, 2000). It is somewhat surprising that transformed cells that express the DL→AS mutant is not more tumorigenic than transformed cells that express E1A 12S wt. Previous results with E1A mutants that are defective in interaction with DYRK1/HAN11 and CtBP1/2 complexes (e.g., dl1135) (Boyd et al., 1993) or with all three protein complexes (mutants 177-9 and 177-E) (Subramanian et al., 1989) revealed that transformed cells that express these mutants were highly tumorigenic both in athymic mice and in syngeneic rats. Thus, E1A mutants defective in interaction with DYRK1/HAN11 and FOXK1/K2 appear to influence the tumorigenic activity rather that the mutant defective in interaction with only CtBP. Thus, interaction of three different protein complexes with E1A C-terminal region may regulate cell transformation and oncogenesis in a non-redundant fashion.

Our results suggest that the interaction of CtBP with E1A enhances viral early and late gene expression and viral replication. Although the precise mechanism for the role of CtBP-mediated effect on viral replication remains to be investigated in detail, two different scenarios could be envisioned. In cells infected with the viral mutant HAdv5-DL→AS the expression of viral early gene products was reduced. We have also observed that in transient reporter based assays the trans-activation of viral E3 and E4 promoters was reduced in cells transfected with genomic E1A carrying DL→AS mutation (not shown). The effect of E1A C-terminus on the trans-activation of viral early genes (that is primarily mediated by the CR3 trans-activation domain of E1A) is unexpected. A previous study (Bruton et al., 2008) has reported that CtBP also interacted with the CR3 trans-activation domain through a motif related to the second CtBP-binding motif known as the RRT motif (Quinlan et al., 2006) retarding the overall trans-activation output. It is possible that high affinity interaction of CtBP with the C-terminus may relieve the negative effect of CtBP on CR3-mediated trans-activation. Alternatively, CtBP interaction with E1A may enhance the ‘epithelialness’ of the target cells by relieving CtBP-mediated repression of cellular epithelial target genes. The change in epithelialness may provide more suitable cellular environment for HAdv5 replication. Both scenarios may be consistent with a previous observation that siRNA-mediated depletion of CtBP1/2 resulted in enhanced viral replication in human cells (Grand et al., 2007)). Our results suggest that CtBP2 depletion enhances viral replication (Fig. 5). The relative enhancement in viral replication may be an underestimate since CtBP1 may also negatively influence virus replication, in the absence of CtBP2, as CtBP1 and CtBP2 are known exhibit functional redundancy under certain conditions (Hildebrand and Soriano, 2002). Detailed genomic studies may be warranted to identify CtBP regulated genes in HAdv5 infected cells and to determine their role in viral gene expression and replication.

• Adenovirus E1A C-terminal region suppresses E1A/Ras co-transformation.

• This E1A region binds with FOXK, DYRK1/HAN11 and CtBP cellular protein complexes.

• We found that E1A-CtBP interaction suppresses immortalization and transformation.

• The interaction enhances viral replication in human cells