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Adenovirus-2 DNA: In Vitro Replication and Analysis of Viral Early Proteins M. GREEN, M. Q. ARENS, T. YAMASHITA, W. S. M. WOLD, AND K. H. BRACKMANN Institute for Molecular Virology, St. Louis University School o[ Medicine, St. Louis, Missouri 63110
years later. During the past 5 years, two unusual features of Ad D N A were discovered: inverted terminal repetitions (Garon et al. 1972; Wolfson and Dressier 1972) and a protein that is linked, probably covalently, to the termini of Ad D N A molecules (Robinson and Bellett 1975). Investigators in a number of laboratories have concentrated on productive infection of permissive human KB cells (or HeLa cells) in suspension culture by group-C Ads (Ad2 and Ad5 in particular), and these systems are now some of the best-understood eukaryotic virus-cell systems (Green and Daesch 1961; Wold et al. 1978). The nucleotide sequence (102 bases) of the inverted terminal repetition of Ad2 D N A has been recently established. The Ad2 D N A molecule has been separated into r and l strands (indicating rightward and leftward transcription, respectively). Cleavage patterns with a variety of restriction endonucleases also have been established. Restriction fragments have been used to map the regions of the genome that code for viral m R N A and proteins and also to map mutations, including three groups of DNA-negative mutants. Most important, studies from several groups have established the overall pattern of D N A replication and the approximate locations of origins and termini of viral D N A replication. Ad replicates with at least two stages of gene expression, "early," i.e., before initiation of viral D N A synthesis, and "late." Early genes encode functions for viral D N A replication, probably cell transformation, perhaps the switch from early to late stages of gene expression, and possibly the block in synthesis of cellular D N A and other macromolecules. Late genes encode mainly virion structural proteins and probably several nonvirion regulatory proteins that may "shut off" some early genes or mediate transport of virion proteins from the cytoplasm into the nucleus where virions are assembled. There is some evidence that an intermediate "early-late" stage of gene expression may exist (e.g., 8-14hr postinfection [p.i.]), where genes directly related to D N A replication may be maximally expressed. The late stage of infection (e.g., 18 hr p.i.) is particularly important for studies of viral D N A replication, because exclusively viral D N A is synthesized at this time (Pina and Green 1969). An infection of semipermissive or nonpermissive cells (e.g., rat cells) may result in stable-cell transfor-
The synthesis of D N A is complex, involving numerous enzymes and other proteins that may function in multienzyme systems. Animal D N A viruses (adenoviruses, papovaviruses, herpesviruses, poxviruses, parvoviruses) provide simple, powerful systems for studying eukaryotic D N A replication. We have chosen adenoviruses (Ads) because cells that replicate Ad genomes are particularly useful models for studying D N A replication and its regulation for several reasons: (1) Host-cell D N A synthesis is blocked late after infection with human Ads, thus permitting the unambiguous analysis of viral D N A synthesis. (2) Isolated nuclei and subnuclear complexes have been prepared from Ad2-, Ad5-, and Adl2-infected cells that synthesize almost exclusively viral D N A sequences in vitro. (3) DNA-negative Ad2, Ad5, and A d l 2 temperature-sensitive (ts) mutants are available that are very useful in understanding D N A replication. (4) With the exception of several virus-coded proteins, Ad D N A replication involves mainly cellular enzymes and other proteins, and thus the study of Ad D N A replication illuminates cellular mechanisms of D N A replication. (5) Of considerable practical importance is the fact that Ad-infected, cultured human cells can be grown in very large quantities, and thus purification and analysis of virus- and cell-coded proteins involved in D N A replication is feasible. In 1963 we reported the isolation of human Ad2, Ad4, Adl2, and Ad18 DNAs as very homogeneous molecules sedimenting at 30S-32S. We concluded at that time that treatment with a proteolytic enzyme was necessary to separate viral D N A from a viral protein component (Green and Pina 1963). During the next few years, we isolated D N A from 28 human Ad serotypes representing five distinct groups, and we showed that representative members of each group had linear, uninterrupted D N A genomes of molecular weight 20-25 x 106 which lacked duplex terminal repetitions, cohesive ends, and circular permutations (Green et al. 1967). Thus, Ad D N A molecules were shown to be unique with regard to the structural features that were associated with the replication of well-studied bacteriophage D N A molecules. These unusual properties presented problems in understanding the replication of the linear Ad D N A molecules, in particular, initiation and termination of D N A synthesis, mechanisms that remain completely obscure 10 755
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756
GREEN ET AL.
mation (10 -6 frequency). Ad-transformed cells retain a portion of the A d genome which includes "transformation genes" in an integrated state, One or more virus-coded "transformation proteins" are believed to maintain cell transformation. A n interesting possibility is that transformation proteins may function in viral D N A replication during the productive infection and may maintain celt transformation by effecting cell D N A replication. Three systems have been utilized to study A d D N A replication: whole cells, isolated nuclei, and subnuclear complexes. Whole cells have been useful for studies identifying origins and termini of replication and for studies with temperature-sensitive (ts) mutants. Isolated nuclei have the advantage that radionucleotide precursors equilibrate rapidly; however, initiation of D N A replication does not occur. Subnuclear complexes of two types have been studied that seem to carry out many steps in viral D N A synthesis. These complexes have the advantage that they are simple and can be easily manipulated in vitro; however, it is not known whether they are representative of the D N A replication machinery in vivo. No attempts have been made as yet to reconstruct D N A replication in vitro using purified proteins. In this paper, we summarize experiments directed at understanding the properties of Ad2-coded early proteins, their possible role in viral D N A replication, and their association with a soluble subnuclear complex that performs several aspects of A d D N A replication in vitro. We present data indicating that the Ad2 D N A terminal protein is synthesized early after infection and is associated with D N A replication in vivo and in vitro. In addition, we show preliminary data consistent with the possibility that the terminal protein (often called the "Bellett protein," and which we denote as CBP [covalently bound protein]) may be coded for by a conserved viral gene or possibly by a cellular gene.
RESULTS Identification of Ad2 Early Proteins In contrast to the replication of mammalian cell DNA, which requires continuous synthesis of cellcoded protein(s), the replication of A d D N A late after infection occurs in the presence of cycloheximide (CH), an inhibitor of protein synthesis (Horwitz et al. 1973; Yamashita and Green 1974). This is possible because viral proteins synthesized early after infection, i.e., in the absence of viral D N A replication, are required for viral D N A synthesis late after infection and can probably substitute for a "labile" cell protein, probably in initiating viral D N A replication. A t least three virus-coded proteins are required for replication of group-C A d D N A , since three DNA-negative complementation groups of Ad5 mutants (H5ts125, H5ts36, H5hrl) have been identified (Williams et al. 1974; Harrison et al. 1977). Three DNA-negative complementation groups of temperature-sensitive mutants, apparently also defective in the initiation of
D N A synthesis, were reported for Ad12 (group A) (Shimojo et al. 1975). Figure 1 shows the two-dimensional resolution of [35S]methionine-labeled polypeptides from the nucleoplasm of infected and mock-infected cells. The polypeptides were subjected to isoelectric focusing (horizontal), followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (vertical), as described by O'Farrell (1975). The arrows (Fig. 1) indicate the polypeptides specific to the infected cells; the absence of these polypeptides from the mockinfected extract is indicated by dashed circles. The virus-coded 73k (73,000 m.w.) polypeptide appears as a horizontal "streak," representing multiple-charge forms of the phosphoprotein. Four or more clustered viral acidic polypeptides of approximately 45k (40k-50k) are reproducibty found. These polypeptides may represent the candidate transformation protein or possibly the known subspecies of the 73k protein. The cluster of polypeptides at about 21k probably represents different forms of the Ad2-induced glycoprotein. The i l k acidic polypeptide is an exclusively nuclear protein found in our soluble D N A replication complex (see below). Other unknown viral polypeptides of l l k - 2 1 k are also observed. With the exception of the 73k polypeptide, it is not known if these polypeptides are virus-coded or cell-coded and virus-induced. Lewis et al. (1976) have identified six virus-coded polypeptides (72k, 44k-50k, 19k, 15.5k, and l l k ) by cell-free translation of early RNA, but it is not known if the polypeptides we observed correspond to any of these. As another means of identifying viral early proteins, we have prepared antisera in rodents against different Ad-transformed rat and hamster cells, and we have used these sera to immunoprecipitate radiolabeled polypeptides from early infected cells. Figure 2 shows the polypeptides precipitated by some of these sera. Table 1 indicates which of the four early-gene blocks are present in each transformed cell line against which antisera were prepared and lists which polypeptides are precipitated by each serum. Immunoprecipitation strongly suggests (but does not prove) that these polypeptides are virus-coded. Of particular interest, a 53k polypeptide is precipitated by all sera except that Table 1. Ad2 Early Proteins Immunoprecipitated by Antisera against Ad-transformed Cells Transformed cell antiserum F17 T2C4 8617 F4 5RK(I) Adl-SV40
Polypeptides immunoprecipitated 53k, 28k, 15k~ 53k, gp20/21k, b 15k,a 13.5k (doublet), 11.5k 53k, 15k," 11.5k, l l k 53k, 15k,a 11.5k 15ka 73k, 53k, 18k, 15k
Viral DNA and RNA in transformedcells block 1 blocks 1, 2, 3, 4 blocks 1, 4 blocks 1, 2, 4 block 1 unknown
aAII sera except the weak 5RK(1) serum also precipitated 18k, 14.5k, and 12k, which may be alternate forms of 15k. bAlternate forms of gp20/21k are 44k and probably 19k.
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ADENOVIRUS
DNA REPLICATION
757
Ad2 INFECTED KB CELL NUCLEI (CH)
MOCK INFECTED KB CELL NUCLEI(CH)
Figure 1. Autoradiographs showing two-dimensional electrophoresis (isoelectric focusing, and SDS-PAGE) of Ad2-induced early and mock-infected nucleoplasmic 35S-labeled proteins. KB cells in suspension were infected with Ad2 at a multiplicity of 500 pfu/cell. After 1 hr for virus adsorption, 25/zg/ml of CH was added to enhance early viral protein synthesis. At 4 hr after infection, 20/zg/ml of Ara C was added to the suspension culture to block viral DNA synthesis and thus the synthesis of late viral proteins. The CH block was removed at 4.5 hr by washing the cells with methionine-free media containing 20/~g/ml Ara C, resuspended in the same media, and labeled with [35S]methionine (25 ~Ci/ml) from 5 to 9 hr after infection. At the end of the labeling period, cells were rapidly harvested and washed with PBS lacking Ca ++ and Mg++; nuclei were then isolated as previously described (Wold et al. 1976), Nuclei were sonicated in sonication buffer, clarified, tyophilized, and dissolved in lysis buffer; aliquots containing 7 • 106 cpm were isoelectric-focused and eiectrophoresed as described by O'Farrel[ (1975). against 5RK(I) cells, and all sera precipitate a 15k polypeptide (difficult to see in this get). These polypeptides are candidate transformation proteins encoded by early-gene-block 1. It is not known whether the 53k polypeptide corresponds to the acidic 40k-50k cluster of polypeptides detected on two-dimensional gels. Figure 2 also shows the immunoprecipitation of the 73k protein (often visible as a doublet) by m o n o specific antisera against purified 73k protein. T h e sev-
erat bands of about 40k-50k are subspecies of the 73k protein, as indicated by peptide map analysis (see beIow).
Studies of a Soluble Ad DNA Replication Complex W e have described the isolation and properties of a soluble subnuclear complex that synthesizes A d 2
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758
GREEN ET AL. Table 2. Cell Enzymes and Early Viral Proteins Associated with the Subnuclear Ad2 DNA Replication Complex Cell enzyme activities DNA polymerase a (major) DNA polymerase y (minor) RNA polymerase DNA ligase
73K--
Early viral proteins 73k SS DNA phosphoprotein (major protein) 40k-50k subspecies of 73k protein 55k covalently linked protein (2 molecules/viral genome) llk nuclear protein (pronounced) 21k glycoprotein (minor) 15k protein (minor) 8.3k protein (minor)
71K~ 53K--
42K ~
26K~
21K-18K--
13K--
~?
:
=
F-17
T2C4
SV-40
8617
73K
Rot
Figure 2. Ad2 early proteins immunoprecipitated by rat antisera to Ad-transformed cells (F17, T2C4, and 8617 ceils), serum from hamsters bearing tumors induced by Adl/SV40 hybrid virus, and guinea pig antiserum to purified Ad2 73k protein. Equal input radioactivity (2• 107cpm) of [3SS]methionine-labeled Ad2-infected cell cytoplasm was incubated with immunoglobulin prepared from the sera described above, and immunoprecipitation was performed by the staphylococcus-A-protein procedure, as described by Kessler (1975). D N A in vitro (Yamashita et al. 1977; Arens et al. 1977; Rho et al. 1977). The properties of this complex are summarized in Table 2. The complex synthesizes exclusively viral D N A by a semiconservative mechanism (Yamashita et al. 1977). Genome-length molecules are formed, and all or almost all portions of both strands are synthesized in vitro (Yamashita et al. 1977; Arens and Yamashita 1978). Termination occurs at or near the right end of the I strand and the left terminus of the r strand in vitro, as it has been shown to occur in vivo (Arens and Yamashita 1978). We have investigated which early polypeptides are associated with the D N A replication complex (Rho et al. 1977). Early proteins were labeled with [35S]methionine in the presence of cytosine arabinoside (Ara C) to prevent the expression of late genes; then they were allowed to become associated with
the replication complex by incubation of cells for 8 hours without Ara C. The crude complex prepared by this protocol had as much endogenous D N A polymerase activity as a complex harvested 18 hours p.i. from cells not labeled or treated with drugs. The complex isolated from cells at 10 hours p.i. (the end of the labeling period) had much less polymerase activity. Very little polymerase was detected in complexes from mock-infected cells. The crude 35S-labeled infected and mock-infected complexes were purified by filtration through Bio-Gel A-50m. The infected-cell complex had the majority of the endogenous D N A polymerase activity in the excluded fraction, whereas the mock-infected complex had little polymerase activity in any fraction. These results suggest that our protocol for labeling early proteins does not adversely affect the formation of the Ad2 D N A synthesizing complex. Figure 3 illustrates the 35S-labeled polypeptides present in a total cell extract of infected and mockinfected cells and in the purified complex. The following early polypeptides are seen in infected-whole-cell extracts (Fig. 3A): DBP (73k single-stranded [SS] DNA-binding protein [DBP]), 21k, 19k, 15k, ll.5k, llk, and 8.3k. The purified complex contained major amounts of DBP and llk, and minor quantities of 21k, 15k, and possibly 8.3k (not shown). These early polypeptides remained associated with the complex after purification through a second Bio-Gel A-50m column (Fig. 3B), suggesting that they may be stably associated with a large complex. The purified complex also contained several major polypeptides of about 50k (Fig. 3). Polypeptides of 40k-50k were also immunoprecipitated by monospecific antiserum against purified DBP (Fig. 2), suggesting that these are subspecies of DBP. To confirm this possibility, the 73k (upper and lower bands) and the 42k and 47k [35S]methionine-labeled polypeptides immunoprecipitated by the anti-DBP serum from cytoplasmic extracts (Fig. 2) and the upper and lower 73k species immunoprecipitated from nuclear extracts (not shown) were subjected to tryptic fingerprint analysis. As shown in Figure 4, the peptide maps of the four 73k species and 47k were very similar. The
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ADENOVIRUS DNA REPLICATION
AJ
: IBMI
DBP-DBP--
759
lar weight increases to about 77k (Jeng et al. 1977). The reason for this change in gel mobility is not known. As shown in Figure 4, the tryptic peptide maps of the 74k and 77k forms of DBP (termed lower and upper forms) are identical. Therefore, again, the modification to DBP that changes its mobility in SDSP A G E is not reflected in the peptide maps.
5OK--
Studies o f the Protein Linked to the 5' T e r m i n i o f Ad D N A
21K--
19K/r 15K--II.5K-IlK J B . 3 K .......
: 15K
IIK 8,.~-
:;, ": L
ii = 9
Figure 3. Autoradiograph of Ad2-induced early polypeptides present in the purified soluble Ad2 DNA replication complex. 35S-Labeled infected (/) and mock-infected (M) cell complexes, and indicated cell fractions, were prepared and equal counts of each fraction were electrophoresed through 27-cm 5-20% gradient SDS-PAGE slab gels. Proteins were visualized by autoradiography. (A) Whole cell extracts; (B) purified soluble Ad2 DNA replication complex passed through two Bio-Gel A-50m columns. The radioactivity (cpm) in each of the fractions was as follows. Cytoplasm (I) 272 • 106; (M)285 x 106. Crude soluble complex: (I)35.5 • 106; (M) 46.5 • 106. Insoluble nuclear material: (I) 51 • 106; (M) 52.2 • 106. Nucleoplasm: (I) 24 • 106; (M) 21.4 • 106. Purified soluble complex (I) 1.1 • 106; (M) 1.3 • 106. (Reprinted, with permission, from Rho et al. 1977).
map of the 42k polypeptide was also highly related, but it was missing some of the peptides of the 73k species. These data are consistent with the results of Rosenwirth et al. (1976) which indicate that 40k-50k subspecies of D B P exist. The association of the 73k DBP and its 40k-50k subspecies with the soluble D N A replication complex is further evidence that DBP functions in viral D N A replication. However, significant quantities of DBP are present in the cytoplasm of infected cells, as indicated both by immunofluorescence studies (Sugawara et al. 1977) and by cell-fractionation procedures. Therefore, a cytoplasmic function for DBP is not excluded. As shown in Figure 4, identical peptide maps were obtained for DBP, 47k, and 42k, extracted from the cytoplasm and the nucleoplasm. Therefore, if DBP does have a cytoplasmic function, this is not reflected in its peptide map. As noted above, DBP is a phosphoprotein. Interestingly, DBP also appears to undergo another form of posttranslational modification. After a short pulse label with either [35S]methionine or 32po4, DBP has an apparent molecular weight of about 74k by SDSP A G E , but after a pulse-chase, the apparent molecu-
Investigators in several laboratories have recently shown that A d D N A contains a protein firmly bound to both 5' termini (i.e., CBP or covalently bound protein) (Robinson et al. 1973; Sharp et al. 1976; Keegstra et al. 1977; Rekosh et al. 1977). Very little is known about CBP and its role in virus replication. CBP could play a structural role, e.g., in packaging the viral D N A into the virion, or it could function in viral D N A replication. Assuming that CBP is virus-coded, if it has a structural role, then it is likely to be coded for by a late viral gene, as are other virion structural proteins. On the other hand, if it functions in D N A replication, then it probably is coded for by an early gene. Furthermore, if the protein functions in D N A replication, it should be associated with newly synthesized viral D N A made in vivo. It should also be present in our soluble Ad2 D N A replication complex and should be associated with D N A synthesized in vitro. An experiment was designed to test whether the Ad2 CBP is synthesized during the early or late stage of virus infection (T. Yamashita et al., in prep.). Ad2-infected cells were treated with hydroxyurea (HU) from 1 to 18 hours p.i. to block the synthesis of viral and cellular DNAs, while permitting the synthesis of early viral proteins and cell-coded proteins. Conditions that inhibit the synthesis of viral DNA, such as treatment of cells with H U or A r a C or infection with DNA-negative, temperature-sensitive mutants at the nonpermissive temperature, are known to inhibit the synthesis of late viral proteins. H U was used because previous studies of A d D N A synthesis (Sussenbach and van der Vliet 1973; Yamaguchi et al. 1977) have shown that H U blocks viral D N A replication, but does not block cellular R N A and protein synthesis, and that viral D N A synthesis begins immediately after removal of HU. H U was removed at 18 hours and CH was added to permit the synthesis of viral D N A and to maintain the inhibition of late viral protein synthesis. Under these conditions, cell and early viral proteins that were synthesized at 1-18 hours p.i., during the period of inhibition of D N A synthesis, should function to permit the synthesis of viral D N A after the removal of HU. Viral D N A synthesis proceeds in the presence of CH under these conditions, as previously reported (Horwitz et al. 1973; Yamashita and Green 1974). According to the experimental protocol described above, cells were treated with H U from 1 to 18 hours p.i.; H U was removed, CH was added, and then cells were labeled with [3H]thymidine for 5 hours. Data
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760
GREEN E T AL. ELECTROPHORESIS
i 84
~
-
~i~
UPPER 73K NUCLEI
UPPER
73K CYTOPLASM
LOWER 73K NUCLEI
LOWER 73K CYTOPLASM
Ad2 EARLY PROTEINS-IMMUNOPRECIPITATION
47K CYTOPLASM
42K
CYTOPLASM
BY ANTI 7 3 K IgG
Figure 4. Tryptic peptide maps of Ad2 73k protein immunoprecipitated from [35S]methionine-labeled nuclear and cytoplasmic extracts of Ad2 early infected cells by anti-73k immunoglobulin. The cytoplasmic upper and lower bands and the 47k and 42k subspecies were isolated from the gel illustrated in Fig. 2 for peptide mapping. Maps were prepared by radioiodination of gel slices containing immunoprecipitated polypeptide with 1251,two-dimensional electrophoresis, and thin-layer chromatography as described by Elder et al. (1977). Samples were spotted in the lower-right portion of the thin-layer plate and electrophoresed from right to left as the first dimension, followed by ascending chromatography from bottom to top as the second dimension. were obtained (not shown) indicating that (1) no detectable late viral proteins were synthesized during the H U block, (2) H U did not affect protein synthesis, (3) CH did not affect the rate of viral D N A synthesis after release of the H U block, and (4) late proteins were not synthesized during the CH block. Cells were harvested, nuclei were isolated, and D N A was extracted in the presence of 4 M guanidine hydrochloride (GdnHCl). The Ad2 [3H]thymidine-labeled D N A was purified by centrifugation in a 5-20% sucrose gradient containing 4 r~ GdnHCI. Most of the [3H]thymidinelabeled D N A sedimented homogeneously as a single peak at a slightly lower position than that of marker Ad2 14C-labeled, protease-treated D N A (not shown). The sedimentation of D N A containing CBP has been shown to be slightly slower than that of D N A treated with proteinase and purified by phenol extraction (Robinson et al. 1973). Treatment of in-vivo-labeled
D N A with proteinase K abolished the difference in sedimentation profile. The 3H-labeled viral D N A prepared from nuclei as described above had a slightly lower density than Ad2[14C]DNA in CsC1-GdnHCI equilibrium density gradients (not shown). This also suggests that the [3H]DNA is attached to CBP, because the density of the C B P - D N A complex is known to be lower than that of free D N A (Robinson et al. 1973). After pretreatment with proteinase K, in-vivolabeled D N A banded with a profile identical to that of marker Ad2 DNA. The presence of CBP on 3H-labeled viral D N A (prepared as described above) from nuclei was assayed by agarose gel electrophoresis (T. Yamashita et al., in prep.). Several investigators have shown that A d D N A containing CBP will not enter agarose gels during electrophoresis (Brown et al. 1975; Sharp et al. 1976; Padmanabhan and Padmanabhan 1977). Therefore,
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A D E N O V I R U S DNA R E P L I C A T I O N Ad2 [3H]DNA (peak fractions pooled from a 4M GdnHCl-sucrose gradient) was analyzed by electrophoresis on 1.4% cylindrical agarose gels. As shown in Figure 5A, most of the D N A remained at the top of the gel. In several experiments, this D N A accounted for 60-90% of the total input of radioactivity. On the other hand, marker [32p]DNA, purified from Ad2 virions and treated with proteinase, migrated into the
10
!20
il
4"8
-
b
15b w
~6 2
5
0
0
7
II
6
10
4"~ ~3 2
2
I 0--
o
N
II 10
~3
3~
2
2
I
I
17
D-~ 55
0
25o
D
x
X
2O E Q. o
15~
0
10 20 3O 4O 5O 6O GEL SLICE NUMBER
761
gel as expected. These data suggest that the [3H]DNA purified in this experiment may contain CBP. To test whether putative CBP was attached to the termini of the D N A molecules, [3H]DNA was mixed with 32p-labeled marker virion DNA, digested with Ec.oRI restriction endonuclease, and analyzed by agarose gel electrophoresis. EcoRI cleaves Ad2 D N A into six fragments, designated A through E on the basis of size. Fragment A and fragment C represent the molecular left and right ends, respectively. As shown in Figure 5B, the EcoRI B, D, and F fragments of [3H]DNA entered the gel readily and comigrated with 32p-labeled CBP-free Ad2 DNA. On the other hand, fragments A and C did not enter the gel, which is evidence for the association of CBP with these fragments. In contrast, when the [3H]DNA was treated with proteinase K following digestion with EcoRI, all of the fragments entered the gel in the same molar ratios as 32P-labeled CBP-free DNA, and there was no radioactivity remaining at the top of the gel (Fig. 5C). To confirm that the radioactivity at the top of the gel (Fig. 5B) was in fact present in the EcoRI A and C fragments, the top fragments of the gel in Figure 4B were cut out, treated with proteinase K, recast into another cylindrical gel, and coelectrophoresed with an EcoRI digest of CBP-free 32p-labeled viral DNA. As shown in Figure 5D, the 3H-labeled A and C fragments both appeared in the positions coincident with the A and C fragments of the marker [32p]DNA. These data provide strong evidence that the D N A synthesized under our experimental conditions, i.e., where early viral protein synthesis was permitted and late protein synthesis was not, contains CBP attached to the terminal fragments. Thus, CBP does not appear to be a late protein; rather, it seems to be either an early virus-coded protein or a cell-coded protein. It was of interest to test whether D N A synthesized in vitro is attached to CBP. The compJex was labeled for 45 minutes by incorporation of [3H]dTFP in a standard incorporation reaction. The [3H]DNA was then purified by velocity centrifugation in 5-20% sucrose gradients containing 4M GdnHC1. The invitro-synthesized D N A sedimented as a homogeneous peak, but slightly slower than that of virion D N A free of DBP (not shown). The in-vitro-labeled D N A also showed considerable spreading toward the bottom of Figure 5. Agarose gel electrophoresis of the DNA-protein complex isolated from Ad2-infected KB cells treated with HU from 1 to 18hr after infection and labeled with [3H]thymidine in the presence of CH from 18 to 23 hr. The DNA-protein complex was extracted from isolated nuclei with GdnHCI and purified by rate-zonal centrifugation in GdnHCl-sucrose density gradients and treated as follows prior to electrophoresis in cylindrical agarose gels: (A) Untreated; (/3) digested with endonuclease EcoRI; (C) digested with EcoRI and then with proteinase K and SDS; (D) top slice from a gel treated and electrophoresed as described in B was digested with proteinase K and SDS prior to electrophoresis. (0) 3H Radioactivity; ((3) virion Ad2 [32p]DNA purified by treatment with proteinase K and phenol extraction.
Downloaded from symposium.cshlp.org on July 8, 2016 - Published by Cold Spring Harbor Laboratory Press
762
GREEN E T AL.
the gradient, a phenomenon characteristic of the DNA-protein complex. When the in-vitro-labeled D N A was treated with proteinase K prior to extraction with GdnHCI, the profiles of the in-vitro-labeled and DBP-free marker D N A s coincided exactly. The in-vitro-labeled D N A also banded at a lower density in CsCI equilibrium gradients than did the marker, i.e., the peak was three fractions higher in the gradient. Treatment with proteinase K prior to centrifugation resulted in a shift in the density of the in-vitro-labeled D N A to that of the CBP-free marker virion DNA. These data indicate that the majority of D N A molecules synthesized in vitro are genome length. The data also suggest that in vitro-labeled D N A may contain CBP. Experiments similar to those described above for in-vivo-labeled Ad2 D N A were performed to demonstrate directly that CBP is attached to both termini of in-vitro-labeled DNA. A replication complex was prepared and the D N A was labeled in vitro with [32p]dTTP in the standard reaction mixture. The D N A was purified by centrifugation in 5-20% sucrose gradients containing 4M GdnHC1. Peak fractions were pooled, dialyzed to remove the GdnHCI, digested with EcoRI, and analyzed by electrophoresis on agarose slab gels (Arens and Yamashita 1978). As shown in Figure 6 (lane 3), fragment C was missing from the in-vitro-labeled D N A and fragment A was present in only low amounts, whereas significant amounts of fragments B, D, E, and F were produced. Much of the radioactivity remained at the top of the gel. If the in-vitro-labeled D N A was not treated with EcoRI, essentially all of the radioactivity remained at the top of the gel, and none of the EcoRI fragments were produced (lane 4). Very similar results were obtained by analysis of viral D N A purified from virions by extraction with 4M GdnHCI, without treatment of proteinase or SDS (Fig. 6, lane 2). Viral D N A treated with proteinase is shown in Figure 6 (lane 1); very little radioactivity is present at the top of the gel and all fragments can be readily seen. To quantitate the radioactivity in each of these fragments, the bands were cut and counted directly. The results are shown in Table 3. Clearly, the amounts of EcoRI fragments A and C are greatly reduced in D N A prepared both in vitro and extracted from virions without treatment of proteinase. These data provide strong evidence that the soluble replication complex contains CBP, and that CBP is attached to the termini of A d D N A synthesized in vitro.
I
Our results described above indicate that CBP is synthesized during early stages of infection. Thus, it may be a product of either a viral early gene or a cell gene. To investigate this question, we have obtained peptide maps of the CBP associated with Ad2 and Ad7 DNA. Ad2 and Ad7 are members of groups C
3
4
ii
i
?,
Figure 6. Agarose slab gel electrophoresisof EcoRI digestof in-vitro-synthesized Ad2 DNA. (1) DNA labeled in vivo and isolated from labeled virions by treatment with proteinase K and SDS and extracted with phenol-chloroform prior to digestion with EcoRI endonuclease; (2) DNA labeled as in 1 and purified as a DNA-protein complex by sedimentation in a sucrose gradient containing 4 M GdnHC1; (3) DNA labeled in vitro for 45 rain, centrifuged in a sucrose density gradient containing GdnHCI to maintain the DNA-protein complex, and digested with EcoRI prior to electrophoresis; (4) DNA prepared as in 3 without digestion with EcoRI prior to electrophoresis. The radioactivity associated with each fragment region is given in Table 3. and B, respectively, of human adenoviruses. The D N A s of these viruses are less than 20% homologous under stringent hybridization conditions. Therefore, if CBP is a virus-coded protein, the Ad2 and Ad7 peptide maps are likely to be dissimilar. In contrast, if CBP is cell-coded, the peptide maps should be identiTable 3. Radioactivity in EcoRI-generated Fragments of Ad2 DNA Synthesized In Vitro and Resolved by Agarose Gel Electrophoresis 32p (epm) Region of gel
Is CBP Coded for by a Viral Early Gene or a Cell Gene?
2
Top EcoRI fragment EcoRI fragment EcoRI fragment EcoRI fragment EcoRI fragment EcoRI fragment total cpm
lane 1 A B C D E F
153 6,730 1,395 1,074 838 689 483 11,362
lane 2
lane 3
lane4
1843 636 1082 47 710 589 378 5285
2664 979 752 232 613 544 284 6068
3633 609 204 162 199 222 142 5164
Data from T. Yamashitaet al. (in prep.).
Downloaded from symposium.cshlp.org on July 8, 2016 - Published by Cold Spring Harbor Laboratory Press
ADENOVIRUS DNA REPLICATION cal (unless a different cell-coded protein is linked to the D N A s of the two serotypes). For these experiments, we used CBP labeled in vitro with 125I because it is difficult to obtain [35S]methionine-labeled CBP. Viral D N A - C B P complexes were extracted from virions using 4 M GdnHC1 and purified either by two equilibrium density centrifugations in CsCI gradients containing 4 M GdnHCI or by two steps of exclusion chromatography on Sepharose 4B. After dialysis, the D N A s were digested with DNase and resolved by SDS-PAGE. The polypeptides were identified in the gel by staining with Coomassie blue. Figure 7 shows the results with three different preparations of Ad2 CBP and one preparation of Ad7 CBP. The preparations contained essentially only two polypeptides, CBP and DNase. However, minor bands corresponding to the Ad2 hexon and the Ad7 fiber were also present. Ad2 CBP and Ad7 CBP had almost the same position in the gel. The CBP bands were cut from the gel, labeled with 125I, and digested with chymotrypsin, and the peptide maps were obtained. These peptide maps were prepared in collaboration with Drs. John Elder and Richard Lerner (Scripps Clinic and Research Foundation). The results are shown in Figure 8. The maps of the Ad2 CBP and Ad7 CBP were identical except for one peptide. Indeed, when small amounts of the Ad2 and Ad7 digests were mixed, all but two of the ~25I-labeled peptides comigrated. Conceivably, the one peptide specific to Ad2 CBP, and the other peptide specific to Ad7 CBP, might represent the portion of the molecule linked to cell DNA, and the difference in position of these peptides could be due to the presence of a few nucleotides linked to the peptide. These results indicate that CBP is the product of either a cell gene or a highly conserved viral early gene. As noted, the CBP preparations before S D S - P A G E contained trace amounts of some virion polypeptides. Thus, it was important to establish that the putative
763
CBP polypeptide band was not a virion protein, especially virion core proteins V and VII, which are known to bind tightly to A d DNA. Figure 9 compares the chymotryptic peptide maps of Ad2 CBP with virion proteins V, VII, and II (hexon). Clearly, the map of CBP is different from the maps of any of these proteins 9 The map of DNase is also completely different (not shown). Therefore, we can be confident that the CBP band does not represent virion protein V, VII, or II, or DNase. The DNAs
of Five Groups of Human Ad Contain CBP
We have shown that the 31 human A d serotypes fall into at least five groups based upon D N A genome homologies (see Wold et al. 1978). If CBP is involved in D N A replication, then it should be associated with the DNAs from serotypes in all five groups. Figure 10 shows the analysis for CBP of D N A s of Ad12 (group A), Ad7 (group B), Ad2 (group C), Ad19 (group D), and Ad4 (group E). In each case, viral D N A was purified from virions lysed with 4 r~ GdnHCl. These DNAs, as well as protease-treated DNAs, were cleaved with EcoRI, and the fragments were resolved by agarose gel electrophoresis. The results indicate that the D N A of each serotype is linked to CBP. In all cases, two fragments are missing (or greatly reduced) from lanes representing CBP-DNA, and D N A can be found at the top of the gel. Where the cleavage maps are known, the missing fragments are identified as the end fragments, i.e., the Ad2 and Ad12 A and C fragments and the Ad7 A and B fragments (the small Ad7 C fragment and Ad2 F fragment have run off the gel). With Ad4, the terminal fragments appear to be A and C (the Ad4 D N A has not been completely digested), and with Ad19 the terminal fragments appear to be B and C. Thus, we conclude that D N A s of serotypes in all five groups are linked to CBP. n
tr~
Ad2 Virion Figure 7. Isolation of Ad2 CBP and Ad7 CBP. Three preparations of Ad2 and one of Ad7 DNA-CBP complexes (1-4 mg of DNA) were isolated from highly purified virions by disruption in 4M GdnHCl and banding twice in CsC1-Gdn gradients or by exclusion chromatography on Sepharose 4B in 4M GdnHCI. Each preparation was digested with pancreatic DNase, electrophoresed in 10-18% linear gradient SDS-PAGE gels, and stained with Coomassie blue. The following samples were applied to the gel: (A) Ad2 virion; (B-D) Ad2 CBP digested with 4/~g DNase; (E) a marker mixture containing bovine serum albumin (68k), ovalbumin (43k), trypsin inhibitor (21.5k), and cytochrome C (12.5k); (F, G)4/~g and 8/zg DNase; (H) Ad7 CBP digested with 8/zg DNase; (I) Ad7 virion.
Ad2-CBP 2 3
9
~"~ DNose ~= 4ug Bug
I
~'~o Ad7Virion
Adenovirus-2 DNA: In Vitro Replication and Analysis of Viral Early Proteins M. GREEN, M. Q. ARENS, T. YAMASHITA, W. S. M. WOLD, AND K. H. BRACKMANN Institute for Molecular Virology, St. Louis University School o[ Medicine, St. Louis, Missouri 63110
years later. During the past 5 years, two unusual features of Ad D N A were discovered: inverted terminal repetitions (Garon et al. 1972; Wolfson and Dressier 1972) and a protein that is linked, probably covalently, to the termini of Ad D N A molecules (Robinson and Bellett 1975). Investigators in a number of laboratories have concentrated on productive infection of permissive human KB cells (or HeLa cells) in suspension culture by group-C Ads (Ad2 and Ad5 in particular), and these systems are now some of the best-understood eukaryotic virus-cell systems (Green and Daesch 1961; Wold et al. 1978). The nucleotide sequence (102 bases) of the inverted terminal repetition of Ad2 D N A has been recently established. The Ad2 D N A molecule has been separated into r and l strands (indicating rightward and leftward transcription, respectively). Cleavage patterns with a variety of restriction endonucleases also have been established. Restriction fragments have been used to map the regions of the genome that code for viral m R N A and proteins and also to map mutations, including three groups of DNA-negative mutants. Most important, studies from several groups have established the overall pattern of D N A replication and the approximate locations of origins and termini of viral D N A replication. Ad replicates with at least two stages of gene expression, "early," i.e., before initiation of viral D N A synthesis, and "late." Early genes encode functions for viral D N A replication, probably cell transformation, perhaps the switch from early to late stages of gene expression, and possibly the block in synthesis of cellular D N A and other macromolecules. Late genes encode mainly virion structural proteins and probably several nonvirion regulatory proteins that may "shut off" some early genes or mediate transport of virion proteins from the cytoplasm into the nucleus where virions are assembled. There is some evidence that an intermediate "early-late" stage of gene expression may exist (e.g., 8-14hr postinfection [p.i.]), where genes directly related to D N A replication may be maximally expressed. The late stage of infection (e.g., 18 hr p.i.) is particularly important for studies of viral D N A replication, because exclusively viral D N A is synthesized at this time (Pina and Green 1969). An infection of semipermissive or nonpermissive cells (e.g., rat cells) may result in stable-cell transfor-
The synthesis of D N A is complex, involving numerous enzymes and other proteins that may function in multienzyme systems. Animal D N A viruses (adenoviruses, papovaviruses, herpesviruses, poxviruses, parvoviruses) provide simple, powerful systems for studying eukaryotic D N A replication. We have chosen adenoviruses (Ads) because cells that replicate Ad genomes are particularly useful models for studying D N A replication and its regulation for several reasons: (1) Host-cell D N A synthesis is blocked late after infection with human Ads, thus permitting the unambiguous analysis of viral D N A synthesis. (2) Isolated nuclei and subnuclear complexes have been prepared from Ad2-, Ad5-, and Adl2-infected cells that synthesize almost exclusively viral D N A sequences in vitro. (3) DNA-negative Ad2, Ad5, and A d l 2 temperature-sensitive (ts) mutants are available that are very useful in understanding D N A replication. (4) With the exception of several virus-coded proteins, Ad D N A replication involves mainly cellular enzymes and other proteins, and thus the study of Ad D N A replication illuminates cellular mechanisms of D N A replication. (5) Of considerable practical importance is the fact that Ad-infected, cultured human cells can be grown in very large quantities, and thus purification and analysis of virus- and cell-coded proteins involved in D N A replication is feasible. In 1963 we reported the isolation of human Ad2, Ad4, Adl2, and Ad18 DNAs as very homogeneous molecules sedimenting at 30S-32S. We concluded at that time that treatment with a proteolytic enzyme was necessary to separate viral D N A from a viral protein component (Green and Pina 1963). During the next few years, we isolated D N A from 28 human Ad serotypes representing five distinct groups, and we showed that representative members of each group had linear, uninterrupted D N A genomes of molecular weight 20-25 x 106 which lacked duplex terminal repetitions, cohesive ends, and circular permutations (Green et al. 1967). Thus, Ad D N A molecules were shown to be unique with regard to the structural features that were associated with the replication of well-studied bacteriophage D N A molecules. These unusual properties presented problems in understanding the replication of the linear Ad D N A molecules, in particular, initiation and termination of D N A synthesis, mechanisms that remain completely obscure 10 755
Downloaded from symposium.cshlp.org on July 8, 2016 - Published by Cold Spring Harbor Laboratory Press
756
GREEN ET AL.
mation (10 -6 frequency). Ad-transformed cells retain a portion of the A d genome which includes "transformation genes" in an integrated state, One or more virus-coded "transformation proteins" are believed to maintain cell transformation. A n interesting possibility is that transformation proteins may function in viral D N A replication during the productive infection and may maintain celt transformation by effecting cell D N A replication. Three systems have been utilized to study A d D N A replication: whole cells, isolated nuclei, and subnuclear complexes. Whole cells have been useful for studies identifying origins and termini of replication and for studies with temperature-sensitive (ts) mutants. Isolated nuclei have the advantage that radionucleotide precursors equilibrate rapidly; however, initiation of D N A replication does not occur. Subnuclear complexes of two types have been studied that seem to carry out many steps in viral D N A synthesis. These complexes have the advantage that they are simple and can be easily manipulated in vitro; however, it is not known whether they are representative of the D N A replication machinery in vivo. No attempts have been made as yet to reconstruct D N A replication in vitro using purified proteins. In this paper, we summarize experiments directed at understanding the properties of Ad2-coded early proteins, their possible role in viral D N A replication, and their association with a soluble subnuclear complex that performs several aspects of A d D N A replication in vitro. We present data indicating that the Ad2 D N A terminal protein is synthesized early after infection and is associated with D N A replication in vivo and in vitro. In addition, we show preliminary data consistent with the possibility that the terminal protein (often called the "Bellett protein," and which we denote as CBP [covalently bound protein]) may be coded for by a conserved viral gene or possibly by a cellular gene.
RESULTS Identification of Ad2 Early Proteins In contrast to the replication of mammalian cell DNA, which requires continuous synthesis of cellcoded protein(s), the replication of A d D N A late after infection occurs in the presence of cycloheximide (CH), an inhibitor of protein synthesis (Horwitz et al. 1973; Yamashita and Green 1974). This is possible because viral proteins synthesized early after infection, i.e., in the absence of viral D N A replication, are required for viral D N A synthesis late after infection and can probably substitute for a "labile" cell protein, probably in initiating viral D N A replication. A t least three virus-coded proteins are required for replication of group-C A d D N A , since three DNA-negative complementation groups of Ad5 mutants (H5ts125, H5ts36, H5hrl) have been identified (Williams et al. 1974; Harrison et al. 1977). Three DNA-negative complementation groups of temperature-sensitive mutants, apparently also defective in the initiation of
D N A synthesis, were reported for Ad12 (group A) (Shimojo et al. 1975). Figure 1 shows the two-dimensional resolution of [35S]methionine-labeled polypeptides from the nucleoplasm of infected and mock-infected cells. The polypeptides were subjected to isoelectric focusing (horizontal), followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (vertical), as described by O'Farrell (1975). The arrows (Fig. 1) indicate the polypeptides specific to the infected cells; the absence of these polypeptides from the mockinfected extract is indicated by dashed circles. The virus-coded 73k (73,000 m.w.) polypeptide appears as a horizontal "streak," representing multiple-charge forms of the phosphoprotein. Four or more clustered viral acidic polypeptides of approximately 45k (40k-50k) are reproducibty found. These polypeptides may represent the candidate transformation protein or possibly the known subspecies of the 73k protein. The cluster of polypeptides at about 21k probably represents different forms of the Ad2-induced glycoprotein. The i l k acidic polypeptide is an exclusively nuclear protein found in our soluble D N A replication complex (see below). Other unknown viral polypeptides of l l k - 2 1 k are also observed. With the exception of the 73k polypeptide, it is not known if these polypeptides are virus-coded or cell-coded and virus-induced. Lewis et al. (1976) have identified six virus-coded polypeptides (72k, 44k-50k, 19k, 15.5k, and l l k ) by cell-free translation of early RNA, but it is not known if the polypeptides we observed correspond to any of these. As another means of identifying viral early proteins, we have prepared antisera in rodents against different Ad-transformed rat and hamster cells, and we have used these sera to immunoprecipitate radiolabeled polypeptides from early infected cells. Figure 2 shows the polypeptides precipitated by some of these sera. Table 1 indicates which of the four early-gene blocks are present in each transformed cell line against which antisera were prepared and lists which polypeptides are precipitated by each serum. Immunoprecipitation strongly suggests (but does not prove) that these polypeptides are virus-coded. Of particular interest, a 53k polypeptide is precipitated by all sera except that Table 1. Ad2 Early Proteins Immunoprecipitated by Antisera against Ad-transformed Cells Transformed cell antiserum F17 T2C4 8617 F4 5RK(I) Adl-SV40
Polypeptides immunoprecipitated 53k, 28k, 15k~ 53k, gp20/21k, b 15k,a 13.5k (doublet), 11.5k 53k, 15k," 11.5k, l l k 53k, 15k,a 11.5k 15ka 73k, 53k, 18k, 15k
Viral DNA and RNA in transformedcells block 1 blocks 1, 2, 3, 4 blocks 1, 4 blocks 1, 2, 4 block 1 unknown
aAII sera except the weak 5RK(1) serum also precipitated 18k, 14.5k, and 12k, which may be alternate forms of 15k. bAlternate forms of gp20/21k are 44k and probably 19k.
Downloaded from symposium.cshlp.org on July 8, 2016 - Published by Cold Spring Harbor Laboratory Press
ADENOVIRUS
DNA REPLICATION
757
Ad2 INFECTED KB CELL NUCLEI (CH)
MOCK INFECTED KB CELL NUCLEI(CH)
Figure 1. Autoradiographs showing two-dimensional electrophoresis (isoelectric focusing, and SDS-PAGE) of Ad2-induced early and mock-infected nucleoplasmic 35S-labeled proteins. KB cells in suspension were infected with Ad2 at a multiplicity of 500 pfu/cell. After 1 hr for virus adsorption, 25/zg/ml of CH was added to enhance early viral protein synthesis. At 4 hr after infection, 20/zg/ml of Ara C was added to the suspension culture to block viral DNA synthesis and thus the synthesis of late viral proteins. The CH block was removed at 4.5 hr by washing the cells with methionine-free media containing 20/~g/ml Ara C, resuspended in the same media, and labeled with [35S]methionine (25 ~Ci/ml) from 5 to 9 hr after infection. At the end of the labeling period, cells were rapidly harvested and washed with PBS lacking Ca ++ and Mg++; nuclei were then isolated as previously described (Wold et al. 1976), Nuclei were sonicated in sonication buffer, clarified, tyophilized, and dissolved in lysis buffer; aliquots containing 7 • 106 cpm were isoelectric-focused and eiectrophoresed as described by O'Farrel[ (1975). against 5RK(I) cells, and all sera precipitate a 15k polypeptide (difficult to see in this get). These polypeptides are candidate transformation proteins encoded by early-gene-block 1. It is not known whether the 53k polypeptide corresponds to the acidic 40k-50k cluster of polypeptides detected on two-dimensional gels. Figure 2 also shows the immunoprecipitation of the 73k protein (often visible as a doublet) by m o n o specific antisera against purified 73k protein. T h e sev-
erat bands of about 40k-50k are subspecies of the 73k protein, as indicated by peptide map analysis (see beIow).
Studies of a Soluble Ad DNA Replication Complex W e have described the isolation and properties of a soluble subnuclear complex that synthesizes A d 2
Downloaded from symposium.cshlp.org on July 8, 2016 - Published by Cold Spring Harbor Laboratory Press
758
GREEN ET AL. Table 2. Cell Enzymes and Early Viral Proteins Associated with the Subnuclear Ad2 DNA Replication Complex Cell enzyme activities DNA polymerase a (major) DNA polymerase y (minor) RNA polymerase DNA ligase
73K--
Early viral proteins 73k SS DNA phosphoprotein (major protein) 40k-50k subspecies of 73k protein 55k covalently linked protein (2 molecules/viral genome) llk nuclear protein (pronounced) 21k glycoprotein (minor) 15k protein (minor) 8.3k protein (minor)
71K~ 53K--
42K ~
26K~
21K-18K--
13K--
~?
:
=
F-17
T2C4
SV-40
8617
73K
Rot
Figure 2. Ad2 early proteins immunoprecipitated by rat antisera to Ad-transformed cells (F17, T2C4, and 8617 ceils), serum from hamsters bearing tumors induced by Adl/SV40 hybrid virus, and guinea pig antiserum to purified Ad2 73k protein. Equal input radioactivity (2• 107cpm) of [3SS]methionine-labeled Ad2-infected cell cytoplasm was incubated with immunoglobulin prepared from the sera described above, and immunoprecipitation was performed by the staphylococcus-A-protein procedure, as described by Kessler (1975). D N A in vitro (Yamashita et al. 1977; Arens et al. 1977; Rho et al. 1977). The properties of this complex are summarized in Table 2. The complex synthesizes exclusively viral D N A by a semiconservative mechanism (Yamashita et al. 1977). Genome-length molecules are formed, and all or almost all portions of both strands are synthesized in vitro (Yamashita et al. 1977; Arens and Yamashita 1978). Termination occurs at or near the right end of the I strand and the left terminus of the r strand in vitro, as it has been shown to occur in vivo (Arens and Yamashita 1978). We have investigated which early polypeptides are associated with the D N A replication complex (Rho et al. 1977). Early proteins were labeled with [35S]methionine in the presence of cytosine arabinoside (Ara C) to prevent the expression of late genes; then they were allowed to become associated with
the replication complex by incubation of cells for 8 hours without Ara C. The crude complex prepared by this protocol had as much endogenous D N A polymerase activity as a complex harvested 18 hours p.i. from cells not labeled or treated with drugs. The complex isolated from cells at 10 hours p.i. (the end of the labeling period) had much less polymerase activity. Very little polymerase was detected in complexes from mock-infected cells. The crude 35S-labeled infected and mock-infected complexes were purified by filtration through Bio-Gel A-50m. The infected-cell complex had the majority of the endogenous D N A polymerase activity in the excluded fraction, whereas the mock-infected complex had little polymerase activity in any fraction. These results suggest that our protocol for labeling early proteins does not adversely affect the formation of the Ad2 D N A synthesizing complex. Figure 3 illustrates the 35S-labeled polypeptides present in a total cell extract of infected and mockinfected cells and in the purified complex. The following early polypeptides are seen in infected-whole-cell extracts (Fig. 3A): DBP (73k single-stranded [SS] DNA-binding protein [DBP]), 21k, 19k, 15k, ll.5k, llk, and 8.3k. The purified complex contained major amounts of DBP and llk, and minor quantities of 21k, 15k, and possibly 8.3k (not shown). These early polypeptides remained associated with the complex after purification through a second Bio-Gel A-50m column (Fig. 3B), suggesting that they may be stably associated with a large complex. The purified complex also contained several major polypeptides of about 50k (Fig. 3). Polypeptides of 40k-50k were also immunoprecipitated by monospecific antiserum against purified DBP (Fig. 2), suggesting that these are subspecies of DBP. To confirm this possibility, the 73k (upper and lower bands) and the 42k and 47k [35S]methionine-labeled polypeptides immunoprecipitated by the anti-DBP serum from cytoplasmic extracts (Fig. 2) and the upper and lower 73k species immunoprecipitated from nuclear extracts (not shown) were subjected to tryptic fingerprint analysis. As shown in Figure 4, the peptide maps of the four 73k species and 47k were very similar. The
Downloaded from symposium.cshlp.org on July 8, 2016 - Published by Cold Spring Harbor Laboratory Press
ADENOVIRUS DNA REPLICATION
AJ
: IBMI
DBP-DBP--
759
lar weight increases to about 77k (Jeng et al. 1977). The reason for this change in gel mobility is not known. As shown in Figure 4, the tryptic peptide maps of the 74k and 77k forms of DBP (termed lower and upper forms) are identical. Therefore, again, the modification to DBP that changes its mobility in SDSP A G E is not reflected in the peptide maps.
5OK--
Studies o f the Protein Linked to the 5' T e r m i n i o f Ad D N A
21K--
19K/r 15K--II.5K-IlK J B . 3 K .......
: 15K
IIK 8,.~-
:;, ": L
ii = 9
Figure 3. Autoradiograph of Ad2-induced early polypeptides present in the purified soluble Ad2 DNA replication complex. 35S-Labeled infected (/) and mock-infected (M) cell complexes, and indicated cell fractions, were prepared and equal counts of each fraction were electrophoresed through 27-cm 5-20% gradient SDS-PAGE slab gels. Proteins were visualized by autoradiography. (A) Whole cell extracts; (B) purified soluble Ad2 DNA replication complex passed through two Bio-Gel A-50m columns. The radioactivity (cpm) in each of the fractions was as follows. Cytoplasm (I) 272 • 106; (M)285 x 106. Crude soluble complex: (I)35.5 • 106; (M) 46.5 • 106. Insoluble nuclear material: (I) 51 • 106; (M) 52.2 • 106. Nucleoplasm: (I) 24 • 106; (M) 21.4 • 106. Purified soluble complex (I) 1.1 • 106; (M) 1.3 • 106. (Reprinted, with permission, from Rho et al. 1977).
map of the 42k polypeptide was also highly related, but it was missing some of the peptides of the 73k species. These data are consistent with the results of Rosenwirth et al. (1976) which indicate that 40k-50k subspecies of D B P exist. The association of the 73k DBP and its 40k-50k subspecies with the soluble D N A replication complex is further evidence that DBP functions in viral D N A replication. However, significant quantities of DBP are present in the cytoplasm of infected cells, as indicated both by immunofluorescence studies (Sugawara et al. 1977) and by cell-fractionation procedures. Therefore, a cytoplasmic function for DBP is not excluded. As shown in Figure 4, identical peptide maps were obtained for DBP, 47k, and 42k, extracted from the cytoplasm and the nucleoplasm. Therefore, if DBP does have a cytoplasmic function, this is not reflected in its peptide map. As noted above, DBP is a phosphoprotein. Interestingly, DBP also appears to undergo another form of posttranslational modification. After a short pulse label with either [35S]methionine or 32po4, DBP has an apparent molecular weight of about 74k by SDSP A G E , but after a pulse-chase, the apparent molecu-
Investigators in several laboratories have recently shown that A d D N A contains a protein firmly bound to both 5' termini (i.e., CBP or covalently bound protein) (Robinson et al. 1973; Sharp et al. 1976; Keegstra et al. 1977; Rekosh et al. 1977). Very little is known about CBP and its role in virus replication. CBP could play a structural role, e.g., in packaging the viral D N A into the virion, or it could function in viral D N A replication. Assuming that CBP is virus-coded, if it has a structural role, then it is likely to be coded for by a late viral gene, as are other virion structural proteins. On the other hand, if it functions in D N A replication, then it probably is coded for by an early gene. Furthermore, if the protein functions in D N A replication, it should be associated with newly synthesized viral D N A made in vivo. It should also be present in our soluble Ad2 D N A replication complex and should be associated with D N A synthesized in vitro. An experiment was designed to test whether the Ad2 CBP is synthesized during the early or late stage of virus infection (T. Yamashita et al., in prep.). Ad2-infected cells were treated with hydroxyurea (HU) from 1 to 18 hours p.i. to block the synthesis of viral and cellular DNAs, while permitting the synthesis of early viral proteins and cell-coded proteins. Conditions that inhibit the synthesis of viral DNA, such as treatment of cells with H U or A r a C or infection with DNA-negative, temperature-sensitive mutants at the nonpermissive temperature, are known to inhibit the synthesis of late viral proteins. H U was used because previous studies of A d D N A synthesis (Sussenbach and van der Vliet 1973; Yamaguchi et al. 1977) have shown that H U blocks viral D N A replication, but does not block cellular R N A and protein synthesis, and that viral D N A synthesis begins immediately after removal of HU. H U was removed at 18 hours and CH was added to permit the synthesis of viral D N A and to maintain the inhibition of late viral protein synthesis. Under these conditions, cell and early viral proteins that were synthesized at 1-18 hours p.i., during the period of inhibition of D N A synthesis, should function to permit the synthesis of viral D N A after the removal of HU. Viral D N A synthesis proceeds in the presence of CH under these conditions, as previously reported (Horwitz et al. 1973; Yamashita and Green 1974). According to the experimental protocol described above, cells were treated with H U from 1 to 18 hours p.i.; H U was removed, CH was added, and then cells were labeled with [3H]thymidine for 5 hours. Data
Downloaded from symposium.cshlp.org on July 8, 2016 - Published by Cold Spring Harbor Laboratory Press
760
GREEN E T AL. ELECTROPHORESIS
i 84
~
-
~i~
UPPER 73K NUCLEI
UPPER
73K CYTOPLASM
LOWER 73K NUCLEI
LOWER 73K CYTOPLASM
Ad2 EARLY PROTEINS-IMMUNOPRECIPITATION
47K CYTOPLASM
42K
CYTOPLASM
BY ANTI 7 3 K IgG
Figure 4. Tryptic peptide maps of Ad2 73k protein immunoprecipitated from [35S]methionine-labeled nuclear and cytoplasmic extracts of Ad2 early infected cells by anti-73k immunoglobulin. The cytoplasmic upper and lower bands and the 47k and 42k subspecies were isolated from the gel illustrated in Fig. 2 for peptide mapping. Maps were prepared by radioiodination of gel slices containing immunoprecipitated polypeptide with 1251,two-dimensional electrophoresis, and thin-layer chromatography as described by Elder et al. (1977). Samples were spotted in the lower-right portion of the thin-layer plate and electrophoresed from right to left as the first dimension, followed by ascending chromatography from bottom to top as the second dimension. were obtained (not shown) indicating that (1) no detectable late viral proteins were synthesized during the H U block, (2) H U did not affect protein synthesis, (3) CH did not affect the rate of viral D N A synthesis after release of the H U block, and (4) late proteins were not synthesized during the CH block. Cells were harvested, nuclei were isolated, and D N A was extracted in the presence of 4 M guanidine hydrochloride (GdnHCl). The Ad2 [3H]thymidine-labeled D N A was purified by centrifugation in a 5-20% sucrose gradient containing 4 r~ GdnHCI. Most of the [3H]thymidinelabeled D N A sedimented homogeneously as a single peak at a slightly lower position than that of marker Ad2 14C-labeled, protease-treated D N A (not shown). The sedimentation of D N A containing CBP has been shown to be slightly slower than that of D N A treated with proteinase and purified by phenol extraction (Robinson et al. 1973). Treatment of in-vivo-labeled
D N A with proteinase K abolished the difference in sedimentation profile. The 3H-labeled viral D N A prepared from nuclei as described above had a slightly lower density than Ad2[14C]DNA in CsC1-GdnHCI equilibrium density gradients (not shown). This also suggests that the [3H]DNA is attached to CBP, because the density of the C B P - D N A complex is known to be lower than that of free D N A (Robinson et al. 1973). After pretreatment with proteinase K, in-vivolabeled D N A banded with a profile identical to that of marker Ad2 DNA. The presence of CBP on 3H-labeled viral D N A (prepared as described above) from nuclei was assayed by agarose gel electrophoresis (T. Yamashita et al., in prep.). Several investigators have shown that A d D N A containing CBP will not enter agarose gels during electrophoresis (Brown et al. 1975; Sharp et al. 1976; Padmanabhan and Padmanabhan 1977). Therefore,
Downloaded from symposium.cshlp.org on July 8, 2016 - Published by Cold Spring Harbor Laboratory Press
A D E N O V I R U S DNA R E P L I C A T I O N Ad2 [3H]DNA (peak fractions pooled from a 4M GdnHCl-sucrose gradient) was analyzed by electrophoresis on 1.4% cylindrical agarose gels. As shown in Figure 5A, most of the D N A remained at the top of the gel. In several experiments, this D N A accounted for 60-90% of the total input of radioactivity. On the other hand, marker [32p]DNA, purified from Ad2 virions and treated with proteinase, migrated into the
10
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-
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2
I
I
17
D-~ 55
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10 20 3O 4O 5O 6O GEL SLICE NUMBER
761
gel as expected. These data suggest that the [3H]DNA purified in this experiment may contain CBP. To test whether putative CBP was attached to the termini of the D N A molecules, [3H]DNA was mixed with 32p-labeled marker virion DNA, digested with Ec.oRI restriction endonuclease, and analyzed by agarose gel electrophoresis. EcoRI cleaves Ad2 D N A into six fragments, designated A through E on the basis of size. Fragment A and fragment C represent the molecular left and right ends, respectively. As shown in Figure 5B, the EcoRI B, D, and F fragments of [3H]DNA entered the gel readily and comigrated with 32p-labeled CBP-free Ad2 DNA. On the other hand, fragments A and C did not enter the gel, which is evidence for the association of CBP with these fragments. In contrast, when the [3H]DNA was treated with proteinase K following digestion with EcoRI, all of the fragments entered the gel in the same molar ratios as 32P-labeled CBP-free DNA, and there was no radioactivity remaining at the top of the gel (Fig. 5C). To confirm that the radioactivity at the top of the gel (Fig. 5B) was in fact present in the EcoRI A and C fragments, the top fragments of the gel in Figure 4B were cut out, treated with proteinase K, recast into another cylindrical gel, and coelectrophoresed with an EcoRI digest of CBP-free 32p-labeled viral DNA. As shown in Figure 5D, the 3H-labeled A and C fragments both appeared in the positions coincident with the A and C fragments of the marker [32p]DNA. These data provide strong evidence that the D N A synthesized under our experimental conditions, i.e., where early viral protein synthesis was permitted and late protein synthesis was not, contains CBP attached to the terminal fragments. Thus, CBP does not appear to be a late protein; rather, it seems to be either an early virus-coded protein or a cell-coded protein. It was of interest to test whether D N A synthesized in vitro is attached to CBP. The compJex was labeled for 45 minutes by incorporation of [3H]dTFP in a standard incorporation reaction. The [3H]DNA was then purified by velocity centrifugation in 5-20% sucrose gradients containing 4M GdnHC1. The invitro-synthesized D N A sedimented as a homogeneous peak, but slightly slower than that of virion D N A free of DBP (not shown). The in-vitro-labeled D N A also showed considerable spreading toward the bottom of Figure 5. Agarose gel electrophoresis of the DNA-protein complex isolated from Ad2-infected KB cells treated with HU from 1 to 18hr after infection and labeled with [3H]thymidine in the presence of CH from 18 to 23 hr. The DNA-protein complex was extracted from isolated nuclei with GdnHCI and purified by rate-zonal centrifugation in GdnHCl-sucrose density gradients and treated as follows prior to electrophoresis in cylindrical agarose gels: (A) Untreated; (/3) digested with endonuclease EcoRI; (C) digested with EcoRI and then with proteinase K and SDS; (D) top slice from a gel treated and electrophoresed as described in B was digested with proteinase K and SDS prior to electrophoresis. (0) 3H Radioactivity; ((3) virion Ad2 [32p]DNA purified by treatment with proteinase K and phenol extraction.
Downloaded from symposium.cshlp.org on July 8, 2016 - Published by Cold Spring Harbor Laboratory Press
762
GREEN E T AL.
the gradient, a phenomenon characteristic of the DNA-protein complex. When the in-vitro-labeled D N A was treated with proteinase K prior to extraction with GdnHCI, the profiles of the in-vitro-labeled and DBP-free marker D N A s coincided exactly. The in-vitro-labeled D N A also banded at a lower density in CsCI equilibrium gradients than did the marker, i.e., the peak was three fractions higher in the gradient. Treatment with proteinase K prior to centrifugation resulted in a shift in the density of the in-vitro-labeled D N A to that of the CBP-free marker virion DNA. These data indicate that the majority of D N A molecules synthesized in vitro are genome length. The data also suggest that in vitro-labeled D N A may contain CBP. Experiments similar to those described above for in-vivo-labeled Ad2 D N A were performed to demonstrate directly that CBP is attached to both termini of in-vitro-labeled DNA. A replication complex was prepared and the D N A was labeled in vitro with [32p]dTTP in the standard reaction mixture. The D N A was purified by centrifugation in 5-20% sucrose gradients containing 4M GdnHC1. Peak fractions were pooled, dialyzed to remove the GdnHCI, digested with EcoRI, and analyzed by electrophoresis on agarose slab gels (Arens and Yamashita 1978). As shown in Figure 6 (lane 3), fragment C was missing from the in-vitro-labeled D N A and fragment A was present in only low amounts, whereas significant amounts of fragments B, D, E, and F were produced. Much of the radioactivity remained at the top of the gel. If the in-vitro-labeled D N A was not treated with EcoRI, essentially all of the radioactivity remained at the top of the gel, and none of the EcoRI fragments were produced (lane 4). Very similar results were obtained by analysis of viral D N A purified from virions by extraction with 4M GdnHCI, without treatment of proteinase or SDS (Fig. 6, lane 2). Viral D N A treated with proteinase is shown in Figure 6 (lane 1); very little radioactivity is present at the top of the gel and all fragments can be readily seen. To quantitate the radioactivity in each of these fragments, the bands were cut and counted directly. The results are shown in Table 3. Clearly, the amounts of EcoRI fragments A and C are greatly reduced in D N A prepared both in vitro and extracted from virions without treatment of proteinase. These data provide strong evidence that the soluble replication complex contains CBP, and that CBP is attached to the termini of A d D N A synthesized in vitro.
I
Our results described above indicate that CBP is synthesized during early stages of infection. Thus, it may be a product of either a viral early gene or a cell gene. To investigate this question, we have obtained peptide maps of the CBP associated with Ad2 and Ad7 DNA. Ad2 and Ad7 are members of groups C
3
4
ii
i
?,
Figure 6. Agarose slab gel electrophoresisof EcoRI digestof in-vitro-synthesized Ad2 DNA. (1) DNA labeled in vivo and isolated from labeled virions by treatment with proteinase K and SDS and extracted with phenol-chloroform prior to digestion with EcoRI endonuclease; (2) DNA labeled as in 1 and purified as a DNA-protein complex by sedimentation in a sucrose gradient containing 4 M GdnHC1; (3) DNA labeled in vitro for 45 rain, centrifuged in a sucrose density gradient containing GdnHCI to maintain the DNA-protein complex, and digested with EcoRI prior to electrophoresis; (4) DNA prepared as in 3 without digestion with EcoRI prior to electrophoresis. The radioactivity associated with each fragment region is given in Table 3. and B, respectively, of human adenoviruses. The D N A s of these viruses are less than 20% homologous under stringent hybridization conditions. Therefore, if CBP is a virus-coded protein, the Ad2 and Ad7 peptide maps are likely to be dissimilar. In contrast, if CBP is cell-coded, the peptide maps should be identiTable 3. Radioactivity in EcoRI-generated Fragments of Ad2 DNA Synthesized In Vitro and Resolved by Agarose Gel Electrophoresis 32p (epm) Region of gel
Is CBP Coded for by a Viral Early Gene or a Cell Gene?
2
Top EcoRI fragment EcoRI fragment EcoRI fragment EcoRI fragment EcoRI fragment EcoRI fragment total cpm
lane 1 A B C D E F
153 6,730 1,395 1,074 838 689 483 11,362
lane 2
lane 3
lane4
1843 636 1082 47 710 589 378 5285
2664 979 752 232 613 544 284 6068
3633 609 204 162 199 222 142 5164
Data from T. Yamashitaet al. (in prep.).
Downloaded from symposium.cshlp.org on July 8, 2016 - Published by Cold Spring Harbor Laboratory Press
ADENOVIRUS DNA REPLICATION cal (unless a different cell-coded protein is linked to the D N A s of the two serotypes). For these experiments, we used CBP labeled in vitro with 125I because it is difficult to obtain [35S]methionine-labeled CBP. Viral D N A - C B P complexes were extracted from virions using 4 M GdnHC1 and purified either by two equilibrium density centrifugations in CsCI gradients containing 4 M GdnHCI or by two steps of exclusion chromatography on Sepharose 4B. After dialysis, the D N A s were digested with DNase and resolved by SDS-PAGE. The polypeptides were identified in the gel by staining with Coomassie blue. Figure 7 shows the results with three different preparations of Ad2 CBP and one preparation of Ad7 CBP. The preparations contained essentially only two polypeptides, CBP and DNase. However, minor bands corresponding to the Ad2 hexon and the Ad7 fiber were also present. Ad2 CBP and Ad7 CBP had almost the same position in the gel. The CBP bands were cut from the gel, labeled with 125I, and digested with chymotrypsin, and the peptide maps were obtained. These peptide maps were prepared in collaboration with Drs. John Elder and Richard Lerner (Scripps Clinic and Research Foundation). The results are shown in Figure 8. The maps of the Ad2 CBP and Ad7 CBP were identical except for one peptide. Indeed, when small amounts of the Ad2 and Ad7 digests were mixed, all but two of the ~25I-labeled peptides comigrated. Conceivably, the one peptide specific to Ad2 CBP, and the other peptide specific to Ad7 CBP, might represent the portion of the molecule linked to cell DNA, and the difference in position of these peptides could be due to the presence of a few nucleotides linked to the peptide. These results indicate that CBP is the product of either a cell gene or a highly conserved viral early gene. As noted, the CBP preparations before S D S - P A G E contained trace amounts of some virion polypeptides. Thus, it was important to establish that the putative
763
CBP polypeptide band was not a virion protein, especially virion core proteins V and VII, which are known to bind tightly to A d DNA. Figure 9 compares the chymotryptic peptide maps of Ad2 CBP with virion proteins V, VII, and II (hexon). Clearly, the map of CBP is different from the maps of any of these proteins 9 The map of DNase is also completely different (not shown). Therefore, we can be confident that the CBP band does not represent virion protein V, VII, or II, or DNase. The DNAs
of Five Groups of Human Ad Contain CBP
We have shown that the 31 human A d serotypes fall into at least five groups based upon D N A genome homologies (see Wold et al. 1978). If CBP is involved in D N A replication, then it should be associated with the DNAs from serotypes in all five groups. Figure 10 shows the analysis for CBP of D N A s of Ad12 (group A), Ad7 (group B), Ad2 (group C), Ad19 (group D), and Ad4 (group E). In each case, viral D N A was purified from virions lysed with 4 r~ GdnHCl. These DNAs, as well as protease-treated DNAs, were cleaved with EcoRI, and the fragments were resolved by agarose gel electrophoresis. The results indicate that the D N A of each serotype is linked to CBP. In all cases, two fragments are missing (or greatly reduced) from lanes representing CBP-DNA, and D N A can be found at the top of the gel. Where the cleavage maps are known, the missing fragments are identified as the end fragments, i.e., the Ad2 and Ad12 A and C fragments and the Ad7 A and B fragments (the small Ad7 C fragment and Ad2 F fragment have run off the gel). With Ad4, the terminal fragments appear to be A and C (the Ad4 D N A has not been completely digested), and with Ad19 the terminal fragments appear to be B and C. Thus, we conclude that D N A s of serotypes in all five groups are linked to CBP. n
tr~
Ad2 Virion Figure 7. Isolation of Ad2 CBP and Ad7 CBP. Three preparations of Ad2 and one of Ad7 DNA-CBP complexes (1-4 mg of DNA) were isolated from highly purified virions by disruption in 4M GdnHCl and banding twice in CsC1-Gdn gradients or by exclusion chromatography on Sepharose 4B in 4M GdnHCI. Each preparation was digested with pancreatic DNase, electrophoresed in 10-18% linear gradient SDS-PAGE gels, and stained with Coomassie blue. The following samples were applied to the gel: (A) Ad2 virion; (B-D) Ad2 CBP digested with 4/~g DNase; (E) a marker mixture containing bovine serum albumin (68k), ovalbumin (43k), trypsin inhibitor (21.5k), and cytochrome C (12.5k); (F, G)4/~g and 8/zg DNase; (H) Ad7 CBP digested with 8/zg DNase; (I) Ad7 virion.
Ad2-CBP 2 3
9
~"~ DNose ~= 4ug Bug
I
~'~o Ad7Virion
