Identification of two distinct regions within the adenovirus minimal origin of replication that are required for adenovirus type 4 DNA replication in vitro.

The adenovirus type 4 origins of replication are located at each end of the linear, protein-linked viral DNA molecule and consist of the terminal 18 bp of the viral genome. The sequence of the first 8 bp of the viral genome varies among different adenovirus serotypes, but the sequence from bp 9 to 18 is conserved in all human serotypes, suggesting that it may be of critical importance to origin function. Using an in vitro system in which purified fractions or crude extracts of adenovirus type 4-infected HeLa cells can support initiation and elongation on linearized plasmid templates containing cloned origin sequences, we examined the effect of single base changes in positions 9 to 18 of the adenovirus origin on DNA replication in vitro. Changes in positions 12 to 16 have little effect, whereas alterations at positions 9, 10, 11, 17, and 18 all reduce the efficiency of initiation of DNA replication by between 50 and 90%. Our results show that the region from bp 9 to 18 contains two sets of bases essential for DNA replication which are separated by 5 bp in which single base changes can be accommodated. The likely role of the region from bp 9 to 18 as containing the recognition sequence for a DNA-protein interaction essential for viral DNA replication is discussed.     

The origin of adenovirus DNA replication: minimal DNA sequence requirement in vivo.

Adenovirus mini-chromosomes which contain two cloned, inverted adenovirus termini replicate in vivo when supplied with non-defective adenovirus as a helper. This system has been used to define the minimum cis acting DNA sequences required for adenovirus DNA replication in vivo. Deletions into each end of the adenovirus inverted terminal repeat (ITR) were generated with Bal31 exonuclease and the resulting molecules constructed into plasmids which contained two inverted copies of the deleted ITR separated by the bacterial neomycin phosphotransferase gene. To determine the effect of the deletion in vivo plasmids cleaved to expose the adenovirus termini were co-transfected with adenovirus type 2 DNA into tissue culture cells. The replicative ability of the molecules bearing adenovirus termini was assayed by Southern blotting of extracted DNA which had been treated with DpnI, a restriction enzyme which cleaves only methylated and therefore unreplicated, input DNA. Molecules containing the terminal 45 bp of the viral genome were fully active whereas molecules containing only 36 bp were in-active in this assay. Therefore sequences required for DNA replication are contained entirely within the terminal 45 bp of the viral genome. Thus, both the previously described highly conserved region (nucleotides 9-18) and the binding site for the cellular nuclear factor I (nucleotides 19-48) are essential for adenovirus DNA replication in vivo.     

Replication of adenovirus mini-chromosomes

We have isolated adenovirus origins of DNA replication from both the right and left ends of the genome, which are functional on linear autonomously replicating mini-chromosomes. The mini-chromosomes contain two cloned inverted adenovirus termini and require non-defective adenovirus as a helper. Replicated molecules are covalently attached to protein, and DNA synthesis is initiated at the correct nucleotide even when the origins are not located at molecular ends. The activity of embedded origins leads to the generation of linear minichromosomes from circular or linear molecules. These observations therefore suggest that sequences within the adenovirus origin of replication position the protein priming event at the adenovirus terminus. Experiments investigating the regeneration of deleted viral inverted terminal repeat sequences show a sequence-independent requirement for inverted sequences in this process. This result strongly suggests that repair results from the formation of a panhandle structure by a displaced single strand. On the basis of these observations we propose a model for the generation of adenovirus mini-chromosomes from larger molecules.     

The adenovirus terminal protein influences binding of replication proteins and changes the origin structure.

The adenovirus terminal protein (TP) is covalently linked to the 5' ends of the adenovirus genome and enhances DNA replication in vitro by increasing template activity. To study the effect of TP in more detail we isolated short origin fragments containing functional TP using anion exchange chromatography. These fragments were highly active as templates for DNA replication in a reconstituted system. Employing band-shift assays we found that the affinity of the precursor terminal protein-DNA polymerase complex for the TP-containing origin was increased 2 to 3-fold. Binding affinities of two other replication stimulating proteins, NFI and Oct-1, were not influenced by the terminal protein. Upon DNaseI footprinting we observed, unexpectedly, that the breakdown pattern had changed at various positions in the origin, notably in the area 3-6 and 41-51 by the presence of TP. Some differences in the footprint pattern of NFI and Oct-1 were also found. Our results indicate that TP induces subtle changes in the origin structure that influence the interaction of other replication proteins.     

Structure and function of the adenovirus origin of replication

Efficient initiation of adenovirus DNA replication requires the presence of specific terminal nucleotide sequences that collectively constitute the viral origin of replication. Using plasmids with deletions or base substitutions in a cloned segment of DNA derived from the terminus of the adenovirus 2 genome, we have demonstrated that the origin contains two functionally distinct regions. The first 18 bp of the viral genome are sufficient to support a limited degree of initiation. However, the presence of a sequence in the region between nucleotides 19 and 67 greatly enhances the efficiency of the initiation reaction. This region contains a specific binding site for a protein present in uninfected cells (KD = 2 X 10(-11) M). The bound protein protects the DNA segment between base pairs 19 and 43 from attack by DNAase I. Studies with deletion mutants indicate that binding of the cellular protein is responsible for the enhancement of initiation.     

Functional homology between the sequence-specific DNA-binding proteins nuclear factor I from HeLa cells and the TGGCA protein from chicken liver.

Nuclear factor I from HeLa cells, a protein with enhancing function in adenovirus DNA replication, and the chicken TGGCA protein are specific DNA-binding proteins that were first detected by independent methods and that appeared to have similar DNA sequence specificity. To test whether they are homologous proteins from different species we have compared (i) their DNA binding properties and (ii) their function in reconstituted adenovirus DNA replication systems. Using deletion and substitution mutants derived from the DNA binding site on the adenovirus 2 inverted terminal repeat, it was found that the two proteins protect the same 24-nucleotide region of both strands against DNase I digestion and that they have identical minimal recognition sequences of 15 bp containing dyad symmetry. Like nuclear factor I, the TGGCA protein enhances the initiation reaction of adenovirus 2 DNA replication in vitro in a DNA recognition site-dependent manner.     

DNA sequences required for the initiation of adenovirus type 4 DNA replication in vitro

In-vivo studies have demonstrated that adenovirus type 2 and adenovirus type 4 have different DNA sequence requirements for the initiation of DNA replication. To investigate the basis of these differences an in-vitro system has been developed which will faithfully initiate adenovirus type 4 DNA replication. A plasmid containing 140 base-pairs of the right terminus of adenovirus type 4 supported initiation of DNA replication in vitro, provided that the plasmid was linearized in such a way as to locate the viral terminal sequences at the molecular ends of the DNA. Initiation by adenovirus type 4-infected cell extracts was also supported by a plasmid containing the complete adenovirus type 2 inverted terminal repeat (ITR). Deletion analysis of both adenovirus types 2 and 4 ITRs revealed that only the terminal 18 base-pairs of the genomes (perfectly conserved between the 2 viruses) were required for initiation in vitro. Thus, initiation was not enhanced by the presence of either the NFI site, the NFIII site or both sites together. Fractionation of a HeLa cell nuclear extract, by ion-exchange chromatography, identified a nuclear factor that stimulated the initiation reaction four- to fivefold. The stimulatory factor did not correspond to either of the cellular proteins NFI or NFIII which stimulate adenovirus type 2 DNA replication in vitro. Initiation in vitro was also supported by single-stranded DNA templates, albeit at a lower efficiency. Studies with synthetic oligonucleotides indicated a surprising specificity for initiation: whereas the strand used as template during initiation in vivo was active as a template for initiation in vitro, the complementary strand was inactive.     

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.     

Restriction endonuclease mapping of adenovirus 35, a type isolated from immunocompromised hosts.

The DNA of adenovirus 35 (Ad35), a type recently associated with infections in immunocompromised hosts, was mapped by the use of BamHI, SmaI, PstI, EcoRI, and HpaI restriction endonucleases. In addition to standard mapping procedures, we used the in vitro adenovirus DNA replication system with origins at both physical ends of the linear molecule to determine the terminal fragments. Deletions of single restriction endonuclease sites in a group of closely related adenovirus isolates from patients with acquired immunodeficiency syndrome helped determine the location of some DNA fragments on the genome.     

Adenovirus sequences required for replication in vivo.

We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occupies the first 18 to 21 bp and includes sequences conserved between all adenovirus serotypes. The adjacent auxillary region extends past nucleotide 36 but not past nucleotide 67 and contains the binding site for nuclear factor I.     

Specific binding of the adenovirus terminal protein precursor-DNA polymerase complex to the origin of DNA replication

Initiation of adenovirus DNA replication is dependent on a complex of the precursor of the terminal protein and the adenovirus-coded DNA polymerase (pTP-pol complex). This complex catalyzes the formation of a covalent linkage between dCMP and pTP in the presence of a functional origin of DNA replication residing in the terminal nucleotide sequence of adenovirus DNA. We have purified the pTP-pol complex of adenovirus type 5 and studied its binding to double-stranded DNA. Using DNA-cellulose chromatography it could be shown that the pTP-pol complex has a higher affinity for adenovirus DNA than for calf thymus or pBR322 DNA. From the differential binding of the pTP-pol complex to plasmids containing adenovirus terminal sequences with different deletions, it has been concluded that a sequence of 14 nucleotide pairs at positions 9-22 plays a crucial role in the binding of pTP-pol to adenovirus DNA. This region is conserved in the DNA's of all human adenovirus serotypes and is obviously an important structural element of the adenovirus origin of DNA replication. Comparative binding studies with adenovirus DNA polymerase and pTP-pol indicated that pTP is responsible for the binding. The nature of the binding of pTP-pol to the conserved sequence will be discussed.     

A novel terminal resolution-like site in the adeno-associated virus type 2 genome.

The adeno-associated virus 2 (AAV) contains a single-stranded DNA genome of which the terminal 145 nucleotides are palindromic and form T-shaped hairpin structures. These inverted terminal repeats (ITRs) play an important role in AAV DNA replication and resolution, since each of the ITRs contains a terminal resolution site (trs) that is the target site for the AAV rep gene products (Rep). However, the Rep proteins also interact with the AAV DNA sequences that lie outside the ITRs, and the ITRs also play a crucial role in excision of the proviral genome from latently infected cells or from recombinant AAV plasmids. To distinguish between Rep-mediated excision of the viral genome during rescue from recombinant AAV plasmids and the Rep-mediated resolution of the ITRs during AAV DNA replication, we constructed recombinant AAV genomes that lacked either the left or the right ITR sequence and one of the Rep-binding sites (RBSs). No rescue and replication of the AAV genome occurred from these plasmids following transfection into adenovirus type 2-infected human KB cells, as expected. However, excision and abundant replication of the vector sequences was clearly detected from the plasmid that lacked the AAV left ITR, suggesting the existence of an additional putative excision site in the left end of the AAV genome. This site was precisely mapped to one of the AAV promoters at map unit 5 (AAV p5) that also contains an RBS. Furthermore, deletion of this RBS abolished the rescue and replication of the vector sequences. These studies suggest that the Rep-mediated cleavage at the RBS during viral DNA replication may, in part, account for the generation of the AAV defective interfering particles.     

The nucleotide sequence of the right-hand terminus of adenovirus type 5 DNA: implications for the mechanism of DNA replication.

The nucleotide sequence of the right-hand terminal 3% of adenovirus type 5 (Ad5) DNA has been determined, using the chemical degradation technique developed by Maxam and Gilbert (1977). This region of the genome comprises the 1003 basepair long HindIII-I fragment and the first 75 nucleotides of the adjacent HindIII-F fragment, extending from the right-hand terminus to the sequences from which the main body of the mRNA of early region 4 is transcribed. One of the origins of adenovirus DNA replication is located within this part of the genome. The sequencing results are discussed in relation to several models proposed for the mechanism of replication of linear DNA molecules, which invariably depend on the presence of specific arrangements of nucleotides at the termini of those linear DNAs.     

Initiation of adenovirus DNA replication.

In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared with those obtained in a soluble nuclear extract competent for viral DNA replication. It was observed that in vitro DNA replication, which is dependent on the exogenously added viral DNA-protein complex as its optimal template, occurs in a manner apparently indistinguishable from the situation in virus-infected cells. This includes the presence of proteinaceous material on the molecular termini of newly initiated viral DNA.     

Possible mechanism of adenovirus generation from a cloned viral genome tagged with nucleotides at its ends

The entire cloned human adenovirus type 5 (Ad5) genome is known to be able to generate infectious virus after transfection into 293 cells when the both ends of the genome are exposed by digestion with appropriate restriction enzymes. However, when one or both ends of the genome are tagged with nucleotides and are not intact, whether the tagged end of the viral genome was remained tagged or corrected to be intact during the generation of viral clones has been unclear and, if such oligonucleotide removal occurs, how does the virus remove these tagged sequences and thereby restore its proper structure? Here, we show in our semi‐quantitative study that the generation efficiency of virus clones decreases depending on the length of nucleotide tags at the both ends and that both the oligonucleotide tags were precisely removed during virus generation with restoration of the proper terminal sequences. Interestingly the viral genome of which one end was tagged, while the other was attached about 12‐kb sequences, did generate intact viral clones at a reduced but significant efficiency. From these results, we here propose a possible mechanism whereby the terminal‐protein‐deoxycytidine complex enters from the enzyme‐cleaved end and reaches deoxyguanine at the initiating position of DNA synthesis in vivo. A replication origin at one end, embedded deeply in double‐stranded DNA, can be activated by two cycles of one‐directional full‐length DNA synthesis initiated by the other exposed replication origin about 30 kilobases away. We also describe new cassette cosmids which can use not only PacI but also BstBI for construction of an adenovirus vector, without reducing construction efficiency.     

The rabbit C family of short, interspersed repeats. Nucleotide sequence determination and transcriptional analysis

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.     

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.     

Genome structure of incomplete particles of adenovirus.

Incomplete particles arising during productive growth of adenovirus were separated from infectious particles by density gradient centrifugation. The DNA contained in particles of low density was characterized by restriction enzyme analysis and by electron microscopy and heteroduplexing techniques. The DNA is heterogeneous in length, ranging in size from 15% of the normal genome to full length. Many individual molecules contain long, inverted terminal repetitions, which consist of the sequences extending from the normal left-hand end of the viral genome inward; the normal right end sequences appear to be missing from these molecules. The region of the genome reiterated in these molecules is that which has been implicated in transformation of rat cells by adenovirus (Gallimore, Sharp, and Sambrook, 1974; Graham, van der Eb, and Heijneker, 1974). A model for adenovirus replication is presented that accounts for the aberrant structures observed.     

The Mre11/Rad50/Nbs1 complex limits adeno-associated virus transduction and replication

Adeno-associated virus (AAV) is a parvovirus with a small single-stranded DNA genome that relies on cellular replication machinery together with functions supplied by coinfecting helper viruses. The impact of host factors on AAV infection is not well understood. We explored the connection between AAV helper functions supplied by adenovirus and cellular DNA repair proteins. The adenoviral E1b55K/E4orf6 proteins induce degradation of the cellular Mre11 repair complex (MRN) to promote productive adenovirus infection. These viral proteins also augment recombinant AAV transduction and provide crucial helper functions for wild-type AAV replication. Here, we show that MRN poses a barrier to AAV and that the helper function provided by E1b55K/E4orf6 involves MRN degradation. Using a fluorescent method to visualize the viral genome, we show an effect at the viral DNA level. MRN components accumulate at AAV replication centers and recognize the viral inverted terminal repeats. Together, our data suggest that AAV is targeted by MRN and has evolved to exploit adenoviral proteins that degrade these cellular factors.