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Tryptic fingerprint analysis of adenovirus types 2, 5 and 12 DNA-Binding proteins

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Tryptic fingerprint analysis of adenovirus types 2, 5 and 12 DNA-Binding proteins

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Each of types 2, 5 and 12 human adenovirus (Ad) induces the synthesis of single-strand-specific DNA-binding proteins at early times after infection of human or monkey cells. The molecular weights of these proteins are: Ad2, 72,000; Ad5, 72,000; and Ad12, 58,000. In addition, each of these viruses produces a collection of two or three lower molecular weight (41,000–47,000), single-strand-specific DNA-binding proteins that appear in variable amounts after infection. Tryptic fingerprints of the peptides derived from [35S]methionine-labeled 72,000-MW (Ad2 and 5), 58,000-MW (Ad12) and 41,000–47,000-MW (Ad2, 5 and 12) DNA-binding proteins have been obtained. The results of this analysis permit the following conclusions: (i) The 72,000-MW (Ad2 and 5) and 58,000-MW (Ad12) proteins synthesized in adenovirus-infected cells are distinct from a 72,000-MW cellular DNA-binding protein found in uninfected or mock-infected cells. (ii) The [35S]methionine-labeled peptides derived from the 41,000–47,000-MW proteins (Ad2, 5 and 12) are similar if not identical to the [35S]methionine-labeled tryptic peptides derived from the larger (Ad2 and 5, 72,000-MW; Ad12, 58,000-MW) DNA-binding proteins. (iii) The [35S]methionine-labeled peptides derived from the adenovirus types 2 and 5, 72,000-MW proteins are quite similar but distinct from the adenovirus type 12, 58,000-MW protein. (iv) The peptide fingerprints of the adenovirus type 2, 72,000-MW protein synthesized in either human or monkey cells are very similar or identical.

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... DBP is the product of an Ad2 early gene (i.e., expressed before initiation of viral DNA replication) located somewhere within map positions 0.59 to 0.72 (25). Analogous viral-coded DBPs of 72K and 58K daltons have been identified in Ad5 (38,39)and Adl2 (32,33)-infected cells, respectively. Singlestranded DBPs of 36 to 48K daltons are also found in Ad2-, Ad5-, and Adl2-infected cells that apparently are subspecies (probably proteolytic degradation products) of the larger DBPs (32,33,37,39). ...
... Analogous viral-coded DBPs of 72K and 58K daltons have been identified in Ad5 (38,39)and Adl2 (32,33)-infected cells, respectively. Singlestranded DBPs of 36 to 48K daltons are also found in Ad2-, Ad5-, and Adl2-infected cells that apparently are subspecies (probably proteolytic degradation products) of the larger DBPs (32,33,37,39). ...
... Chinnadurai, Z. Gilead, and M. Green, unpublished data). Tryptic peptide analyses (32) indicated that Ad2 and Ad5 DBPs are similar in composition but are quite distinct from Adl2 DBP. If all adenovirus serotypes encode DBPs that have the same function in viral DNA replication, it is possible that the DBPs may contain certain structural similarities that confer a common antigenic determinant to the DBP molecule. ...
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High-titer monospecific antiserum against highly purified adenovirus 2 (Ad2) single-stranded DNA binding protein (DBP) was used to study, by indirect immunofluorescence (IF), the synthesis of DBP in Ad2-infected human cells and adenovirus-transformed rat, hamster, and human cell lines. In infected cells the synthesis of DBP was first detected in the cytoplasm at 2 to 4 h postinfection and reached a maximum intensity at 6 h postinfection. At this time DBP began to accumulate in the nucleus, where it reached maximum intensity at about 14 h postinfection. The cytoplasmic IF was diffuse, whereas nuclear IF appeared as dots that coalesced into large globules as infection progressed. In cells treated with 1-beta-d-arabinofuranosylcytosine to inhibit viral DNA synthesis, strong nuclear IF was observed in the form of dots, but the large fluorescent globules were not observed. The Ad2 (oncogenic group C) anti-DBP serum reacted very strongly by IF with Ad5 (group C)-infected, to a lesser extent with Ad7 and Ad11 (group B)-infected, and weakly with Ad12 and Ad18 (group A)-infected KB cells (treated with 1-beta-d-arabinofuranosylcytosine). These results may indicate that Ad2 DBP is closely related immunologically to DBPs induced early after infection by adenovirus serotypes in oncogenic group C, moderately related to DBPs of serotypes in oncogenic group B, and perhaps distantly related to DBPs of serotypes in oncogenic group A. The following adenovirus-transformed cell lines were examined for DBP synthesis by IF with the Ad2 anti-DBP serum: six rat cell lines (T2C4, F17, 8662, 8638, 8617, and F161) transformed by Ad2 virus, three hamster cell lines transformed by Ad2 virus (Ad2HT1) and Ad2-simian virus 40 hybrid virus (ND1HK1 and ND4HK4), and one rat (5RK) and one human (293-31) cell line transformed by transfection with Ad5 DNA. T2C4 and 8662 appeared weakly positive, whereas Ad2HT1 and ND4HK1 were strongly positive. The other transformed cell lines did not produce DBP detectable by IF. Thus, some but not all transformed cell lines produce DBP, which indicates that DBP is not required for maintenance of cell transformation and that transformed cells can express "nontransforming" viral genes as protein.
... Recently an Adl2 DBP of 58K to 60K has been described (29). Ad2 and Ad5 DBPs of 41K to 45K are also I Present address: Department of Microbiology, Faculty of Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyohu, Kyoto, Japan 606. synthesized, but peptide analyses (20), genetic studies (29), and immunoprecipitation studies (27a) indicate that these are derived from the larger DBP (possibly degradation products). Ad2 DBP has been shown to be viral coded by cell-free translation of hybridization-purified Ad2 early mRNA into a 72K polypeptide with a peptide map similar to that of DBP isolated from infected cells, and the DBP gene has been mapped in Ad2 EcoRI-B fragment (Ad2 map position, 0.59 to 0.71) (16). ...
... The 35S-labeled DBP band was not present in lanes representing pulse (lane B)or pulse-chase (lane D)-labeled, mock-infected cell extracts. Analyses of proteins labeled with 32P04 indicated that DBP, and no other Ad2-induced polypeptide, was associated with the phosphate label (in Fig. 1 Fig. 1, there are no apparent virus-specific 32P-labeled polypeptides of 41K to 45K daltons, i.e., representing the DBP subspecies of these apparent molecular weights (20). Apparently the 32P-labeled DBP subspecies were present in insufficient amounts to be observed in the autoradiogram, because in other experiments these polypeptides were shown to be phosphorylated (data not shown). ...
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The adenovirus type 2-coded single-stranded DNA binding protein (DBP) was shown to be a phosphoprotein and to exist in at least two forms that differ in mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After a 30-min pulse with [35S]methionine or 32PO4, 35S- or 32P-labeled DBP had a nominal molecular weight of 74,000 whereas after a 30-min label followed by a 20-h chase, 35S- and 32P-labeled DBP had a nominal molecular weight of 77,000. Both large and small forms of 35S- and 32P-labeled DBP bound to single-stranded DNA-cellulose columns and were eluted by 0.4 to 0.6 M NaCl; both forms also were immunoprecipitated by antiserum against adenovirus type 1-simian virus 40-induced tumor cells (this antiserum contains antibodies against DBP) and by monospecific antiserum against 95 to 99% purified DBP. With highly purified 32P-DBP labeled 7 to 10 h postinfection, it was shown that the 32P radioactivity was firmly associated with protein material (i.e., not contaminating nucleic acids or phospholipids) and had properties expected of a phosphoester of an amino acid; paper electrophoresis of acid hydrolysates of this preparation identified phosphoserine but not phosphothreonine. Phosphoserine but not phosphothreonine was also identified in acid hydrolysates of another preparation of 32P-DBP labeled for 30 min, chased for 20 h, and then immunoprecipitated by adenovirus type 1-simian virus 40 antiserum.
... Apart from the Adl2 virion proteins, little is known at present about the Adl2 gene products and their functions. Among the early Adl2-specific proteins which have been charac- 21 22 ESCHE, SCHILLING, AND DOERFLER terized recently are a tumor-specific antigen (18,19) and a DNA-binding protein (32,33). ...
... In this report, we cell-free systems directed by early viral RNA from productively infected cells, additional proteins of 61K, 37K, 14K, and 1lK molecular weights were observed with preselected viral i-68 K RNA isolated from Adl2-infected BHK-21 cells. j 61 K The early 61K protein had an electrophoretic 50 K mobility very similar to the Adl2-coded DNA-42 K binding protein discovered in vivo by Rosen-39 K wirth et al. (32). The reticulocyte in vitro translation system was programmed with (B) 15pgofpreselected lateAd12RNA prepared from infected KB cells, (C) no added RNA, (D) 10 pg of viral RNA prepared from Ad12-infected BHK-21 cells at 12 h postinfection and preselected once by hybridization to Adl2 DNA; (E) immunoprecipitated polypeptides from the cell-free system programmed with preselected early Adl2 RNA isolated from infected BHK-21 cells. ...
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The early and late gene products of human adenovirus type 12 (Ad12), as well as the viral proteins synthesized in an Ad12-transformed cell line, were identified by translation of viral mRNA in an in vitro protein-synthesizing system. Cytoplasmic RNA was isolated from permissive KB or nonpermissive BHK cells infected with Ad12 and from Ad12-transformed HA12/7 cells. Virus-specific RNA was selected by hybridization to Ad12 DNA covalently bound to cellulose. Viral RNA was then translated in a fractionated rabbit reticulocyte cell-free system or in wheat germ S-30 extracts. The proteins synthesized were characterized by immunoprecipitation and subsequent electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. RNA prepared from KB cells late after infection with Ad12 elicited the synthesis of most of the structural polypeptides of the virion and at least two presumably nonstructural Ad12 proteins. When viral RNA isolated early after infection of KB cells with Ad12 was translated in vitro, 10 polypeptides were observed: E-68K, E-50K, E-42K, E-39K, E-34K, E-21K, E-19K, E-13K, E-12K, and E-10K. Ad12-specific RNA was also isolated from the Ad12-transformed hamster cell line HA12/7, which contains several copies of the Ad12 genome integrated in the host genome. The RNA codes for at least seven polypeptides with molecular weights very similar to those of the early viral proteins.
... Several Ad2 early polypeptides have been identified that are approximately the same molecular weight as CBP. These include four to six 40K-50K polypeptides coded by early region Ela (map position 1.5 to 4.5) (9, 10; D. Halbert, D. Spector, and H. Raskas, personal communication), a 53K polypeptide coded by region Elb (map position 4.5 to 11) (Halbert et al., personal communication), and any of the several 40K-50K subspecies of the 73K single-stranded DNA binding protein coded by region E2 (map position 62 to 68) (9,15). The Ela-40K-50K polypeptides are synthesized in vivo and migrate to a specific position on O'Farrell-type two-dimensional gels (6; M. Green, W. S. M. Wold, K. Brackmann, and M. A. Cartas, Virology, in press). ...
... The CBP map is also different from the tryptic [35S]Met-peptide maps of any of the major virion structural polypeptides, such as potential contaminant polypeptides II and V (Fig. 3). The 42K, 47K, and 73K polypeptides are all clearly related, consistent with reports from other laboratories (15) that several 40K-50K subspecies of 73K exist. It Met-labeled polypeptide bands were excised from dried gels: Ad2 CBP (Fig. 1); El-53K, E2-73K, E2-47K, and E2-42K (Fig. 2); E1-40-50K from a two-dimensional gel (Green et We conclude that, although CBP is synthesized during early stages of infection (18), it does not correspond to any of the known virus-coded early polypeptides. ...
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A polypeptide of 55,000 daltons (55K) is linked, probably covalently, to the K' termini of adenovirus type 2 DNA. The 55K polypeptide is synthesized during early stages of infection (T. Yamashita, M. Arens, and M. Green, J. Virol. 30: 497-507, 1979) and thus may function in viral DNA replication, gene regulation, or cell transformation. Several virus-coded early polypeptides have been identified that could correspond to the terminal 55K, including the E1-40K-50K and E1-53K candidate transformation polypeptides and the E2-42K/47K/73K single-stranded DNA-binding polypeptide. We show here that two-dimensional tryptic [35S]methionine-peptide maps of the terminal 55K differ completely from [35S]methionine-peptide maps of four related E1-40K-50K polypeptides, the E1-53K, and the related E2-42K, E2-47K, and E2-73K polypeptides. We conclude that the terminal 55K polypeptide does not correspond to any of the known virus-coded early polypeptides.
... Early after adenovirus infection a singlestrand-specific, DNA-binding protein appears (15,17,18), which varies in molecular weight from 60,000 for type 12 (14,15) to about 72,000 (72K) for adenovirus types 2 and 5 (14,18). The function of this protein is unknown, but its similarity to the gene 32 protein of bacteriophage T4 (5) suggested that the adenovirus DNA-binding protein may also be associated with replication of the viral DNA (17,18). ...
... Early after adenovirus infection a singlestrand-specific, DNA-binding protein appears (15,17,18), which varies in molecular weight from 60,000 for type 12 (14,15) to about 72,000 (72K) for adenovirus types 2 and 5 (14,18). The function of this protein is unknown, but its similarity to the gene 32 protein of bacteriophage T4 (5) suggested that the adenovirus DNA-binding protein may also be associated with replication of the viral DNA (17,18). ...
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Studies have been done to characterize further H5ts125, an adenovirus type 5 conditionally lethal, temperature-sensitive (ts) mutant defective in initiation of DNA synthesis and to investigate whether the single-strand-specific DNA-binding (72,000 molecular weight) protein is coded by the mutated viral gene. When H5ts125-infected cells were labeled with [35S]methionine at 32 degrees C and then incubated without isotope at 39.5 degrees C, the mutant's nonpermissive temperature, the 72,000 molecular weight polypeptide was progressively degraded. Immunofluorescence examination of cells infected with wild-type virus, H5ts125, and H5ts149 (a second, unique DNA-minus mutant) showed that immunologically reactive DNA-binding protein was barely detectable in H5ts125-infected cells at 39.5 degrees C, whereas this protein was present in wild-type- and H5TS149-infected cells, that the protein made at 32 degrees C in H5ts125-infected cells lost its ability to bind specific DNA-binding protein antibody when the infected cells were shifted to 39.5 degrees C, and that if H5ts125-infected cells were shifted from the restrictive temperature to 32 degrees C, even in the presence of cycloheximide to stop protein synthesis, immunologically reactive DNA-binding protein reappeared.
... A comparison of the proteins present in cells infected by some of the other herpesviruses from primitive hosts would also be of interest. In this context we should note the results of Rosenwirth et al. (1976) who, based on tryptic peptide analysis, found almost identical 72K DNA-binding proteins in adenovirus type 2-and type 5-infected cells and similarities between these two proteins and the 58K DNA-binding protein of adenovirus type 12. ...
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Previously, we have shown a common antigen of several herpesviruses (pseudorabies virus, equine abortion virus and bovine mammillitis virus) to be antigenically related to the major DNA-binding proteins of herpes simplex virus types 1 and 2. In this study we have purified the cross-reacting polypeptide from cells infected with pseudorabies virus, equine abortion virus and bovine mammillitis virus and shown the cross-reacting protein to be a major DNA-binding protein for each virus. Tryptic peptide analysis of the cross-reacting DNA-binding proteins of all five viruses has shown structural similarities. The proteins thus were shown to share common antigenic sites, to have similar biological properties and to have a highly conserved amino acid sequence. This unexpected similarity between proteins from diverse herpes viruses suggests an essential and fundamental role of the major DNA-binding protein in herpes virus replication.
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It has been shown that both HSV1- and HSV2-induced “immediate early” polypeptides bind, with a range of affinities, to native calf thymus DNA in vitro. The biological relevance of this observation is reflected in cell fractionation studies, which demonstrate that HSV1-and HSV2-induced immediate early polypeptides migrate to the nucleus and there bind strongly to chromatin from infected cells.
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A procedure has been developed for the purification of adenovirus type 2 DNA-binding protein (DBP) from nuclei of infected HeLa cells. This procedure routinely yields 0.2 to 0.6 mg of protein per 10⁹ cells that is greater than 98% DBP. Binding protein so prepared does not precipitate at low ionic strength, interacts with both single- and double-stranded DNA, and complements Ad5 ts125 function in an in vitro DNA synthesizing system dependent upon exogenous DBP. An examination of the hydrodynamic properties of Ad2 DBP indicated that DBP undergoes a concentration-dependent self-association process. In high ionic strength solutions (1.0 M NaCl), self-association is a limited process observed at DBP concentrations above about 0.1 mg/mL; the product is a unit having a molecular weight of a trimer. At low ionic strengths (0.1 M NaCl), self-association is more extensive and is observed at lower protein concentrations. Our findings suggest that units other than the 72,000 molecular weight monomer may interact with DNA in the cell. Purified Ad2 DBP was digested with several proteolytic enzymes to determine if smaller DNA-binding products could be generated that resemble the 48,000 molecular weight species observed in extracts of infected cells. Digestion of purified DBP with Pronase or chymotrypsin produced relatively stable fragments with molecular weights of 45,000 and 53,000, respectively. Trypsin cleavage produced a 51,000 molecular weight fragment that upon continued incubation was further digested to produce a 35,000-M/sub r/ peptide. The production of the 35,000-M/sub r/ peptide by trypsin cleavage of the 51,000-M/sub r/ fragment was not observed if a sufficient amount of DNA was added to the DBP solution prior to trypsin digestion. This result indicates that bound DNA protects a trypsin-sensitive site(s) in the 51,000-M/sub r/ fragment, and it suggests that the 51,000-M/sub r/ fragment contains at least a part of the binding site for single-stranded DNA.
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Immunization in rabbits with intact, highly purified adenovirus type 2 (Ad2) virions, yielded antisera with high titers of antibodies against the 72 000 dalton DNA-binding protein (DBP). This was established by rocket immunoelectrophoresis when an anti-intact Ad2-antiserum was analyzed against fractions from an ion-exchange chromatogram of soluble antigens remaining after virus isolation from virus-infected HeLa cells. The high anti-DBP titer did not reflect the composition of the immunogen, since no DBP was detectable within virions. An antiserum raised in response to mildly disrupted virions showed no specificity against the DBP, but contained antibodies against the same structural proteins as the anti-intact Ad2-antiserum, when analyzed by the immunoblotting technique. These findings indicate that the nonpermissive rabbit as an experimental host permits early gene expression of a human adenovirus.
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At early times, adenovirus-2-infected HeLa cells have been shown to contain four induced polypeptides with apparent molecular weights of 70–75,000 (E1), 45–48,000, 19–20,000 (E2), and 10–11,000 (E3). In this communication, a combination of high multiplicity infection, separation of infected cells into cytoplasmic and nuclear fractions, and high resolution SDS-polyacrylamide gel electrophoresis has allowed the characterization of three other early species: 42,000 (E1A), 17,000 (E2A) and 14,000 (E2B). In addition, further fractionation of cytoplasmic and nuclear fractions revealed an association of E2 and E2A with cell plasma membranes and E3 with nuclear particulate matter. Pulsechase data, behavior of early proteins on SDS-hydroxylapatite columns, and fractionation with ammonium sulfate also have served to further characterize the early proteins. The 45–48K protein was not seen in our system.
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Early in lytic infection of KB cells with adenovirus type 5 (Ad5) a viral-coded single-strand-specific DNA-binding protein with a molecular weight of 72,000 is synthesized. The function of this protein in viral DNA synthesis was studied using two different methods for specific inactivation. First, DNA synthesis was examined in KB cells infected with an early temperature-sensitive mutant, H5ts125, which produces a thermolabile DNA-binding protein. At the nonpermissive temperature (40°) the start of the first and subsequent rounds of DNA replication was inhibited. However, viral DNA synthesis in vitro using a system of isolated nuclei obtained from H5ts125-infected KB cells was not thermosensitive compared to wild type. In this in vitro system, replicating molecules were normally converted to mature DNA at 40° without reinitiation of new replication rounds. These results show that thermal inactivation of the H5ts125 DNA-binding protein destroys its capacity to function in the initiation of new replication rounds but does not impair chain growth. As a second method of inactivation we studied viral DNA synthesis in vitro in the presence of antibody against the purified DNA-binding protein. A 50–60% inhibition of the rate of viral DNA synthesis was observed while cellular DNA synthesis was not affected. Density-labeling experiments showed that the antibody inhibits the duplication of both complementary strands, presumably by a decrease of fork movement in replicating adenovirus DNA. The results suggest that the Ad5 DNA-binding protein functions in initiation of DNA replication as well as in elongation of nascent viral DNA chains.
Chapter
The human adenoviruses types 2 and 5 (Ad2 and Ads) contain a linear, non-permuted double-stranded DNA of 23 × 105 dalton molecular weight (Green et al., 1967). The ends of the molecule have a 110 to 140 base pairs long inverted repetition (Wolfson and Dressler, 1972; Garon et al., 1972; Roberts et al., 1974), which allows formation of single-stranded circles when the DNA is denatured and renatured at low concentration.
Article
Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein synthesized early after injection of human cells with adenovirus type 2 (Ad2) showed that polypeptides of 11,000 (11K), 12K, 14K, 15K, 19K, 21K, 24K, and 72k molecular weight were present in infected but not in mock-infected cells. These polypeptides corresponded in electrophoretic mobility to the following polypeptides synthesized in vitro by using mRNA complementary to specific regions of the Ad2 genome: 11K, 19K, 21K (91.5 to 96.8 map units), 14K (78.2 to 83.4 map units), 72K (62.4 to 67.9 map units), and 15K (4.9 to 11.0 map units). Polypeptides of 25K, 17K, 15.5K, and 13K were also synthesized in vitro, but have not yet been detected in infected cells. In addition, six adeno-specific polypeptides of 38 to 50K molecular weight could be discerned in infected cells if two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis was used to compare extracts from infected and mock-infected cells. Partial protease digestion showed these 38 to 50K polypeptides to be related in sequence to each other and to the 40 to 50K polypeptide made in vitro (1.3 to 4.0 map units).
Article
The group C adenoviruses code for a single-strand specific DNA-binding protein of molecular weight 72,000 daltons which is synthesized at early times after productive viral infection. Experiments were designed to determine whether this single-strand specific DNA-binding protein was expressed in adenovirus tumors and transformed cells. Two independently derived preparations of anti-sera from hamsters bearing group C adenovirus tumors were tested for antibody against the single-stranded DNA-binding proteins. One antiserum contained antibodies that reacted with these DNA-binding proteins, while the second antiserum did not contain detectable levels of antibody. Five adenovirus type 2 transformed rat cell lines were tested for the presence of the single-stranded specific DNA-binding proteins. Two of the five transformed cells expressed detectable levels of this protein. These results indicate that the group C adenovirus single-strand specific DNA-binding proteins are expressed in some, but not all, adenovirus tumors and transformed cell lines. Those transformed cell lines (type 2) containing a portion of the adenovirus genome designated by the Eco R-I-B restriction enzyme fragment express the single-strand specific DNA-binding proteins. Those cell lines missing this Eco R-I-B fragment do not contain this viral protein. Other experiments have located the structural gene of the signle-strand specific DNA-binding protein in the Eco-R-I-B DNA fragment, indicating that when this gene is present in a transformed cell, it is expressed.
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One adenovirus type 2 (Ad2) early protein, with an apparent molecular weight of 14,000 in sodium dodecyl sulfate-polyacrylamide gels (E14K), was purified to homogeneity. Purification involved fractionation of cytoplasmic extracts, precipitation at low pH, and DEAE-cellulose, phosphocellulose, and hydroxylapatite chromatography. The yield was around 12 microgram of purified protein per 10(9) HeLa cells. The two Ad2 DNA binding proteins with molecular weights of 75,000 and 45,000 (E75K and E45K) were purified by the same procedure. Tryptic peptide analyses indicated that the E14K protein is unrelated to the DNA binding proteins. The purified E14K protein has a high content of basic amino acids and a sedimentation coefficient of 5.5S in the native state, corresponding to a molecular weight of around 95,000. Pulse-chase experiments suggest that the E14K polypeptide is a primary translation product. Immunoprecipitation with a monospecific antiserum against the E14K protein revealed that it is exclusively localized in the cytoplasm of infected cells. E14K started to be synthesized at 2 hpostinfection, with a maximal rate of synthesis at 4 to 6 h postinfection. Immunoprecipitation of cell extracts from four different Ad2-transformed hamster embryo cell lines revealed that only one (Ad2HE4) of them expresses this protein. The adenovirus-simian virus 40 hybrid virus (Ad2ND1) does not express this protein, suggesting that the gene for the E14K protein is located in the part of the Ad2 genome which is deleted in this hybrid virus.
Article
As documented throughout this book, the adenoviruses have proved to be useful models for probing the basic mechanisms of macromolecular synthesis in eukaryotic cells. Much of the work on adenovirus DNA replication has been motivated by the idea that analysis of the replication of simple viral chromosomes may provide insights useful for understanding the complex mechanisms involved in the replication of eukaryotic chromosomes. Progress in this area has been rapid in recent years. Many of the general features of the adenovirus DNA replication pathway are now reasonably well understood from analysis of viral DNA synthesis in vivo. In addition, a cell-free replication system dependent on exogenous adenovirus DNA templates has been developed. This system has provided a means to explore the molecular mechanisms involved in initiation and chain elongation and to purify and characterize replication proteins. This chapter summarizes the available information on adenovirus DNA replication with particular emphasis on current developments. Additional information may be found in several recent reviews of the subject (Winnacker, 1978; Flint and Broker, 1980; Kelly, 1982; Challberg and Kelly, 1982).
Article
We have studied the polypeptides associated with the expression of the transforming region of the Ad5 genome by immunoprecipitating antigens (using the double antibody and protein A-Sepharose techniques) from cells infected with wild-type (wt) Ad5 or transformation-defective host range (hr) mutants and from cells transformed by Ad5. Three different antisera were used: P antiserum specific for early viral products (Russell et al., 1967) and two different hamster tumor antisera. Immunoprecipitation of antigens from wt-infected KB cells followed by SDS-polyacrylamide gel electrophoresis of precipitated proteins revealed that a major polypeptide having a molecular weight of approximately 58,000 was detected with all three antisera and with both the double antibody and the protein A-Sepharose techniques, while P antiserum also precipitated polypeptides of molecular weights 72,000, 67,000 and 44,000, which probably represent the DNA binding protein and related polypeptides, respectively. With the double antibody technique, in addition to the proteins mentioned above, P antiserum and the hamster tumor antisera precipitated a 10,500 dalton polypeptide which was not detected when the protein A-Sepharose procedure was used. Using either the double antibody or the protein A-Sepharose technique, we found that hr mutants from complementation group II failed to induce the synthesis of the 58,000 dalton protein, whereas mutants from complementation group I produced normal or near normal amounts. Using the double antibody technique, we found that the 10,500 dalton protein was absent or made in reduced amounts by group I mutants. A 58,000 dalton protein was detected in a number of different Ad5-transformed cell lines, including the 293 human line, the 14b hamster line and several transformed rat cell lines. This observation and the fact that transformation negative group II mutants fail to induce the synthesis of a 58,000 dalton polypeptide suggest that this protein is one of the Ad5-specific products necessary for cell transformation.
Article
The protein covalently bound to the 5' termini of adenovirus type 2 DNA has been purified from virus labeled with [35S]methionine, using exclusion chromatography of disrupted virions to isolate the DNA-protein complex, which is then digested with DNase. The terminal protein isolated from mature virus is most effectively labeled if the cells are exposed to [35S]methionine during the "intermediate" period of 13 to 21 h postinfection, suggesting that the protein is synthesized during this interval. The tryptic peptides of the terminal protein were compared with those of several known adenovirus-coded proteins and found to be unrelated. In particular, the terminal protein is not related to the 38-50K early proteins encoded by the leftmost 4.4% of the adenovirus genome, one region essential for the transforming activity of the virus. Neither is it related to the 72K single-strand-specific DNA binding protein, the minor virion component IVa2, or the major capsid component hexon.
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We have identified adenovirus type 2 (Ad2)-induced early polypeptides (EPs) and have attempted to determine which EPs are coded by each of the four early gene blocks. [35S]methionine-labeled EPs were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cycloheximide pretreatment followed by labeling in hypertonic medium (210 to 250 mM NaCl) facilitated the detection of EPs. Seven major (reproducible bands in autoradiograms) EPs were detected with molecular weights of 74,000 (74K), 21K, 19K, 15K, 13.5K, 11.5K, and 11K. Minor (weaker bands) EPs of 55K, 52K, 42K, 18K, 12K, 8.8K, and 8.3K were also often seen. To identify and map the genes for virus-coded EPs, we prepared antisera against five lines of adenovirus-transformed cells that retain different fractions of the viral genome. The lines were F17, 8617, F4, and T2C4 transformed by Ad2 virions and 5RK (clone I) transformed by transfection with the Ad5 HsuI-G fragment (map position 0 to 8). The early gene blocks retained and expressed (in part) as RNA in these cells were as follows: 5RK(I), block 1 (70% of left 8% of genome); F17, block 1; 8617, blocks 1 and 4; F4 blocks 1, 2, and 4; T2C4, blocks 1, 2, 3, and 4. The following major EPs were immunoprecipitated: 15K by all antisera; 53K and 14.5K by F17, T2C4, 8617, and F4 antisera; 11.5K by T2C4, 8617, and F4 antisera; 44K, 42K, 19K, and 13.5K by T2C4 antisera; 11K by 8617 antisera. Minor EPs of 28K, 18K, and 12K were precipitated by all antisera except 5RK(I). The 53K and 15K EPs were precipitated also from Ad2 early infected monkey cells by the F17 antiserum and by sera from hamsters bearing tumors induced by Ad1-simian virus 40. The relationships between some of the immunoprecipitated EPs were investigated by the partial proteolysis procedure. All 53K EPs are the "same" (i.e., highly related), all 15K EPs are the "same," and all 11.5K EPs are the "same." The 15K EP is highly related to the 14.5 K EP. Although less certain, all 28K EPs appeared related, as did all 18K EPs. The T2C4-specific 44K EP is probably a dimer of the 21K glycopolypeptide. The T2C4-specific 13.5K EP and the 8617-specific 11K EP appear unrelated to any other polypeptides. These immunoprecipitation data provide evidence that early gene block I (map position 1 to 11) may encode major 53K, 15K, and 14.5K polypeptides, and minor 28K, 18K, and 12K polypeptides, and that all or some of the gene for 15K and 14.5K lies within map position 1 to 8. The surprisingly complex pattern of polypeptides coded by early gene block I raises the possibility that some polypeptides may be coded by overlapping "spliced" mRNA's. The possible block locations of the genes for the 21K, 13.5K, and 11.5K polypeptides are discussed.
Article
Serum from hamsters bearing group C adenovirus-induced tumors can be divided into two classes: first, a broad spectrum serum that contains antibodies to several early adenovirus proteins, immunoprecipitated from virus-infected cell extracts, with molecular weights of 72,000, 58,000, 44,000 and 17,000 daltons; and second, a narrow spectrum serum that contains antibodies to the 58,000 dalton protein from virus-infected cell extracts. Both types of sera have been used to immunoprecipitate specifically the 58,000 dalton protein from a type 2 adenovirus-transformed hamster cell line and a type 2 adenovirus-SV40 nondefective hybrid (Ad2+ND-1) transformed hamster cell line. In addition, the broad spectrum serum immunoprecipitates or co-precipitates a late adenovirus protein of 120,000 daltons from virus-infected, but not virus-transformed cells. Peptide maps of the 120,000 dalton antigen and the virus hexon structural protein (120,000 daltons) demonstrate that these proteins are closely related. The 72,000 dalton antigen has been shown to be the adenovirus single-strand-specific DNA binding protein. Peptide maps of this 72,000 dalton antigen demonstrate that it contains all the peptides found in the 44,000 dalton antigen. The 72,000 dalton antigen contains two additional peptide fragments not detected in the 44,000 dalton protein, indicating that this 44,000 dalton antigen is a proteolytic breakdown product of the 72,000 dalton protein. The 58,000 dalton adenovirus tumor antigen has a peptide map which is completely distinct from the 120,000, 72,000 and 44,000 dalton proteins. These data demonstrate that the 58,000 dalton antigen is chemically distinct from the 72,000-44,000 dalton early adenovirus proteins.
Article
The type 5 adenovirus single-strand-specific DNA-binding protein can be labeled with 32PO4 added as inorganic phosphate to the medium of infected monkey or human cells. The DNA binding protein in the infected cell can be isolated as a phosphoprotein at early and late times after infection. Most or all of the [32P]phosphate can be removed from the DNA-binding protein by alkaline phosphatase or hydrolysis at alkaline pH. The dephosphorylated protein retains the ability to bind specifically to single-stranded DNA. H5ts125 is a mutation in the structural gene of the 72,000 MW single-strand-specific DNA-binding protein. When cells infected with this viral mutant at the permissive temperature are shifted to the nonpermissive temperature, the phosphorylation of the 72,000 MW protein decreased rapidly (within 30 min) even though approximately normal (wild type) levels of this protein (labeled with 35S]methionine for 30 min) were detected in H5ts125-infected cells shifted to the nonpermissive temperature. An in vitro protein kinase assay utilizing nuclei from infected or uninfected cells has been developed. This in vitro assay can detect the phosphorylation of at least three adenovirus-induced phosphoproteins (100,000 MW; 72,000 MW; 36,000 MW) that are also observed when adenovirus-infected cells are labeled with inorganic [32P] phosphate in vivo. The protein kinase activity detected in this in vitro assay is not dependent upon or stimulated by the addition of exogenous cyclic AMP or cyclic GMP. The 72,000 M W protein phosphorylated in vitro by this phosphokinase activity has been shown to be the adenovirus single-strand-specific DNA-binding protein by a specific immunoprecipitation test.
Article
DNA-binding protein was characterized by previous investigators as a single-stranded DNA-binding protein analogous to the gene 32 protein of phage T4 (Van der Vliet & Levine, 1973; Sugawara et al., 1977). In the studies presented here the interactions between natural and synthetic polynucleotides and the DNA-binding protein of adenovirus 2-infected HeLa cells have been examined. Polynucleotide melting techniques revealed a tight yet dissociable binding to the helix structure of double-stranded DNA. In addition, binding and filter binding competition experiments at high DNA to protein ratios revealed a specific binding to double-stranded DNA termini with a dissociation constant of 1 × 10−9 to 2 × 10−9m. The ability of DNA-binding protein to bind to heat-denatured viral DNA was confirmed but the binding to double-stranded DNA termini was more specific on a molar basis. DNA-binding protein can recognize both flush and staggered ends of double-stranded DNA molecules.
Article
The animal viruses with DNA genomes have been classified into five groups—parvoviruses, papovaviruses, adenoviruses, herpesviruses, and poxviruses—in order of increasing complexity (Melnick, 1974). Each of these virus groups have unique features which provide favorable experimental material for the investigator. The papovaviruses and adenoviruses have been especially useful in the study of DNA structure and replication. The availability of large quantities of viral DNA and the comparatively small genome size of these viruses have permitted experimental approaches leading to a detailed understanding of the primary (Dahr et al., 1974a, b; Fiers et al., 1974; Roberts et al., 1974), secondary, and tertiary (Vinograd and Lebowitz, 1966) structure of these DNA molecules. Restriction enzymes have been employed to generate defined fragments of these genomes which in turn have been used to study DNA replication and transcription and to localize viral mutants on the physical map of these genomes (Tables 2 and 3). With both the adenoviruses and papovaviruses the outlines of a DNA replication scheme are now available, the replicative intermediates have been visualized and several viral and cellular functions involved in DNA replication have been characterized (Levine, 1974).
Article
An adenovirus type 2 (Ad2) DNA-binding protein was purified by sequential DNA-cellulose, Sephadex G-200, and DEAE-Sephadex chromatography, with a yield of 120 mug of binding protein (95 to 99% homogeneity) starting with 2 X 10(9) infected cells. By omitting the Sephadex G-200 step, 400 to 600 mug of 95% pure binding protein was obtained. To obtain high yields of highly purified binding protein, it was necessary to include deoxycholate and Nonidet P-40 at selected stages during the preparation. The highly purified binding protein appeared to have retained its native stage as indicated by: (i) binding to single-stranded but not native Ad2 DNA, (ii) almost complete precipitation by immunoglobulin G from hamsters immunized by extracts of tumors induced by Ad2-simian virus 40 hybrid viruses, and (iii) identical sedimentation coefficient with binding protein obtained from DNA-cellulose chromatography only. Zonal centrifugation in sucrose gradients and gel filtration revealed that purified binding protein has a sedimentation coefficient of 3.4S and a Stokes radius of 5.2 nm. Based on these two values, a molecular weight of 73,000 was calculated, in agreement with the estimate from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A frictional ratio of 1.88 was calculated, suggesting that the Ad2 DNA-binding protein does not have a typical globular protein structure.
Article
The adenovirus-type-5-coded single-strand-specific DNA-binding protein was purified from infected human KB cells and characterized by sucrose gradient centrifugation and gel filtration. The protein has a sedimentation coefficient of 3.3 S and a diffusion coefficient of 4.4 × 10−7 cm/s, which corresponds to a molecular weight of 68000 for the native protein. This is close to the peptide molecular weight of 72000 and indicates that the protein is a monomer. The frictional coefficient ratio is 1.82 which suggests that the DNA-binding protein is highly anisometric. The binding of the protein to various nucleic acids was studied by filtration through nitrocellulose filters and sucrose gradient centrifugation. The binding to single-stranded DNA is a fast process which does not require bivalent cations or sulfhydryl groups and occurs over a broad pH range. The binding to synthetic polydeoxyribonucleotides is at least 10-fold less efficient than that to natural single-stranded DNA. At low protein-to-DNA ratios the binding to single-stranded DNA is cooperative. A maximal protein to DNA ratio of 33 (w/w) can be obtained which corresponds to 1 protein molecule for about 7 nucleotides. Sedimentation and electron microscopic studies indicate that the DNA-binding protein keeps single-stranded DNA in an extended configuration. The saturated nucleoprotein complex is slightly more resistant to nuclease digestion than single-stranded DNA.
Article
DNA tumor viruses have contributed immense wealth of knowledge in the past few years regarding the eukaryotic cellular processes involving replication, transcription, and translation. Adenoviruses (Ad) in particular have played a pioneering and significant role in the understanding of the mechanisms of many of these biological processes mainly due to the interaction of viral proteins with the host proteins during the virus life cycle. The development of the first cell-free system to study Ad DNA replication (Challberg and Kelly 1979; for reviews, see Challberg and Kelly 1989; Stillman 1989; Hay and Russell 1989) was pivotal to our current understanding of eukaryotic DNA replication.
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Infection of African green monkey kidney cells with type 5 adenovirus leads to the synthesis of two infected, cell-specific proteins with approximate molecular weights of 72,000 and 48,000, that bind specifically to single-stranded but not double-stranded DNA. The production of these two proteins was studied after infection with two DNA-negative adenovirus mutants belonging to different complementation groups (H5 ts36 and H5 ts 125). Both DNA binding proteins were detected in cells infected with either mutant at the permissive temperature (32 C) AND ALSO IN H5 ts36-infected cells at the nonpermissive temperature (39.5 C). In H5 ts125-infected cells at 39.5 C, however, less than 5% of the normal wild-type level of these DNA binding proteins was detectable. When H5 ts125-infected cells were labeled with radioactive leucine at 32 C and subsequently shifted to 39.5 C in the presence of unlabeled leucine (chase), the level of DNA binding proteins found in these infected cells was markedly reduced compared to cultures not shifted to 39.5 C. These data suggest that the DNA binding proteins themselves were temperature sensitive. This conclusion was confirmed by experiments in which the DNA binding proteins were eluted from DNA cellulose with buffers of increasing temperatures (thermal elution). The H5 ts 125 proteins were shown to elute at lower temperatures than either wild-type or H5 ts36 proteins. These results are taken to indicate that the H5 ts125 mutant codes for a DNA binding protein that is thermolabile for continued binding to single-stranded DNA.
Article
1. The human adenoviruses types 2, 5 and 12 code for the production of a single strand specific DNA binding protein. The molecular weights of these proteins were 72,000 for types 2 and 5 and 60,000 for type 12. In all three cases proteolytic breakdown fragments of these binding proteins (48,000 MW) were also observed.
Article
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 48,000. 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.
Article
During productive infection of human cells with types 2 or 5 adenovirus a number of proteins specific for the infected cells, but not found in the intact virion, can be detected by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS). None of these proteins, however, has been isolated in a native state and therefore their functions remain obscure. The observation that replicating adenovirus DNA contained extensive single stranded regions led the authors to look for proteins specific for infected cells that bind only to single stranded DNA and may be involved directly in DNA replication. Such a class of proteins has now been found in a number of prokaryotic system as well as in mammalian cells, and they are known, at least in the case of T4 gene 32 protein, to be required for DNA replication and genetic recombination. To eliminate the large excess of adenovirus coat proteins, synthesised in human cells, which are known to bind to DNA, African green monkey cells were used as hosts. In these cells viral DNA synthesis occurs at a normal rate but late viral capsid proteins are not produced or are synthesised in very small amounts. Two adenovirus infected cell specific DNA binding proteins with molecular weights of 72,000 and 48,000 were isolated. Neither protein was present in the purified virion and the synthesis of these proteins was not blocked by inhibitors of DNA replication. Both proteins bound preferentially to single stranded DNA and to the replicative intermediates of adenovirus DNA, while no binding to native viral DNA was detectable.
Article
Radioisotope labeling has been used to analyze the replication of SV40 DNA in primary African green monkey kidney cells. The incorporation of [3H]thymidine into viral-specific DNA begins at about 15 hours after infection and reaches a maximum rate at 30 hours.Sedimentation of viral DNA through neutral sucrose gradients indicates that little or no supercoiled viral DNA (20 to 21 s) is synthesized during the first five minutes of labeling with [3H]thymidine (at 30 hr after infection). Instead, a viral DNA form that sedimented at 24 to 25 s is observed. The 3H-labeled 25 s DNA can be chased into viral supercoiled DNA by the addition of unlabeled thymidine to the culture. This viral precursor DNA, unlike mature viral closed circular DNA, is completely denaturable in alkali and bands at a lighter density than closed circular SV40 DNA in an ethidium bromide-cesium chloride equilibrium gradient.Benzoylated-naphthoylated DEAE-cellulose column chromatography was used to fractionate mature (21 s) and replicating (25 s) SV40 DNA. Viral supercoiled DNA elutes from such a column between 0.55 and 0.6 m-NaCl. Under these same conditions, replicating SV40 DNA remains bound to the column and can be eluted later with caffeine. Electron micrographs of viral DNA obtained from the caffeine-eluted fraction of the column show replicating SV40 molecules with two branch points, three branches and no visible ends (circles). About 75% of these molecules have completed 90 to 95% of their replication.
Article
Eighty-eight temperature-sensitive (ts) mutants of adenovirus 12, which did not replicate at 38° but did replicate well at 31° were isolated. Thirty-four of these mutants were selected and classified into 13 groups (groups A to M) by complementation tests. Analyses of the defects in viral replication at the nonpermissive temperature (38°) revealed that mutants in 4 groups (groups A, B, C, and D) were defective in the production of hexon, fiber, and penton base, mutants in group E were defective in the production of both fiber and penton base, mutants in group F were defective in the production of both hexon and penton base, a mutant in group G was defective in the production of both hexon and fiber, mutants in group H were defective in the production of only hexon, mutants in group I were defective in the production of only fiber, mutants in group J were defective in the production of only penton base and mutants in 3 groups (groups K, L, and M) were not defective in the synthesis of hexon, fiber, and penton base. These defectiveness were detected by serological reaction and polyacrylamide gel electrophoresis. Immunofluorescent examinations of cells infected with these mutants as well as the examination of heat stability of virions produced at the permissive temperature showed differences among these groups.
Article
Analysis of (35)S-methionine-labeled extracts of adenovirus 2-infected KB cells revealed 22 virus-induced polypeptide components. Most proteins of the virion were easily detected in extracts of whole cells labeled for short periods between 15 and 30 h after infection; however, several virion components were conspicuously absent. Radioactivity appeared in two of these virion components during a chase in nonradioactive medium, and this appearance was paralleled by a decrease in the radioactivity associated with two nonvirion adenovirus-induced proteins, results which imply precursor-product relationships for these components. Comparison of one of the chasable adenovirus-induced components (designated P-VII; mass of 20,000 daltons) and the major core protein (VII; mass of 18,500 daltons) of the virion showed that they have four common methionine-containing tryptic peptides; P-VII has an additional methionine residue which is not found in the major core protein. We propose that at least two of the adenovirus 2 virion components are derived by the cleavage of higher molecular weight precursor polypeptides.