Identification of families of overlapping polypeptides coded by early “transforming” gene region 1 of human adenovirus type 2

VIROLOGY

97,275~286 (1979)

Identification of Families of Overlapping Polypeptides Coded By Early “Transforming” Gene Region 1 of Human Adenovirus Type 2 MAURICE Institute

GREEN,’ WILLIAM S. M. WOLD, KARL H. BRACKMANN, MARIA A. CARTAS

AND

for Molecular Virology, St. Louis University School of Medicine, 5681 Park Avenue, St. Louis, Missmwi 69110 Accepted May 22, 1979

Antisera against four lines of adenovirus e-transformed rat cells, including F1’7cells which contain only early gene region 1 (map position 1.5- 11) (the transforming region), immunoprecipitate major polypeptides of 53,000 (53K) and 15,000(15K) daltons (Gilead, Y.-H. Jeng, W. S. M. Wold, K. Sugawara, H. M. Rho, M. L. Harter, and M. Green, 1976, Nature (London) 264,263-266; W. S. M. Wold and M. Green, 1976,J. Virol. 30,297-310). We show here that the 53Ks precipitated by three of these antisera as well as Adl-SV40 induced ham ster tumor sera all have identical two-dimensional tryptic [%]Met-peptide maps. Similarly, all 15Ks precipitated by these antisera have identical maps. Tryptic and chymotryptic maps demonstrate that most if not all of the [35S]Met-peptides of 15K are shared by 53K. Recent tryptic peptide maps (D. Halbert, D. Spector, H. J. Raskas, W. S. M. Wold, and M. Green, unpublished data) prove that the 53K corresponds to a 53K translated in vitro from a 22s RNA derived from map positions 4.5-11 (D. Halbert, D. Spector, and H. J. Raskas, J. Viral., in press). Thus, both the 53K and 15K are coded within map position 4.5-11. Four [35S]Met-labeled polypeptides of 40K-50K, specific to Ad2 early infected human cells, were isolated in O’Farrell-type 2D gels. These polypeptides are coded within map position 1.5-4.5 (D. Halbert, D. Spector, and H. J. Raskas, J. Virol., in press; D. Halbert, D. Spector, H. J. Raskas, W. S. M. Wold, and M. Green, unpublished data). As expected, our tryptic and chymotryptic peptide maps of these four polypeptides indicate no relationship to 53K or 15K. Two of the 40K-50K polypeptides are highly related, the other two are highly related, and all four are partially related. In addition to 53K and 15K, the F17 antiserum precipitated a 28K polypeptide and minor bands of 14K-16K, 18K-20K, and llK-12K. Tryptic and chymotryptic maps of these polypeptides show that (1) 14K-16K and 18K-20K polypeptides are members of the 53W15K family, (2) llK- 12K polypeptides are related to each other but are unrelated to any other polypeptides, and (3) 23K is unrelated to any other polypeptides. We conclude that early region 1 codes for two (53W15K family, 40K-50K family) or possibly more (e.g., llK-12K family) families of polypeptides; the polypeptides included in each family overlap in amino acid sequence and therefore may be translated from overlapping spliced mRNAs. The possible contribution of these polypeptides to the phenotype of Ad2transformed cells is discussed. INTRODUCTION

(Flint et al., 1975; Craig et al., 1975; Pettersson et al., 1976; Bmtner et al., 1976; Early gene region 1 of human adenovirus Chow et ~2.) 1977; Neuwald et al., 1977). At type 2 (Ad2) (and closely related Ad5) is an important model to understand eucaryotic least two areas in region 1, mp 1.5-4.5 and gene expression and viral cell transforma- 4.5- 11, code for two or more overlapping tion (reviewed by Wold et al., 1978). Region spliced mRNAs (Kitchingman et al., 1977; 1 is located within map position (mp) 1.5- Berk and Sharp, 1978a; Spector et al., 1978; 11 on the conventional map of 100 units L. Chow, T. Broker, and J. Lewis, personal communication). Because region 1 is probably transcribed mainly by cell mechanisms, mapping the genes in region 1 and analysis 1 To whom reprint requests should be addressed. 275

004%6822/79/120275-12$02.00/O Copyright All rights

Q 1979 by Academic Press, Inc. of reproduction in any form reserved.

276

GREEN

of their mRNA and polypeptide products may provide insights into the organization and expression of cellular genes. Region 1 is of further interest because it contains the DNA sequences for Ad2 cell transformation (Gallimore et al., 1974; van der Eb et al., 1977). All Ad2 transformed cells synthesize mRNA from this region (Flint et al., 1975; Chinnadurai et al., 1976; Bachenheimer and Darnell, 1976; Chinnadurai et al., 1978), as well as proteins that are immunologically related to several Ad2 early proteins (Gilead et al., 1976; Levinson and Levine, 1977; Johansson et al., 1978; Wold and Green, 1979). These observations, and the isolation of Ad2 transformation-defective mutants (Graham et al., 1978) that map in region 1 (Frost and Williams, 1978), suggest that proteins coded by this region play a role in Ad2 cell transformation. Two approaches were used to identify these proteins and to map their genes. First, in vitro translation of viral mRNA selected by hybridization to DNA restriction fragments mapped several polypeptides of 38K to 51K (Lewis et al., 1976; Lupker et al., 1977; Harter and Lewis, 1978; Halbert et al., in press) and 14K (Lupker et al., 1977), and 28K (Halbert et al., in press) within mp 1.5-4.5, a 15K (Lewis et al., 1976; Halbert et al., in press) and a 52K (Halbert et al., in press) within mp 4.5-11, and a 12K within mp 9.5-11 (Pettersson and Mathews, 1977). A second approach to identify and map region 1 polypeptides was immunoprecipitation of [35S]Met-labeled polypeptides using antisera against Ad2 or Ad5 transformed cells that retain only region 1. We precipitated 53K and 15K polypeptides with antiserum against F17 cells (Gilead et al., 1976), a line of Ad2 transformed rat cells (Gallimore, 1974). Antisera against three other AdB-transformed cell lines (T2C4, 8617, F4) that contain region 1 also precipitated the 53K and 15K polypeptides as major components (Wold and Green, 1979). Levinson and Levine (1977) identified an Ad5-specific polypeptide of 58K. In this paper, we present [35S]Metpeptide maps showing that antisera against Fl?, T2C4, and 8617 cells immunoprecipitate indistinguishable 53K and indistinguishable 15K polypeptides. These

ET AL.

same polypeptides were also immunoprecipitated by a serum from hamsters bearing tumors induced by Ad1 -SV40 nondefective hybrid virus (Gilead et al., 1976). We further show that most of 15K sequences are shared with 53K. Peptide maps also show that 53K and 15K are unrelated to four 40-50K polypeptides, isolated by two-dimensional gel electrophoresis and that are coded within mp 1.5-4.5 (D. Halbert, D. Spector, H. J. Raskas, W. S. M. Weld, and M. Green, unpublished data) and that have been shown by partial proteolysis to be related (Harter and Lewis, 1978). We show that all four 40K-50K polypeptides are partially related and form two families of highly related polypeptides. We have also mapped a 28K polypeptide, several minor 14K-16K and 18K-20K polypeptides, and three llK-12K polypeptides that are immunoprecipitated by the F17 antiserum. The 14K-16K and 18K-20K polypeptides are highly related to the 15K. The llK- 12K polypeptides are related to each other. The llK-12K and 28K are not related to each other or to any other F17-specific polypeptides. These results together with RNA data suggest that two areas in region 1 contain “overlapping genes” that code for partially related RNAs and polypeptides. The possible roles that these polypeptides play in cell transformation and the possible location of the genes for 53K and 15K are discussed. MATERIALS

AND METHODS

Preparation of [35S]Met-labeled Ad2 infected cell proteins. KB cells were grown

in Eagle’s minimal essential medium (MEM) containing 5% horse serum, and Ad2 stocks were prepared as described (Green and Wold, 1978). Two protocols were used to label cells: labeling in the presence of arabinosylcytosine (ara C), or pretreatment with cycloheximide followed by labeling in the presence of ara C. Ara C inhibits viral DNA replication and therefore inhibits the transition to the late stage of infection. Cells were infected with 500 plaque-forming units (PFU) of Ad2 per cell (Wold et al., 1976) and treated with cycloheximide (25 pg/ml) from l-4 hr postinfection to enhance subsequent early protein synthesis

PEPTIDE

MAPS OF Ad2 TRANSFORMING

(Harter et al., 1976). The cycloheximide block was removed by centrifuging and washing cells twice with Met-free MEM. Cells were resuspended at 5 x lo5 cells/ml in Met-free MEM containing 20 pg/ml of ara C and 5% horse serum, and labeled with [35S]Met (20 #Zi/ml, 570 Cilmmol, New England Nuclear) from 5-14 hr postinfection. Cells were harvested by centrifugation, washed twice with cold PBS lacking Ca2+ and Mg2+ containing 1 mlkf phenylmethylsulfonyl fluoride (PMSF) to inhibit protease activity and suspended in cold isotonic high pH buffer containing 1 mM PMSF. Cells were lysed with 0.5% Nonidet P-40 (NP-40), nuclei were removed by centrifugation, and the cytoplasm was clarified at 100,000 g (Wold et al., 1976). Immunoprecipitation of Ad2 early proteins. Clarified cytoplasm (2 x 10’ cpm)

REGION

PROTEINS

277

11 hr postinfection as described above. Cells were harvested and sonicated in 50 ~1 of 10 m&f Tris-HCl (pH 7.4), 5 m&f MgC&, 50 pg/ml of pancreatic RNase for 10 min in a Raytheon DF-101 sonic oscillator at full power. The suspension was incubated with DNase I (20 pg) for 30 min at 4”, lyophilized, and dissolved in 300 ~1 of lysis buffer (O’Farrell, 1975); log cpm of acid-precipitable polypeptides was obtained. Aliquots of 2 x lo8 cpm were isoelectric focused on 12 x 0.4cm cylindrical gels prepared with pH 3.510 and pH 5-7 ampholytes. The gels were then electrophoresed on slab gels in the second dimension as described above. Peptide maps. Bands of [35S]Met-labeled polypeptides were identified on dried gels by autoradiography and peptide mapped essentially as described by Elder et al., (1977). The cut out bands were washed with 10% methanol, dried, and digested with 100 ~1 of freshly prepared trypsin or chymotrypsin (50 pg/ml, Worthington) in 50 mM NH4HC03 at 37” for 2 hr, and further incubated with 0.5 ml of HZ0 overnight. The lyophilized supernatants (lOOO-25,000 cpm) were dissolved in 10 ~1 of electrophoresis buffer (acetic acid: formic acid: water, 15:5:80), spotted on a 10 x lo-cm cellulosecoated thin layer plate, and electrophoresed on a DeSaga/Brinkmann thin layer electrophoresis apparatus at 1000 V for 15-20 min. Ascending chromatography was performed in the second dimension in butanol: pyridine: acetic acid: water (32.5:25:5:20). Plates were coated with molten 0.4% PPO in 2methylnaphthalene (Bonner and Stedman, 1978), and autoradiographed at -70”. With the 40K-50K or 53K polypeptides, 10,000 cpm of radioactivity required about 4 days of autoradiography. With the 15K polypeptide, 1000 cpm required about 3 weeks autoradiography.

was incubated with 20 ~1 of F17 antiserum (Gilead et al., 1976) in 150 mil4 NaCl, 50 mM Tris-HCl (pH 7.4), 5 m&f EDTA, 0.5% NP-40,1 mM PMSF, and 0.1% bovine serum albumin in 1.0 ml for 14 hr at 4”. After incubation with 200 ~1 of “formalin-fixed” 10% suspension of Staphylococcus aureus containing protein A (Kessler, 1975) for 1 hr at 4”, the immunoprecipitate-bacteria complex was centrifuged for 90 set in a Beckman Model B Microfuge and washed four times with 0.5 ml of 500 mM NaCl, 10 mM Tris-HCl (pH 7.4), 0.5% NP-40, and 1% deoxycholate. Immunoprecipitated polypeptides were eluted with 2% sodium dodecyl sulfate (SDS), 1% P-mercaptoethanol, 0.1% bromophenol blue, and 10% glycerol at 37” for 10 min. Acrylamide gel electrophoresis. Immunoprecipitates were electrophoresed on a linear lo-18% SDS-polyacrylamide slab gel (30 x 15 x 0.15 cm) at 12 mA/gel for 15 hr (Wold and Green, 1979). Gels were stained with Coomassie blue, dried, and RESULTS autoradiographed using Kodak X-Omat R film to visualize immunoprecipitated pro- Identijkation of Ad24nduced Early Potyteins. peptides Immunoprecipitated by F17 Two-dimensional electrophoresis of Ad2 infected cell potypeptides. The procedure of

O’Farrell (1975) was used. One hundred milliliters of Ad2 infected and mock infected KB cells was pretreated with cycloheximide from 1 to 4 hr postinfection, and labeled with 50 @J/ml of [35S]Met from 4 to

Antiserum

Figure 1 shows the [35S]Met-labeled polypeptides precipitated by F17 antiserum. As reported previously, (Gilead et al., 1976; Wold and Green, 1979) prominent polypeptides with apparent molecular weights of

GREEN ET AL.

278 lmmunoprecipitoti0~ Ad2 Condidole Transformation Proteins

ing. These polypeptides were not precipitated from infected cell extracts by normal rat serum (Fig. l), nor from mock infected cell extracts by Fl’i’ antiserum or normal rat serum (not shown).

of

Chymotryptic and Tryptic [35S]Met-Peptide Maps of .%‘K and 15K Immunoprecipitated by 4’17 Antiserum

EI-53K-

-29

El-26K-

El-16KEI-l6K\ El-ISKEl-14SK-

,/lb*, - 14.3

F-17 Norm. Rat

FIG. 1. Polyacrylamide gel electropherogram of Ad2 early [%]Met-labeled polypeptides immunoprecipitated by F17 antiserum. The cells were pretreated with cycloheximide prior to labeling. About 27,000 and 4500 cpm of immunoprecipitate by F17 and by normal rat serum, respectively, were electrophoresed and autoradiographed as described under Materials and Methods. Molecular weight markers are pbosphorylase b (94K), bovine serum albumin (t%K), ovalbumin (43K), carbonic anhydrase (29K), and lysozyme (14.3K).

53K and 15K were precipitated. The F17 antiserum also precipitated a 28K polypeptide, two or more minor 18K-20K polypeptides, two or more minor 14-16K polypeptides, and three llK- 12K polypepdides. Although 53K and 15K were reproducibly visualized as major bands in many other experiments, the weak 18K-20K, 14K16K, llK-12K bands, and the stronger 28K band, were not observed in all experiments. That is, occasionally we did not detect the 28K, other times not the llKl2K, and still other times not the minor 18K-20K and/or the 14K-16K polypeptides. These same polypeptides were observed regardless of whether cells were treated with cycloheximide prior to label-

The 53K and 15K bands were cut from the gel in Fig. 1 and mapped. The chymotryptic maps are shown in Fig. 2a. Of interest, the 5 major [35S]Met-peptides of 15K are shared by 53K (the common peptides are numbered). Tryptic maps of the 53K and 15K are shown in Fig. 2b. The 15K map has 5 peptides that are common to the 53K map. These results indicate that most of the 15K sequences are shared by 53K. As expected, the chymotryptic and tryptic maps of 53K are different from the corresponding maps of the 73K single-stranded DNA binding protein and the 42K and 47K subspecies (data in Green et al., 1978) that are coded within mp 62-68. Tryptie [35S]Met-Peptide Maps of 53K and 15K Polypeptides Immunoprecipitated by Antisera against Two AdZ-Transformed Rat Cells (TZC4, 8617) and by Ad1 -SV.@-Induced Hamster Tumor Serum We have reported that antisera against three AdZ-transformed rat cell lines, T2C4, 8617, and F4, all immunoprecipitate 53K and 15K polypeptides as major components (Wold and Green, 1979). Sera from hamsters bearing tumors induced by Ad1 - SV40 (Ad1 is closely related to Ad2) nondefective hybrid virus also immunoprecipitate 53K and 15K polypeptides (Gilead et al., 1976). To test whether all 53Ks and all 15Ks are in fact the same polypeptides and that they do not mask any additional unrelated polypeptides, we did peptide mapping experiments as described above. As shown in Figs. 3a and b, tryptic peptide maps of the 53Ks and the 15Ks that are precipitated by the Adl-SV40, T2C4, and 8617 antisera are highly related to each other and to the F17-specific 53K and 15K. There is no evidence for additional unrelated polypeptides that may comigrate with the 53K and

PEPTIDE MAPS OF Ad2 TRANSFORMING -

15K MAPS OF Ad2

-

279

ELECTROPHORESIS

53K CHYMOTRYPTIC

REGION PROTEINS

53K AND 15K POLYPEPTIDES

ELECTtXMfORESk3

b

t

53K TRYPTIC

15K

MAPS OF Ad2 53K AND 15K POLYPEPTIDES

FIG. 2. Chymotryptic (a) and tryptic (b) [93]Met-peptide maps of Ad2 52K and 15K polypeptides immunoprecipitated by F1’7 antiserum. The bands of 53K and 15K, in gels similar to that shown in Fig. 1, were digested with chymotrypsin or trypsin, spotted in the lower right corner of thin-layer plates, and electronhoresed from right to left in the first dimension. Ascending chromatography was performed in the second dimension from bottom to top.

15K in electropherograms. The common precipitation of the same homogeneous 53K and 15K polypeptides by four different antisera is consistent with a possible role for these polypeptides in cell transformation. Chymotryptic and Tryptic [35S]Met-Peptide Maps of Four Ad&Induced Early Polypeptides of .JOK-50K Resolved by Two-Dimensional Gel Electrophoresis An important question is whether 53K is related to the 38K-51K polypeptides mapped at mp 1.5-4.5 by cell-free translation (Lewis et al., 1976; Lupker et al., 1977; Halbert et al., in press). Harter and Lewis (1978) recently reported that these 38K51K polypeptides are synthesized in viva and migrate to specific positions in O’Farrell-type two-dimensional (ZD) gels. We

isolated and mapped four polypeptides located at the same position in 2D gels of infected cells, and we have confirmed by peptide mapping (D. Halbert, D. Spector, H. J. Raskas, W. S. M. Wold, and M. Green, unpublished data) that these are related to a 41K polypeptide translated in vitro from a spliced RNA derived from mp O-4.5 (Halbert et al., in press). Figure 4 shows this region (30K to 60K molecular weight and pH 5 to 7 isoelectric point) of 2D gel electropherograms of Ad2 early infected and mock-infected [35S]Met-labeled polypeptides. The infected cell extract contained four prominent polypeptides of about 40K-50K (labeled A-D) that were absent from the mock-infected extract, as observed in about 10 separate experiments. The chymotryptic and tryptic maps of each of these four polypeptides are shown in Figs.

GREEN ET AL.

280

53K (AdI

53K i TX41 TRYPTIC

MAPS

POLYPEPTIDES

p&

15K (AdI

15K (T2C4) TRYPTIC

MAPS

53Kt8617)

W-40)

OF 53K

)_%

-

W-40)

15K(8617)

OF 15K POLYPEPTIDES

FIG. 3. Tryptic [YS]Met-peptide maps of 53K (a) and 15K (b) polypeptides immunopreeipitated by T2C4, Adl-SV40, and 8617 antisera. See the legend to Fig. 2 for procedures.

5a and b, respectively. With both proteases, the maps of A and B are very similar, as are the maps of C and D. A and B maps have six peptides (numbered) that are present in C and D. Thus, A and B are highly related, C and D are highly related, and A and B are partially related to C and D. Of interest, the chymotryptic and tryptic maps of the 40K-50K polypeptides (Fig. 5) are very different from the chymotryptic and tryptic maps of 53K and 15K (Figs. 2 and 3), indicating little or no relationship. The chymotryptic and tryptic maps of the 40K-50K polypeptides are also unrelated to the ‘73K DNA binding protein and its 42K47K subspecies (data in reference Green et al., 1978). Alkaline

Ad2

Acid

INF EARLY

Chymotryptic and Tryptic [35S]Met-Peptide Maps of 28K, 18K, l6K, and 11.5K, and 11.3K Polypeptides Immunoprecipitated by F17 Antiserum

The other polypeptides (28K, UK-20K, 14K- 16K, 1lK- 12K) shown in Fig. 1 were peptide mapped to investigate the relationship to each other and to the 53K, 15K, and 40K-50K polypeptides. Chymotryptic and tryptic peptide maps are shown in Figs. 6a and b, respectively. We conclude that (1) 18K and 16K both are highly related to 15K, (2) 11.3K and l1.5K are related to each other (and to the 12K, not shown) but are not related to any other polypeptides, and (3) 28K is not related to any other polypepAcid

Alkaline

MOCK

IN!?

FIG. 4. 2D electropherograms of [%]Met-labeled polypeptides from Ad2 early infected and mockinfected cells. About 2 x lo8 cpm was analyzed as described under Materials and Methods. An enlarged region of 30 to 60K molecular weight and pH about 4.4-6 isoelectric point is shown.

PEPTIDE

MAPS OF Ad2 TRANSFORMING

tides. We have previously shown by partial proteolysis that the 14.5K polypeptide is related to 15K (Wold and Green, 1979). We have not peptide mapped the approximately 19K band shown in Fig. 1. DISCUSSION

We have shown that antiserum against F17 cells immunoprecipitated polypeptides of 53K, 28K, 15K, and minor bands of 18K20K, 14K-16K, and llK-12K from Ad2 early infected cells. Peptide maps establish that (1) most of 15K sequences are common to 53K, (2) the same 53K and the same 15K polypeptides are precipitated by antisera against four different transformed cells (F17, T2C4,8617, Adl-SV40), (3) 53K and 15K are unrelated to four 40K-50K polypeptides that probably are coded within mp 1.5-4.5, (4) the four 40K-50K polypeptides are all related, (5) the minor bands of 18K20K and 14K-16K are related to 15K, (6) three llK- 12K polypeptides are related to each other but are unrelated to any other polypeptides, and (7) 28K is unrelated to any other polypeptides. We note that our peptide maps are relevant only to Metcontaining polypeptides, and it is possible that some of these polypeptides (especially 28K and llK-12K) may share peptides lacking Met. We do not know why the F17 antiserum, or antisera against four other lines of Ad2- or Ad5-transformed rat cells (Wold and Green, 1979), failed to immunoprecipitate the 40K-50K polypeptides. Harter and Lewis (1978) have reported that four or five polypeptides that correspond to our 40K-50K polypeptides are related, using the partial proteolysis procedure. Proposed Location of 5SK and l5K Genes Recently Halbert et al. (in press) have determined that a 53K polypeptide is translated in vitro from a 22 S RNA. This RNA is coded within mp 4.5-11. Recent tryptic peptide maps indicate that the 53K immunoprecipitated by the F17, T2C4, 8617, and Adl-SV40 antisera corresponds to the 53K translated in vitro. (D. Halbert, D. Spector, H. J. Raskas, W. S. M. Wold, and M. Green, unpublished data).

REGION

PROTEINS

281

Therefore, the 15K, 53K, and the minor 18K-20K and 14K-16K polypeptides immunoprecipitated by our antisera must be coded within mp 4.5-11. The 15K is immunoprecipitated by an antiserum against 5RK cells (clone I) (Wold and Green, 1979), a rat line transformed by transfection with HsuI-G (mp O-8) fragment (van der Eb et al., 1977), suggesting that 15K is coded, at least in part, by sequences within mp 4.58.0. Therefore, the N-terminal portion of 53K must also be coded within mp 4.5-8.0. The gene for a 12K polypeptide (virion protein IX) maps at about mp 9.5-10.5; this suggests (but does not require) that the 53K gene lies within 4.5-9.5. Since 4.2% of the genome is required to code a 53K polypeptide, the genetic content of this region is sufficient for the 53K and 15K genes. Halbert et al. (in press) have translated a 28K polypeptide from a 9 S spliced mRNA coded by mp 1.5-4.5. If their in vitro 28K corresponds to our 28K, then the 28K must be translated mainly in a different reading frame from the four 40K-50K polypeptides, because our 28K and the 40K-50K polypeptides share no [35S]Met-containing peptides. There is little information available on where the llK-12K gene(s) might be located. It is possible that the llK-12K polypeptides are related to the 12K virion protein IX that is coded by mp 9.5-10.5. Region 1 May Contain Two or More Sets of Overlapping Genes Recent studies with the small transforming DNA tumor viruses SV40 and polyoma provide a precedent to the interpretation of our findings. The SV40 early (transforming) region (0.65-o. 17) contains at least two “overlapping genes” (e.g., see Prives et al., 1977; Thimmappaya and Weissman, 1977; Volkaert et al., 1978; Crawford et al., 1978; Paucha and Smith, 1978; Berk and Sharp, 1978b; Sleigh et al., 1978). For convenience, we use the term “overlapping gene” to indicate that a single DNA region codes for two or more distinct partially related mRNAs and polypeptides; whether these overlapping polypeptides have different functions is not considered. One gene is located at mp 0.65-0.55 and

GREENETAL.

282

a A

CHYMOTRYPTIC MAPS OF 40-50K

-

POLYPEPTIDES

EL.EcrRoPHoREsfs 8

b A

6 5

TRYPTIC

MAPS OF 40-50K

POLYPEPTIDES

PEPTIDE MAPS OF Ad2 TRANSFORMING

codes for little t antigen (15K-20K). Little t is coded by a 19.5 S mRNA that spans most of the early region; this mRNA contains a termination codon at mp 0.55 so that only the 5’ portion is translated. The other gene is located at mp 0.65-0.59 and 0.54-0.17 and codes for big T antigen (90K-100K). The 19 S mRNA that codes for T is thought to be spliced, lacking the intervening DNA sequences at mp 0.590.54, including the termination codon at 0.55, so that most of the RNA is translated. T and t have the same N-terminal sequence. However, the C-terminal sequence of t coded by mp 0.59-0.55 is absent from T. Similar studies with polyomavirus strongly indicate an analogous organization of early genes (It0 et al., 1977; Hutchinson et al., 1978; Schaffhausen et al., 1978). Although the evidence is not yet as complete, Ad2 early region 1 may also contain overlapping genes. mp 1.5-4.5 and 4.5-11 each code for two or three overlapping RNAs, most with spliced structures (Kitchingman et al., 1977; Berk and Sharp, 1978a; Spector et al., 1978; Chow et al., personal communication). Our peptide maps of four 40K-50K polypeptides (see Fig. 4A-D), coded by mp 1.5-4.5 (Lewis et al., 1976; Lupker et al., 1977; Harter and Lewis, 1978), indicate that A and B are highly related, that C and D are highly related, and that all four are partially related. These polypeptides may be coded by two to four overlapping genes at mp 1.5-4.5. If this is the case, then these genes may be read in the same frame at the 5’ end but in different frames at the 3’ end, because mp 1.5-4.5 read in one frame cannot code for more than about 40K daltons of protein. Perhaps the reading frame is changed by RNA splicing. Alternatively, the four 40K-50K polypeptides may differ from each other by some type of unknown posttranslational modification which affects their isoelectric point and their apparent molecular weight. Peptide maps of 53K and 15K indicate that they are highly related and

REGION PROTEINS

283

therefore may be coded by two overlapping genes within mp 4.5-11.0. The minor 18K-20K and 14K-16K polypeptides are related to 15K and thus may also form part of the 53W15K family. However, since most of these are minor bands, the possibility that they are proteolysis products of 53K must be considered. More work is necessary to prove that the related 40K-50K polypeptides, and the related 53K and 15K polypeptides, are coded by different overlapping genes, i.e., that each polypeptide has a separate biological function. However, the strong analogy between Ad2 region 1 and the papovavirus early region tends to support an overlapping gene model. Of particular interest, adenovirus and papovavirus early genes probably are expressed by host mechanisms, which raises the possibility that overlapping genes may be a general feature of eucaryotic cells. Possible Roles of 5sK, l5K, and 40-5OK Polypeptides in Cell Trunsforwuhn Cells have been transformed by transfection with purified HsuI-G (mp O-8) (van der Eb et al., 1977) and HpuI-E (mp O-4.5) (van der Eb et al., 1978) fragments, indicating that information to transform these cells lies within mp O-8 and mp O-4.5, respectively. There is evidence (see Introduction) that viral-coded transformation proteins may function in Ad2 cell transformation, The 40K-50K (and possibly the 28K) polypeptides coded by mp 1.5-4.5 are candidate transformation proteins. Since the 53K and 15K are coded within mp 4.511, they do not play a role in HpuI-E transformation. Similarly, intact 53K cannot be involved in HsuI-G transformation, although 15K may be. Nevertheless, it is possible that 53K and/or 15K play a role in the transformation of cells by virions. For example, cells transformed by HpuI-E and HsuI-G show minimal properties associated with in vitro transformation as compared to cells transformed by virions (van der Eb

FIG. 5. Chymotryptic (a) and tryptic (b) maps of four 40K-50K polypeptides isolated by 2D electrophoresis of [W]Met-labeled Ad2 early infected cell extracts. Polypeptides A-D were cut from the gel in Fig. 4, treated with chymotrypsin or trypsin, and mapped as described in Fig. 2.

GREEN ET AL.

284 -,,, ‘. “‘.‘i‘Y :j. ;

a i *k 1c

!

aa.

hamster tumor serum. This work was supportedby Grants ROl-CA-21824, ROl-AI-01725, and partially by R23-CA-24710. M.G. is recipient of a Research Career Award 5K5-AI-4739 from NIH. REFERENCES

b

FIG. 6. Chymotryptic (a) and tryptic (h) [%]Metpeptide mapsof 18K, 16K, 15K, 28K, 11.5K, and 11.3K polypeptides immunoprecipitated by F17 antiserum. See legend to Fig. 2 for procedures.

et al., 1977; Gallimore et al., 1977). All of four virion-transformed cells (F17, T2C4, 8617, F4) that we examined synthesize polypeptides immunologically related to both 53K and 15K (Wold and Green, 1979). These as well as other cell lines appear to retain most of early region 1, not just sequences within mp O-4.5. This suggests that sequences within 4.5-11, perhaps through the functions of 53K and 15K, may contribute to the phenotype of virion transformed cells. The F17, T2C4, 8617, and F4 antisera all immunoprecipitate 28K, llK12K, and minor 14K-16K and 18K-22K species. Therefore, these polypeptides may also be important in cell transformation. ACKNOWLEDGMENTS We thank H. Thornton for assistance with cell culture and preparation of the F17 antiserum and L. Young for technical assistance. MG thanks J. H. Elder and R. A, Lerner for instruction in peptide mapping and R. Gilden for a gift of the Adl-SV40

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