JOURNAL
Vol. 66, No. 1
OF VIROLOGY, Jan. 1992, p. 480-488 0022-538X/92/010480-09$02.00/0 Copyright X) 1992, American Society for Microbiology
Functional Changes in Temperature-Sensitive Mutants of the Adenovirus Single-Stranded DNA-Binding Protein Are Accompanied by Structural Alterations MASAYOSHI TSUJI AND GEOFFREY R. KITCHINGMAN* Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101-0318 Received 5 August 1991/Accepted 9 October 1991
Adenovirus requires the virus-encoded single-stranded DNA-binding protein (DBP) to replicate its DNA. We have previously shown (M. Tsuji, P. C. van der Vliet, and G. R. Kitchingman, J. Biol. Chem. 266:1617816187, 1991) that the inability of three temperature-sensitive (ts) mutant DBPs (Ad2+ NDlts23, Ad2ts111A, and Ad5ts125) to support DNA replication at the nonpermissive temperature was associated with impaired ability to bind to DNA. In this study, we examined these mutant proteins for structural alterations that might be linked to the functional changes. All three ts mutants, but not the wild-type protein, showed different proteolytic cleavage patterns before and after heating at 40°C (the nonpermissive temperature), suggesting a possible conformational change during heating. The Ad2+NDlts23 and Ad2ts111A DBPs have single amino acid changes located in a putative zinc finger subdomain (positions 282 and 280). In the presence of zinc ions, these ts mutants showed significantly increased resistance to inactivation at 40°C. Surprisingly, however, the stabilizing effect of zinc was also observed with the Ad5ts125 DBP, which contains a mutation located more than 100 amino acids from the zinc finger. Other related metal ions, such as cobalt, cadmium, and mercury, did not protect the ts DBPs from inactivation at 40°C. These results indicate that functional changes of the ts DBPs in DNA replication and DNA binding are accompanied by structural alterations in the protein and that zinc and the metal-binding subdomain may play an important role in the structure and/or function of the DBP.
One of the viral proteins expressed during the early phase of adenovirus infection is a single-stranded (ss) DNA-binding protein (DBP). This protein is produced in large quantities and is localized predominantly in the nuclei of infected cells. The DBP performs multiple functions during the infectious cycle, including support of viral DNA replication (reviewed in reference 10), regulation of viral gene expression at both the transcriptional (24) and the posttranscriptional (3) levels, determination of host range (14), and involvement in the virus assembly process (25). The protein binds preferentially to ssDNA with no apparent sequence specificity and binds to both double-stranded DNA (8, 29, 32) and RNA (1, 5, 30). The apparent molecular weight of the DBP, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), is 72,000 (13), but the protein is actually composed of 529 amino acids, which gives a calculated molecular weight of 59,049 (16). This discrepancy is probably due to a high degree of phosphorylation (13) and to the proline-rich nature of the protein (16), leading to the anomalous migration in SDS-containing gels. Biochemical studies have revealed the presence of two structurally and functionally distinct domains in the protein. Mild chymotrypsin digestion splits the protein into highly phosphorylated 26-kDa amino-terminal and nonphosphorylated 44-kDa carboxyl-terminal fragments (13). The carboxyl-terminal domain is capable of binding to ssDNA (13) and RNA (5) and is fully functional in supporting DNA replication in vitro (2, 9). Genetic studies have identified amino acid residues important for nuclear localization (21) and for host range (16) in the amino-terminal domain of the protein. Three highly conserved regions (designated CR1, CR2, and CR3) within the carboxyl-terminal domain have *
been found by comparative sequence analysis (12) among the DBPs of various human adenovirus serotypes. Some of the mutations introduced in CR2 (amino acids 322 to 330) and CR3 (amino acids 464 to 475) substantially decrease the protein's ability to bind to ssDNA and to support adenoassociated virus DNA replication in vivo (22, 23) without affecting its in vivo stability, phosphorylation, or subcellular localization. Thus, these two regions of the DBP may be directly involved in DNA binding. Three temperature-sensitive (ts) adenovirus mutants (Ad2+ND1ts23, Ad2tslllA, and Ad5tsl25) that are defective in DNA replication at the nonpermissive temperature have been isolated, identifying two other regions of the carboxy-terminal domain important for the functions of the protein. The Ad2+NDlts23 (17) and Ad2tslllA (27) mutations are in close proximity (a Leu-to-Phe change at amino acid 282 and a Gly-to-Val change at amino acid 280, respectively), and these amino acids are flanked by -His-X-Cysand -Cys-X-His-, which has the potential to coordinate a zinc ion (37) in a fashion similar to that found in "zinc finger" proteins (4, 7). These mutant proteins appear to be stable and properly phosphorylated in vivo at the nonpermissive temperature (27, 31). The Ad5tsl25 DBP (6), which has a Pro-to-Ser change at amino acid 413 (16), has been used to identify most of the DBP's functions in RNA metabolism, as well as its function in DNA replication. The mutant protein produced in vivo at the nonpermissive temperature is poorly phosphorylated (19) and rapidly degraded (11, 34), suggesting that the mutation is in a region that is important for the protein's stability. Interestingly, however, functional defects caused by this mutation can be corrected phenotypically by secondary mutations at amino acid 347, 352, or 508 (15, 26). Collectively, these DBP mutants identify four regions important for proper functioning of the protein. We have previously demonstrated that the three ts mutant
Corresponding author. 480
VOL. 66, 1992
TEMPERATURE-SENSITIVE ADENOVIRUS DNA-BINDING PROTEINS
DBPs lose the ability to bind to oligonucleotides when heated at the nonpermissive temperature, suggesting that their inability to support DNA replication results from impaired DNA-binding activity (33). A key question that remained to be answered was whether the mutations are located in the sites of the protein that directly interact with ssDNA, thereby causing a functional change without altering the overall protein structure, or whether heating at the nonpermissive temperature induces a structural alteration that affects DNA binding and perhaps interaction with the adenovirus DNA polymerase (20). To address this question, we examined the proteolytic cleavage patterns of the three ts mutant proteins before and after heating at the nonpermissive temperature and found differences that were indicative of a possible conformational change. We also investigated a possible role of zinc ion and the putative metal-binding subdomain (amino acids 273 to 286) in the functions of the DBP because both the Ad2+NDlts23 and Ad2ts1llA mutations are located within this region. In the presence of zinc, these two mutant proteins, as well as the Ad5tsl25 DBP, retain DNA-binding activity over significantly extended periods at the nonpermissive temperature. MATERIALS AND METHODS DBPs. The wild-type and three ts mutant DBPs were purified (29) from KB cells infected with AdSdl301, Ad2+NDlts23, Ad2tslllA, and AdStsl25 with the modifications described previously (33). Proteolytic digestions. DBP preparations were diluted to 0.5 mg/ml in TEMG buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 2 mM 2-mercaptoethanol [2-ME], 10% glycerol) containing 100 mM NaCl. These samples were either digested directly or heated to 40°C for 30 min prior to digestion. Reactions were set up on ice by mixing 2 R1 of DBPs (1 ,ug) and 4 ,ul of proteolytic enzymes (1 ng/,lp) in the buffers described below. Tosylsulfonyl phenylalanyl chloromethylketone-treated trypsin and tosyl lysine chloromethyl ketone-treated chymotrypsin (both from Sigma) digestions were performed in buffer P (40 mM Tris-HCI [pH 8.0], 1 mM EDTA, 2 mM 2-ME, 10% glycerol) containing 0.5 M NaCl at 30°C for 15 and 20 min, respectively, and stopped with 0.5 p.1 of 100 mM phenylmethylsulfonyl fluoride. Staphylococcus aureus V-8 protease (Pierce) digestions were done in 100 mM phosphate buffer (pH 7.8) at 30°C for 30 min and terminated by heating at 90°C for 5 min. Thermolysin (Sigma) digestions were done in 20 mM Tris-HCl (pH 8.0-2 mM CaCl2-1.4 mM 2-ME-5% glycerol at 30°C for 15 min and stopped with 2 ,ul of 50 mM EDTA. Papain (Sigma) digestions were performed in buffer P at 30°C for 15 min and stopped with 1 pl of 50 mM iodoacetamide. Proteinase K (Bethesda Research Laboratories) digestions were also performed in buffer P at 30°C for 15 min and stopped with 0.5 ,ul of 100 mM phenylmethylsulfonyl fluoride. Proteolytic fragments were separated by SDS-PAGE (18) and visualized with the Bio-Rad Silver Stain kit. Gel mobility shift assay. Details of the gel shift assay using an 84-mer DNA have been described previously (33). Briefly, the wild-type and ts DBPs, either untreated or treated as described below, were mixed with specified amounts of 32P-labeled 84-mer DNA. Samples were incubated on ice for at least 60 min and then electrophoresed through a 1% agarose gel in TBE (45 mM Tris-borate, 1 mM EDTA [pH 8.3]). Gels were partially dehydrated and exposed to Kodak XAR film with Cronex Lightning-Plus intensifying screens.
481
Stabilizing effect of DNA. The DBPs (200 ng) in TEMG buffer containing 20 mM NaCl and 200 jig of bovine serum albumin per ml were either heated to 40°C for various periods and then mixed on ice with 5 ng of 32P-labeled 84-mer DNA or mixed first with 5 ng of 32P-labeled 84-mer DNA on ice and then incubated at 40°C for various periods. Free and protein-bound DNAs were separated at 4°C by the gel mobility shift assay. Stabilizing effect of antibody. A monoclonal antibody against DBP (B6 antibody in reference 28) was purified from hybridoma culture supernatants by affinity chromatography by using an anti-mouse immunoglobulin G (IgG)-Sepharose 4B column. Four sets of reactions were carried out with 200 ng of DBPs in TEMG buffer containing 20 mM NaCl and 200 ,ug of bovine serum albumin per ml. In the first, DBPs alone were incubated at 40°C for various periods and then mixed with 5 ng of 32P-labeled 84-mer DNA. In the second and third sets of reactions, DBPs were mixed with 200 ng of either unimmunized mouse IgG (Sigma) or purified B6 IgG, the mixtures were heated at 40°C for various periods, and 5 ng of 32P-labeled 84-mer DNA was added. In the fourth assay, DBPs were heated at 40°C for various periods and then mixed with 200 ng of B6 IgG and S ng of 32P-labeled 84-mer DNA. Reactions were analyzed at 4°C by the gel mobility shift assay. Effects of various metal ions on DNA-binding activity of DBP. Solutions (15 p.l) containing 250 ng of DBP, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM 2-ME (omitted when CoS04 was used), 20 mM NaCl, 0.2 mg of bovine serum albumin per ml, 10% glycerol, and either zinc acetate, CoS04, CdCl2, HgCl2, MnCl2, or MgCl2 at 2 mM were incubated at 40°C for the periods indicated in the figures. Samples were chilled on ice, and EDTA was added to a final concentration of 2.5 mM. DNA-binding activity was tested at 4°C with 5 ng of 32P-labeled 84-mer DNA in the gel mobility shift assay. EM. Samples (10 ,ul) for electron microscopy (EM) contained 0.1 ,ug of M13mpl8 ssDNA and 4 jig of DBPs (either unheated or heated at 40°C for 30 min) in 10 mM Tris-HCl (pH 8.0-1 mM EDTA-2 mM 2-ME-20 mM NaCI. The samples were either diluted with 90 p.l of 50% formamide in 100 mM Tris-HCI (pH 8.0-10 mM EDTA, mixed with 2.5 ,ul of 0.5% cytochrome c, and spread on distilled water or diluted with 90 p,l of 0.25 M ammonium acetate, mixed with 2.5 ,u1 of 1% cytochrome c, and spread onto a 0.025 M ammonium acetate hypophase. Materials on the surface were picked up with carbon-coated grids, stained with alcoholic uranyl acetate, rotary shadowed with platinum, and examined by a Philips 301 EM. RESULTS Proteolytic digestion patterns. We previously demonstrated (33) that ts DBPs Ad2+NDlts23, Ad2tslllA, and AdStsl25 have wild-type activity at 30°C in both the oligonucleotide-binding and the in vitro DNA replication assays but irreversibly lose both activities by heating at 40°C. The mechanism underlying this change could be either a small structural change confined to the DNA-binding site in the protein or a global conformational change that makes the protein no longer functional. One method of distinguishing between these two possibilities is examination of the mutant DBPs before and after heating at 40°C for susceptibility to proteolytic enzymes, as the accessibility of an enzyme to a potential cleavage site would differ as a protein undergoes a
conformational change.
482
J. VIROL.
TSUJI AND KITCHINGMAN
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The wild-type and three mutant DBPs, either unheated or heated at 40°C for 30 min, were digested separately with six proteolytic enzymes having a range of substrate specificities, from highly specific (trypsin and V-8 protease) to nonspecific (proteinase K). With each of the six enzymes, all three ts mutant proteins yielded at least one characteristic band that was different between the heated and unheated samples, while the wild-type protein produced identical digestion patterns under the two conditions (Fig. 1). For chymotrypsin, V-8 protease, trypsin, and thermolysin, lower-molecular-weight peptide fragments were produced when the unheated but not heated DBP was used as a substrate, suggesting that a cleavage site was rendered inaccessible by heating. With papain and proteinase K, digestion of the DBP to lower-molecular-weight fragments was observed following heating, indicating increased accessibility to these enzymes. Thus, the ts mutants, but not the wild-type protein, probably undergo a conformational change at the nonpermissive temperature. This does not necessarily mean that the proteins are totally denatured: the ts DBPs that had been heated to 40°C yielded specific cleavage fragments (Fig. 1), whereas those heated to 90°C for 10 min did not have such specific fragments, presumably because of a more random conformation (data not shown). The unheated ts mutant DBPs and those heated at 30°C for 30 min had digestion patterns similar to that of wild-type DBP (data not shown), suggesting that the mutations do not significantly alter the
proteins' overall structures as long as they are produced and kept at the permissive temperature. Interestingly, digestion of the unheated ts DBPs with the less specific proteolytic enzymes papain and proteinase K yielded a predominant cleavage fragment with a molecular weight of approximately 39,000. The production of such a fragment suggests the presence of a rigid, compactly folded region within the protein, probably corresponding to a part of the carboxylterminal domain. Loss of this fragment with heating to 40°C indicates that the mutations are located at sites that are crucial for maintenance of the compactly folded structure. Stabilization of ts DBPs by DNA and antibody. If a structural change in the mutant proteins is the cause of their ts phenotype, interaction of the DBP with other macromolecules might interfere with conformational changes, protecting the proteins from functional inactivation at the nonpermissive temperature. Two macromolecules, an 84-mer ssDNA and an anti-DBP monoclonal antibody (B6), were chosen to test this hypothesis. The wild-type and three ts DBPs were heated at 40°C for various periods and then mixed with 32P-labeled 84-mer DNA or first mixed with 32P-labeled 84-mer DNA and then incubated at 40°C. The DBP-DNA complex and free DNA were separated at 4°C by agarose gel electrophoresis (Fig. 2). The ts DBPs existing free in solution were all rapidly inactivated at 40°C, but the proteins that had bound to the 84-mer DNA remained associated with the DNA at 40°C for
VOL. 66, 1992
TEMPERATURE-SENSITIVE ADENOVIRUS DNA-BINDING PROTEINS
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as long as 80 min, indicating that binding to DNA rendered the ssDNA-binding activity of the ts DBPs resistant to thermal inactivation. To examine the effect of antibody, DBPs alone or mixed with either unimmunized mouse IgG or purified anti-DBP antibody were heated at 40°C for various periods and their DNA-binding activities were examined at 4°C by the gel mobility shift assay using 32P-labeled 84-mer DNA (Fig. 3). Binding of antibody to the DBP resulted in a further shift of
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the mobility of the DNA band. The B6 anti-DBP monoclonal antibody did not interfere with binding of DBP to DNA. Thus, the epitope recognized by the antibody, which has been mapped between amino acids 170 and 270 of the DBP (23), does not appear to be the site that directly interacts with ssDNA. The ts DBPs bound to the antibody retained their DNA-binding activity at 40°C for significantly longer periods than did the unbound DBPs. The antibody had no effect when it was added to the ts DBPs after they had been incubated at 40°C for 30 min. EM of DBP-M13 DNA complexes. We previously showed that the ts DBPs lost the ability to bind to oligonucleotides after heating at 40°C but retained some binding activity to larger ssDNA molecules, such as M13 DNA (33). This observation led us to speculate that the ts DBPs, when
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Vol. 66, No. 1
OF VIROLOGY, Jan. 1992, p. 480-488 0022-538X/92/010480-09$02.00/0 Copyright X) 1992, American Society for Microbiology
Functional Changes in Temperature-Sensitive Mutants of the Adenovirus Single-Stranded DNA-Binding Protein Are Accompanied by Structural Alterations MASAYOSHI TSUJI AND GEOFFREY R. KITCHINGMAN* Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101-0318 Received 5 August 1991/Accepted 9 October 1991
Adenovirus requires the virus-encoded single-stranded DNA-binding protein (DBP) to replicate its DNA. We have previously shown (M. Tsuji, P. C. van der Vliet, and G. R. Kitchingman, J. Biol. Chem. 266:1617816187, 1991) that the inability of three temperature-sensitive (ts) mutant DBPs (Ad2+ NDlts23, Ad2ts111A, and Ad5ts125) to support DNA replication at the nonpermissive temperature was associated with impaired ability to bind to DNA. In this study, we examined these mutant proteins for structural alterations that might be linked to the functional changes. All three ts mutants, but not the wild-type protein, showed different proteolytic cleavage patterns before and after heating at 40°C (the nonpermissive temperature), suggesting a possible conformational change during heating. The Ad2+NDlts23 and Ad2ts111A DBPs have single amino acid changes located in a putative zinc finger subdomain (positions 282 and 280). In the presence of zinc ions, these ts mutants showed significantly increased resistance to inactivation at 40°C. Surprisingly, however, the stabilizing effect of zinc was also observed with the Ad5ts125 DBP, which contains a mutation located more than 100 amino acids from the zinc finger. Other related metal ions, such as cobalt, cadmium, and mercury, did not protect the ts DBPs from inactivation at 40°C. These results indicate that functional changes of the ts DBPs in DNA replication and DNA binding are accompanied by structural alterations in the protein and that zinc and the metal-binding subdomain may play an important role in the structure and/or function of the DBP.
One of the viral proteins expressed during the early phase of adenovirus infection is a single-stranded (ss) DNA-binding protein (DBP). This protein is produced in large quantities and is localized predominantly in the nuclei of infected cells. The DBP performs multiple functions during the infectious cycle, including support of viral DNA replication (reviewed in reference 10), regulation of viral gene expression at both the transcriptional (24) and the posttranscriptional (3) levels, determination of host range (14), and involvement in the virus assembly process (25). The protein binds preferentially to ssDNA with no apparent sequence specificity and binds to both double-stranded DNA (8, 29, 32) and RNA (1, 5, 30). The apparent molecular weight of the DBP, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), is 72,000 (13), but the protein is actually composed of 529 amino acids, which gives a calculated molecular weight of 59,049 (16). This discrepancy is probably due to a high degree of phosphorylation (13) and to the proline-rich nature of the protein (16), leading to the anomalous migration in SDS-containing gels. Biochemical studies have revealed the presence of two structurally and functionally distinct domains in the protein. Mild chymotrypsin digestion splits the protein into highly phosphorylated 26-kDa amino-terminal and nonphosphorylated 44-kDa carboxyl-terminal fragments (13). The carboxyl-terminal domain is capable of binding to ssDNA (13) and RNA (5) and is fully functional in supporting DNA replication in vitro (2, 9). Genetic studies have identified amino acid residues important for nuclear localization (21) and for host range (16) in the amino-terminal domain of the protein. Three highly conserved regions (designated CR1, CR2, and CR3) within the carboxyl-terminal domain have *
been found by comparative sequence analysis (12) among the DBPs of various human adenovirus serotypes. Some of the mutations introduced in CR2 (amino acids 322 to 330) and CR3 (amino acids 464 to 475) substantially decrease the protein's ability to bind to ssDNA and to support adenoassociated virus DNA replication in vivo (22, 23) without affecting its in vivo stability, phosphorylation, or subcellular localization. Thus, these two regions of the DBP may be directly involved in DNA binding. Three temperature-sensitive (ts) adenovirus mutants (Ad2+ND1ts23, Ad2tslllA, and Ad5tsl25) that are defective in DNA replication at the nonpermissive temperature have been isolated, identifying two other regions of the carboxy-terminal domain important for the functions of the protein. The Ad2+NDlts23 (17) and Ad2tslllA (27) mutations are in close proximity (a Leu-to-Phe change at amino acid 282 and a Gly-to-Val change at amino acid 280, respectively), and these amino acids are flanked by -His-X-Cysand -Cys-X-His-, which has the potential to coordinate a zinc ion (37) in a fashion similar to that found in "zinc finger" proteins (4, 7). These mutant proteins appear to be stable and properly phosphorylated in vivo at the nonpermissive temperature (27, 31). The Ad5tsl25 DBP (6), which has a Pro-to-Ser change at amino acid 413 (16), has been used to identify most of the DBP's functions in RNA metabolism, as well as its function in DNA replication. The mutant protein produced in vivo at the nonpermissive temperature is poorly phosphorylated (19) and rapidly degraded (11, 34), suggesting that the mutation is in a region that is important for the protein's stability. Interestingly, however, functional defects caused by this mutation can be corrected phenotypically by secondary mutations at amino acid 347, 352, or 508 (15, 26). Collectively, these DBP mutants identify four regions important for proper functioning of the protein. We have previously demonstrated that the three ts mutant
Corresponding author. 480
VOL. 66, 1992
TEMPERATURE-SENSITIVE ADENOVIRUS DNA-BINDING PROTEINS
DBPs lose the ability to bind to oligonucleotides when heated at the nonpermissive temperature, suggesting that their inability to support DNA replication results from impaired DNA-binding activity (33). A key question that remained to be answered was whether the mutations are located in the sites of the protein that directly interact with ssDNA, thereby causing a functional change without altering the overall protein structure, or whether heating at the nonpermissive temperature induces a structural alteration that affects DNA binding and perhaps interaction with the adenovirus DNA polymerase (20). To address this question, we examined the proteolytic cleavage patterns of the three ts mutant proteins before and after heating at the nonpermissive temperature and found differences that were indicative of a possible conformational change. We also investigated a possible role of zinc ion and the putative metal-binding subdomain (amino acids 273 to 286) in the functions of the DBP because both the Ad2+NDlts23 and Ad2ts1llA mutations are located within this region. In the presence of zinc, these two mutant proteins, as well as the Ad5tsl25 DBP, retain DNA-binding activity over significantly extended periods at the nonpermissive temperature. MATERIALS AND METHODS DBPs. The wild-type and three ts mutant DBPs were purified (29) from KB cells infected with AdSdl301, Ad2+NDlts23, Ad2tslllA, and AdStsl25 with the modifications described previously (33). Proteolytic digestions. DBP preparations were diluted to 0.5 mg/ml in TEMG buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 2 mM 2-mercaptoethanol [2-ME], 10% glycerol) containing 100 mM NaCl. These samples were either digested directly or heated to 40°C for 30 min prior to digestion. Reactions were set up on ice by mixing 2 R1 of DBPs (1 ,ug) and 4 ,ul of proteolytic enzymes (1 ng/,lp) in the buffers described below. Tosylsulfonyl phenylalanyl chloromethylketone-treated trypsin and tosyl lysine chloromethyl ketone-treated chymotrypsin (both from Sigma) digestions were performed in buffer P (40 mM Tris-HCI [pH 8.0], 1 mM EDTA, 2 mM 2-ME, 10% glycerol) containing 0.5 M NaCl at 30°C for 15 and 20 min, respectively, and stopped with 0.5 p.1 of 100 mM phenylmethylsulfonyl fluoride. Staphylococcus aureus V-8 protease (Pierce) digestions were done in 100 mM phosphate buffer (pH 7.8) at 30°C for 30 min and terminated by heating at 90°C for 5 min. Thermolysin (Sigma) digestions were done in 20 mM Tris-HCl (pH 8.0-2 mM CaCl2-1.4 mM 2-ME-5% glycerol at 30°C for 15 min and stopped with 2 ,ul of 50 mM EDTA. Papain (Sigma) digestions were performed in buffer P at 30°C for 15 min and stopped with 1 pl of 50 mM iodoacetamide. Proteinase K (Bethesda Research Laboratories) digestions were also performed in buffer P at 30°C for 15 min and stopped with 0.5 ,ul of 100 mM phenylmethylsulfonyl fluoride. Proteolytic fragments were separated by SDS-PAGE (18) and visualized with the Bio-Rad Silver Stain kit. Gel mobility shift assay. Details of the gel shift assay using an 84-mer DNA have been described previously (33). Briefly, the wild-type and ts DBPs, either untreated or treated as described below, were mixed with specified amounts of 32P-labeled 84-mer DNA. Samples were incubated on ice for at least 60 min and then electrophoresed through a 1% agarose gel in TBE (45 mM Tris-borate, 1 mM EDTA [pH 8.3]). Gels were partially dehydrated and exposed to Kodak XAR film with Cronex Lightning-Plus intensifying screens.
481
Stabilizing effect of DNA. The DBPs (200 ng) in TEMG buffer containing 20 mM NaCl and 200 jig of bovine serum albumin per ml were either heated to 40°C for various periods and then mixed on ice with 5 ng of 32P-labeled 84-mer DNA or mixed first with 5 ng of 32P-labeled 84-mer DNA on ice and then incubated at 40°C for various periods. Free and protein-bound DNAs were separated at 4°C by the gel mobility shift assay. Stabilizing effect of antibody. A monoclonal antibody against DBP (B6 antibody in reference 28) was purified from hybridoma culture supernatants by affinity chromatography by using an anti-mouse immunoglobulin G (IgG)-Sepharose 4B column. Four sets of reactions were carried out with 200 ng of DBPs in TEMG buffer containing 20 mM NaCl and 200 ,ug of bovine serum albumin per ml. In the first, DBPs alone were incubated at 40°C for various periods and then mixed with 5 ng of 32P-labeled 84-mer DNA. In the second and third sets of reactions, DBPs were mixed with 200 ng of either unimmunized mouse IgG (Sigma) or purified B6 IgG, the mixtures were heated at 40°C for various periods, and 5 ng of 32P-labeled 84-mer DNA was added. In the fourth assay, DBPs were heated at 40°C for various periods and then mixed with 200 ng of B6 IgG and S ng of 32P-labeled 84-mer DNA. Reactions were analyzed at 4°C by the gel mobility shift assay. Effects of various metal ions on DNA-binding activity of DBP. Solutions (15 p.l) containing 250 ng of DBP, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM 2-ME (omitted when CoS04 was used), 20 mM NaCl, 0.2 mg of bovine serum albumin per ml, 10% glycerol, and either zinc acetate, CoS04, CdCl2, HgCl2, MnCl2, or MgCl2 at 2 mM were incubated at 40°C for the periods indicated in the figures. Samples were chilled on ice, and EDTA was added to a final concentration of 2.5 mM. DNA-binding activity was tested at 4°C with 5 ng of 32P-labeled 84-mer DNA in the gel mobility shift assay. EM. Samples (10 ,ul) for electron microscopy (EM) contained 0.1 ,ug of M13mpl8 ssDNA and 4 jig of DBPs (either unheated or heated at 40°C for 30 min) in 10 mM Tris-HCl (pH 8.0-1 mM EDTA-2 mM 2-ME-20 mM NaCI. The samples were either diluted with 90 p.l of 50% formamide in 100 mM Tris-HCI (pH 8.0-10 mM EDTA, mixed with 2.5 ,ul of 0.5% cytochrome c, and spread on distilled water or diluted with 90 p,l of 0.25 M ammonium acetate, mixed with 2.5 ,u1 of 1% cytochrome c, and spread onto a 0.025 M ammonium acetate hypophase. Materials on the surface were picked up with carbon-coated grids, stained with alcoholic uranyl acetate, rotary shadowed with platinum, and examined by a Philips 301 EM. RESULTS Proteolytic digestion patterns. We previously demonstrated (33) that ts DBPs Ad2+NDlts23, Ad2tslllA, and AdStsl25 have wild-type activity at 30°C in both the oligonucleotide-binding and the in vitro DNA replication assays but irreversibly lose both activities by heating at 40°C. The mechanism underlying this change could be either a small structural change confined to the DNA-binding site in the protein or a global conformational change that makes the protein no longer functional. One method of distinguishing between these two possibilities is examination of the mutant DBPs before and after heating at 40°C for susceptibility to proteolytic enzymes, as the accessibility of an enzyme to a potential cleavage site would differ as a protein undergoes a
conformational change.
482
J. VIROL.
TSUJI AND KITCHINGMAN
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The wild-type and three mutant DBPs, either unheated or heated at 40°C for 30 min, were digested separately with six proteolytic enzymes having a range of substrate specificities, from highly specific (trypsin and V-8 protease) to nonspecific (proteinase K). With each of the six enzymes, all three ts mutant proteins yielded at least one characteristic band that was different between the heated and unheated samples, while the wild-type protein produced identical digestion patterns under the two conditions (Fig. 1). For chymotrypsin, V-8 protease, trypsin, and thermolysin, lower-molecular-weight peptide fragments were produced when the unheated but not heated DBP was used as a substrate, suggesting that a cleavage site was rendered inaccessible by heating. With papain and proteinase K, digestion of the DBP to lower-molecular-weight fragments was observed following heating, indicating increased accessibility to these enzymes. Thus, the ts mutants, but not the wild-type protein, probably undergo a conformational change at the nonpermissive temperature. This does not necessarily mean that the proteins are totally denatured: the ts DBPs that had been heated to 40°C yielded specific cleavage fragments (Fig. 1), whereas those heated to 90°C for 10 min did not have such specific fragments, presumably because of a more random conformation (data not shown). The unheated ts mutant DBPs and those heated at 30°C for 30 min had digestion patterns similar to that of wild-type DBP (data not shown), suggesting that the mutations do not significantly alter the
proteins' overall structures as long as they are produced and kept at the permissive temperature. Interestingly, digestion of the unheated ts DBPs with the less specific proteolytic enzymes papain and proteinase K yielded a predominant cleavage fragment with a molecular weight of approximately 39,000. The production of such a fragment suggests the presence of a rigid, compactly folded region within the protein, probably corresponding to a part of the carboxylterminal domain. Loss of this fragment with heating to 40°C indicates that the mutations are located at sites that are crucial for maintenance of the compactly folded structure. Stabilization of ts DBPs by DNA and antibody. If a structural change in the mutant proteins is the cause of their ts phenotype, interaction of the DBP with other macromolecules might interfere with conformational changes, protecting the proteins from functional inactivation at the nonpermissive temperature. Two macromolecules, an 84-mer ssDNA and an anti-DBP monoclonal antibody (B6), were chosen to test this hypothesis. The wild-type and three ts DBPs were heated at 40°C for various periods and then mixed with 32P-labeled 84-mer DNA or first mixed with 32P-labeled 84-mer DNA and then incubated at 40°C. The DBP-DNA complex and free DNA were separated at 4°C by agarose gel electrophoresis (Fig. 2). The ts DBPs existing free in solution were all rapidly inactivated at 40°C, but the proteins that had bound to the 84-mer DNA remained associated with the DNA at 40°C for
VOL. 66, 1992
TEMPERATURE-SENSITIVE ADENOVIRUS DNA-BINDING PROTEINS
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as long as 80 min, indicating that binding to DNA rendered the ssDNA-binding activity of the ts DBPs resistant to thermal inactivation. To examine the effect of antibody, DBPs alone or mixed with either unimmunized mouse IgG or purified anti-DBP antibody were heated at 40°C for various periods and their DNA-binding activities were examined at 4°C by the gel mobility shift assay using 32P-labeled 84-mer DNA (Fig. 3). Binding of antibody to the DBP resulted in a further shift of
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FIG. 3. Gel shift assay showing stabilization of ts DBPs by an anti-DBP antibody (Ab). Four sets of experiments were carried out. DBPs alone were incubated at 40°C for the indicated periods (A). DBPs mixed with unimmunized mouse IgG were heated at 40°C (B). DBPs bound to B6 anti-DBP antibody were heated at 40°C (C). DBPs were incubated alone at 40°C for the indicated periods and then mixed with B6 antibody (D). DNA-binding activity was measured at 4°C by the gel mobility shift assay with 32P-labeled 84-mer DNA. Lanes E contained 32P-labeled 84-mer DNA only. Inc., incubation; wt, wild type.
the mobility of the DNA band. The B6 anti-DBP monoclonal antibody did not interfere with binding of DBP to DNA. Thus, the epitope recognized by the antibody, which has been mapped between amino acids 170 and 270 of the DBP (23), does not appear to be the site that directly interacts with ssDNA. The ts DBPs bound to the antibody retained their DNA-binding activity at 40°C for significantly longer periods than did the unbound DBPs. The antibody had no effect when it was added to the ts DBPs after they had been incubated at 40°C for 30 min. EM of DBP-M13 DNA complexes. We previously showed that the ts DBPs lost the ability to bind to oligonucleotides after heating at 40°C but retained some binding activity to larger ssDNA molecules, such as M13 DNA (33). This observation led us to speculate that the ts DBPs, when
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