Adenovirus DNA-binding protein in cells infected with wild-type 5 adenovirus and two DNA-minus, temperature-sensitive mutants, H5ts125 and H5ts149.

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.     

Initiation of adenovirus DNA replication: detection of covalent complexes between nucleotide and the 80-kilodalton terminal protein

We have previously shown that the 5'-terminal deoxycytidine residue of each nascent adenovirus 5 DNA strand synthesized in vitro is covalently linked to the 80-kilodalton (kd) terminal protein precursor via a phosphodiester bond to a serine residue in the protein. When extracts prepared from adenovirus 5-infected cells are incubated with [alpha-33P]dCTP as the only added deoxynucleoside triphosphate, complexes consisting of nucleotide covalently linked to the 80-kd protein can be detected. The nucleotide moieties present in such complexes include d(pC) and d(pCpA), the 5'-terminal nucleotide and dinucleotide of adenovirus 5 DNA, respectively, as well as some longer oligonucleotides. The formation of these complexes requires the presence of adenovirus DNA containing the attached 55-kd terminal protein and ATP. Extracts from H5ts125-infected cells which are defective in DNA replication catalyze complex formation to the same extent as extracts prepared from wild-type infected cells; thus, the presence of the adenovirus-coded 72-kd DNA-binding protein is apparently not required. Most, if not all, of the 80-kd protein-nucleotide complexes that are formed are noncovalently bound to the input viral DNA. These observations are consistent with the protein-priming model for the initiation of adenovirus DNA replication.     

Analysis of the adenovirus type 5 terminal protein precursor and DNA polymerase by linker insertion mutagenesis.

A series of adenovirus type 5 precursor terminal protein (pTP) and DNA polymerase (Ad pol) genes with linker insertion mutations were separately introduced into the vaccinia virus genome under the control of a late vaccinia virus promoter. The recombinant viruses were used for overexpression of the mutant genes in HeLa cells. In total, 22 different mutant pTP and 10 different Ad pol vaccinia virus recombinants were constructed, including some that expressed carboxyl-terminus-truncated forms of both proteins and one that produced the mutant H5ts149 Ad pol. To investigate the structure-function relationships of both proteins, extracts from cells infected with the recombinant viruses were tested for in vitro complementation of the initiation and elongation steps in adenovirus DNA replication. The results were in accordance with those of earlier in vivo experiments with these insertion mutants and indicate that multiple regions of both proteins are essential for adenovirus DNA replication. The carboxyl termini of both pTP and Ad pol were shown to be essential for proper functioning of these proteins during initiation of adenovirus DNA replication. Three different DNA replication-negative pTP mutants were shown to have residual activity in the initiation assay, suggesting not only that pTP is required for initiation but also that it may play a role in DNA replication after the deoxycytidylation step.     

Gene product of region E4 of adenovirus type 5 modulates accumulation of certain viral polypeptides.

An adenovirus type 5 mutant, designated H5ilE4I, was constructed in which region E4 was replaced by a cloned cDNA. The cDNA was a copy of an mRNA which exclusively contains open translational reading frames 6 and 7. The phenotype of the mutant was compared with that of the previously characterized E4 mutant H2dl808 and wild-type adenovirus 5. Although the H5ilE4I mutant lacked at least five E4 genes, it was nondefective for growth in HeLa cells. The defects in viral DNA replication, late protein synthesis, and shutoff of host cell protein synthesis associated with the phenotype of the H2dl808 mutant were not observed in HeLa cells infected with the H5ilE4I mutant. However, differences were observed regarding the time of onset of viral DNA replication and the accumulation of the hexon polypeptide as well as the 72-kilodalton adenovirus-specific DNA-binding protein. The results thus indicate that open reading frame 6 or 7 or both contain all genetic information required for viral replication in tissue culture cells, whereas another E4 gene modulates the accumulation of certain viral polypeptides. The early onset of viral DNA replication in H5ilE4I-infected cells may be an indirect effect of the enhanced expression of the 72-kilodalton DNA-binding protein.     

Resistance of Adenoviral DNA Replication to Aphidicolin Is Dependent on the 72-Kilodalton DNA-Binding Protein

Aphidicolin is a highly specific inhibitor of DNA polymerase α and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33°C) to nonpermissive (41°C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase α and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase α came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion.     

Temperature-sensitive mutants of adenovirus single-stranded DNA-binding protein. Inability to support DNA replication is associated with an altered DNA-binding activity of the protein.

The adenovirus single-stranded DNA-binding protein (DBP) is an essential factor in viral DNA replication. Three temperature-sensitive (ts) adenoviruses (Ad2+ND1ts23, Ad2ts111A, and Ad5ts125) are known to have single amino acid substitutions in their DBPs that result in defective DNA replication at the nonpermissive temperature. To elucidate the mechanism(s) involved in the ts phenotype, we purified the three mutant DBPs and studied their DNA-binding properties and their ability to support DNA replication in an in vitro system. The results confirm that the three ts DBPs were incapable of supporting DNA replication at the nonpermissive temperature (40 degrees C). The defect was found at both the initiation and elongation steps of DNA replication. The 2-fold stimulation of pTP.dCMP formation by the DBP was lost by prior heating of the ts DBPs. The pronounced effect of the DBP on the early elongation process was severely diminished, but not abolished, by prior heating to 40 degrees C. The functional change at 40 degrees C was irreversible, as the ts DBPs preincubated at 40 degrees C were no longer active when assayed at 30 degrees C. Upon heating to 40 degrees C, all three ts DBPs lost their ability to bind to oligonucleotides, although they still retained some binding activity for large single-stranded DNAs such as M13 DNA. Thus, the inability of these three ts DBPs to support DNA replication is attributable to their altered DNA-binding properties.     

Characterization of an adenovirus early protein required for viral DNA replication: A single strand specific DNA binding protein

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. 2. Analysis of the methionine containing tryptic peptides of these proteins indicate that the types 2 and 5 proteins are similar and clearly distinguishable from the type 12 protein. The peptide maps of these three viral proteins are clearly different from a similar protein found in mock infected cells. 3. Temperature sensitive mutants of type 5 (H5ts125) and type 12(H12tsA275) adenoviruses fail to produce these proteins at the nonpermissive temperature. H5ts125 infected cells grown at the permissive temperature produce a 72,000 MW protein that is thermolabile, for continued binding to DNA, when compared to type 5 wild type adenovirus 72,000 MW protein. An analysis of the phenotype of this adenovirus mutant indicates that it codes for a viral function at early times after infection that is required for viral DNA replication. 4. The in vitro translation of adenovirus specific m-RNA results in the synthesis of a small amount of a 72,000 MW protein that binds to single stranded DNA just like the authentic adenovirus DNA binding proteins produced in infected cells. 5. Adenovirus anti-Tumor antigen (T) anti-serum from hamsters carrying independently derived adenovirus tumors, have been tested for the presence of antibody to purified DNA binding proteins. One antiserum is positive for these antibodies while the other is negative. These results indicate that some, but not all, adenovirus tumors contain large enough levels of the DNA binding proteins to elicit an antibody response. 6. The type 5 adenovirus temperature sensitive mutant, H5ts125, that codes for a thermolabile DNA binding protein, was complemented or suppressed at the nonpermissive temperature, for the replication of adenovirus DNA, by SV40. SV40tsA temperature sensitive mutants, defective in SV40 DNA replication, do not suppress or complement H5ts125 at the nonpermissive temperature.     

Purification and functional characterization of adenovirus ts111A DNA-binding protein. Fluorescence studies of protein-nucleic acid binding

The adenovirus single-stranded DNA (ssDNA)-binding protein (DBP) is necessary for the elongation step in viral DNA replication. In an attempt to characterize the putative ssDNA-binding domain of the DBP, we purified and characterized the Ad2ts111A DBP, which contains a glycine-to-valine substitution at amino acid 280. This mutation is adjacent to that in the previously studied Ad2+ND1ts23. Ad2+ND1ts23 exhibits a temperature-sensitive defect in DNA replication, and its DBP has previously been shown to bind ssDNA with reduced affinity. Ad2ts111A DBP, like Ad2+ND1ts23, does not support adenovirus DNA replication in vitro at elevated temperatures. However, the Ad2ts111A DBP binds ssDNA more tightly than does Ad2+ND1ts23 and is not temperature sensitive in this function. To determine the nucleic acid-binding properties of DBP, we applied spectrofluorometric techniques, which had not been used previously to study adenovirus DBP. Using the homopolynucleotide poly(1,N6)-ethenoadenylic acid (poly(r epsilon A], we have determined that the binding site size is approximately 16 nucleotides. In 20 mM NaCl, the Ad2wt, Ad2ts111A, and Ad2+ND1ts23 DBP proteins all bound stoichiometrically to poly(r epsilon A) with overall apparent affinities above 108 M-1. Based on titrations carried out at higher salt concentrations, however, the stability of these complexes did appear to increase in the order Ad2+ND1ts23 less than Ad2ts111A less than Ad2wt. By these techniques, we have confirmed also that the DBP of another temperature-sensitive mutant, H5ts107, like the Ad2ts111A DBP, retains its ability to bind ssDNA even at a restrictive temperature utilizing the salt concentration compatible with adenovirus DNA replication in vitro. The H5ts107 DBP, which contains an amino acid substitution at position 413, is defective for in vitro replication at nonpermissive temperature but is not temperature sensitive for binding to ssDNA. In summary, our results indicate that the replication defects of the Ad2ts111A are similar to those of H5ts107 and cannot be attributed to defective, nonspecific ssDNA binding by the DBP. It appears that ssDNA binding by itself is not sufficient to account for the role of DBP in adenovirus DNA replication.     

Adenovirus DNA-binding protein forms a multimeric protein complex with double-stranded DNA and enhances binding of nuclear factor I.

The 72-kilodalton adenovirus DNA-binding protein (DBP) binds to single-stranded DNA as well as to RNA and double-stranded DNA and is essential for the replication of viral DNA. We investigated the binding of DBP to double-stranded DNA by gel retardation analysis. By using a 114-base-pair DNA fragment, five or six different complexes were observed by gel retardation. The mobility of these complexes is dependent on the DBP concentration, suggesting that the complexes arise by sequential binding of DBP molecules to the DNA. In contrast to binding to single-stranded DNA, the binding of DBP to double-stranded DNA appears to be noncooperative. DBP binds to linear DNA as well as to circular DNA, while linear DNA containing the adenovirus terminal protein was also recognized. No specificity for adenovirus origin sequences was observed. To study whether the binding of DBP could influence initiation of DNA replication, we analyzed the effect of DBP on the binding of nuclear factor I (NFI) and NFIII, two sequence-specific origin-recognizing proteins that enhance initiation. At subsaturating levels of NFI, DBP increases the rate of binding of NFI considerably, while no effect was seen on NFIII. This stimulation of NFI binding is specific for DBP and was not observed with another protein (NFIV), which forms a similar DNA-multimeric protein complex. In agreement with enhanced NFI binding, DBP stimulates initiation of adenovirus DNA replication in vitro especially strongly at subsaturating NFI concentrations. We explain our results by assuming that DBP forms a complex with origin DNA that promotes formation of an alternative DNA structure, thereby facilitating the binding of NFI as well as the initiation of DNA replication via NFI.     

Functional characterization of thermolabile DNA-binding proteins that affect adenovirus DNA replication.

The human adenovirus type 2 (Ad2) mutant Ad2ts111 has previously been shown to contain two mutations which result in a complex phenotype. Ad2ts111 contains a single base change in the early region 1B (E1B) 19,000-molecular-weight (19K) coding region which yields a cyt deg phenotype and another defect which maps to the E2A 72K DNA-binding protein (DBP) coding region that causes a temperature-sensitive DNA replication phenotype. Here we report that the defect in the Ad2ts111 DBP is due to a single G----T transversion that results in a substitution of valine for glycine at amino acid 280. A temperature-independent revertant, Ad2ts111R10, was isolated, which reverts back to glycine at amino acid 280 yet retains the cyt and deg phenotypes caused by the 19K mutation. We physically separated the two mutations of Ad2ts111 by constructing a recombinant virus, Ad2ts111A, which contained a wild-type Ad2 E1B 19K gene and the gly----val mutation in the 72K gene. Ad2ts111A was cyt+ deg+, yet it was still defective for DNA replication at the nonpermissive temperature. The Ad2ts111 DBP mutation is located only two amino acids away from the site of the mutation in Ad2+ND1ts23, a previously sequenced DBP mutant. Biochemical studies of purified Ad2+ND1ts23 DBP showed that this protein was defective for elongation but not initiation of replication in a cell-free replication system consisting of purified Ad polymerase, terminal protein precursor, and nuclear factor I. Ad2+ND1ts23 DBP bound less tightly to single-strand DNA than did Ad2 DBP, as shown by salt gradient elution of purified DBPs from denatured DNA cellulose columns. This decreased binding to DNA was probably due to local conformational changes in the protein at a site that is critical for DNA binding rather than to global changes in protein structure, since both the Ad2+ND1ts23 and Ad2 DBPs showed identical cleavage patterns by the protease thermolysin at various temperatures.     

Adenovirus DNA replication in vitro: a protein linked to the 5' end of nascent DNA strands.

Soluble nuclear extracts prepared from adenovirus-infected HeLa cells supported adenovirus DNA replication with exogenous DNA-protein complex as template, but protease-treated, phenol-extracted DNA was less active. Replication was enhanced when creatine phosphate and creatine phosphokinase were included in the reaction mixture, rendering the reaction independent of exogenous ATP. Genomic-length, newly synthesized DNA strands were first observed 30 min after initiation of replication and continued to increase in amount for at least 4 h. Thus, the rate of replication is consistent with previous estimates of the rate of replication in vivo. Nascent DNA strands bound to benzoylated, naphthoylated DEAE-cellulose due to their association with protein. The 5' termini of nascent DNA strands were resistant to the 5'- to 3'-specific T7 exonuclease, and the 3' termini of nascent strands were sensitive to the 3'- to 5'-specific exonuclease III. These results suggest that a protein becomes covalently linked to the 5' termini of nascent DNA strands replicated in vitro. Nuclear extracts prepared from adenovirus type 2-infected cells also supported replication of DNA-protein complex prepared from the unrelated type 7 adenovirus. The limited sequence homology between these two viruses at the origin of replication further defines recognition sequences at the origin. These results are discussed in terms of a model for adenovirus DNA replication in which the terminal protein and sequences within the inverted terminal repetition are involved in the formation of an initiation complex that is able to prime DNA replication.     

Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay.

A rapid and quantitative nitrocellulose filter-binding assay is described for the detection of nuclear factor I, a HeLa cell sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication. In this assay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolymeric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment containing a functional nuclear factor I binding site compared with a control DNA fragment to which nuclear factor I did not bind specifically. This specific DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay allowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells and reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA binding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That this fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its function in the initiation of adenovirus DNA replication. Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide binding per mol of nuclear factor I-binding site.     

Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovrius defective in viral DNA synthesis.

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.     

The function(s) provided by the adenovirus-specified, DNA-binding protein required for viral late gene expression is independent of the role of the protein in viral DNA replication

The adenovirus type 2 (Ad2) host range mutant Ad2hr400 grows efficiently in cultured monkey cells at 37 degrees C, but is cold sensitive for plaque formation and late gene expression at 32.5 degrees C. After nitrous acid mutagenesis of an Ad2hr400 stock, cold-resistant variants were selected in CV1 monkey cells at 32.5 degrees C. One such variant, Ad2ts400, was also temperature sensitive (ts) for growth in both CV1 and HeLa cells. Marker rescue analysis has been used to show that the two phenotypes, cold resistant and temperature sensitive, are due to two independent mutations, each of which resides in a different segment of the gene encoding the 72-kilodalton DNA binding protein (DBP). The cold-resistant mutation (map coordinates 63.6 to 66) is a host range alteration that enhances the ability of the virus to express late genes and grow productively in monkey cells at 32.5 degrees C. The temperature-sensitive mutation is in the same complementation group and maps to the same segment of the DBP gene (map coordinates 61.3 to 63.6) as the well-characterized DBP mutant Ad5ts125. Like Ad5ts125, Ad2ts400 is unable to replicate viral DNA or to properly shut off early mRNA expression at the nonpermissive temperature. Two sets of experiments with Ad2ts400 suggest that DBP contains separate functional domains. First, when CV1 cells are coinfected at the nonpermissive temperature with Ad2 plus Ad2ts400 (Ad2 allows DNA replication and entry into, but not completion of, the late phase of infection), normal late gene expression and productive growth occur. Second, temperature shift experiments show that, although DNA replication is severely restricted at the nonpermissive temperature in ts400-infected monkey cells, late gene expression occurs normally. These results indicate that the DBP activity required for normal late gene expression in monkey cells is functional even when the DBP's DNA replication activity is disrupted.