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 nonpermissive 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, transcription-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. These cells are human diploid lung fibroblasts were prepared as described previously (4); 50 jig was and thus are reasonable model cells for a natural adenovirus hybridized to a uniformly32P-labeled M13 probe specific for infection. In a previous study (32), the growth of pm975 was El, E2, E3, and E4 (38). The hybrids were digested with 100 compared with that of wild-type Ad2. However, pm975 was U of S1 nuclease (Bethesda Research Laboratories) and derived from d1309 (33) and thus has an altered DNA analyzed on 5% polyacrylamide-8 M urea gels for 1,600 Vh sequenceih E3 (28). The replication of d1309 is indistinguish- (El and E2) or 800 Vh (E3 and E4). The gels were dried and able from that of wild-type virus in HeLa cells (28; unpub- exposed to X-ray film. lished data), and it was used as the control virus in most of Western analysis. Cells (106) were lysed and digested with this work. To determine whether the reduced viral replica- micrococcal nuclease-RNase A-DNase I in a volume of 75,ul tion of pm975 in arrested human cells was due to alterations as described previously (17). The extracts were then mixed in E3 or El, replication through two cycles of infection was with 0.25 volumes of 4x Laemmli gel sample buffer, boiled compared with that of d1309 (Fig. 1). Since the growth and for 10 min, and electrophoresed on 12% polyacrylamide gels ultimate yield of pm975 is reduced compared with d1309, it is (30). The proteins were transferred to nitrocellulose as the ElA mutation of pm975 that is responsible for the described previously (47), and the filter wasincubated with reduced replication in arrested WI-38 cells. Thus we con- 9.6% nonfat dry milk to block nonspecific protein binding. clude that the 243-aa protein encoded by the ElA 12S The filter was then incubated for 12 h with two antisera, one mRNA is required for some aspect of efficient viral replica- directed against the adenovirus E2 72-kilodalton (kd) DNA tion in these cells. Though the pm975 mutant is far from binding protein (a gift of L. T. Feldman) and the other being absolutely defective in these cells, its reduced growth directed against the E1B 21-kd protein (47). The first-stage provides an explanation for the selective pressure leading to antibody was detected with the Immun-Blot system (a the evolution of the 243-aa protein. horseradish peroxidase-coupled antibody; Bio-Rad Labora- DNA synthesis in pm975-infected G0-arrested human cells. tories). DNA synthesis was examined in G0-arrested WI-38 cells at DNA synthesis analyses. At the indicated times, 50,uCi of various times after infection. Cells were mock infected or [3H]thymidine (25 Ci/mmol; Amersham Corp.) was added to infected with Ad2, pm975, or d11500, an adenovirus mutant 5 ml of medium on an infected plate. At the end of the which does not express the ElA 13S mRNA or its transla- labeling period the plates were rinsed with phosphate-buff- tion product, the 289-aa protein (32) (Fig. 2A). The cells ered saline, and then 1 ml of lysis buffer (0.1 M NaCl, 5 mM were labeled with [3H]thymidine for 12-h intervals and EDTA, 10 mM Tris [pH 8.0], 0.5% sodium dodecyl sulfate, harvested at the conclusion of each 12-h labeling, and the 1 mg of pronase per ml) was added. The plates were incorporation of [3H]thymidine into trichloroacetic acid-pre- incubated for 2 to 12 h at 37°C, and the lysed cell extract was cipitable molecules was determined. DNA synthesis in the then extracted twice with phenol-chloroform. To measure Ad2-infected cells was much higher than that of pm975- or total [3H]thymidine incorporation, 50,ul of the extract was d11500-infected cells, and the peak of maximal synthesis was spotted on Whatman GF/A glass filters, dried, washed with earlier in the wild-type infection. Similar results were ob- 744 SPINDLER, ENG, AND BERK J. VIROL. tained with d1309 used as the wild-type control (data not shown). To determine what fraction of the total labeled DNA was viral or cellular, we performed the experiment shown in Fig. 2B. Due to the high guanosine-plus-cytosine content of Ad2 and AdS DNA (49), it can be separated from host cell DNA by buoyant density centrifugation. Figure 2B shows the analysis of DNA synthesized at 24 to 36 h postinfection (p.i.), at a time when wild-type (dl309) DNA synthesis was maximal. Mutant d1312 has a large ElA deletion so that no ElA products are synthesized (28). The extent of viral DNA replication in pm975-infected cells, determined from the the peak of viral DNA, was total counts incorporated into I 33% that observed in d1309-infected cells. The greatly re- 510 Io duced viral DNA synthesis of d11500 and d1312 is not unexpected, since both of these mutants lack the ElA 289-aa protein, which is responsible for inducing transcription from a. early viral genes required for viral DNA synthesis. De- creased transcription of the early regions would be expected to delay or prevent the onset of viral DNA replication. However, decreased viral DNA replication in the pm975- infected cells was surprising since pm975 expresses the 289-aa protein and can induce early transcription in these cells as well as wild-type virus does (see below). Analysis of other DNA synthesis labeling periods also showed reduced viral DNA replication of pm975 relative to d1309 (data not shown). In a repeat experiment of that shown in Fig. 2, the synthesis of pm975 viral DNA was 19% of wild-type in Go-arrested WI-38 cells. To determine whether this defect in viral DNA replication 48 72 96 l2 was due to the WI-38 cells themselves or to their being HOURS POST-INFECTION strictly Go arrested, we repeated the experiment shown in Fig. 2B with actively replicating WI-38 cells (Fig. 3A) or FIG. 1. Growth of d1309 and pm975 in Go-arrested human cells. HeLa cells in exponential growth (Fig. 3B). The growing G0-arrested WI-38 cells were infected at a multiplicity of infection of 0.05 (105 PFU/60-mm dish) with d1309 (0) or pm975 (0). Virus WI-38 cells were labeled at 24 to 48 h p.i., the HeLa cells particles were harvested at the indicated times, and virus yield was were labeled at 16 to 20 h p.i., and the DNAs were analyzed titrated in a plaque-forming assay on HeLa cells. in buoyant density gradients. In both of these actively replicating cells, the pm975 viral replication occurred at nearly wild-type levels. During these labeling periods, little h p.i. (at the onset of viral DNA synthesis in these cells; see or no viral DNA replication occurred in HeLa cells infected above) and analyzed in an Si nuclease analysis (Fig. 4). araC with d11500 or d1312, or in WI-38 cells infected with d1312. was included to block viral DNA replication so that tran- Viral DNA replication did occur in d11500-infected WI-38 scription was measured from the same number of templates cells to ca. 25 to 30% the level observed with d1309. This in d1309- and pm975-infected cells. It is unlikely that treat- probably resulted from the delayed expression of early viral ment with araC affected the quantities of early mRNAs functions, a phenomenon which has been observed previ- expressed in these cells compared with cells not treated with ously in d11500-infected HeLa cells (32). These results araC, since araC treatment did not affect the expression of indicate that the observed defect in viral replication of early viral proteins (see below). pm975 in G0-arrested WI-38 cells is a function of their being The isolated RNA was hybridized to single-stranded, Go arrested rather than a function of the cell type. uniformly 32P-labeled M13 probes specific for El, E2, E3, Early mRNAs and proteins in pm975-infected G0-arrested and E4. Synthesis of all the early region mRNAs from human cells. The reduced viral DNA synthesis in G0-arrested pm975-infected cells was equal to or greater than that seen in cells infected with pm975 compared with dl309 could have d1309-infected cells. The M13 probe for E2 was derived from been due to reduced expression of viral early proteins which Ad2, and thus, in addition to the major protected band (1,600 are directly involved in DNA replication. Since ElA func- nucleotides), smaller bands were observed which resulted tions are required for the normal induction of early mRNA from nonhomologies between the Ad2 probe and the RNA synthesis in HeLa cells, we determined whether the pm975 from the Ad5-derived viruses (d1309 and pm975). E4 RNA mutation affected early mRNA synthesis in G0-arrested from the pm975-infected cells showed a pattern of Si- human cells. In HeLa cells, only the ElA 289-aa protein protected fragments similar to that from d1309-infected HeLa translated from the 13S ElA mRNA is required for the cells during a later stage of infection (cf. Fig. 4B, lanes 5 and induction of transcription from the other early regions (4, 8, 6). That is, with respect to E4 mRNA, the pm975-infected 29, 33, 41). However, we considered the possibility that in WI-38 cells seemed more advanced in the infection at 24 h nontransformed, G0-arrested cells, the 243-aa protein also p.i. than did the d1309-infected WI-38 cells. Minor bands might be required for wild-type levels of early mRNA whose sizes are not identified in Fig. 4 (for example, A and synthesis. B, lanes 3) are derived from mRNAs which are synthesized Cytoplasmic RNAs from G0-arrested WI-38 cells infected late in infections in the presence of araC (15). We conclude with d1309 or pm975 in the presence of araC were isolated 24 that early mRNA synthesis occurs at normal levels in the VOL. 53, 1985 Ad2 DNA REPLICATION STIMULATED BY ElA PROTEIN 745

04 04

24 48 72 96 20 25 30 35 HOURS P. 1. FRACTION NUMBER FIG. 2. DNA synthesis in G0-arrested WI-38 cells. (A) Total DNA synthesis in infected cells. Incorporation of [3H]thymidine into trichloroacetic acid-precipitable molecules in G0-arrested cells which were mock infected (A) or infected with Ad2 (0), pm975 (0), or d11500 (O) was measured by labeling for 12-h time periods ending at 12, 24, 36, 48, 72, or 96 h p.i. as described in the text. (B) Viral and cell-specific DNA synthesis. Total DNA samples labeled with [3H]thymidine from 24 to 36 h p.i. in an experiment similar to that shown in (A) were analyzed by equilibrium buoyant density centrifugation. G0-arrested WI-38 cells were infected with d1309 (0), which is isogenic to pm975 except that d1309 is wild type in ElA, pm975 (0), d11500 (I), or d1312 (A). V and C, Positions in the gradients of viral and chromosomal DNA, respectively. The bottom of the gradient is fraction 1. pm975-infected GO-arrested cells, and thus the 243-aa protein Late viral protein synthesis. Finally, we examined whether is not required for transcription induction in these cells. in addition to viral DNA replication, late protein synthesis We examined translation of two early region proteins in (after DNA replication) was altered in the GO-arrested WI-38 GO-arrested cells infected with d1309 and pm975. Protein cells. Infected cells were labeled at various times with extracts were prepared at 12, 24, or 36 h p.i. in the presence [35S]methionine, and the pattern of late viral protein synthe- or absence of araC for both viruses and analyzed by Western sis was examined (Fig. 6). The shutoff of host protein immunoblotting (Fig. 5). Antisera specific for the E2 72-kd synthesis was not as complete in the arrested WI-38 cells as DNA binding protein and the E1B 21-kd protein were used in the HeLa cells (cf. lanes 1 to 8 with lanes 9 and 10). There to detect these proteins in the infected cells. The steady- was no significant difference in the population or quantity of state level of the 72-kd protein was somewhat higher in the late proteins produced by pm975- or d1309-infected cells. pm975-infected cells, both in the presence and absence of Indeed, the pattern of pm975-infected late viral proteins araC. The proteins migrating at ca. 45 kd are commonly appears at the 24-h time point, earlier than with d1309. Thus, observed degradation products of the DNA-binding protein. we conclude that decreased viral growth in pm975-infected The level of 21-kd protein also was higher in the pm975- cells is not due to any observable defect in late protein infected cells; it is only barely detectable at 36 h in the synthesis. It is interesting that although the pm975 mutant d1309-infected cells. These higher levels of early region exhibits reduced viral DNA synthesis in these cells, late proteins in pm975-infected cells may be due directly to proteins are synthesized to higher levels earlier than are higher transcription levels (see above) or to increased trans- those in wild-type infected cells. Evidently, the amount of lation efficiency or protein stability. In any case, the defect viral DNA synthesized in pm975-infected cells is adequate in pm975 growth in the arrested WI-38 cells cannot be due to for transcription of normal amounts of late viral mRNAs. decreased levels of these early region proteins, since they Induction of cellular DNA synthesis in GO-arrested rat cells. are present at levels equal to or greater than the dl309 early The results above indicate that the principle function of the proteins. A comparison of the ElA proteins in these WI-38 243-aa ElA protein is to stimulate maximal viral DNA cells infected with either virus was not possible since the replication in GO-arrested permissive human cells. This concentration of ElA protein in these cells was below the finding raised the question of the importance of this protein level of detection by this methodology (data not shown) (47). in stimulating cellular DNA synthesis in GO-arrested nonper- 746 SPINDLER, ENG, AND BERK J. VIROL.

A V 1 8 _ dl 309e

pm 975 6

_0 I ', 0- o 4r

IU a- 25aaim-20 10 15 20 IS 20 25 FRACTION NUMBER FRACTION NUMBER FIG. 3. DNA synthesis in growing WI-38 and HeLa cells. (A) Just-confluent WI-38 cells grown in 10% fetal bovine serum were infected and labeled with [3H]thymidine from 24 to 48 h p.i., and the DNA was analyzed by equilibrium buoyant density centrifugation. (B) Exponentially growing HeLa cells grown in 7% newborn bovine serum were infected, labeled with [3H]thymidine from 16 to 20 h p.i., and analyzed as in (A). Symbols are as described in the legend to Fig. 2B.

A 23 C4 £2 E3 E 4 2 3 C 4 5 6 C l 2 3 C C 4 5 6 1050- IV _ -1600 _a _ __ -750

375- 0 _0 750- C. _ 300- 0 _ a 614- -.e 476- -AVN 'I..sU 190- 0 S S 405- 0 _ * 350- a a _ FIG. 4. S1 analysis of early region RNAs in virus-infected, G0-arrested WI-38 cel!s. Cytoplasmic RNAs were prepared from G0-arrested WI-38 cells 24 h p.i. with d1309 or pm975 in the presence of araC. RNAs were analyzed by using uniformly labeled M13 probes specific for El and E2 (A) or E3 and E4 (B) (38) and S1 nuclease. Lanes 1 and 4 are the analyses of RNA from d1309-infected WI-38 cells; lanes 2 and 5 are from pm975-infected WI-38 cells. RNA from HeLa cells infected with d1309 for 45 h in the presence of araC (lanes 3 and 6) or tRNA (C) were analyzed as controls. The numbers indicate the sizes (in nucleotides) of the major protected exons. Exposures were for 14 to 19 h without intensifying screens, except lanes 1 and 2 in A were exposed for 7 days with a screen. VOL. 53, 1985 Ad2 DNA REPLICATION STIMULATED BY ElA PROTEIN 747

gene structure which dictates synthesis of the two closely pm 975 dl 309 related proteins is conserved among various adenoviruses 12 24 36 12 24 36 (49), suggesting a biological selection for the smaller 243-aa + - + _ + - araC protein. In growing human cells (HeLa and WI-38) the 243-aa protein is dispensable, since a mutant lacking it (pm975) replicates virus to wild-type levels (33). However, 72 kd- .....b^_ the 243-aa ElA protein is necessary for growth of adenovirus to wild-type levels in G0-arrested human cells (32; this work). In addition, the 243-aa protein is required for full transformation of rodent cells by adenovirus. Mutant pm975 transforms cells at a much lower frequency than does wild type, and those cells that do form foci are only partially transformed since they are defective for anchorage-independent growth (32). 21 kd- Various steps in the life cycle of the pm975 mutant (lacking the ElA 243-aa protein) in G0-arrested WI-38 cells were investigated. Both early mRNA synthesis and early 2 3 4 5 6 7 8 9 10 11 12 protein synthesis (before the onset of viral DNA replication) were comparable to wild-type levels in these cells. No FIG. 5. Analysis of early viral proteins in G0-arrested WI-38 quantitative or qualitative difference was found in the syn- cells. Early proteins of G0-arrested WI-38 cells were analyzed by and wild- Western immunoblot analysis at 12 h (lanes 1, 2, 7, and 8), 24 h thesis of adenovirus late proteins between pm975 (lanes 3, 4, 9, and 10), or 36 h (lanes 5, 6, 11, and 12) p.i. by pm975 type virus. Thus we feel that none of these stages in the viral (lanes 1 to 6) or d1309 (7 to 12), in the presence or absence of araC, life cycle are altered sufficiently to affect viral yield in the as indicated. The filter was incubated with anti-E2 72-kd serum and G0-arrested cells. anti-ElB 21-kd serum (see the text). The positions of the E2 72-kd Decreased pm975 viral DNA replication was observed in and E1B 21-kd proteins are shown. the G0-arrested cells at all times between 24 and 72 h p.i., relative to wild-type virus. At 24 to 48 h p.i. pm975 viral DNA was synthesized at 20 to 30% the level of wild type. In missive rodent cells. Wild-type adenovirus induces cellular DNA synthesis in serum-starved, G0-arrested rodent cells (7, 44, 50), but a mutant which expresses neither ElA protein, d1312, is defective in this induction (6). To identify -J which of the two ElA proteins is responsible for the induc- I, tion, we serum starved confluent plates of the rat cell line, di 309 pm 975 u LO CREF cells (13), and mock infected them or infected them -0 with d1309 (wild-type control), dl312, pm975, or d11500. The 24 36 48 60 24 36 48 60 E cells were labeled with [3H]thymidine for 12-h intervals, and -205 Kd incorporation into total DNA was measured (Fig. 7A). At 36 -fI -116 h p.i., d11500 and d1309 induced a four- to fivefold stimula- -97 tion of DNA synthesis relative to mock- or d1312-infected -m -68 cells, whereas pm975 stimulated DNA synthesis only two- EZ fold. In two additional experiments, d11500 and d1309 stimulated DNA synthesis two- to threefold, whereas DNA v4 ^- ..- synthesis in pm975-infected cells was not stimulated relative -45 to mock-infected cells (data not shown). The DNA obtained -36 from the 24- to 36-h p.i. labeling shown in Fig. 7A was - 29 analyzed on buoyant density gradients (Fig. 7B). Labeled DNA from all of the infections sedimented in the position of cellular DNA. The slight shoulder observed for the DNA -2 4 from d1309-infected cells was not observed in two additional - 20 experiments (data not shown). We conclude that wild type and d11500 induced cellular DNA synthesis in these Go- - 4 arrested rodent cells, whereas pm975 is less effective in this -ix induction. Thus the 243-aa ElA protein expressed by d11500 has an activity which stimulates G0-arrested CREF cells to enter S phase and synthesize cellular DNA. This activity 12 34 56 789 10 appears to be greater for the 243-aa ElA protein than for the 289-aa ElA protein. FIG. 6. Late viral protein synthesis in G0-arrested cells. Go- arrested WI-38 cells were infected with d1309 (lanes 1 to 4) or pm975 (lanes 5 to 8) and labeled with [355]methionine for 2 h at 24 h (lanes DISCUSSION 1 and 5), 36 h (lanes 2 and 6), 48 h (lanes 3 and 7), or 60 h (lanes 4 and 8) p.i. The total cell lysates were analyzed on a sodium dodecyl The two proteins encoded by adenovirus ElA are closely sulfate-polyacrylamide gel. Lane 9 contains an extract of cells that related, yet it has been shown that only the 289-aa protein were labeled 36 h after mock infection. Lane 10 is an extract of has the ability to induce transcription from the other early Ad2-infected HeLa cells at 24 h p.i. The roman numerals represent adenovirus genes (12, 32) or nonviral genes (16), whereas the adenovirus structural proteins (1, 49). The sizes of molecular weight 243-aa protein has much lower levels of this activity. The standards are indicated in kd. 748 SPINDLER, ENG, AND BERK J. VIROL.

0 0~ 0- 2

24 48 72 96 HOURS P. 1. FRACTION NUMBER FIG. 7. DNA synthesis of adenovirus in GO-arrested rodent cells. (A) Total DNA synthesis in infected CREF cells. Incorporation of [3H]thymidine to trichloroacetic acid-precipitable molecules in GO-arrested CREF cells which were mock infected (A) or infected with d1309 (0), d11500 (O), pm975 (0), or d1312 (A) was measured by labeling for 12-h time periods ending at 12, 24, 36, 48, 72, or 96 h p.i. as described in the text. (B) Cellular DNA synthesis in infected CREF cells. DNA samples labeled with [3H]thymidine from 24 to 36 h p.i. from the experiment shown in (A) were analyzed by buoyant density centrifugation. Symbols are as in (A). V and C, Positions of adenovirus DNA and cellular DNA in the gradients, respectively. The viral position was determined from a parallel gradient containing only adenovirus DNA. light of the apparent synthesis of early viral mRNAs, and data). Thus, we conclude that the 243-aa protein does not act early and late viral proteins in these G0-arrested cells, we to alter the levels of the cellular origin binding protein. conclude that the decreased viral DNA synthesis observed Infection by adenovirus induces the human 70-kd heat in the absence of the ElA 243-aa protein is responsible, at shock protein (36). A correlation between constitutive ex- least in part, for the decreased yield of pm975 virus in pression of the heat shock gene and ability to support early G0-arrested human cells. However, we have not analyzed viral gene expression in the absence of ElA has been the efficiency of virion morphogenesis from viral DNA and observed (27). We examined the levels of heat shock protein virion structural proteins. This step in the production of in G0-arrested WI-38 cells with an antiserum specific for the infectious virus might, conceivably, also be defective in human heat shock protein (a gift of J. R. Nevins). Little pm975-infected cells, as it is for polyomavirus mutants difference was seen in the level of heat shock protein in defective in middle and small t antigen (14). No defect in G0-arrested cells either mock infected or infected with viral DNA synthesis was observed in actively growing wild-type (d1309 and Ad2), pm975 (without 243-aa protein), WI-38 or HeLa cells. The ElA 243-aa protein thus is d11500 (without 289-aa protein), or d1312 (without 243- and required only for productive adenovirus infection of cells 289-aa protein) (unpublished data). Thus we are unable to arrested in a Go state, and its absence is manifested as correlate decreased pm975 viral growth with induction of the reduced viral DNA synthesis. heat shock gene in WI-38 cells. Study of adenovirus DNA replication has been facilitated Mutant pm975 is partially defective, even though it ex- by a soluble in vitro DNA replication system (9; for review, presses the ElA 289-aa protein which contains all the aa see reference 10). Recently, a protein present in uninfected sequence of the 243-aa protein. The additional 46-aa se- cells has been shown to bind to a specific sequence at the quence inserted into the middle of the 289-aa protein relative adenovirus origin of replication (34, 40). In collaboration to the 243-aa protein (39) apparently causes it to lose some with P. J. Rosenfeld and T. J. Kelly, Jr., we attempted to activity which the 243-aa protein retains, at least at the determine whether this cellular DNA replication factor was concentrations expressed in GO-arrested WI-38 cells and present in lower amounts in G0-arrested versus growing cells transformed by adenovirus infection. However, at WI-38 cells; no difference was found (unpublished data). We higher cellular concentrations, as is usually found in cells also examined whether more of this protein was present transformed with DNA by the Graham-van der Eb method after infection of arrested WI-38 cells by wild-type virus than (18, 24), the 289-aa protein may be able to express this after infection by pm975; again, no difference was seen in the 243-aa protein-specific activity. This may explain the obser- extracts of cells infected by the two viruses (unpublished vation that a cDNA clone of the Ad2 ElA 13S mRNA able to VOL. 53, 1985 Ad2 DNA REPLICATION STIMULATED BY ElA PROTEIN 749 express only the 289-aa protein can cooperate with the of Health. A.J.B. was supported by a Faculty Research Award from EJ-ras gene in the DNA-mediated transformation of primary the American Cancer Society. mammalian cells (E. Ruley, personal communication). Induction of cellular DNA synthesis was observed in LITERATURE CITED G0-arrested rodent cells infected by wild-type virus and the 1. Anderson, C. W., P. R. Baum, and R. F. Gesteland. 1973. Processing of adenovirus 2-induced proteins. J. Virol. 12: mutant dll500, which expresses only the 243-aa ElA protein 241-252. (Fig. 7). The mutant which expresses only the 289-aa pro- 2. Babiss, L. E., P. B. Fisher, and H. S. Ginsberg. 1984. Deletion tein, pm975, induced much less cellular DNA synthesis than and insertion mutations in early region la of type 5 adenovirus did dllS00 and wild type. Thus, the 243-aa protein is suffi- that produce cold-sensitive or defective phenotypes for trans- cient to induce cellular DNA synthesis to wild-type levels in formation. J. Virol. 49:731-740. these G0-arrested rat cells, whereas the 289-aa protein has 3. Baker, C. C., and E. B. Ziff. 1981. Promoters and heterogeneous less of this activity. 5' termini of the messenger RNAs of adenovirus serotype 2. J. The ElA 243-aa protein is required for complete transfor- Mol. Biol. 149:189-221. mation by adenovirus (33) and maximal viral DNA repli- 4. Berk, A. J., F. Lee, T. Harrison, J. Williams, and P. A. Sharp. in G0-arrested and it induces cellular DNA 1979. Pre-early adenovirus 5 gene product regulates synthesis of cation cells, early viral messenger RNAs. Cell 17:935-944. synthesis in G0-arrested rodent cells. Somewhat similar 5. Bouck, N., N. Beales, T. Shenk, P. Berg, and G. diMayorca. observations have been made for the middle and small t 1978. New region of the simian virus 40 genome required for antigens of polyomavirus and the small t antigen of simian efficient viral transformation. Proc. Natl. Acad. Sci. U.S.A. virus 40 (SV40). Polyomavirus mutants defective in the 75:2473-2477. expression of middle and small t antigen are defective for 6. Braithwaite, A. W., B. F. Cheetham, P. Li, C. R. Parish, L. K. transformation of nonpermissive rat cells (21). Although Waldron-Stevens, and A. J. D. Bellett. 1983. Adenovirus-in- these proteins are not essential for viral DNA replication, duced alterations of the cell growth cycle: a requirement for nor are they known to directly participate in viral DNA expression of ElA but not of E1B. J. Virol. 45:192-199. after infection of mouse cells with 7. Braithwaite, A. W., J. D. Murray, and A. J. D. Bellett. 1981. synthesis, permissive Alterations to controls of cellular DNA synthesis by adenovirus these mutants, viral DNA replication is reduced several-fold infection. J. Virol. 39:331-340. compared with wild-type polyomavirus (37). Small t antigen 8. Carlock, L. R., and N. C. Jones. 1981. Transformation-defective of SV40 also is implicated in the process of transformation mutant of adenovirus type 5 containing a single altered Ela and is required for maximal viral DNA replication in Go- mRNA species. J. Virol. 40:657-664. arrested permissive CV-1 cells (43). Small t mutants of SV40 9. Challberg, M. D., and T. J. Kelly, Jr. 1979. Adenovirus DNA are defective in the transformation of cells to anchorage-in- replication in vitro. Proc. Natl. Acad. Sci. U.S.A. 76:655-659. dependent growth (5, 45) and are defective in the transfor- 10. Challberg, M. D., and T. J. Kelly, Jr. 1982. Eukaryotic DNA mation of arrested cells but not actively growing cells (31). replication: viral and plasmid model systems. Annu. Rev. AMP concentra- Biochem. 51:901-934. Theophylline increases intracellular cyclic 11. Courtois, G., and A. Berk. 1984. Adenovirus ElA protein tions leading to the Go arrest of CV-1 cells. SV40 small t activation of an integrated viral gene. EMBO J. 3:1145-1149. mutants synthesize much less viral DNA in theophylline- 12. Ferguson, B., N. Jones, J. Richter, and M. Rosenberg. 1984. treated CV-1 cells than does wild-type SV40 (43). In the case Adenovirus Ela gene product expressed at high levels in of each of these DNA virus-transforming proteins, the Escherichia coli is functional. 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