Adenovirus 2 lp+ locus codes for a 19 kd tumor antigen that plays an essential role in cell transformation

Cell, Vol. 33, 759-766,

July 1983. Copyright

0 1983 by MIT

009.T8674/83/070759-08

$02.00/O

Adenovirus 2 /p+ Locus Codes for a 19 kd Tumor Antigen That Plays an Essential Role in Cell Transformation G. Chinnadurai Institute for Molecular Virology St. Louis University Medical Center 3681 Park Avenue St. Louis, Missouri 631 IO

Summary Adenovirus 2 large plaque (/p) mutants produce large clear plaques on human KB cells. These mutants are shown to be defective in inducing transformation of the established rat embryo cell line 3Y 1. The /p mutation was localized within one of the two transforming early gene blocks, Elb (map position 4.5 to 11.2) which codes for two major T antigens of 53 kd and 19 kd by marker transfer. The mutational defects in mutants lp3 and lp5 were analyzed by DNA sequence analysis and by analysis of viral El proteins. These results reveal that lp3 and lp5 mutations map within the 19 kd tumor antigen coding region. Mutant lp3 has a single base pair change at the N terminus of 19 kd polypeptide, resulting in the substitution of valine for alanine. Mutant $5 has two mutational changes, one of which results in the substitution of tyrosine for aspartic acid near the Nterminal region. The second mutation changes the termination codon into a leucine codon, increasing the size of the 19 kd tumor antigen. These results provide direct genetic evidence for an essential role of the 19 kd tumor antigen in cell transformation and indicate that the N-terminal region of the 19 kd tumor antigen is an essential functional domain for the induction of cell transformation. Introduction The transforming genes of human adenovirus (Ad) have been localized within the left 12% of the viral genome by analysis of viral sequences that are expressed in Adtransformed cell lines (Gallimore et al., 1974; Flint et al., 1976; Green et al., 1981) and by in vitro transformation experiments using transfection with restriction fragments of the viral DNA (Graham et al., 1974). The transforming region (El) of Ad2 (and closely related Ad5) is comprised of two transcriptional units, Ela and El b. Ela maps between map positions (mp) 1.3 and 4.4, and El b between mp 4.5 and 11.2 (reviewed in Tooze, 1980). By in vitro translation, the Ela region has been shown to code for polypeptides ranging from 28 to 53 kd (Esche et al., 1980; Lewis et al., 1976; Hatter and Lewis, 1978; Halbert et al., 1979; Ricciardi et al., 1981). The El b region codes for a 53 kd polypeptide and a 19 kd polypeptide (Esche et al., 1980; Halbert et al., 1979). The Elb 53 and 19 kd polypeptides constitute the major adenovirus T antigens (Gilead et al., 1976; Levinson and Levine, 1977). The 53 kd tumor antigen appears to be coded by a 22s mRNA, whereas the 19 kd tumor antigen is coded by both the

22s mRNA and the 13s mRNA (see Figure 4). The 53 kd and 19 kd tumor antigens are distinct; they have different peptide maps (Brackmann et al., 1980) and DNA sequence analysis reveals that the 19 kd polypeptide is translated from the first open reading frame and the 53 kd polypeptide from the second open reading frame (Bos et al., 1981; Gingeras et al., 1982). van der Eb and his colleagues have shown that transfection with the left 4.5% (Hpa I-E fragment) of the Ad5 genome which contains the Ela coding sequences transforms rat embryo cells in vitro to a “partially transformed” state (Houweling et al., 1980) but at a lower frequency than the leftmost 8% (Hind Ill-G fragment) of the viral genome does (van der Eb et al., 1977). Similarly, with the Ad12 system, a DNA fragment (Act I-H fragment) representing the left 4.5% of the viral genome induces partial transformation (Shiroki et al., 1979) while the left 6.8% (Hind Ill-G fragment) induces complete transformation (Shiroki et al., 1977). These results indicate that the DNA sequences containing Ela and the portion of El b that contains the complete sequences coding for the El b 19 kd polypeptide and the N terminus of the Elb 53 kd polypeptide are sufficient to induce a fully transformed phenotype. Viral mutants with lesions in El have proved useful in analysis of Ad-induced cellular transformation (Graham et al., 1978; Jones and Shenk, 1979a; Solnick and Anderson, 1982; Ho et al., 1982). Two complementation groups (hrl and hrll) of Ad5 host range mutants defective in cell transformation have been isolated (Harrison et al., 1977; Ho et al., 1982). Mutants in hrl and hrll groups have been mapped within Ela and El b, respectively (Galos et al., 1980; Ho et al., 1982). The hrl Ela mutants (i.e., hrl-hr5) induce “abortive transformation” in that they induce small transformed foci from which permanent cell lines cannot be easily established (Graham et al., 1978). The hrl coldsensitive (cs) mutants have a cs transformation phenotype, and temperature-shift experiments indicate that Ela function is required, indirectly or directly, for maintenance of transformation (Ho et al.,, 1982). One of the Ad5 deletion mutants isolated by Jones and Shenk (1979a), which maps entirely within Ela (d/312), has also been shown to be transformation defective. It is difficult to judge the effect of hrl and d/312 on transformation because these mutants are defective in the normal expression of El b (Berk et al., 1979; Jones and Shenk, 197913). However, the Ad2 Ela mutant hr440, which expresses normal amounts of El b, has been shown to be transformation defective (Solnick and Anderson, 1982) indicating that Ela gene products are needed for transformation. Shiroki et al. (1981) have shown that Ad5 d/31 3, which has a large deletion between mp 3.8 and 11 (i.e., retains most of the Ela but deletes most of El b), transforms rat 3Yl cells to a partially transformed state. All available El b mutants fall under the complementation group hrll, some members of which are defective in the Elb 53 kd tumor antigen (Lassam et al., 1979). These mutants are defective in transformation when used as virus

Cell 760

particles (Graham et al., 1978; Ho et al., 1982) but can transform as does wild-type virus when viral DNA is used in the transfection assay (D. T. Rowe and F. L. Graham, personal communication). These results, in conjunction with the observation that the left 8% of the viral genome can induce fully transformed phenotype (Graham et al., 1974; van der Eb et al., 1977) indicate that the El b 53 kd tumor antigen may not play an essential role in DNAmediated cell transformation. In the studies reported here I have identified a class of Elb transformation-defective mutants designated /p mutants and have shown that the /p+ locus codes for the 19 kd tumor antigen. These studies provide direct genetic evidence that a specific Ad-coded protein, the 19 kd tumor antigen, has an essential role in cell transformation.

I

HONH,

DNAPofB201.2 Cotransfectron

We previously isolated four /p mutants (lpi-lp4) that produce large clear plaques on human KB cells, in contrast with the small, fuzzy-edged plaques produced by wild type. Those mutants were isolated by mutagenesis of Ad2 virus particles, and one of them (1~3) was localized within the left 40% of the viral genome (Chinnadurai et al., 1979). Subsequently we isolated three other /p mutants by local mutagenesis of the left 15% of the viral genome. The scheme for isolation of these mutants is shown in Figure IA. The left 15% (Xho I-C fragment) of the Ad2 genome was cloned with plasmid vector pBR322. The cloned DNA was mutagenized with hydroxylamine (Chu et al., 1979) and cotransfected with terminal DNA-protein complex from an Ad2 Ela-El b host range deletion mutant d/201.2 (Brusca and Chinnadurai, 1981) in human KB cells. KB cells are nonpermissive for d/201.2; therefore, only viable recombinants between the mutagenized DNA and the genome of d/201.2 will yield plaques. Three /p mutants were isolated from about 200 plaques examined. The plaque morphology of wild type and mutant lo5 is shown in Figure 1B. These results indicated that the /p mutation is within the left 15% of the viral genome.

Physical Mapping of the /p Mutants Since mutagenesis of the left 15% of the viral genome produced /p mutants, we assumed that a likely region for the mutation would be the early gene block El that encompasses the left 11.2% of the genome. Consistent with this expectation, cotransfection of the left 9.4% (Bgl II-E fragment) of mutant lo5 DNA with d/201.2 DNA protein complex generated /p progeny viruses in pilot studies. In order to map precisely the /p mutation within the El region, we constructed recombinant plasmids in which segments of wild-type sequences were fused with /p sequences. This reconstruction results in sequences with overlapping homology with the sequences on both sides of the deleted region (mp -2.0-7.0) in the d/201.2 genome. These overlapping sequences are essential for marker rescue of d/201.2 by homologous in vivo recombination as illustrated

I” KB ceils

In vrvo recombrnatron

Results Isolation of Ad2 /p Mutants by Local Mutagenesis

at 70”

l

.

-

lp5-7

B

wt

lP5

Figure 1. Isolation of Ad2 /p Mutants (A) Scheme for isolation of Ad2 /p mutants by local mutagenesis, About 5 pg of plasmid DNA (pGC341) was linearized with restriction endonuclease Sal I and mutagenized with 0.05 M hydroxyfamine at 70% for 13 min (Chu et al., 1979). The plasmid DNA was then cotransfected into human KB cells along with 1 pg of DNA-terminal protein complex (Chinnadurai et al., 1978) from d/201.2 and 4 wg of tamer salmon sperm DNA. Progeny virus was collected afler 4-8 days and plaque assayed on KB cells. (B) Plaque morphology of Ad2 weld type and /p5 on KB cells. Cells were stained with neutral red and photographed with Polaroid type 55 film on day 12.

in Figure IA. In these constructs we USed DNAs from both lo3 (isolated by mutagenesis of virus particles) and lo5 (isolated by local mutagenesis). The structures of the constructs are shown in Figure 2. When construct I, containing wild-type DNA from mp w-0.5-4.5 and lp3 or lo5 DNA from mp 4.5-9.4, was used for marker rescue of d/201.2, all the progeny virus were of /p phenotype, indicating that the lo mutation is located within the El b region (i.e., between mp 4.5 and 9.4). When

Transforming 761

I

Gene of Adenovirus

2

/--

---\ 94

\

PGC

212

180111, ,0--L--_--.,

construct I

,/-----,\ Figure 2. Structure Rescue of di201.2

canstruct LL of Ad2 Wild-Type-/p

DNA Constructs

Used for Marker

The map positions of the cleavage sites on the viral DNAs are shown above the restriction enzymes. The wild-type sequences are shown with the open bars and the /p sequences are shown with the filled bars. For construct I, pGC212 DNA (in which the left 7.9% of the viral genome. i.e., the Hind IIIG fragment, has been cloned between the Eco RI and Hind III sites of pBR322) was cleaved with Hpa I and Barn HI. The excised DNA segment containcng viral DNA and vector DNA was replaced wrth a viral DNA fragment from mp 4.5 (Hpa I site) to mp 9.4 (Bgl II sate) of DNAs from lo3 or /pS. In construct IIt pGC212 DNA was cleaved with Kpn I and Barn HI and the wild type DNA from mp 5.8 (Kpn I site) to mp 7.9 and part of the vector DNA was replaced with an 77~3or lp5 DNA fragment from mp 5.8 to 9.4.

construct Ii, containing lp3 or lo5 DNA, was used in the marker rescue of d/201.2, all the progeny virus were of wild-type phenotype. These results indicate that the mutations causing the /p phenotype in lp3 and lp5 are between mp 4.5 and 5.8. DNA Sequence Analysis of $ Mutants Guided by the marker rescue studies, we focused DNA sequence analysis between mp 4.5 and 6.5 using the construct I wild-type and /p plasmid DNAs. The DNA sequence analysis of Ad5 and Ad2 carried out by Bos et al. (1981) and Gingeras et al. (1982) and the N-terminal amino acid analysis of the 19 kd polypeptide (Anderson and Lewis, 1980) have indicated that this region contains the coding sequences for El b 19 kd tumor antigen and the N terminus of El b 53 kd tumor antigen. We sequenced about 680 bp from position 1568 to 2250 of wild-type, lp3, and lp5 DNAs. Sequencing gels showing mutated regions of lp3 and lp5 DNAs are shown in Figure 3. The effect of mutations on the 19 kd polypeptide is diagramatically illustrated in Figure 4. The wild-type sequence of this region was entirely in accordance with the Ad2 sequence published by Gingeras et al. (1982). In the sequenced region, lp3 revealed a single base pair change at position 1718, where there is a C to T transition. In the case of lp5, alterations at two sites were observed-G to T transversions at positions 1954 and 2237. In both lp3 and lp5, the observed mutational change is within the sequence coding for the 19 kd tumor antigen. The G to T transversion of lp5 at position 2237 is in the N-terminal region of the 53 kd tumor antigen, where it causes an amino acid substitution of isoleucine for methionine. It is noteworthy that the lp3 and lp5 mutations are outside the two open reading frames identified by Gingeras et al. (1982) on the I strand of the El region. In lp3, the single base pair change results in the substitution of valine for alanine at the N terminus of the 19 kd polypeptide. In the case of lp5, the first mutational change results in the substitution of tyrosine for

Figure 3. DNA Sequence

Analysis

The altered bases are indicated boxed. (a) lp3; (b) and (c) lp5.

of the Ad2

/p Mutants

by the stars, and the altered triplets are

aspartic acid. The second mutation changes the normal termination codon of the 19 kd polypeptide into a leucine codon. Published results (Bos et al., 1981; Gingeras et al., 1982) indicate that the 19 kd polypeptide could be coded by both a 13s and a 22s mRNA (Figure 4). Thus the loss of normal termination of the 19 kd polypeptide of lp5 would result in two polypeptides related to the 19 kd polypeptide, but of higher molecular weight. When translation is initiated on the 22s mRNA, the altered 19 kd polypeptide would terminate at position 2272, resulting in 12 extra amino

Cell 762

A ---

Figure 4. Organization of the Ad2 El b Sequences Coding for the 53 kd and 19 kd Tumor Antigens

4

5 I

6 l

ATG ATG 16991711 2016 w 1

7 I

8 I

9 I

10 I

11

12

I

--.

(A) Organizatron of El b coding for the two tumor antigens, drawn according to the data of Gtngeras et al. (1982). (6) Structure of 19 kd tumor antrgen coded by wild type and /p mutants. Sites of the mutations In the 19 kd polypeptide of the /p mutants and the corresponding region on the wrldtype sequences are denoted by triangles, The triplets and amino acid sequence of the wild-type and mutant 19 kd polypeptide are also shown. The extra amino acids on the altered 19 kd polypeptide coded by lp.5 are rndrcated by the filled bar or by the bar with slanted lines.

t-nap position

TGA 2236 i

22s 19 kd

I

1699 1711

>

53

kd

2236 2249

13s 19 kd

B

acids. When translation is initiated on the 13s mRNA, in which nucleotide 2249 is juxtaposed with nucleotide 3589 by splicing (Perricaudet al al., 1980) the translation would terminate at position 3617, resulting in 14 extra amino acids.

A

B III a -

b

c

53 kd

Analysis of El b Proteins Coded by the /p Mutants Other investigators have established that the Elb region codes for a 53 kd and a 19 kd tumor antigen by in vitro translation (Halbert et al., 1979; Esche et al., 1980). In addition to these polypeptides, an El b 20 kd polypeptide has been identified both by analysis of in vivo synthesized proteins by immunoprecipitation and by in vitro translation (Green et al., 1982). We have analyzed the Elb proteins coded by Ad2 wild type, 1~3, and lo5 by in vitro translation and by immunoprecipitation of in vivo synthesized proteins. For in vitro translation, early mRNA was isolated and selected by hybridization to a cloned DNA fragment from the El b region (mp 5.1-9.4) and translated in a reticulocyte lysate system (Pelham and Jackson, 1978). The autoradiogram produced by SDS-polyacrylamide gel electrophoresis is shown in Figure 5A. mRNA from cells infected with wild type coded a major 19 kd polypeptide and a minor 53 kd polypeptide, in accordance with published results. The 53 kd polypeptide is very faint in Figure 5A and is often poorly translated in vitro in our experiments and in those of other investigators. The mRNA from cells infected with lp3 synthesized 53 kd and 19 kd polypeptides indistinguishable by size from the polypeptides synthesized from the wild-type mRNA. However, mRNA from cells infected with lp5 did not code for the 19 kd polypeptide. Instead, a 21 kd polypeptide was synthesized in addition

-21 kd - 19 kd 1

2

3

4

N F17

N

Frgure 5. SDS-Polyacrylamide Gel Electrophoretic peptides Coded by Ad2 Wild Type and /p Mutants

F17

N

Analysis

F17 of El b Poly

(A) In vitro translation. Lane 1, no RNA; lane 2; wild-type RNA, lane 3; $3 RNA; lane 4, lp5 RNA. (B) lmmunoprecipitation of in viva synthesized polypeptides wrth normal rat serum (N) or F17 antiserum (F17). (a) Wildtype-infected cells; (b) /p3-Infected cells; (c) /p5-infected cells.

to the 53 kd polypeptide. We have shown that the /p5coded 21 kd polypeptide is related to the wild-type 19 kd polypeptide by tryptic peptide mapping. As seen in Figure 6, wild-type 19 kd polypeptide produced a pattern (designated X) similar to the in vivo and in vitro synthesized 19 kd polypeptide that has been observed by others (Brackmann et al., 1980; Halbert and Raskas, 1982). The 21 kd polypeptide coded by lo5 produced the characteristic 19 kd pattern and in addition produced a unique peptide (designated Z). This is expected because the predicted

Transforming 763

Gene of Adenovirus

2

21 kd polypeptide coded by lo5 contains an additional methionine-containing tryptic peptide. The relatedness of the /@-coded 21 kd polypeptide and the wild-type 19 kd polypeptide has also been established by immunoprecipitation with sera raised against synthetic peptides predicted from the 19 kd polypeptide coding region (M. Green, personal communication). The observed size of the altered 19 kd polypeptide coded by lo5 is in good agreement with the predicted molecular weights of 22.07 or 22.23 kd. We cannot distinguish whether the in vitro synthesized 21 kd polypeptide is coded by the 22s mRNA, the 13s mRNA, or both, because the difference between the two polypeptides would be only two amino acids and would not be distinguished by SDS-polyacrylamide gel electrophoresis. The El b polypeptides synthesized by cells infected with Ad2 wild type or/p mutants were analyzed by immunoprecipitation using antiserum prepared against an Ad2-transformed cell line F17 (Figure 58). The F17 antiserum has been extensively characterized by Green, Weld, and coworkers and has been shown to contain antibodies to El b proteins (Gilead et al., 1976; Green et al., 1979; Wold and Green, 1979). In cells infected with wild-type virus, we detected three major polypeptides specific for infected cells. The sizes of the three polypeptides are 53, 20, and 19 kd. The 53 kd and the 20 kd polypeptides are related to one another, and the 19 kd polypeptide is distinct from them. These three polypeptides have been described in detail by Green et al. (1982). Cells infected with lp3 also synthesized three polypeptides similar to those observed in cells infected with wild type. In cells infected with lp5, the 53 kd and the 20 kd polypeptides were similar to those observed in wild-type-infected cells. As expected, the 19 kd polypeptide was absent; instead, low amounts of the 21 kd polypeptide related to it were observed. Although

the 21 kd polypeptide is a major in vitro translation product, only a small amount of it was seen in vivo. It may be that the modified protein is more labile in vivo, or the 21 kd polypeptide coded by lo5 might adopt an altered conformation and therefore be recognized less efficiently by F17 antibodies, In addition to El b proteins, we analyzed the Ela-coded proteins by in vitro translation using mRNAs complementary to the leftmost 4.5% of the viral genome. No differences were observed between the wild type and the mutants (results not shown).

Transformation of Rat 3Yl Cells by /p Mutants Since we have localized the /p mutation within the Elb region coding for the 19 kd tumor antigen, we wanted to examine the role of this protein in inducing cell transformation of the established rat embryo fibroblast cell line, 3Yl (Kimura et al., 1975) which is very sensitive to transformation by human adenoviruses (Shiroki et al., 1977, 1981). As seen in Table I, when 3Yl cells were infected with different Ad2 /p mutants, lo5 was completely transformation defective, lp3 and lp4 showed greatly reduced transformation efficiency (i.e. about one tenth that of wild type), and Ipl and lp2 were partially transformation defective (about half that of wild type). In addition, transfection with the DNAs of the transformation-defective mutants lp3 or lp5 did not result in significant transformation. These results indicate that the El b 19 kd tumor antigen coded by the /p+ locus plays an essential role in cell transformation, The observed effect on cell transformation is not due to excessive cell killing by the /p mutants, since the cloning efficiency of wild-type-infected and mutant-infected cells are the same (results not shown).

Discussion

A

We have identified a class of Ad2 mutants that is defective in inducing cell transformation. These viable mutants produce large clear plaques on human KB cells and are therefore designated large plaque (/p) mutants. They resemble the cytocidal (cyt) mutants of Ad12 isolated by

B

c

Table 1. Transformation mutants

I

* E Figure 6. Two-Dimensional Labeled Polypeptides

Tryptic

Peptide

Analysis

of %Methionine-

El b-specific RNA was selected, translated in vitro, and the 19 kd polypeptide of wild-type (A) and 21 kd pclypeptide /p5(B) were subjected to tryptic peptide mapping as described by Brackmann et al. (1960). The pattern designated Y IS observed less frequently and is probably the result of partial digestlon with trypsin. E Indicates the direction of electrophoresis. and C Indicates the directlon of chromatography.

of Rat 3Yl Cells with Ad2 Wild Type and /p

Virus”

Viral DNAb

WT

42

15

IP7

15

ND

lP2

16

ND

/P3

4

3

lP4

4

ND

iP5 No virus or viral DNA

0 0

1 1

Values expressed are foci per plate. a Cells were Infected with Ad2 wild type or /p mutants at lo-20 pfu/cell. D One microgram of viral DNA was transfected on 5 X 18 cells contained In a 25 cm2 bottle. ND not determined.

Cell 764

Takemori and coworkers (1968). Ad12 cyt mutants produce large plaques on human embryonic kidney cells. These mutants are also defective in cell transformation and are poorly oncogenic (or nononcogenic) in newborn hamsters. We have localized the Ad2 /p mutation within the section of early region El b coding for a 19 kd tumor antigen by marker transfer, by DNA sequence analysis, and by analysis of viral proteins. Comparable data for Ad12 cyt mutants are not available. Mapping studies of Ad12 cyt mutants should reveal whether the /p+ locus of Ad2 and the cyt+ of locus of Ad12 are similar. Our results demonstrate that the Elb 19 kd tumor antigen plays an essential role in inducing cell transformation We do not know the role of this polypeptide during productive infection. Since 19 kd polypeptide is synthesized in higher abundance during late stages of viral infection (Esche et al., 1980) it may play a role in viral DNA replication or in late gene expression. Conclusions that can be drawn from DNA transfection studies complement our results on cell transformation. lmmunoprecipitation studies (Schrier et al., 1979) have revealed that cells fully transformed by the Ad5 Hind Ill-G fragment (mp O-7.9) do not synthesize the Ad5 Elb 53 kd (also referred to as 65 kd) tumor antigen, but do express Elb 19 kd tumor antigen. Ad5 Hpa I-E fragment (mp O4.5) transformed cells (partially transformed) do not express Elb 19 kd tumor antigen; therefore, the partially transformed state of the Hpa I-E transformed cells may be due to the absence of the 19 kd tumor antigen. By transfection of cloned El DNA with random insertional mutations generated by the bacterial transposon Tn5, McKinnon et al. (1982) have shown that the N-terminal region of El b plays an essential role in transformation. These results are consistent with the observation that all Ad2- or Ad5transformed cell lines examined express the 19 kd tumor antigen (Matsuo et al., 1982). The functional domains of the 19 kd tumor antigen which may be essential for inducing cell transformation can be inferred from the DNA sequence analysis data of Ad2 (Gingeras et al., 1982) and Ad5 (Bos et al., 1981) and from our analysis of Ad2 /p mutants. Comparison of the sequences of Ad2 and Ad5 regions coding for 19 kg tumor antigen reveals that the C-terminal region exhibits variations leading to amino acid substitutions, but the N terminus is highly conserved between the two serotypes. This suggests that the N terminus of the 19 kd tumor antigen contains important functional information. This view is strengthened by the observation that lp3 DNA contains a single base pair change, resulting in a single amino acid substitution at the N terminus which greatly reduces the transforming potential of this mutant. Although lp5 mutation causes a single amino acid substitution in the 53 kd polypeptide, it is likely that the transformation-defective phenotype is due to the mutations in 19 kd polypeptide. DNA sequence analysis of Ad2 and Ad5 has revealed a number of amino acid substitutions at the N terminus of 53 kd polypeptide. The DNA transfection studies indicate that the 53 kd polypeptide may not play an essential role

in cell transformation. We do not know whether the mutation that causes the single amino acid substitution within the COrMNed region of the 19 kd tumor antigen or the one that causes the increased size of the protein in lp5 is the critical mutation. However, it appears that the mutation at position 1954 can alone cause an /p phenotype because DNA constructs containing the mutation at position 2237 rescued wild-type-like progeny during marker rescue of d/201.2 Results obtained by DNA transfection studies (Houweling et al., 1980; Shiroki et al., 1979) and by the use of an Ad5 El b mutant d/313 (Shiroki et al., 1981) indicate that the Ela region alone can induce partial transformation. These transformed cells are more fibroblastic than epitheloid in shape and grow more slowly than completely transformed cells. We have observed that mutant lp5 is transformation defective in spite of the presence of an intact Ela region. We attribute this discrepancy to the assay conditions. Under our conditions transformed foci were formed under an agar overlay; partially transformed cells may not form visible foci under these conditions. Others also have failed to detect transformation by transfection of DNA fragments containing only intact Ela sequences under stringent conditions (McKinnon et al., 1982). How the 19 kd tumor antigen causes the transformed phenotype is a major issue. It is noteworthy that the Ad2 19 kd tumor antigen is associated with plasma membrane (Persson et al., 1982) as are other transformation proteins such as polyoma middle T antigen (Ito, 1979) and pp60”“, coded by avian sarcoma virus (Willingham et al., 1979; Levinson et al., 1980). It remains to be determined whether the Ad 19 kd tumor antigen is an analog of these transforming proteins. It has also been observed that Ad genes which lie outside the El region play a role in the transformation process when infection is performed with virus particles. Ad5 N-group ts mutants at mp 18.0-22.5 have been shown to be defective in the initiation of transformation (Williams et al., 1974). On the other hand, Ad5 ts725, which is defective in the 72 kd single-stranded DNA binding protein encoded by the E2a region, enhances transformation (Ginsberg et al., 1974). Possible interactions between these proteins and the 19 kd tumor antigen remain to be investigated. Experimental

Procedures

Viral Mutants HZ lpi-lp4 were isolated previously by mutagenesis of Ad2 virus particles with hydroxylamine (Chinnadurai et at., 1979). H2 lpWp7 were isolated by in vitro mutagenesrs of the cloned left 15% (Xho I-C fragment) of the viral genome with hydroxylamine (Chu et al.. 1979). All mutants were grown in human KB cells. Cloning of DNA Fragments and DNA Sequence Analysis The plasmid vector pBR322 and the host Escherichia coli strain HBlOl were used in the cloning of all DNA fragments. Manipulatron of DNA fragments and cloning were carried out by standard protocols. DNA sequence analysis of the wild-type rp3, and $5 DNAs were carried out using cloned DNA fragments by the chemical degradation method of

Maxam and Gilbert (1980). For sequence labeled fragments were used.

analysis,

both 5’. and 3’.end-

Analysis of Viral Proteins For in vitro translation, total cytoplasmic RNA was isolated (Chinnadurai et al., 1976) from cultures of KB spinner cells that were infected with wild type, lp3. or $5. Cells were infected at an input multiplicity of 50-100 pfu/ cell and maintained in media containing 25 AS/ml of cycloheximide or 20 fig/ml of cytosine arabinoside for 15 hr. Virus-specific RNA was selected by a modification (suggested by Jim Lewis) of the fitter hybridization-elution method developed by Ricciardi et al. (1979). Hybridizations were carried out using cloned DNA fragments from mp 0 to 4.5 (Ela) and 5.1 to 9.4 (El b). The mRNAs were translated in a reticulocyte lysate system (Pelham and Jackson, 1978) purchased from New England Nuclear, and the poly peptides were analyzed on 14% SDS-polyacrylamide gels. For immunoprecipitation of Ad2 El proteins, cells were infected and maintained in cycloheximide or cytosine arabinoside as described above, and labeled with 35S-methionine (IO &i/ml) for 4 hr in the presence of 20 pg/ml of cytosine arabinoside. Whole cell extracts were prepared, and Ad2 El b proteins were immunoprecipitated using an antiserum prepared against the Ad2transformed rat cell line F17 (Gilead et al., 1976; Green et al., 1979; Wold and Green, 1979). The immunoprecipitates were analyzed on 14% SDS-polyacrylamide gels. Transformation Assays Confluent cultures of 3Y 1 cells were trypsinized (1 or 2 days after confluence), and about I@ cells were plated per 25 cm’ bottle. After 2 hr. cells were infected with Ad2 wild type or /p mutants at an input multiplicity of lo-20 pfu/cell. Infected ceils were maintained in Ca”-free Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum for about 1 week. Subsequently, the liqurd medium was replaced with 10 ml of semisolid medium containing 0.3% agarose. The cells were fed with another 5 ml of agarose overlay medium on the third week. After 4-5 weeks, agarose overlays were removed and the transformed foci were counted by staining with Giemsa. Transformation assays with viral DNAs were carried out essentially as described above. Flasks (25 cm’) containing about 5 X IO5 cells were transfected with 1 pg of viral DNA along with 9 pg of carrier salmon sperm DNA. DNA transfections were carried out by the calcium method of Graham and van der Eb (1973). Acknowledgments I thank M. Green, J. Lewis, W. S. M. Wold, J. Brusca, and S. Bolten for their advrce and help. This work was supported by research grants from the National Cancer Institute. I am an Established Investigator of the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

March 4, 1983; revised April 29, 1983

gel electrophoresis 6779.

and immunoprecipitation.

J. Biol. Chem. 255, 6772-

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