Initiation Of Viral DNA Replication Research Articles

Human Papillomavirus DNA Replication: INTERACTIONS BETWEEN THE VIRAL E1 PROTEIN AND TWO SUBUNITS OF HUMAN DNA POLYMERASE α/PRIMASE

Papovaviruses are valuable models for the study of DNA replication in higher eukaryotic organisms, as they depend on host factors for replication of their DNA. In this study we investigate the interactions between the human papillomavirus type 11 (HPV-11) origin recognition and initiator protein E1 and human polymerase alpha/primase (pol alpha/primase) subunits. By using a variety of physical assays, we show that both 180- (p180) and 70-kDa (p70) subunits of pol alpha/primase interact with HPV-11 E1. The interactions of E1 with p180 and p70 are functionally different in cell-free replication of an HPV-11 origin-containing plasmid. Exogenously added p180 inhibits both E2-dependent and E2-independent cell-free replication of HPV-11, whereas p70 inhibits E2-dependent but stimulates E2-independent replication. Our experiments indicate that p70 does not physically interact with E2 and suggest that it may compete with E2 for binding to E1. A model of how E2 and p70 sequentially interact with E1 during initiation of viral DNA replication is proposed.

Colocalization of baculovirus IE-1 and two DNA-binding proteins, DBP and LEF-3, to viral replication factories.

We have recently identified a DNA-binding protein (DBP) from the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) which can destabilize double-stranded DNA (V. S. Mikhailov, A. L. Mikhailova, M. Iwanaga, S. Gomi, and S. Maeda, J. Virol. 72:3107-3116, 1998). DBP was found to be an early gene product that was not present in budded or occlusion-derived virions. In order to characterize the localization of DBP during viral replication, BmNPV-infected BmN cells were examined by immunostaining and confocal microscopy with DBP antibodies. DBP first appeared as diffuse nuclear staining at 4 to 6 h postinfection (p.i.) and then localized to several specific foci within the nucleus at 6 to 8 h p.i. After the onset of viral DNA replication at around 8 h p.i., these foci began to enlarge and eventually occupied more than half of the nucleus by 14 h p.i. After the termination of viral DNA replication at about 20 h p.i., the DBP-stained regions appeared to break down into approximately 100 small foci within the nucleus. At 8 h p.i., the distribution of DBP as well as that of IE-1 or LEF-3 (two proteins involved in baculovirus DNA replication) overlapped well with that of DNA replication sites labeled with bromodeoxyuridine incorporation. Double-staining experiments with IE-1 and DBP or IE-1 and LEF-3 further confirmed that, between 8 and 14 h p.i., the distribution of IE-1 and LEF-3 overlapped with that of DBP. However, IE-1 localized to the specific foci prior to DBP or LEF-3 at 4 h p.i. In the presence of aphidicolin, an inhibitor of DNA synthesis, immature foci containing IE-1, LEF-3, and DBP were observed by 8 h p.i. However, the subsequent enlargement of these foci was completely suppressed, suggesting that the enlargement depended upon viral DNA replication. At 4 h p.i., the number of IE-1 foci correlated with the multiplicity of infection (MOI) between 0.4 and 10. At higher MOIs (e.g., 50), the number of foci plateaued at around 15. These results suggested that there are about 15 preexisting sites per nucleus which are associated with the initiation of viral DNA replication and assembly of viral DNA replication factories.

Replicative functions of minute virus of mice NS1 protein are regulated in vitro by phosphorylation through protein kinase C.

NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002-8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1(P)) supported RCR under these conditions, dephosphorylated NS1 (NS1(O)) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1(O), are enriched in protein kinase C (PKC), and, when present together, reactivate NS1(O) for replication. One of these components, containing atypical PKC, was sufficient to restore NS1(O) helicase activity. The requirement of NS1(O) reactivation for characteristic PKC cofactors such as Ca2+/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro.

The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication.

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase alpha-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.

Distinct roles of two binding sites for the bovine papillomavirus (BPV) E2 transactivator on BPV DNA replication.

The modulation of DNA replication by transcription factors was examined by using bovine papillomavirus type 1 (BPV). BPV replication in vivo requires two viral proteins: E1, an origin-binding protein, and E2, a transcriptional transactivator. In the origin, E1 interacts with a central region flanked by two binding sites for E2 (BS11 and BS12), of which only BS12 has been reported to be essential for replication in vivo. Using chemical interference and electrophoretic mobility shift assays, we found that the binding of E2 to each site stimulates the formation of distinct E1-origin complexes. A high-mobility C1 complex is formed by using critical E2 contacts to BS12 and E1 contacts to the dyad symmetry element. In contrast, interaction of E2 with the BS11 element on the other origin flank promotes the formation of the lower-mobility C3 complex. C3 is a novel species that resembles C2, a previously identified complex that is replication active and formed by E1 alone. The binding of E1 greatly differs in the C1 and C3 complexes, with E1 in the C1 complex limited to the origin dyad symmetry region and E1 in the C3 complex encompassing the region from the proximal edge of BS11 through the distal edge of BS12. We found that the presence of both E2-binding sites is necessary for wild-type replication activity in vivo, as well as for maximal production of the C3 complex. These results show that in the normal viral context, BS11 and BS12 play separate but synergetic roles in the initiation of viral DNA replication that are dependent on their location within the origin. Our data suggest a model in which the binding of E2 to each site sequentially stimulates the formation of distinct E1-origin complexes, leading to the replication-competent complex.

The Human Papillomavirus Type 18 (HPV18) Replication Protein E1 Is a Transcriptional Activator When Interacting with HPV18 E2

The human papillomavirus type 18 E1 and E2 proteins are both required for the initiation of viral DNA replication. Whereas E2 is the major viral transcription regulator, E1 is the replication initiator protein. They interact with each other and with the origin sequences to initiate viral DNA replication. We show that the HPV18 E1 and E2 proteins, when bound to an origin sequence cloned upstream of a heterologous promoter, synergistically activate transcription. This synergy required binding of E2 to at least two binding sites, but was partially independent of E1 binding to the origin of replication. Transcriptional activation was observed even in the absence of replication of the target DNA. Only homologous E1 and E2 proteins binding to homologous origin sequences from BPV1 or HPV18 viruses could synergistically activate transcription. We show that the HPV18 E1 protein can activate transcription when targeted to the DNA by fusion of the complete polypeptide with the BPV1 E2 C-terminus dimerization/DNA binding domain, implying that HPV18 E1 is an intrinsic transcriptional activator, though less potent than E2. The interaction between E1 and E2 may form a transcriptionally active complex during initiation of viral DNA replication.

Characterization of the DNA-binding activity of the E1 and E2 proteins and the E1/E2 complex of human papillomavirus type 33.

The E1 and E2 proteins of papillomaviruses are essential for the initiation of viral DNA replication. We have purified the E2 protein of human papillomavirus type 33 (HPV-33) by immunoaffinity chromatography. The purified E2 protein bound with high affinity to all four consensus binding sites of HPV-33 (Kd approximately equal to 2 x 10(-10)M). A putative E2 binding site differing at one position in the second stem of the palindrome was not bound by E2. The E1 protein of HPV-33 purified by affinity chromatography using glutathione S-transferase as tag displayed specific DNA-binding activity in footprint analyses protecting HPV-33 nucleotides 7896 to 7909/1 to 18 from DNasel digestion. Hypersensitive sites at position 6 on the sense and position 1 on the antisense strand were observed in the middle of the protected region. An E1/E2 complex protected the E1 binding site and E2 binding sites from DNasel digestion suggesting that both proteins retain DNA-binding activity in the complex.

Unwinding of the box I element of a herpes simplex virus type 1 origin by a complex of the viral origin binding protein, single-strand DNA binding protein, and single-stranded DNA.

The herpes simplex virus type 1 (HSV-1) genome contains three origins of replication: oriL and two copies of oriS. These origins contain specific sequences, box I and box II, linked by an AT-rich segment, that are recognized by an HSV-1-encoded origin binding protein (UL9 protein) which also possesses DNA helicase activity. Despite its intrinsic helicase activity, the UL9 protein is unable to unwind oriS or the box I element of oriS, either in the presence or absence of the HSV-1-encoded single-strand DNA binding protein, ICP8. However, a complex of the UL9 protein and ICP8 can unwind box I if it contains a 3' single-stranded tail at least 18 nt in length positioned downstream of box I. These findings suggest a model for the initiation of HSV-1 DNA replication in which a complex consisting of the UL9 protein bound to box I, and ICP8 bound to single-stranded DNA generated at the A+T rich linker, perhaps as a consequence of transcription, unwinds an HSV-1 origin of replication to provide access to the replication machinery with the consequent initiation of viral DNA replication. This mode of unwinding is distinct from that observed for other animal viruses--e.g., simian virus 40 or bovine papilloma virus--in which the initiator protein, T antigen, or E1 protein alone, unwinds elements of the origin sequence, and the single-strand DNA binding protein serves only to keep the separated strands apart.

Modulation of Bovine Papillomavirus DNA Replication by Phosphorylation of the Viral E1 Protein

E1 is the DNA replication origin recognition protein for bovine papillomavirus (BPV), and it carries out enzymatic functions required for initiation of viral DNA replication. Cellular mechanisms likely play a role in regulating BPV DNA replication. We are investigating the role of phosphorylation of E1 on viral replicationin vivoand on E1 activityin vitro.Serine 109 is a phosphoacceptorin vivoand is targeted by protein kinase A and protein kinase Cin vitro.A viral genome carrying a serine 109 to alanine mutation replicates more efficiently than wild-typein vivoin a transient replication assay. Furthermore, purified mutant protein, while having wild-type levels of ATPase activity, is able to bind more origin-containing DNA than wild-type E1. Phosphorylation therefore appears to play a selective role in modulating a specific E1 function during viral DNA replication.

Activation of Replication Origins in ϕ29-related Phages Requires the Recognition of Initiation Proteins to Specific Nucleoprotein Complexes

Protein p6 of Bacillus subtilis phage phi29 activates the initiation of viral DNA replication by forming a multimeric nucleoprotein complex at the origins of replication, located at both ends of the linear genome. This activation requires a precise positioning of the protein p6 array with respect to the initiation site. To investigate this activation mechanism, we have purified the phi29 protein p6 counterparts from the related phages Nf and GA-1 and analyzed the formation of complexes with DNA. In the homologous protein p6-DNA complexes the phi29 and Nf protein arrays showed an identical positioning, different than that of the GA-1 protein array. In contrast, in the heterologous complexes the protein showed a different arrangement except in the case of the Nf protein-phi29 DNA complex. We have also purified the proteins involved in the initiation of replication (terminal protein and DNA polymerase) from phages Nf and GA-1 and measured the ability of the different p6 proteins to activate homologous and heterologous replication origins. The results obtained indicate that the activation requires not only the formation of a specific nucleoprotein complex but also its specific recognition by the proteins involved in the initiation of DNA replication.

The initiator protein E1 binds to the bovine papillomavirus origin of replication as a trimeric ring-like structure.

The replication initiator protein E1 binds to the origin of replication of bovine papillomavirus in several forms. E1 can bind to its recognition sequence as a monomer together with the viral transcription factor E2, or as a trimeric E1 complex. The trimerization of E1 is mediated by the sequence-specific binding of E1 to DNA, and results in an E1 complex that is linked topologically to the DNA because the three molecules of E1 form a ring-like structure that encircles the DNA. These results demonstrate that E1 utilizes unusual mechanisms for sequence-specific binding to DNA and for the generation of a structure that encircles the DNA. We believe that these forms of E1 bound to the origin of replication represent intermediates in a transition in the function of E1, from a sequence-specific origin of replication recognition protein to a form of E1 that is competent for the initiation of viral DNA replication.

Domains of the E1 Protein of Human Papillomavirus Type 33 Involved in Binding to the E2 Protein

Papillomavirus E1 and E2 proteins are essential for the initiation of viral DNA replication. We have now analyzed the interaction of E1 and E2 of human papillomavirus type 33, which is associated with cervical carcinoma. When synthesized in insect cells using the baculovirus expression system, the E1 and E2 proteins interacted efficiently at 4°. A monoclonal antibody recognizing E1 amino acids 584–600 inhibited the binding of E2 and vice versa, indicating that these amino acids are involved in E2 binding. To confirm this result, a mutational analysis of E1 was performed. The E2 binding activity of E1 deletion and point mutant proteins was assayed using glutathione S-transferase E1 fusion proteins andin vitrotranslated proteins. At 4°, the C-terminal portion of E1 including amino acids 312–644 was sufficient for E2 binding. Introduction of C-terminal deletions or a point mutation at position 586 (Pro → Glu) resulted in the loss of the E2 binding activity. A second more N-terminally located binding domain (E1 amino acids 312–450) became active when the assays were performed at 22°. The monoclonal antibody still inhibited E2 binding at this temperature, indicating that both E2 binding domains are engaged in the context of the full-length protein.

Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator.

Papillomavirus genomes are maintained as multicopy nuclear plasmids in transformed cells. To address the mechanisms by which the viral DNA is stably propagated in the transformed cells, we have constructed a cell line CH04.15 expressing constitutively the viral proteins E1 and E2, that are required for initiation of viral DNA replication. We show that these viral proteins are necessary and sufficient for stable extrachromosomal replication. Using the cell line CH04.15, we have shown that the bovine papillomavirus-1 (BPV-1) minimal origin of replication (MO) is absolutely necessary, but is not sufficient for stable extrachromosomal replication of viral plasmids. By deletion and insertion analysis, we identified an additional element (minichromosome maintenance element, MME) in the upstream regulatory region of BPV-1 which assures stable replication of the MO-containing plasmids. This element is composed of multiple binding sites for the transcription activator E2. MME appears to function in the absence of replication but requires E1 and E2 proteins for activity. In contrast to, for example, Epstein-Barr virus oriP, stably maintained BPV-1 plasmids are not subject to once-per-cell cycle replication as determined by density labelling experiments. These results indicate that papillomavirus episomal replicators replicate independently of the chromosomal DNA of their hosts.

Regulation of African cassava mosaic virus complementary-sense gene expression by N-terminal sequences of the replication-associated protein AC1

Fragments of the African cassava mosaic virus (ACMV) genome, cloned upstream of the beta-glucuronidase (GUS) reporter gene in an expression cassette, were analysed for their ability to direct complementary-sense gene expression in tobacco protoplasts by measuring GUS activity. Five arbitrary domains (A-E) have been designated that contribute to the expression of AC1 (replication-associated protein) and AC4. Consistent with earlier reports, AC1 gene expression was negatively regulated (80% reduction in activity) by its own protein product, and suppression was mimicked by truncated versions of AC1 comprising the N-terminal 57 amino acids. AC1 also suppressed AC4 gene expression to a similar extent. Nucleotide sequences responsible for suppression were mapped to domain A, a 92 bp fragment located immediately upstream of the AC1 initiation codon encompassing the consensus TATA box and transcription start point. Complementary-sense gene expression also decreased by 30-40% in the presence of AV1 (coat protein) although other DNA A-encoded proteins (AV2, AC2, AC3 and AC4) had no effect. The results are discussed in the light of recent advances concerning the initiation of viral DNA replication and the control of gene expression.

In vivo functional relationships among terminal proteins of Bacillus subtilis φ29-related phages

Gene 3 of the Bacillus subtilis phage φ29 encodes the terminal protein (TP), which acts as a primer in the initiation of viral DNA replication. We have developed an in vivo functional assay for the φ29 TP based on the ability of TP-producing B. subtilis non-suppressor (su −) cells to support DNA replication of a φ29 sus3 mutant phage. This trans-complementation assay has been used to study in vivo functional relationships between the TP of φ29 and related phages. Our results demonstrate that φ29 TP functionally substitutes the TP of phage PZA, whereas replication of phage Nf DNA cannot take place in vivo using the φ29 TP as a primer.

E1 protein of human papillomavirus type 1a is sufficient for initiation of viral DNA replication.

Previous studies on transient replication of papillomaviruses have shown an absolute requirement for the viral E1 and E2 proteins in DNA replication. Here we demonstrate that for human papillomavirus type 1a (HPV-1a) DNA, the E1 protein alone is sufficient for in vivo replication of plasmids containing the viral origin of replication. Replication was origin-specific and required the presence of a DNA sequence containing a putative E1 binding site, but the E2 binding sites were dispensable. In the presence of the E1 protein, E2 stimulated replication of plasmids containing the E1 and E2 binding sites, but no stimulation was observed when the origin plasmids lacked E2 binding sites. Conversely, in the presence of E1 alone, the E2 binding sites did not affect replication. Plasmids containing the replication origins of HPV-6b, HPV-18, and bovine papillomavirus type 1 (BPV-1) also replicated efficiently in the presence of the HPV-1a E1 and E2 proteins. However, plasmids containing the origins of HPV-6b and HPV-18 failed to replicate in the presence of HPV-1a E1 alone, whereas a plasmid containing the BPV-1 origin replicated to lower levels than the HPV-1a origin-containing plasmid. These results suggest that replication from papillomaviral origins in the presence of E1 alone is presumably dependent on the strength of E1-origin interactions. Additionally, E1-dependent replication is stimulated by the E2 protein in the presence of E2 binding sites.

The Complete DNA Sequence of Autographa californica Nuclear Polyhedrosis Virus

The complete nucleotide sequence of the genome of clone 6 of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been determined. The molecule comprises 133,894 base pairs and has an overall A + T content of 59%. Our analysis suggests that the virus encodes some 154 methionine-Initiated, and potentially expressed, open reading frames (ORFs) of 150 nucleotides or greater. These ORFs are distributed evenly throughout the virus genome on either strand. The ORFs are arranged as adjacent, nonoverlapping reading frames separated by short intergenic regions. Based on the primary nucleotide sequence, predictions have been made concerning the functions of certain genes, the sites for initiation of viral DNA replication, the regulation of early and late gene transcription, and factors that may affect the AcNPV gene translational efficiency. The genome sequence date confirm, with minor differences, the information obtained for other AcNPV clones. It is proposed that clone C6 is considered the archetype AcNPV for comparison purposes.

Different Oligomeric Forms of Protein Phosphatase 2A Activate and Inhibit Simian Virus 40 DNA Replication

The ability of simian virus 40 (SV40) large T antigen to catalyze the initiation of viral DNA replication is regulated by its phosphorylation state. Previous studies have identified the free catalytic subunit of protein phosphatase 2A (PP2Ac) as the cellular phosphatase which can remove inhibitory phosphoryl groups from serines 120 and 123. The catalytic C subunit exists in the cell complexed with a 65-kDa A subunit and one of several B subunits. To determine if any of the holoenzymes could activate T antigen, we tested the ability of the heterodimeric AC and two heterotrimeric ABC forms to stimulate T-antigen function in unwinding the origin of SV40 DNA replication. Only free catalytic subunit C and the heterotrimeric form with a 72-kDa B subunit (PP2A-T72) could stimulate T-antigen-dependent origin unwinding. Both the dimeric form (PP2A-D) and the heterotrimer with a 55-kDa B subunit (PP2A-T55) actively inhibited T-antigen function. We found that PP2A-T72 activated T antigen by dephosphorylating serines 120 and 123, while PP2A-D and PP2A-T55 inactivated T antigen by dephosphorylating the p34cdc2 target site, threonine 124. Thus, alterations in the subunit composition of PP2A holoenzymes have significant functional consequences for the initiation of in vitro SV40 DNA replication. The regulatory B subunits of PP2A may play a role in regulating SV40 DNA replication in infected cells as well.

Different oligomeric forms of protein phosphatase 2A activate and inhibit simian virus 40 DNA replication.

The ability of simian virus 40 (SV40) large T antigen to catalyze the initiation of viral DNA replication is regulated by its phosphorylation state. Previous studies have identified the free catalytic subunit of protein phosphatase 2A (PP2Ac) as the cellular phosphatase which can remove inhibitory phosphoryl groups from serines 120 and 123. The catalytic C subunit exists in the cell complexed with a 65-kDa A subunit and one of several B subunits. To determine if any of the holoenzymes could activate T antigen, we tested the ability of the heterodimeric AC and two heterotrimeric ABC forms to stimulate T-antigen function in unwinding the origin of SV40 DNA replication. Only free catalytic subunit C and the heterotrimeric form with a 72-kDa B subunit (PP2A-T72) could stimulate T-antigen-dependent origin unwinding. Both the dimeric form (PP2A-D) and the heterotrimer with a 55-kDa B subunit (PP2A-T55) actively inhibited T-antigen function. We found that PP2A-T72 activated T antigen by dephosphorylating serines 120 and 123, while PP2A-D and PP2A-T55 inactivated T antigen by dephosphorylating the p34cdc2 target site, threonine 124. Thus, alterations in the subunit composition of PP2A holoenzymes have significant functional consequences for the initiation of in vitro SV40 DNA replication. The regulatory B subunits of PP2A may play a role in regulating SV40 DNA replication in infected cells as well.

The NFIII/OCT-1 binding site stimulates adenovirus DNA replication in vivo and is functionally redundant with adjacent sequences.

The inverted terminal repeat (ITR) of adenovirus type 5 (Ad5) is 103 bp in length and contains the origin of DNA replication. Cellular transcription factors NFI/CTF and NFIII/OCT-1 bind to sites within the ITR and participate in the initiation of viral DNA replication in vitro. The ITR also contains multiple copies of two conserved sequence motifs that bind the cellular transcription factors SP1 and ATF. We have analyzed a series of viruses that carry deletions at the left terminus of Ad5. A virus carrying a deletion of the NFIII/OCT-1, SP1, and ATF sites within the ITR (mutant dl309-44/107) was wild type for virus growth. However, the deletion of these elements in addition to sequences immediately flanking the ITR (mutant dl309-44/195) resulted in a virus that grew poorly. The analysis of growth parameters of these and other mutants demonstrate that the NFIII/OCT-1 and adjacent SP1 sites augment the accumulation of viral DNA following infection. The function of these elements was most evident in coinfections with a wild-type virus, suggesting that these sites enhance the ability of a limiting trans-acting factor(s), that stimulates viral DNA replication, to interact with the ITR. The results of these analyses indicate functional redundancy between different transcription elements at the left terminus of the Ad5 genome and demonstrate that the NFIII/OCT-1 site and adjacent SP1 site, previously thought to be nonessential for adenovirus growth, play a role in viral DNA replication in vivo.