Nucleotide sequence of the gene encoding adenovirus type 2 DNA binding protein.

We have determined the nucleotide sequence of the gene encoding adenovirus type 2 (Ad2) DNA binding protein (DBP). From the nucleotide sequence the complete amino acid sequence of Ad2 DBP has been deduced. A comparison of the amino acid sequences of Ad2 and Ad5 DBP, both 529 residues long, reveals that the C-terminal 354 residues of both sequences are identical. Within the N-terminal 175 amino acid residues Ad2 and Ad5 show nine differences. The site of mutation in Ad2 ND1ts23, a mutant with a temperature-sensitive DNA replication, was mapped at the nucleotide level. A single nucleotide alteration in the DBP gene, resulting in a leucine leads to phenylalanine substitution at position 282 in the amino acid sequence is responsible for the temperature-sensitive character of this mutant. Previously, we localized the mutation of another DBP mutant with a temperature-sensitive DNA replication (H5ts125) at position 413 in the amino acid sequence of the DBP molecule (Nucleic Acids Res. 9 (1981) 4439-4457). These mapping data are discussed in relation to the structure and function of the DBP molecule.     

Adenovirus type 5 DNA binding protein stimulates binding of DNA polymerase to the replication origin

The adenovirus (Ad) DNA-binding protein (DBP) is essential for the elongation phase of Ad DNA replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. DBP also enhances the rate of initiation, with the highest levels obtained at low concentrations of Ad DNA polymerase (Pol). Here, we show that stimulation of initiation depends on the template conformation. Maximal stimulation, up to 15-fold, is observed on double-stranded or viral TP-containing origins. The stimulation is reduced on partially single-stranded origins and DBP does not enhance initiation any more once the origin is completely unwound. This suggests a role for DBP in origin unwinding that is comparable to its unwinding capacity during elongation. However, mutant DBP proteins defective in unwinding and elongation can still enhance initiation on ds templates. DBP also stimulates the binding of nuclear factor I (NFI) to the origin and lowers the K(m) for coupling of the first nucleotide to the precursor terminal protein by Pol. Mobility shift experiments reveal that DBP stimulates the binding of Pol on double-stranded origin and nonorigin DNA but not on single-stranded DNA. This effect is specific for DBP and is also seen with other DNA Pols. Our results suggest that, rather than by origin unwinding, DBP enhances initiation by modulating the origin conformation such that DNA Pol can bind more efficiently.     

Presence of protein at the termini of intracellular adenovirus type 5 DNA.

Adenovirus type 5 contains linear double-stranded DNA with protein covalently attached to the ends of the molecules. The presence of protein at the termini of intracellular viral DNA in adenovirus type 5-infected cells was investigated at different stages during the replication process. The intracellular viral DNA was isolated from the nuclei by lysis in 4 M guanidine hydrochloride. Electrophoresis on agarose gels of HsuI restriction enzyme fragments and sucrose gradient centrifugation were used to detect protein on intracellular viral DNA. After uncoating parental DNA still contains protein attached to the termini of the viral genome. Replicating and mature progeny viral DNA can also be isolated in the form of DNA-protein complexes. These complexes exhibit the same properties as the DNA-protein complex isolated from purified virions. These results suggest that the protein at the termini of intracellular viral DNA is identical to the protein attached to the 5'-ends of the DNA extracted from virions and that it is possibly involved in the replication of viral DNA.     

DNA binding properties of the adenovirus DNA replication priming protein pTP

The precursor terminal protein pTP is the primer for the initiation of adenovirus (Ad) DNA replication and forms a heterodimer with Ad DNA polymerase (pol). Pol can couple dCTP to pTP directed by the fourth nucleotide of the viral genome template strand in the absence of other replication proteins, which suggests that pTP/pol binding destabilizes the origin or stabilizes an unwound state. We analyzed the contribution of pTP to pTP/pol origin binding using various DNA oligonucleotides. We show that two pTP molecules bind cooperatively to short DNA duplexes, while longer DNA fragments are bound by single pTP molecules as well. Cooperative binding to short duplexes is DNA sequence independent and most likely mediated by protein/protein contacts. Furthermore, we observed that pTP binds single-stranded (ss)DNA with a minimal length of approximately 35 nt and that random ssDNA competed 25-fold more efficiently than random duplex DNA for origin binding by pTP. Remarkably, short DNA fragments with two opposing single strands supported monomeric pTP binding. pTP did not stimulate, but inhibited strand displacement by the Ad DNA binding and unwinding protein DBP. These observations suggest a mechanism in which the ssDNA affinity of pTP stabilizes Ad pol on partially unwound origin DNA.     

Gene organization of the transforming region of adenovirus type 7 DNA

The sequence of the leftmost 11% of the weakly oncogenic human adenovirus type 7 (Ad7) DNA has been determined. This part of the Ad7 viral genome encompasses early region E1 which has been shown to be involved in the process of cell transformation in vitro (Dijkema et al., 1979). From the nucleotide sequence and determined coordinates of the E1 mRNAs, we are able to predict the primary structure of the polypeptides encoded by the transforming region of Ad7. The organization of the E1 region of Ad7 and of other adenovirus serotypes (Bos et al. 1981) leads to the proposal of a novel mechanism for gene regulation at the translational level in which protein synthesis can initiate at either the first or the second AUG triplet available in mRNA. The differences between the large E1b-specific tumor antigens of adenovirus types 12, 7 and 5 may explain the differences in oncogenicity of these viruses.     

The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates

The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails. DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails. Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed. Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule. This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II). During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.     

Structure of the plasmodium knowlesi gene coding for the circumsporozoite protein

The gene that codes for the surface antigen of Plasmodium knowlesi sporozoites (CS protein) is unsplit and present in the genome in only one copy. The CS protein, as deduced from DNA sequence analysis of the structural gene, has an unusual structure with the central 40% of the polypeptide chain present as 12 tandemly repeated amino acid peptide units flanked by regions of highly charged amino acids. The protein has an amino-terminal hydrophobic amino acid signal sequence and a hydrophobic carboxy-terminal anchor sequence. The coding sequence of the gene has an AT content of 53%, compared with 70% AT in the 5′ and 3′ flanking sequences, and is contained entirely within an 11 kb Eco RI genomic DNA fragment. This genomic fragment expresses the CS protein in E. coli, indicating that the parasite promoter and ribosome binding site signals can be recognized in E. coli.     

Immunofluorescence study of the adenovirus type 2 single-stranded DNA binding protein in infected and transformed cells.

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.     

Conserved region 3 of the adenovirus type 5 DNA-binding protein is important for interaction with single-stranded DNA

The adenovirus-encoded single-stranded DNA-binding protein (DBP) functions in viral DNA replication and several aspects of RNA metabolism. Previous studies (G. A. M. Neale and G. R. Kitchingman, J. Biol. Chem. 264:3153-3159, 1989) have defined three highly conserved regions in the carboxy-terminal domain of the protein (amino acids 178 to 186, 322 to 330, and 464 to 475) that may be involved in the binding of the protein to single-stranded DNA. We examined the role of conserved region 3 (464 to 475) by constructing nine classes of point mutants with from one to four amino acid changes. The point mutants were tested for their ability to assist adeno-associated virus DNA replication. All nine differed from wild-type DBP; seven were essentially nonfunctional, whereas two had 55 and 145%, respectively, of the wild-type DBP helper activity. Three of the mutants were found to be temperature sensitive, with significantly greater helper activity at 33 degrees C than at 37 degrees C. All nine mutants produced essentially wild-type levels of protein. One monoclonal antibody against the DBP, termed 2/4, did not immunoprecipitate the mutant DBPs as well as wild-type DBP, indicating either that the antibody recognized sequences around CR3 or that the conformation of the protein around the epitope recognized by 2/4 had changed. Two of the three temperature-sensitive DBP mutants bound to single-stranded DNA-cellulose with the same affinity as wild-type DBP at 4 degrees C; the remaining mutants all showed reduced affinity. These results demonstrated that many of the residues within conserved region 3 of the DBP are important for interaction of the protein with nucleic acid.     

Adeno-associated virus helper activity of adenovirus DNA binding protein

The requirement for the adenovirus (Ad) single-stranded DNA binding protein (DBP) in the expression of adeno-associated virus (AAV) proteins was studied by specific immunofluorescent staining of infected cells and in vitro translation of RNA from infected cells. The Ad5 mutant ts125, which carries a mutation in the DBP gene, helped AAV as efficiently as the Ad5 wild type (WT) did at both the permissive (32 degrees C) and nonpermissive (40.5 degrees C) temperatures in HeLa and KB cells. Furthermore, at 40.5 degrees C ts125 was as efficient as Ad5WT was in inducing the expression of AAV proteins in a line of Detroit 6 cells which is latently infected with AAV. However, little if any AAV protein was synthesized when coinfections were carried out with Ad5WT in CV-C cells, a monkey cell line that is highly restrictive for human Ad replication unless the cells are also infected with simian virus 40. On the other hand, AAV protein was efficiently produced in CV-C cells in coinfections with the Ad5 mutant hr404, whose growth is unrestricted in CV-C cells and whose mutation also maps in the DBP gene. Finally, preparations of cytoplasmic RNA extracted from CV-C cells infected with AAV and Ad5WT or from CV-C cells infected with AAV, Ad5WT, and simian virus 40 were each capable of directing the in vitro synthesis of abundant amounts of AAV proteins in a rabbit reticulocyte lysate system. These results indicate that the abnormal DBP of ts125 still retains its helper function for AAV replication, but that the molecular feature of the DBP which relates to the monkey cell host range restriction of Ad's may also account for the observed block to AAV protein translation in CV-C cells.     

Refined structure of the gene 5 DNA binding protein from bacteriophage fd

The three-dimensional structure of the gene 5 DNA binding protein (G5BP) from bacteriophage fd has been determined from a combination of multiple isomorphous replacement techniques, partial refinements and deleted fragment difference Fourier syntheses. The structure was refined using restrained parameter least-squares and difference Fourier methods to a final residual of R = 0.217 for the 3528 statistically significant reflections present to 2.3 A resolution. In addition to the 682 atoms of the protein, 12 solvent molecules were included. We describe here the dispositions and orientations of the amino acid side-chains and their interactions as visualized in the G5BP structure. The G5BP monomer of 87 peptide units is almost entirely in the beta-conformation, organized as a three-stranded sheet, a two-stranded beta-ribbon and a broad connecting loop. There is no alpha-helix present in the molecule. Two G5BP monomers are tightly interlocked about an intermolecular dyad axis to form a compact dimer unit of about 55 A X 45 A X 36 A. The dimer is characterized by two symmetry-related antiparallel clefts that traverse the monomer surfaces essentially perpendicular to the dyad axis. From the three-stranded antiparallel beta-sheet, formed from the first two-thirds of the sequence, extend three tyrosine residues (26, 34, 41), a lysine (46) and two arginine residues (16, 21) that, as indicated by other physical and chemical experiments, are directly involved in DNA binding. Other residues likely to share binding responsibility are arginine 80 extending from the beta-ribbon and phenylalanine 73 from the tip of this loop, but as provided, however, by the opposite monomer within each G5BP dimer pair. Thus, both symmetry-related DNA binding sites have a composite nature and include contributions from both elements of the dimer. The gene 5 dimer is clearly the active binding species, and the two monomers within the dyad-related pair are so structurally contiguous that one cannot be certain whether the isolated monomer would maintain its observed crystal structure. This linkage is manifested primarily as a skeletal core of hydrophobic residues that extends from the center of each monomer continuously through an intermolecular beta-barrel that joins the pair. Protruding from the major area of density of each monomer is an elongated wing of tenuous structure comprising residues 15 through 32, which is, we believe, intimately involved in DNA binding. This wing appears to be dynamic and mobile, even in the crystal and, therefore, is likely to undergo conformational change in the presence of the ligand.     

Nature of the single-stranded DNA in replicating adenovirus type 5 DNA.

Single-stranded regions in replicating adenovirus type 5 DNA were isolated and hybridized in solution to the separated strands of adenovirus 2 or 5 DNA. The results showed that the two strands of adenovirus 5 DNA are exposed to almost the same extent during replication, suggesting that displacement synthesis may start from either end of the viral DNA.     

Characterization of type 5 adenovirus fiber protein.

Type 5 adenovirus fiber protein was purified and subjected to chemical characterization. Equilibrium sedimentation ultracentrifugation analysis indicated that the intact fiber has a molecular weight of approximately 183,000. Denaturation and chemical analyses implied that the fiber consists of three polypeptide chains, each of about 61,000 mol wt. Mapping of tryptic peptides and electrophoretic separation of the constituent chains suggested that the intact fiber consists of two identical and one unique polypeptide chains.