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An Adenovirus Early Region 1A Protein Is Required for Maximal Viral DNA Replication in Growth-Arrested Human Cells KATHERINE R

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An Adenovirus Early Region 1A Protein Is Required for Maximal Viral DNA Replication in Growth-Arrested Human Cells KATHERINE R JOURNAL OF VIROLOGY, Mar. 1985, p. 742-750 Vol. 53, No. 3 0022-538X/85/030742-09$02.00/0 Copyright X 1985, American Society for Microbiology An Adenovirus Early Region 1A Protein is Required for Maximal Viral DNA Replication in Growth-Arrested Human Cells KATHERINE R. SPINDLER, CAROL Y. ENG, AND ARNOLD J. BERK* Department of Microbiology and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90024 Received 30 August 1984/Accepted 26 November 1984 Two closely related adenovirus early region 1A proteins are expressed in transformed cells. The smaller of these, which is 243 amino acids in length, is required for the transformation of primary rat cells and for the transformation of immortalized rat cells to anchorage-independent growth. This protein is not required for productive infection of exponentially growing HeLa cells but is required for maximal replication in growth (G0)-arrested human lung fibroblasts (WI-38 cells). To determine the function of this protein in viral replication in these G0-arrested cells, we compared viral early mRNA, early protein, and late protein synthesis after infection with wild type or a mutant which does not express the protein. No differences were found. However, viral DNA synthesis by the mutant was delayed and decreased to 20 to 30% that of wild type in these cells. Viral DNA synthesis was much less defective in growing WI-38 cells, and in the transformed human HeLa cell line it occurred at wild-type levels. Furthermore, the mutant which can express only the 243-amino-acid early region 1A protein induced cellular DNA synthesis in GO-arrested rat cells to the same level as wild-type virus. A mutant which can express only the 289-amino-acid early region 1A protein induced less cellular DNA synthesis in GO-arrested rat cells. We propose that the early region 1A 243-amino-acid protein alters the physiology of arrested permissive cells to allow maximal viral DNA replication. In nonpermissive rodent cells, the 243-amino-acid protein drives G0-arrested cells into S phase. This activity is probably important for the immortalization of primary cells. The human adenoviruses have a complex gene structure in Transformation of rodent cells by an adenovirus mutant early region 1A (E1A): at early times, two mRNAs are which cannot express the ElA 243-aa protein, mutant pm975, transcribed which have identical 5' and 3' sequences but are occurs at greatly reduced frequency compared with that by different at internal sequences due to differential splicing wild type and is only partial: transformed foci are defective (49). The larger message, 13S, encodes a protein predicted to in their ability to form colonies in soft agar (anchorage-inde- be 289 amino acids (aa's) in length; the smaller 12S mRNA pendent growth) (32). However, although the importance of encodes a 243-aa protein translated in the same reading the ElA 243-aa protein in transformation is obvious, its role frame but lacking 46 internal aa's unique to the 289-aa in the productive infection of human cells is more subtle. In protein (3, 39). This structure of overlapping mRNAs and HeLa cells, pm975 replication is comparable with wild-type proteins is found in adenoviruses of human subgroup A replication (33), indicating that the 243-aa protein is dispen- (adenovirus type 12 [Adl2]), subgroup B (Ad7), and sub- sable for viral replication in those cells. However, the group C (Ad2 and Ad5) (49) and is therefore likely to be mutant does not replicate to the level of wild type in growth functionally important for the growth of adenoviruses; both (G0)-arrested human WI-38 cells which are nontransformed ElA proteins are probably important, since they are evolu- diploid secondary lung fibroblasts (22, 23). The rate of virus tionarily maintained. production and total yield is reduced compared with that of Various functions of the adenovirus ElA gene products wild-type infected cells (32). Thus the 243-aa protein is have been identified either in the productive infection of required for maximum viral replication in G0-arrested per- permissive human cells or in the transformation of nonper- missive cells. missive rodent cells. The ElA region alone is able to In the present work we have further characterized this immortalize rodent primary embryo cells (25) and in con- function of the 243-aa protein in G0-arrested cells. We found junction with other oncogenes is able to transform these that lack of the 243-aa protein leads to decreased viral DNA cells (42). Both of the ElA proteins are required for com- synthesis in G0-arrested human cells but has little effect on plete transformation of nonpermissive rodent cells after the expression of early or late viral genes. Expression of the infection by adenovirus (2, 8, 20, 24, 25, 28, 32, 46). In 243-aa protein appears to alter the physiology of resting productive infections of permissive cells, the 289-aa ElA human cells, allowing more viral DNA replication to occur. protein induces transcription from the other early regions, Such a function for the 243-aa protein would explain the E1B, E2, E3, E4, and Li (4, 8, 12, 29, 33, 35, 41). Moreover, selective pressure for the conservation of the smaller ElA the ElA 289-aa protein is able to induce transcription of protein. In natural infections human adenoviruses normally integrated adenovirus DNA in stably transformed cells (11), encounter noncycling, terminally differentiated epithelial and it is able to induce viral or nonviral genes introduced cells (49). Since expression of the 243-aa protein results in into cells by infection or transfection (16, 19, 26, 48). In several-fold greater yield of infectious virus in such arrested contrast, the 243-aa ElA protein has little, if any, transcrip- tion-inducing activity (32, 48). cells, there should be a strong selective pressure for its expression. Earlier work (6) has shown that ElA functions are re- * Corresponding author. quired for the induction of cellular DNA synthesis which is 742 VOL. 53, 1985 Ad2 DNA REPLICATION STIMULATED BY ElA PROTEIN 743 observed when G0-arrested rodent cells are infected by cold 5% trichloroacetic acid-95% ethanol, and counted by adenovirus (7, 44, 50). Here we found that d11500, which scintillation counting. expresses only the 243-aa ElA protein, stimulated cellular The proportion of [3H]thymidine label in cellular and viral DNA synthesis in G0-arrested cells to the same extent as did DNA was determined by buoyant density centrifugation as wild-type virus, whereas pm975, which expresses only the follows: 0.5 ml of the cell extract was brought to 8.0 ml in 10 289-aa ElA protein, induced lower levels of cellular DNA mM Tris-hydrochloride [pH 8.5]-1 mM EDTA-0.1% synthesis. The relation between this activity of the 243-aa Sarkosyl (CIBA-GEIGY Corp.)-1.7035 g of CsCl per ml and protein and its function in the immortalization of rodent centrifuged in a Beckman 50 Ti rotor at 35,000 rpm at 18°C pritnary embryo cells is discussed. for 40 h. Fractions (0.2 ml) were collected from the gradient, a samnple of each fraction was spotted on GF/A filters, and MATERIALS AND METHODS scintillation was counted as above. Late viral proteins. At the indicated times, 60-mm plates of Cells and viruses. Human diploid lung WI-38 cells (ATCC infected cells were washed three times with phosphate-buff- CCL 75) were obtained from the American Type Culture ered saline, and 23 ,uCi of [35S]methionine (1,100 Ci/mmol; Collection and maintained in Dulbecco nmodified Eagle me- Amersham Corp.) per ml was added in 3 ml of methionine- dium plus 10% fetal bovine serum. CREF cells were main- free Dulbecco modified Eagle medium. After 2 h of incuba- tained as described previously (13). For experiments with tion at 37°C, the plates were washed three times with G0-arrested cells, the medium on confluent cells was changed phosphate-buffered saline and scraped. Cells (5 x 105) were to Dulbecco modified Eagle medium plus 0.2% calf serum. mixed with 80,ul of 2x Laemmli gel sample buffer (30), and After 4 days of incubation in low serum, the medium was the extract from 5 x 104 cells was electrophoresed on a 15% removed and saved, and the cells were infected with the acrylamide-0.087% bisacrylamide gel (1). The gel was fixed, appropriate virus at a multiplicity of infection of 5 (except in dried, and exposed to X-ray film. the growth experiment; see the legend to Fig. 1); the saved, conditioned medium was replaced on the cells. RESULTS Ad2, AdS, and d1309 (wild type in ElA) (28) were used as Growth of pm975 in arrested human cells. The adenovirus wild-type controls. Ad2/5 pm975 does not make ElA 12S mutant pm975 does not express the ElA 12S mRNA because mRNA or the ElA 243-aa protein (33). Ad2 d11500 does not of a splice site mutation and therefore does not express the synthesize the ElA 13S mRNA or the ElA 289-aa protein ElA 243-aa protein. Mutant pm975 does not replicate as (32). d1312 has a large deletion removing most of ElA (28). efficiently as wild-type Ad2 in strictly G0-arrested cells (32). Where indicated, 1-f-arabinofuranosylcytosine (araC) was We found that WI-38 cells would be strictly Go arrested (as included in infections at 20 p.g/ml and replenished in the measured by [3H]thymidine incorporation; see below) if medium every 12 h (17). grown in reduced serum (0.2%) for 4 days after reaching Hybridization andS1 nuclease analysis. Cytoplasmic RNAs confluence.
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