VIROLOGY
67,14-23
(1975)
Isolation
and Characterization
of Adenovirus
DNA Binding BRIGITTE
ROSENWIRTH,’
KAZUKO HIROTO Accepted
Type 12
Proteins
SHIROKI,2 SHIMOJ02 April
ARNOLD
J. LEVINE,3
AND
3, 1975
Infection of African green monkey kidney cells with type 12 adenovirus results in the production of two single strand specific DNA binding proteins. The molecular weights of these proteins are 60,000 and 48,ooO. Both proteins are synthesized in the absence of viral DNA replication and neither protein appears to correspond to any polypeptide detected in mature adenovirus virions. Temperature sensitive mutants from three different early, DNA negative, complementation groups (tsA, tsB, and tsC) have been tested for the production of these proteins at permissive and nonpermissive temperatures. Mutants of the tsB and tsC classes produce both DNA binding proteins at 32 and at 40”. TsA mutants produce both proteins at 32” but neither DNA-binding protein can be detected when these mutants are grown at 40”. The properties of adenovirus DNA binding proteins produced by types 2,5, and 12 adenoviruses are compared.
Because of the specificity of binding to single stranded DNA and the presence of large single stranded regions of DNA in replicating adenovirus DNA molecules (Sussenbach, van der Vliet, Ellens, and Janz, 1972; van der Eb, 1973) it was felt that the function of these proteins might be related to adenovirus DNA replication. Two different, DNA negative, temperature sensitive mutants of type 5 adenovirus, H5ts36 and H5ts125, were examined for the production of these proteins at nonpermissive temperature. H5ts36 infected cells produced normal levels of both DNA binding proteins at 39.5”, while H5ts125 infected cells at 39.5” contained a little or none of the 72,000 and 48,000 MW proteins (van der Vliet, Levine, Ensinger, and Ginsberg, 1975). Both of these proteins obtained from HMs125 infected cells at 32” (permissive temperature) were thermolabile in their continued binding to single stranded DNA when compared with the adenovirus wild-type 5 proteins. Recent peptide maps of the 72,000 and 48,000 MW proteins have shown that the smaller protein contains a subset of peptides all of which are found in the larger protein (Ro-
INTRODUCTION
African green monkey kidney cells infected with type 5 adenovirus synthesize two proteins, found preferentially in infected cells, that bind to single stranded DNA (van der Vliet and Levine, 1973). The molecular weights of these proteins, as determined by SDS-polyacrylamide gel electrophoresis were 72,000 and 48,000. These proteins were produced in the absence of cellular or viral DNA synthesis and have been shown to bind to single stranded DNA but not double stranded DNA or RNA. Similar proteins have been detected in Adenovirus types 2 and 5 infected human and monkey cells (van der Vliet and Levine, 1973; Levine et al., 1974). Even though these DNA-binding proteins are made in very large amounts neither the 72,000 nor the 48,000 MW proteins were detected in mature adenovirus virions (van der Vliet and Levine, 1973). ’ Present address: Institute of Virology, University of Cologne, Cologne, Germany. ‘The Institute of Medical Science, P. 0. Takanawa, Tokyo 108, Japan. 3 Department of Biochemical Sciences, Princeton University, Princeton, New Jersey 08540. Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
14
ADENOVIRUS
TYPE
12 DNA
senwirth et al., 1975). It appears that the 48,000 MW protein is a proteolytic breakdown product of the larger DNA binding protein. These experiments demonstrated that type 5 adenovirus codes for a 72,000 MW single stranded specific DNA binding protein. The adenovirus gene responsible for this protein is identified by the H5ts125 mutant. The properties of this mutant permit the conclusion that the 72,000 MW DNA binding protein is an early adenovirus protein required for viral DNA replication. This paper describes the isolation and characterization of a similar set of single strand specific DNA binding proteins from adenovirus type of 12 infected cells. MATERIALS
AND
METHODS
Cells and virus stocks. Adenovirus type 12 was obtained from H. Shimojo and W. Russell. The virus was grown in human embryo kidney cells (HEK) or in KB cells. The infectivity was assayed by a plaque assay described previously (Shiroki and Shimojo, 1971; Shiroki et al., 1972). Type 12 adenovirus mutants H12tsA275, H12tsB221, and H12tsC295 were isolated as described by Shiroki et al. (1972). These mutants were classified into separate complementation groups and defined as DNA negative mutants by Shiroki and Shimojo (1974). The mutants were named according to Ginsberg et al. (1973). Isolation of DNA binding proteins. African green monkey kidney (AGMK) cells were infected with type 12 adenovirus or the appropriate mutant at multiplicities of l-10 PFU/cell (van der Vliet and Levine, 1973). Proteins were labeled with either [3H]leucine (5 pCi/ml, 30 mCi/mg), [‘Clleucine (1 pCi/ml, 2.2 mCi/mg) (NEN), or 35S-methionine (5 x lo6 cpm/ ml) prepared as described by Crawford and Gesteland (1973) and Anderson, Baum, and Gesteland (1973) in leucine free or methionine free medium. The leucine free medium was supplemented with 1 mg/liter of unlabeled leucine and 2% calf serum, penicillin, and streptomycin as described (van der Vliet and Levine, 1973). Infected cells were labeled from 6 to 24 hr after infection at 37”, 5-22 hr at 40”, and 7-30 hr
BINDING
PROTEINS
15
at 32”. These cells were harvested, after washing the monolayers with phosphate buffered saline, by scraping the cells off of the petri dish surface with a rubber policeman in hypotonic buffer (0.02 A4 Tris-HCl, pH 7.6; 0.01 M NaCl, 1.5 mMMgCl,, and 2 mM mercaptoethanol). This cell extract was frozen at -20” (and in some cases stored) and thawed. The cellular debris was centrifuged for 20 min at 15,000 g. The supernatant containing the DNA binding proteins was adjusted to yield a final concentration of 10% glycerol (10% v/v), 5 mM EDTA, 150 mM NaCl, and 500 pg of bovine serum albumin (buffer A). This extract was frozen, thawed, and centrifuged to remove slight turbidity and then applied to a DNA-cellulose column containing single stranded calf thymus DNA and eluted at a flow rate of 2 ml/hr as described (van der Vliet and Levine, 1973; Alberts and Herrick, 1971). All procedures were performed at 4”. Isolation
of type 12 adenovirus DNA.
Type 12 adenovirus was grown in KB cells and the virus purified as described (Rosenwirth et al., 1974). The DNA was extracted (van der Vliet and Levine, 1973) and shown to be pure by sedimentation through a sucrose gradient. One-half the DNA sample was denatured at alkaline pH by the addition of 20 mM NaOH for 10 min at room temperature. The sample was chilled and neutralized by the addition of 400 mM Tris-HCl. Binding of proteins to DNA. Two hundred microliters of 35S-labeled DNA binding proteins from the 1 M NaCl elution of the DNA cellulose column was dialyzed against buffer and added to 50 ~1 of single stranded (15 pug/ml) or double stranded (20 pug/ml) type 12 adenovirus DNA. This solution was incubated at 4” for 1 hr and then centrifuged through a linear 5-20% sucrose gradient (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl) in an SW 50.1 rotor at 40,000 rpm for 150 min at 4”. SDS-gel electrophoresis. SDS-polyacrylamide gel electrophoresis of the DNA binding proteins in 10% gels and autoradiography of 35S-labeled proteins was performed as described by Anderson et al. (1973). All scintillation counting was done as in van der Vliet and Levine (1973).
16
ROSENWIRTH RESULTS
Isolation of Adenovirus Binding Proteins
ET AL. 6ooor(o.3M
Type
10.5M
il.OM
12 DNA
Infection of AGMK cells with either types 2 or 5 adenovirus results in the production of an adenovirus specific DNA binding protein that is required for viral DNA replication (van der Vliet et al., 1975). Types 2 and 5 belong to the group C human adenovirus (Green, 1970) and are closely related. Type 12 adenovirus on the other hand belongs to the more oncogenic group A human adenovirus (Green, 1970) and is more distantly related to types 2 or 5. It was of some interest then to determine if type 12 adenovirus produced a DNA binding protein analogous to types 5 or 2 and to compare the properties of this binding protein from these two different groups of human adenoviruses. 0 5 10 15 20 25 30 To do this, ten petri dishes of confluent FRACTION NlMBER AGMK cells were infected with type 12 FIG. 1. Elution of adenovirus infected cell proteins adenovirus and labeled with [3H]leucine. A from a single stranded DNA cellulose column. Conflusecond series of ten petri dishes of AGMK ent monolayers of AGMK cells were infected with cells were mock infected and labeled with type 12 adenovirus and the proteins were labeled with [‘%]leucine. Twenty-four hours later both [aH]leucine. A set of mock infected AGMK cells were labeled with [“Cjleucine. At 22 hr after infection sets of cultures were harvested, mixed mixed, and the protogether, and the protein extract was pre- these cultures were harvested, pared as described in Methods. These teins extracted as described in Methods s. The extract labeled proteins were passed over a DNA was applied to a single stranded DNA cellulose column and eluted stepwise with 0.3, 0.5, and 1.0 M cellulose column containing single stranded NaCl. Fractions of 1 ml were collected and 50 ~1 calf thymus DNA. About 95-98s of the la- samples were counted. Top panel: (04) [3H]leubeled proteins pass through this column in tine labeled proteins from adenovirus type 12 infected the 0.15 M salt wash. Proteins were eluted cells. (m- - 4) [%]leucine labeled proteins from from the column in a stepwise fashion (van mock infected cells. Bottom panel: (A-A) ratio of der Vliet and Levine, 1973) employing in- 3H cpmPC cpm. creasing (0.3, 0.5 and 1.0 M NaCl) salt 0.3 M, 0.5 M, the region between 0.5 M and washes. The elution profile of the [3H]leutine infected cell proteins and the [ l*C ]leu- 1.0 M and the 1.0 M NaCl eluates were tine mock infected cell proteins is pre- concentrated and analysed by SDS-polyacgel electrophoresis. The latter sented in Fig. 1. Between the 0.5 and 1.0 rylamide three fractions (0.5, 0.5-1.0, and 1.0 M M elution steps proteins preferentially found in the infected cell extract were de- eluates) were each found to contain the same two proteins preferentially synthetected as indicated by a two- to threefold increase in the 3H-cpm to “C-cpm ratio at sized in adenovirus type 12 infected cells. that molarity. This result is quantitatively Figure 2 presents a representative profile of gel of the [3H]leuand qualitatively similar to that found an SDS-polyacrylamide with type 5 adenovirus infected cell ex- tine labeled (infected cells) and [‘%]leucells) proteins, tracts (van der Vliet and Levine, 1973). To tine labeled (uninfected characterize the proteins eluted from this eluted from the region of a DNA cellulose DNA cellulose column samples from the column -_-_--. ~. hetween -. . 0.5 and 1.0 M NaCl. The
o’
r
3000
ADENOVIRUS
TYPE
T
1
750
E2000 i? I.? 1000
500
250
0
” 5nI
12 DNA
E 0” u s
0
75.0
50.0
25.0
A 1
OO
IO
20
FRACTION
30
40
L-;0
NUMBER
Fm. 2. SDS polyacrylamide gel profile of the infected cell specific DNA binding proteins. A sample from the region of the DNA cellulose column between 0.5-1.0 M NaCl (fractions 17-20) were concentrated (van der Vliet and Levine, 1973) and electrophoresed on an SDS-polyacrylamide gel. Top panel: (04) [3H]leucine labeled proteins from adenovirus type 12 infected cells. (W- - 4) [“Clleucine labeled proteins from mock infected cells. Bottom panel: (A-A) the ratio of sH cpm/“C cpm.
molecular weights of these proteins were 60,000 and 48,000. From experiment to experiment the ratio of the 60,000 and 46,000 MW proteins varied and in some preparations only one or the other protein was present. The 48,000 MW protein(s) has been shown to contain a subset of peptides observed in the 60,000 MW protein and is therefore most likely proteolytic breakdown product of the larger protein (Rosenwirth et al., 1975). The type 5 adenovirus DNA binding proteins have molecular weights of 72,000 and 48,000 (van der Vliet and Levine, 1973) and in this respect the large molecular weight proteins from types 5 and 12 differ. In order to obtain a better comparison of types 2, 5, and 12 adenovirus DNA binding proteins, these proteins were prepared from AGMK cells infected with each of these viruses and labeled with 35S-methionine.
BINDING
PROTEINS
17
The 35S-labeled material that eluted from a single stranded DNA cellulose column between the 0.5-1.0 M NaCl wash was analysed on SDS-polyacrylamide slab gels. 3”S-methionine labeled adenovirus type 2 virion proteins were included as a reference in adjacent wells of the slab gel. To increase the resolution of this technique autoradiographs of these slab gels were prepared. These results are presented in Fig. 3. These results demonstrate that the 72,000 MW protein from adenovirus types 2 and 5 infected cells and the 60,000 MW protein from adenovirus type 12 infected cells were the major components present in this eluate fraction of the DNA cellulose column. None of these proteins correspond to those polypeptides found in the mature adenovirus virion. With the higher resolution of this method the 48,000 MW protein species of adenovirus types 2, 5, or 12 have been resolved into two or more discrete bands. These bands are the proteolytic breakdown products of the 72,000 and the 60,000 MW protein (Rosenwirth et al., 1975). Two proteins labeled with 35S-methionine are present in the 0.5-1.0 M NaCl eluted fraction of the DNA cellulose column when a mock-infected cell extract of AGMK cells was employed (Fig. 3). The fact that mock infected cells contain two proteins of molecular weights 72,000 and 48,000 is consistent with the possibility that the proteins of the same molecular weights in adenovirus types 2 and 5 infected cells are induced cellular proteins. This is not the correct interpretation however because (1) the peptide maps of adenovirus types 2, 5, and 12 DNA binding proteins are not similar to the peptide maps of the mock infected cell 72,000 MW protein (Rosenwirth et al., 1975) and (2) the 72,000 MW protein produced by H5ts125 at 32” is in fact a thermolabile protein for its continued binding to single stranded DNA (van der Vliet et al., 1975). These slab gels of the adenovirus type 12 DNA binding proteins demonstrate that the 60,000 MW protein is not detected in mature adenovirus (type 2) virions and that the 48,000 MW species is in fact two or more resolvable proteins.
18
ROSENWIRTH
ET A
Fw. 3. SDS-polyacrylamide gel electrophoresis of YS-methionine labeled DNA binding proteins from adenovirus types 2, 5, 12, and mock infected cells. Yj-methionine labeled DNA binding proteins from mock infected and types 2, 5, and 12 adenovirus infected AGMK cells were prepared as in Fig. 1. Slab gels of the 1 M labeled purified NaCl eluted proteins from the DNA cellulose column were run along with “S-methionine adenoviurs type 2 virion proteins. From left to right-Adenovirus types 52, 12, and mock infected cell proteins.
The Adenovirus Type 12 DNA Binding Proteins Bind Only to Single Stranded DNA The type 5 adenovirus binding proteins bind to single stranded DNA but not double stranded DNA in solution (van der Vliet and Levine, 1973). To investigate the specificity of the adenovirus type 12 DNA binding proteins, a 35S-methionine labeled mixture of the 60,000 and 48,000 MW proteins from the fraction eluted from a DNA cellulose column between 0.5-1.0 M NaCl (as in Fig. l), was mixed with either double or single stranded 32P-labeled adenovirus type 12 DNA as described in Methods. This mixture was sedimented through a sucrose gradient to fractionate
the DNA, protein, and DNA-protein complexes (Fig. 4). No detectable binding of these proteins was observed to double stranded DNA while about 22% of the labeled protein bound to single stranded DNA. Over 90% of the labeled protein in this preparation is comprised of the 60,000 and 48,000 MW infected cell specific DNA binding proteins (see Fig. 2) and so it may be concluded that one or both of these proteins binds preferentially to single stranded DNA in solution. DNA
Binding Proteins Produced by Adenovirus Type 12 Temperature Sensitive Mutants The type 5 adenovirus DNA binding proteins cannot be detected in extracts of a
ADENOVIRUS
TYPE
12 DNA
1600
7600
6OOr
FRACTION
NUMBER
FIG. 4. Binding of adenovirus type 12 proteins to single stranded adenovirus DNA. 8”S-methionine labeled DNA binding proteins (from the 0.5-1.0 M NaCl region of a DNA cellulose column) were mixed with single or double stranded adenovirus type 12 DNA ( sT-labeled). About 90% of the ‘%-label was in the 60,000 or the 48,000 MW protein (see Fig. 3). The mixture of protein and DNA were then sedimented through a sucrose gradient to determine if the protein bound to DNA. Top panel: Binding protein plus double stranded DNA. Bottom panel: Binding protein plus single stranded DNA. (O----O) 3*Plabeled adenovirus type 12 DNA. (m---m) 35Slabeled protein.
temperature sensitive, DNA negative, mutant (H5ts125) when infection is carried out at 40” (van der Vliet et al., 1975). The binding protein itself appears to be temperature sensitive for continued DNA binding and so the simplest interpretation is that the mutant protein is denatured at the nonpermissive temperature and either fails to bind to DNA (the way in which the protein is isolated and detected) or is degraded by proteases. Adenovirus type 12 temperature sensitive mutants have been placed into three different, DNA negative, complementation groups called A, B, and C (Shiroki and Shimqio, 1972, 1974). A temperature sensitive mutant from each
BINDING
PROTEINS
19
group was tested for its ability to produce a detectable adenovirus type 12 DNA binding protein. Confluent monolayers of AGMK cells were infected with H12tsA275, H12tsB221 and H12tsC295 at 40” and labeled with [3H]leucine. An equal number of cultures were mock infected and labeled with (‘%]leucine at 40”. Twenty-two hours after infection the mutant and the mock infected cultures were harvested. Each of these three mutant cultures ( 3H-labeled) was mixed with the ‘“C-labeled mock infected cells. Protein extracts were prepared and passed over the DNA cellulose columns as described. In each case the fractions between the 0.5-1.0 M NaCl eluate were analysed ,on SDS-polyacrylamide gels to detect the DNA binding proteins. These results are presented in Fig. 5. The DNA binding proteins were detected in normal quantities in extracts of H12tsB221 and H12tsC295 infected cells grown at 40”. Little or no DNA binding proteins could be detected in H12tsA275 infected cells grown at 40”. A second experiment was performed to determine if the H12tsA275 mutant could produce these DNA binding proteins at 32” (permissive temperature). In this case, a set of four AGMK cell cultures were infected with adenovirus type 12 wild-type, H12tsA275, H12tsB221, and H12tsC295. One-half of each of these four sets was incubated at 32” and labeled with [3H]leutine while the other half was kept at 40” and labeled with [‘%]leucine. These cells were harvested and mixed in the following combinations: (1) H12 wt, 32 and 40”; (2) H12tsA275, 32 and 40”; (3) H12tsB221, 32 and 40”; and (4) H12tsC295, 32 and 40”. Protein extracts were prepared and chromatographed through a DNA cellulose column as usual. The proteins eluting between the 0.5-1.0 M NaCl salt washes were then analysed by SDS-polyacrylamide gel electrophoresis. These results are presented in Fig. 6. Adenovirus type l:! wildtype, H12tsB221, and H12tsC295 all produced the DNA binding proteins at 32 and 40”. The H12tsA275 mutant produced these DNA binding proteins at 32” but there was a great reduction in the detectable levels of 60,000 and 48,000 MW proteins made at 40” by this mutant.
20
ROSENWIRTH 2OOOr
7500
DISCUSSION
2 B 1 T 26.0 ,I
0IQOOOr
I
C 6000 -
E
6000-
-1500
" 2000-
100.0
r-
I
L E
4000 k I" R2000 0 100.0
300
E 0”
5oos j; C)
75.0 0 f SQO 3 25.0 0
m
20 FRACTION
FIG.
5. Production
I---r--
30
40
50
NUMBER
of the adenovirus
type
ET AL.
12 DNA
Infection of AGMK cells with type 12 adenovirus results in the production of two DNA binding proteins that are preferentially found in the infected cell extracts. The molecular weights of these proteins are 60,000 and 48,000. The smaller molecular weight protein can be resolved into two or more species by autoradiography of slab gel SDS-polyacrylamide electrophoresis. Analysis of the peptide maps of the 60,000 and 48,000 MW proteins indicates that the smaller proteins contain a subset of peptides found in the larger protein (Rosenwirth et al., 1975) and are likely proteolytic breakdown products of the 60,000 MW DNA binding protein. This does not eliminate the possibility that the smaller proteins are functionally significant. The adenovirus type 12 temperature sensitive mutants can be placed into three early, DNA negative, complementation groups (Shiroki and Shimojo, 1974). Adenovirus type 12 wild-type, H12tsB221, and H12tsC295 all produce these DNA binding proteins at 40” (nonpermissive temperature) and 32” (permissive temperature). H12tsA275 infected cells produce detectable binding proteins at 32” but not when this mutant is grown at 40”. The observabinding proteins in HlZtsA, H12tsB, and H12tsC infected cells at 40”. Confluent cultures of AGMK cells were infected with H12tsA275, H12tsB221 and H12tsC295 and incubated at 40”. Similar cultures were mock infected and kept at 40”. The mutant infected cultures were labeled with [3H]leucine while the mock infected cultures were labeled with [l’C]leutine. These cultures were harvested, each mutant culture was mixed with mock infected cells and the proteins were extracted as described in Methods. These extracts were fractionated on three separate DNA cellulose columns (as in Fig. 1) and the 1 M NaCl eluted proteins were electrophoresed on polyacrylamide gels. A-H12ts.4275 infected cells labeled with [3H]leucine (04) mixed with mock infected cells labeled with [‘“Clleucine (m---m). B-The ratio of 3H cpm/“C cpm (A-A). C-H12tsB221 infected cells labeled with [3H]leucine (O--O) mixed with mock infected cells labeled with [l’C]leutine (W---m). D-The ratio of 3H cpm/*‘C cpm (A-A). E-H12tsC295 infected cells labeled with IJH]leucine (0 ---O) mixed with mock infected cells labeled with [“Clleucine (m- - mm). F-Ratio of W cpm/“C cpm (A-A).
ADENOVIRUS
TYPE
4000
2000 A
3000 E ”
15Ca L
2000
i 0
1000
,’
Y 1000
500 r
0
0
12.0
B
0 \ RI 4.0 f
6.0 IA
01
I
1
I
1
6000
4000
6ooo
3000
E 04oa 5
2000 f
2000
E e ”
1000
0
0
6.0r
f
2.J+!
@,ooo
6000
g 6000
4000
g f
4000
2000
0 ”
0
6.0 -
5
H 2.0 0 0
I IO
1 20 FRACTION
I 30 NUMBER
I 40
50
:
12 DNA
BINDING
PROTEINS
21
tion that DNA binding proteins are made with HlBtsB or H12tsC mutants grown at 40”, demonstrates that these binding proteins are produced in the absence of viral DNA synthesis. Neither of these mutants synthesize viral DNA at the nonpermissive temperature (Shiroki and Shimojo, 1974). This result is expected because adenovirus late proteins are not produced in AGMK cells (White, Scharff, and Maizel, 1969). The fact that H12tsA275 infected cells at 40” do not contain detectable levels of the 60,000 or 48,000 MW DNA binding proteins is consistent with the possibility that the H12tsA adenovirus gene induces or codes for this set of proteins. A similar observation has also been made with the H5ts125 mutant of type 5 adenovirus (van der Vliet et al., 1975). This mutant does not produce detectable DNA binding proteins at 40” but synthesizes these proteins at 32”. In this case it has been shown that the adenovirus type 5 DNA binding proteins made by the H5ts125 mutant are themselves thermolabile for DNA binding. It has been concluded that type 5 adenovirus codes for this DNA binding protein FIG. 6. Production of adenovirus type 12 DNA binding proteins by wildtype virus, H12tsA, H12tsA, H12tsB. and H12tsC at 32 and 40”. Confluent cultures of AGMK cells were infected with type 12 adenovirus, H12tsA275, H12tsB221, and H12tsC295 at 40” and labeled with [“Clleucine or at 32” and labeled with [3H]leucine. These cells were harvested (at times indicated in Methods), and each mutant culture at 32 and 40” mixed, protein extracts prepared, and chromatographed over DNA cellulose columns. The 0.5 -1 .O M NaCl region of the DNA cellulose column was then electrophoresed in SDS-polyacrylamide gels. A-H12tsA275 infected cells at 40” labeled with [“Clleucine (W---B) mixed with H12tsA275 infected cells at 32” and labeled with [3H]leucine (O---O). B-Ratio of 3H cpm/“C cpm (A-A). C-H12tsB221 infected cells at 40” labeled with [“Clleucine (W-B) mixed with H12tsB221 infected cells at 32” labeled with [Hlleucine (O----O). D-Ratio of 3H cpm/“C cpm (A-A). E-HlPtsC295 infected cells at 40” labeled with [“Clleucine (W - -a) mixed with H12tsC295 infected cells.at 32” labeled with [3H]leucine (O---O). F-Ratio of 3H cpm/“C cpm (A-A). G-Adenovinrs type 12 wild-type virus infected cells at 40° labeled with [Wlleucine (W- - -m) mixed with wild-type virus infected cells at 32” labeled with [3H]leucine (O----O). H-Ratio of sH cpm/“C cpm (A-A).
22
ROSENWIRTH
(van der Vliet et al., 1975). It appears likely that adenovirus type 12 codes for a similar functional entity. If that is correct, then the H12tsA275 mutant at 40” either produces a denatured protein that is degraded by proteases or a protein that no longer binds to single stranded DNA in a DNA cellulose column. It is of some interest to compare the DNA binding proteins made by types 2, 5, and 12 adenovirus. Types 2 and 5 are closely related viruses sharing a number of physical, immunological, chemical, and biological properties (Green, 1970). Type 12 virus on the other hand contains a smaller molecular weight DNA of lower G+C content and is highly oncogenic when compared to adenoviruses types 2 and 5 (Green, 1970; Tooze, 1973). The molecular weight of the DNA binding proteins produced by both types 2 and 5 is 72,000 as compared with 60,000 MW of type 12 virus. The types 2 and 5 proteins have very similar peptide maps which are distinctly different from the peptide map of the type 12 60,000 MW protein (Rosenwirth et al., 1975). Both the 72,000 and the 60,000 MW proteins bind to single stranded DNA from a variety of sources, but not to double stranded DNA or RNA (van der Vliet and Levine, 1973). The 72,000 MW protein (from type 5) and the 60,000 MW protein (from type 12) are frequently accompanied by a collection of discrete proteolytic breakdown products with an average molecular weight of 48,000 independent of virus type. The temperature sensitive mutant H5ts125 does not produce a 72,000 MW DNA binding protein at 40”. The protein detected at 32” is thermolabile for continued DNA binding when compared to the type 5 wild-type protein. Analogously the type 12 mutant H12tsA275 does not produce a detectable 60,000 MW protein at 40” but does when the virus infection is performed at 32”. The phenotype of each of these independently derived mutants is that they fail to synthesize viral DNA at the nonpermissive temperature. It is likely then that adenoviruses produce a single strand specific DNA binding protein that is required for viral DNA replication. The
ET AL.
replicative intermediate of adenovirus contains large regions of single stranded DNA and these proteins may well function at those sites. ACKNOWLEDGMENTS
The authors thank A. K. Teresky, C. Kaminski, and S. Wyckoff for technical assistance. One of us (B.R.) was supported by the Deutsche Forschungsgemeinschaft postdoctoral fellowship. This research was supported by a Grant from the American Cancer Society E-591 and NC1 CA11049-07. REFERENCES
ALBERTS, B. M. and HERRICK, G. (1971). DNA cellulose chromatography. In “Methods in Enzymology” (L. Grossman and K. Moldave, 21, p. 198, Academic Press. ANDERSON,C. W., BAUM, P. R., and GESTELAND,R. F. (1973). The processing of adenovirus 2 induced proteins. J. Viral. 12, 241-252. CRAWFORD,L. V. and GESTELAND, R. F. (1973). Synthesis of polyoma proteins in uitro. J. Mol. Biol.. 74, 627-634.
GINSBERG,H. S., WILLIAMS, J. F., DOERFI.ER,W. H., and SHIMOJO,H. (1973). Proposed nomenclature for mutants of adenoviruses. J. Virol. 12, 663-664. GREEN, M. (1970). Oncogenic Viruses. Ann. Reu. Biochem.
39, 701-756.
LEVINE, A. J., VAN DER VLIET. P. C., ROSENWIRTH,B., RABEK, J., FRENKEL, G., and ENSINGER,M. (1975). Adenovirus infected cell specific DNA-binding proteins. Cold Spring Harbor Symp. Quant. Biol., 559466.
ROSENWIRTH.B., TJIA, S., WESTPHAL. M., and DOERFLER, W. (1974). Incomplete particles of adenovirus. II. Kinetics of formation and polypeptide composition of adenovirus type 2. Virology 60, 431-437. ROSENWIRTH.B., ANDERSON,C. W., LEVINE, A. J., and GESTELAND, R. (19751. In preparation. SHIROKI, K. and SHIMOJO, H. (1971). Transformation of green monkey kidney cells by SV40 genome: The establishment of transformed cell lines and the replication of human adenovirns and SV40 in transformed cells. Virology 45, 163-171. SHIROKI, K., IRISAWA, J., and SHIMOJO, H. (19721. Isolation and a preliminary characterization of temperature-sensitive mutants of adenovirus 12. Virology 49, 1-11. SHIROKI, K. and SHIMOJO, H. (1974). Analysis of adenovirus 12 temperature sensitive mutants defective in viral DNA replication. Cold Spring Harbor Symp. Quant. Biol., in press. TOOZE, J. (1973). The molecular biology of tumor viruses, Cold Springs, Harbor Lab.
ADENOVIRUS
TYPE 12 DNA BINDING
DER EB, A. (1973). Intermediates in Type 5 adenovirus DNA replication. Virology 51, 11-23. 11-23. VANDERVLIET, P. C., LEVINE, A. J., (1973). DNA-binding proteins specific for cells infected by adenovirus. Nature New Biol. 246, 170-174. VAN
PROTEINS
23
P. C., LEVINE, A. J., ENSINGER,M., and GINSBERG,H. S. (1975). Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovirus defective in viral DNA synthesis. J. Viral. 15, 348-354.
VAN DER VLIET,
67,14-23
(1975)
Isolation
and Characterization
of Adenovirus
DNA Binding BRIGITTE
ROSENWIRTH,’
KAZUKO HIROTO Accepted
Type 12
Proteins
SHIROKI,2 SHIMOJ02 April
ARNOLD
J. LEVINE,3
AND
3, 1975
Infection of African green monkey kidney cells with type 12 adenovirus results in the production of two single strand specific DNA binding proteins. The molecular weights of these proteins are 60,000 and 48,ooO. Both proteins are synthesized in the absence of viral DNA replication and neither protein appears to correspond to any polypeptide detected in mature adenovirus virions. Temperature sensitive mutants from three different early, DNA negative, complementation groups (tsA, tsB, and tsC) have been tested for the production of these proteins at permissive and nonpermissive temperatures. Mutants of the tsB and tsC classes produce both DNA binding proteins at 32 and at 40”. TsA mutants produce both proteins at 32” but neither DNA-binding protein can be detected when these mutants are grown at 40”. The properties of adenovirus DNA binding proteins produced by types 2,5, and 12 adenoviruses are compared.
Because of the specificity of binding to single stranded DNA and the presence of large single stranded regions of DNA in replicating adenovirus DNA molecules (Sussenbach, van der Vliet, Ellens, and Janz, 1972; van der Eb, 1973) it was felt that the function of these proteins might be related to adenovirus DNA replication. Two different, DNA negative, temperature sensitive mutants of type 5 adenovirus, H5ts36 and H5ts125, were examined for the production of these proteins at nonpermissive temperature. H5ts36 infected cells produced normal levels of both DNA binding proteins at 39.5”, while H5ts125 infected cells at 39.5” contained a little or none of the 72,000 and 48,000 MW proteins (van der Vliet, Levine, Ensinger, and Ginsberg, 1975). Both of these proteins obtained from HMs125 infected cells at 32” (permissive temperature) were thermolabile in their continued binding to single stranded DNA when compared with the adenovirus wild-type 5 proteins. Recent peptide maps of the 72,000 and 48,000 MW proteins have shown that the smaller protein contains a subset of peptides all of which are found in the larger protein (Ro-
INTRODUCTION
African green monkey kidney cells infected with type 5 adenovirus synthesize two proteins, found preferentially in infected cells, that bind to single stranded DNA (van der Vliet and Levine, 1973). The molecular weights of these proteins, as determined by SDS-polyacrylamide gel electrophoresis were 72,000 and 48,000. These proteins were produced in the absence of cellular or viral DNA synthesis and have been shown to bind to single stranded DNA but not double stranded DNA or RNA. Similar proteins have been detected in Adenovirus types 2 and 5 infected human and monkey cells (van der Vliet and Levine, 1973; Levine et al., 1974). Even though these DNA-binding proteins are made in very large amounts neither the 72,000 nor the 48,000 MW proteins were detected in mature adenovirus virions (van der Vliet and Levine, 1973). ’ Present address: Institute of Virology, University of Cologne, Cologne, Germany. ‘The Institute of Medical Science, P. 0. Takanawa, Tokyo 108, Japan. 3 Department of Biochemical Sciences, Princeton University, Princeton, New Jersey 08540. Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
14
ADENOVIRUS
TYPE
12 DNA
senwirth et al., 1975). It appears that the 48,000 MW protein is a proteolytic breakdown product of the larger DNA binding protein. These experiments demonstrated that type 5 adenovirus codes for a 72,000 MW single stranded specific DNA binding protein. The adenovirus gene responsible for this protein is identified by the H5ts125 mutant. The properties of this mutant permit the conclusion that the 72,000 MW DNA binding protein is an early adenovirus protein required for viral DNA replication. This paper describes the isolation and characterization of a similar set of single strand specific DNA binding proteins from adenovirus type of 12 infected cells. MATERIALS
AND
METHODS
Cells and virus stocks. Adenovirus type 12 was obtained from H. Shimojo and W. Russell. The virus was grown in human embryo kidney cells (HEK) or in KB cells. The infectivity was assayed by a plaque assay described previously (Shiroki and Shimojo, 1971; Shiroki et al., 1972). Type 12 adenovirus mutants H12tsA275, H12tsB221, and H12tsC295 were isolated as described by Shiroki et al. (1972). These mutants were classified into separate complementation groups and defined as DNA negative mutants by Shiroki and Shimojo (1974). The mutants were named according to Ginsberg et al. (1973). Isolation of DNA binding proteins. African green monkey kidney (AGMK) cells were infected with type 12 adenovirus or the appropriate mutant at multiplicities of l-10 PFU/cell (van der Vliet and Levine, 1973). Proteins were labeled with either [3H]leucine (5 pCi/ml, 30 mCi/mg), [‘Clleucine (1 pCi/ml, 2.2 mCi/mg) (NEN), or 35S-methionine (5 x lo6 cpm/ ml) prepared as described by Crawford and Gesteland (1973) and Anderson, Baum, and Gesteland (1973) in leucine free or methionine free medium. The leucine free medium was supplemented with 1 mg/liter of unlabeled leucine and 2% calf serum, penicillin, and streptomycin as described (van der Vliet and Levine, 1973). Infected cells were labeled from 6 to 24 hr after infection at 37”, 5-22 hr at 40”, and 7-30 hr
BINDING
PROTEINS
15
at 32”. These cells were harvested, after washing the monolayers with phosphate buffered saline, by scraping the cells off of the petri dish surface with a rubber policeman in hypotonic buffer (0.02 A4 Tris-HCl, pH 7.6; 0.01 M NaCl, 1.5 mMMgCl,, and 2 mM mercaptoethanol). This cell extract was frozen at -20” (and in some cases stored) and thawed. The cellular debris was centrifuged for 20 min at 15,000 g. The supernatant containing the DNA binding proteins was adjusted to yield a final concentration of 10% glycerol (10% v/v), 5 mM EDTA, 150 mM NaCl, and 500 pg of bovine serum albumin (buffer A). This extract was frozen, thawed, and centrifuged to remove slight turbidity and then applied to a DNA-cellulose column containing single stranded calf thymus DNA and eluted at a flow rate of 2 ml/hr as described (van der Vliet and Levine, 1973; Alberts and Herrick, 1971). All procedures were performed at 4”. Isolation
of type 12 adenovirus DNA.
Type 12 adenovirus was grown in KB cells and the virus purified as described (Rosenwirth et al., 1974). The DNA was extracted (van der Vliet and Levine, 1973) and shown to be pure by sedimentation through a sucrose gradient. One-half the DNA sample was denatured at alkaline pH by the addition of 20 mM NaOH for 10 min at room temperature. The sample was chilled and neutralized by the addition of 400 mM Tris-HCl. Binding of proteins to DNA. Two hundred microliters of 35S-labeled DNA binding proteins from the 1 M NaCl elution of the DNA cellulose column was dialyzed against buffer and added to 50 ~1 of single stranded (15 pug/ml) or double stranded (20 pug/ml) type 12 adenovirus DNA. This solution was incubated at 4” for 1 hr and then centrifuged through a linear 5-20% sucrose gradient (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl) in an SW 50.1 rotor at 40,000 rpm for 150 min at 4”. SDS-gel electrophoresis. SDS-polyacrylamide gel electrophoresis of the DNA binding proteins in 10% gels and autoradiography of 35S-labeled proteins was performed as described by Anderson et al. (1973). All scintillation counting was done as in van der Vliet and Levine (1973).
16
ROSENWIRTH RESULTS
Isolation of Adenovirus Binding Proteins
ET AL. 6ooor(o.3M
Type
10.5M
il.OM
12 DNA
Infection of AGMK cells with either types 2 or 5 adenovirus results in the production of an adenovirus specific DNA binding protein that is required for viral DNA replication (van der Vliet et al., 1975). Types 2 and 5 belong to the group C human adenovirus (Green, 1970) and are closely related. Type 12 adenovirus on the other hand belongs to the more oncogenic group A human adenovirus (Green, 1970) and is more distantly related to types 2 or 5. It was of some interest then to determine if type 12 adenovirus produced a DNA binding protein analogous to types 5 or 2 and to compare the properties of this binding protein from these two different groups of human adenoviruses. 0 5 10 15 20 25 30 To do this, ten petri dishes of confluent FRACTION NlMBER AGMK cells were infected with type 12 FIG. 1. Elution of adenovirus infected cell proteins adenovirus and labeled with [3H]leucine. A from a single stranded DNA cellulose column. Conflusecond series of ten petri dishes of AGMK ent monolayers of AGMK cells were infected with cells were mock infected and labeled with type 12 adenovirus and the proteins were labeled with [‘%]leucine. Twenty-four hours later both [aH]leucine. A set of mock infected AGMK cells were labeled with [“Cjleucine. At 22 hr after infection sets of cultures were harvested, mixed mixed, and the protogether, and the protein extract was pre- these cultures were harvested, pared as described in Methods. These teins extracted as described in Methods s. The extract labeled proteins were passed over a DNA was applied to a single stranded DNA cellulose column and eluted stepwise with 0.3, 0.5, and 1.0 M cellulose column containing single stranded NaCl. Fractions of 1 ml were collected and 50 ~1 calf thymus DNA. About 95-98s of the la- samples were counted. Top panel: (04) [3H]leubeled proteins pass through this column in tine labeled proteins from adenovirus type 12 infected the 0.15 M salt wash. Proteins were eluted cells. (m- - 4) [%]leucine labeled proteins from from the column in a stepwise fashion (van mock infected cells. Bottom panel: (A-A) ratio of der Vliet and Levine, 1973) employing in- 3H cpmPC cpm. creasing (0.3, 0.5 and 1.0 M NaCl) salt 0.3 M, 0.5 M, the region between 0.5 M and washes. The elution profile of the [3H]leutine infected cell proteins and the [ l*C ]leu- 1.0 M and the 1.0 M NaCl eluates were tine mock infected cell proteins is pre- concentrated and analysed by SDS-polyacgel electrophoresis. The latter sented in Fig. 1. Between the 0.5 and 1.0 rylamide three fractions (0.5, 0.5-1.0, and 1.0 M M elution steps proteins preferentially found in the infected cell extract were de- eluates) were each found to contain the same two proteins preferentially synthetected as indicated by a two- to threefold increase in the 3H-cpm to “C-cpm ratio at sized in adenovirus type 12 infected cells. that molarity. This result is quantitatively Figure 2 presents a representative profile of gel of the [3H]leuand qualitatively similar to that found an SDS-polyacrylamide with type 5 adenovirus infected cell ex- tine labeled (infected cells) and [‘%]leucells) proteins, tracts (van der Vliet and Levine, 1973). To tine labeled (uninfected characterize the proteins eluted from this eluted from the region of a DNA cellulose DNA cellulose column samples from the column -_-_--. ~. hetween -. . 0.5 and 1.0 M NaCl. The
o’
r
3000
ADENOVIRUS
TYPE
T
1
750
E2000 i? I.? 1000
500
250
0
” 5nI
12 DNA
E 0” u s
0
75.0
50.0
25.0
A 1
OO
IO
20
FRACTION
30
40
L-;0
NUMBER
Fm. 2. SDS polyacrylamide gel profile of the infected cell specific DNA binding proteins. A sample from the region of the DNA cellulose column between 0.5-1.0 M NaCl (fractions 17-20) were concentrated (van der Vliet and Levine, 1973) and electrophoresed on an SDS-polyacrylamide gel. Top panel: (04) [3H]leucine labeled proteins from adenovirus type 12 infected cells. (W- - 4) [“Clleucine labeled proteins from mock infected cells. Bottom panel: (A-A) the ratio of sH cpm/“C cpm.
molecular weights of these proteins were 60,000 and 48,000. From experiment to experiment the ratio of the 60,000 and 46,000 MW proteins varied and in some preparations only one or the other protein was present. The 48,000 MW protein(s) has been shown to contain a subset of peptides observed in the 60,000 MW protein and is therefore most likely proteolytic breakdown product of the larger protein (Rosenwirth et al., 1975). The type 5 adenovirus DNA binding proteins have molecular weights of 72,000 and 48,000 (van der Vliet and Levine, 1973) and in this respect the large molecular weight proteins from types 5 and 12 differ. In order to obtain a better comparison of types 2, 5, and 12 adenovirus DNA binding proteins, these proteins were prepared from AGMK cells infected with each of these viruses and labeled with 35S-methionine.
BINDING
PROTEINS
17
The 35S-labeled material that eluted from a single stranded DNA cellulose column between the 0.5-1.0 M NaCl wash was analysed on SDS-polyacrylamide slab gels. 3”S-methionine labeled adenovirus type 2 virion proteins were included as a reference in adjacent wells of the slab gel. To increase the resolution of this technique autoradiographs of these slab gels were prepared. These results are presented in Fig. 3. These results demonstrate that the 72,000 MW protein from adenovirus types 2 and 5 infected cells and the 60,000 MW protein from adenovirus type 12 infected cells were the major components present in this eluate fraction of the DNA cellulose column. None of these proteins correspond to those polypeptides found in the mature adenovirus virion. With the higher resolution of this method the 48,000 MW protein species of adenovirus types 2, 5, or 12 have been resolved into two or more discrete bands. These bands are the proteolytic breakdown products of the 72,000 and the 60,000 MW protein (Rosenwirth et al., 1975). Two proteins labeled with 35S-methionine are present in the 0.5-1.0 M NaCl eluted fraction of the DNA cellulose column when a mock-infected cell extract of AGMK cells was employed (Fig. 3). The fact that mock infected cells contain two proteins of molecular weights 72,000 and 48,000 is consistent with the possibility that the proteins of the same molecular weights in adenovirus types 2 and 5 infected cells are induced cellular proteins. This is not the correct interpretation however because (1) the peptide maps of adenovirus types 2, 5, and 12 DNA binding proteins are not similar to the peptide maps of the mock infected cell 72,000 MW protein (Rosenwirth et al., 1975) and (2) the 72,000 MW protein produced by H5ts125 at 32” is in fact a thermolabile protein for its continued binding to single stranded DNA (van der Vliet et al., 1975). These slab gels of the adenovirus type 12 DNA binding proteins demonstrate that the 60,000 MW protein is not detected in mature adenovirus (type 2) virions and that the 48,000 MW species is in fact two or more resolvable proteins.
18
ROSENWIRTH
ET A
Fw. 3. SDS-polyacrylamide gel electrophoresis of YS-methionine labeled DNA binding proteins from adenovirus types 2, 5, 12, and mock infected cells. Yj-methionine labeled DNA binding proteins from mock infected and types 2, 5, and 12 adenovirus infected AGMK cells were prepared as in Fig. 1. Slab gels of the 1 M labeled purified NaCl eluted proteins from the DNA cellulose column were run along with “S-methionine adenoviurs type 2 virion proteins. From left to right-Adenovirus types 52, 12, and mock infected cell proteins.
The Adenovirus Type 12 DNA Binding Proteins Bind Only to Single Stranded DNA The type 5 adenovirus binding proteins bind to single stranded DNA but not double stranded DNA in solution (van der Vliet and Levine, 1973). To investigate the specificity of the adenovirus type 12 DNA binding proteins, a 35S-methionine labeled mixture of the 60,000 and 48,000 MW proteins from the fraction eluted from a DNA cellulose column between 0.5-1.0 M NaCl (as in Fig. l), was mixed with either double or single stranded 32P-labeled adenovirus type 12 DNA as described in Methods. This mixture was sedimented through a sucrose gradient to fractionate
the DNA, protein, and DNA-protein complexes (Fig. 4). No detectable binding of these proteins was observed to double stranded DNA while about 22% of the labeled protein bound to single stranded DNA. Over 90% of the labeled protein in this preparation is comprised of the 60,000 and 48,000 MW infected cell specific DNA binding proteins (see Fig. 2) and so it may be concluded that one or both of these proteins binds preferentially to single stranded DNA in solution. DNA
Binding Proteins Produced by Adenovirus Type 12 Temperature Sensitive Mutants The type 5 adenovirus DNA binding proteins cannot be detected in extracts of a
ADENOVIRUS
TYPE
12 DNA
1600
7600
6OOr
FRACTION
NUMBER
FIG. 4. Binding of adenovirus type 12 proteins to single stranded adenovirus DNA. 8”S-methionine labeled DNA binding proteins (from the 0.5-1.0 M NaCl region of a DNA cellulose column) were mixed with single or double stranded adenovirus type 12 DNA ( sT-labeled). About 90% of the ‘%-label was in the 60,000 or the 48,000 MW protein (see Fig. 3). The mixture of protein and DNA were then sedimented through a sucrose gradient to determine if the protein bound to DNA. Top panel: Binding protein plus double stranded DNA. Bottom panel: Binding protein plus single stranded DNA. (O----O) 3*Plabeled adenovirus type 12 DNA. (m---m) 35Slabeled protein.
temperature sensitive, DNA negative, mutant (H5ts125) when infection is carried out at 40” (van der Vliet et al., 1975). The binding protein itself appears to be temperature sensitive for continued DNA binding and so the simplest interpretation is that the mutant protein is denatured at the nonpermissive temperature and either fails to bind to DNA (the way in which the protein is isolated and detected) or is degraded by proteases. Adenovirus type 12 temperature sensitive mutants have been placed into three different, DNA negative, complementation groups called A, B, and C (Shiroki and Shimqio, 1972, 1974). A temperature sensitive mutant from each
BINDING
PROTEINS
19
group was tested for its ability to produce a detectable adenovirus type 12 DNA binding protein. Confluent monolayers of AGMK cells were infected with H12tsA275, H12tsB221 and H12tsC295 at 40” and labeled with [3H]leucine. An equal number of cultures were mock infected and labeled with (‘%]leucine at 40”. Twenty-two hours after infection the mutant and the mock infected cultures were harvested. Each of these three mutant cultures ( 3H-labeled) was mixed with the ‘“C-labeled mock infected cells. Protein extracts were prepared and passed over the DNA cellulose columns as described. In each case the fractions between the 0.5-1.0 M NaCl eluate were analysed ,on SDS-polyacrylamide gels to detect the DNA binding proteins. These results are presented in Fig. 5. The DNA binding proteins were detected in normal quantities in extracts of H12tsB221 and H12tsC295 infected cells grown at 40”. Little or no DNA binding proteins could be detected in H12tsA275 infected cells grown at 40”. A second experiment was performed to determine if the H12tsA275 mutant could produce these DNA binding proteins at 32” (permissive temperature). In this case, a set of four AGMK cell cultures were infected with adenovirus type 12 wild-type, H12tsA275, H12tsB221, and H12tsC295. One-half of each of these four sets was incubated at 32” and labeled with [3H]leutine while the other half was kept at 40” and labeled with [‘%]leucine. These cells were harvested and mixed in the following combinations: (1) H12 wt, 32 and 40”; (2) H12tsA275, 32 and 40”; (3) H12tsB221, 32 and 40”; and (4) H12tsC295, 32 and 40”. Protein extracts were prepared and chromatographed through a DNA cellulose column as usual. The proteins eluting between the 0.5-1.0 M NaCl salt washes were then analysed by SDS-polyacrylamide gel electrophoresis. These results are presented in Fig. 6. Adenovirus type l:! wildtype, H12tsB221, and H12tsC295 all produced the DNA binding proteins at 32 and 40”. The H12tsA275 mutant produced these DNA binding proteins at 32” but there was a great reduction in the detectable levels of 60,000 and 48,000 MW proteins made at 40” by this mutant.
20
ROSENWIRTH 2OOOr
7500
DISCUSSION
2 B 1 T 26.0 ,I
0IQOOOr
I
C 6000 -
E
6000-
-1500
" 2000-
100.0
r-
I
L E
4000 k I" R2000 0 100.0
300
E 0”
5oos j; C)
75.0 0 f SQO 3 25.0 0
m
20 FRACTION
FIG.
5. Production
I---r--
30
40
50
NUMBER
of the adenovirus
type
ET AL.
12 DNA
Infection of AGMK cells with type 12 adenovirus results in the production of two DNA binding proteins that are preferentially found in the infected cell extracts. The molecular weights of these proteins are 60,000 and 48,000. The smaller molecular weight protein can be resolved into two or more species by autoradiography of slab gel SDS-polyacrylamide electrophoresis. Analysis of the peptide maps of the 60,000 and 48,000 MW proteins indicates that the smaller proteins contain a subset of peptides found in the larger protein (Rosenwirth et al., 1975) and are likely proteolytic breakdown products of the 60,000 MW DNA binding protein. This does not eliminate the possibility that the smaller proteins are functionally significant. The adenovirus type 12 temperature sensitive mutants can be placed into three early, DNA negative, complementation groups (Shiroki and Shimojo, 1974). Adenovirus type 12 wild-type, H12tsB221, and H12tsC295 all produce these DNA binding proteins at 40” (nonpermissive temperature) and 32” (permissive temperature). H12tsA275 infected cells produce detectable binding proteins at 32” but not when this mutant is grown at 40”. The observabinding proteins in HlZtsA, H12tsB, and H12tsC infected cells at 40”. Confluent cultures of AGMK cells were infected with H12tsA275, H12tsB221 and H12tsC295 and incubated at 40”. Similar cultures were mock infected and kept at 40”. The mutant infected cultures were labeled with [3H]leucine while the mock infected cultures were labeled with [l’C]leutine. These cultures were harvested, each mutant culture was mixed with mock infected cells and the proteins were extracted as described in Methods. These extracts were fractionated on three separate DNA cellulose columns (as in Fig. 1) and the 1 M NaCl eluted proteins were electrophoresed on polyacrylamide gels. A-H12ts.4275 infected cells labeled with [3H]leucine (04) mixed with mock infected cells labeled with [‘“Clleucine (m---m). B-The ratio of 3H cpm/“C cpm (A-A). C-H12tsB221 infected cells labeled with [3H]leucine (O--O) mixed with mock infected cells labeled with [l’C]leutine (W---m). D-The ratio of 3H cpm/*‘C cpm (A-A). E-H12tsC295 infected cells labeled with IJH]leucine (0 ---O) mixed with mock infected cells labeled with [“Clleucine (m- - mm). F-Ratio of W cpm/“C cpm (A-A).
ADENOVIRUS
TYPE
4000
2000 A
3000 E ”
15Ca L
2000
i 0
1000
,’
Y 1000
500 r
0
0
12.0
B
0 \ RI 4.0 f
6.0 IA
01
I
1
I
1
6000
4000
6ooo
3000
E 04oa 5
2000 f
2000
E e ”
1000
0
0
6.0r
f
2.J+!
@,ooo
6000
g 6000
4000
g f
4000
2000
0 ”
0
6.0 -
5
H 2.0 0 0
I IO
1 20 FRACTION
I 30 NUMBER
I 40
50
:
12 DNA
BINDING
PROTEINS
21
tion that DNA binding proteins are made with HlBtsB or H12tsC mutants grown at 40”, demonstrates that these binding proteins are produced in the absence of viral DNA synthesis. Neither of these mutants synthesize viral DNA at the nonpermissive temperature (Shiroki and Shimojo, 1974). This result is expected because adenovirus late proteins are not produced in AGMK cells (White, Scharff, and Maizel, 1969). The fact that H12tsA275 infected cells at 40” do not contain detectable levels of the 60,000 or 48,000 MW DNA binding proteins is consistent with the possibility that the H12tsA adenovirus gene induces or codes for this set of proteins. A similar observation has also been made with the H5ts125 mutant of type 5 adenovirus (van der Vliet et al., 1975). This mutant does not produce detectable DNA binding proteins at 40” but synthesizes these proteins at 32”. In this case it has been shown that the adenovirus type 5 DNA binding proteins made by the H5ts125 mutant are themselves thermolabile for DNA binding. It has been concluded that type 5 adenovirus codes for this DNA binding protein FIG. 6. Production of adenovirus type 12 DNA binding proteins by wildtype virus, H12tsA, H12tsA, H12tsB. and H12tsC at 32 and 40”. Confluent cultures of AGMK cells were infected with type 12 adenovirus, H12tsA275, H12tsB221, and H12tsC295 at 40” and labeled with [“Clleucine or at 32” and labeled with [3H]leucine. These cells were harvested (at times indicated in Methods), and each mutant culture at 32 and 40” mixed, protein extracts prepared, and chromatographed over DNA cellulose columns. The 0.5 -1 .O M NaCl region of the DNA cellulose column was then electrophoresed in SDS-polyacrylamide gels. A-H12tsA275 infected cells at 40” labeled with [“Clleucine (W---B) mixed with H12tsA275 infected cells at 32” and labeled with [3H]leucine (O---O). B-Ratio of 3H cpm/“C cpm (A-A). C-H12tsB221 infected cells at 40” labeled with [“Clleucine (W-B) mixed with H12tsB221 infected cells at 32” labeled with [Hlleucine (O----O). D-Ratio of 3H cpm/“C cpm (A-A). E-HlPtsC295 infected cells at 40” labeled with [“Clleucine (W - -a) mixed with H12tsC295 infected cells.at 32” labeled with [3H]leucine (O---O). F-Ratio of 3H cpm/“C cpm (A-A). G-Adenovinrs type 12 wild-type virus infected cells at 40° labeled with [Wlleucine (W- - -m) mixed with wild-type virus infected cells at 32” labeled with [3H]leucine (O----O). H-Ratio of sH cpm/“C cpm (A-A).
22
ROSENWIRTH
(van der Vliet et al., 1975). It appears likely that adenovirus type 12 codes for a similar functional entity. If that is correct, then the H12tsA275 mutant at 40” either produces a denatured protein that is degraded by proteases or a protein that no longer binds to single stranded DNA in a DNA cellulose column. It is of some interest to compare the DNA binding proteins made by types 2, 5, and 12 adenovirus. Types 2 and 5 are closely related viruses sharing a number of physical, immunological, chemical, and biological properties (Green, 1970). Type 12 virus on the other hand contains a smaller molecular weight DNA of lower G+C content and is highly oncogenic when compared to adenoviruses types 2 and 5 (Green, 1970; Tooze, 1973). The molecular weight of the DNA binding proteins produced by both types 2 and 5 is 72,000 as compared with 60,000 MW of type 12 virus. The types 2 and 5 proteins have very similar peptide maps which are distinctly different from the peptide map of the type 12 60,000 MW protein (Rosenwirth et al., 1975). Both the 72,000 and the 60,000 MW proteins bind to single stranded DNA from a variety of sources, but not to double stranded DNA or RNA (van der Vliet and Levine, 1973). The 72,000 MW protein (from type 5) and the 60,000 MW protein (from type 12) are frequently accompanied by a collection of discrete proteolytic breakdown products with an average molecular weight of 48,000 independent of virus type. The temperature sensitive mutant H5ts125 does not produce a 72,000 MW DNA binding protein at 40”. The protein detected at 32” is thermolabile for continued DNA binding when compared to the type 5 wild-type protein. Analogously the type 12 mutant H12tsA275 does not produce a detectable 60,000 MW protein at 40” but does when the virus infection is performed at 32”. The phenotype of each of these independently derived mutants is that they fail to synthesize viral DNA at the nonpermissive temperature. It is likely then that adenoviruses produce a single strand specific DNA binding protein that is required for viral DNA replication. The
ET AL.
replicative intermediate of adenovirus contains large regions of single stranded DNA and these proteins may well function at those sites. ACKNOWLEDGMENTS
The authors thank A. K. Teresky, C. Kaminski, and S. Wyckoff for technical assistance. One of us (B.R.) was supported by the Deutsche Forschungsgemeinschaft postdoctoral fellowship. This research was supported by a Grant from the American Cancer Society E-591 and NC1 CA11049-07. REFERENCES
ALBERTS, B. M. and HERRICK, G. (1971). DNA cellulose chromatography. In “Methods in Enzymology” (L. Grossman and K. Moldave, 21, p. 198, Academic Press. ANDERSON,C. W., BAUM, P. R., and GESTELAND,R. F. (1973). The processing of adenovirus 2 induced proteins. J. Viral. 12, 241-252. CRAWFORD,L. V. and GESTELAND, R. F. (1973). Synthesis of polyoma proteins in uitro. J. Mol. Biol.. 74, 627-634.
GINSBERG,H. S., WILLIAMS, J. F., DOERFI.ER,W. H., and SHIMOJO,H. (1973). Proposed nomenclature for mutants of adenoviruses. J. Virol. 12, 663-664. GREEN, M. (1970). Oncogenic Viruses. Ann. Reu. Biochem.
39, 701-756.
LEVINE, A. J., VAN DER VLIET. P. C., ROSENWIRTH,B., RABEK, J., FRENKEL, G., and ENSINGER,M. (1975). Adenovirus infected cell specific DNA-binding proteins. Cold Spring Harbor Symp. Quant. Biol., 559466.
ROSENWIRTH.B., TJIA, S., WESTPHAL. M., and DOERFLER, W. (1974). Incomplete particles of adenovirus. II. Kinetics of formation and polypeptide composition of adenovirus type 2. Virology 60, 431-437. ROSENWIRTH.B., ANDERSON,C. W., LEVINE, A. J., and GESTELAND, R. (19751. In preparation. SHIROKI, K. and SHIMOJO, H. (1971). Transformation of green monkey kidney cells by SV40 genome: The establishment of transformed cell lines and the replication of human adenovirns and SV40 in transformed cells. Virology 45, 163-171. SHIROKI, K., IRISAWA, J., and SHIMOJO, H. (19721. Isolation and a preliminary characterization of temperature-sensitive mutants of adenovirus 12. Virology 49, 1-11. SHIROKI, K. and SHIMOJO, H. (1974). Analysis of adenovirus 12 temperature sensitive mutants defective in viral DNA replication. Cold Spring Harbor Symp. Quant. Biol., in press. TOOZE, J. (1973). The molecular biology of tumor viruses, Cold Springs, Harbor Lab.
ADENOVIRUS
TYPE 12 DNA BINDING
DER EB, A. (1973). Intermediates in Type 5 adenovirus DNA replication. Virology 51, 11-23. 11-23. VANDERVLIET, P. C., LEVINE, A. J., (1973). DNA-binding proteins specific for cells infected by adenovirus. Nature New Biol. 246, 170-174. VAN
PROTEINS
23
P. C., LEVINE, A. J., ENSINGER,M., and GINSBERG,H. S. (1975). Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovirus defective in viral DNA synthesis. J. Viral. 15, 348-354.
VAN DER VLIET,
