Induction of the Bovine Papillomavirus Origin "Onion Skin"-Type DNA Replication at High E1 Protein Concentrations In Vivo (original) (raw)
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Replication of bovine papillomavirus type 1 DNA initiates within an E2-responsive enhancer element
Journal of Virology, 1990
When bovine papillomavirus transforms cells in vitro, it maintains its genome as a multicopy nuclear plasmid. Plasmid DNA extracted from such transformed cells was analyzed by the two-dimensional gel electrophoresis technique of Brewer and Fangman (B. Brewer and W. Fangman, Cell 51:463-471, 1987). The replication intermediates detected in these assays were found to be the sums of the oligomeric and monomeric forms of the replicating plasmids. The multimeric DNAs were shown by field inversion gel electrophoresis and partial restriction digestion to be head-to-tail concatemers of the monomeric forms. Furthermore, the multimers progressed in size by steps of one monomer, indicating that they did not arise by replication segregation mistakes of the unit length, which would predict a ladder spaced by integrals of two monomers. To map the plasmid DNA replication origin, the replication intermediates of the monomers were isolated by successive sucrose gradient centrifugation and then exami...
In a subclone of 1ID13 mouse fibroblasts latently infected with bovine papillomavirus type 1 (BPV-1) DNA, the viral genome occurred as a mixture of extrachromosomal circular monomers and oligomers. Multiple copies were also associated with the host cell genome, predominantly at a single site in a head-to-tail tandem array. We examined the replicative intermediates of extrachromosomal forms of BPV-1 DNA by using two-dimensional gel electrophoresis. The results obtained indicate that initiation of DNA replication occurred near the center of the EcoRI-BamHI 5.6-kilobase fragment. In some molecules, however, this fragment was replicated from one end to the other by means of a single fork initiated elsewhere. Termination also occurred within this fragment. The EcoRI-BamHI 2.3-kilobase fragment replicated as a DNA molecule containing a termination site for DNA replication and also by means of a single fork traversing the fragment from one end to the other. Thus, replication forks proceeded through these fragments in different manners, apparently depending on whether they were part of a monomer, a d'imer, a trimer, or higher oligomers. These observations lead to the conclusion that initiation of DNA replication in BPV-1 DNA takes place at or close to plasmid maintenance sequence 1. From this point, replication proceeds bidirectionally and termination occurs approximately 1800 opposite the origin. The results obtained are consistent with one or more replication origins being quiescent in BPV-1 DNA oligomers.
Journal of virology, 1995
The viral elements required for the initiation of replication of bovine papillomavirus type 1 DNA include the origin region and two trans-acting factors, the E1 and E2 proteins. We now report that the replication efficiency of a DNA molecule which contains these three elements is modulated by other viral sequences. By measuring the extent of replication of deleted viral genomes in transfected mouse cells, we identified sequences required for maximal efficiency. Addition of these sequences to a construct carrying only the minimal origin region increased its replication. Among these cis-active elements, we identified a 69-bp fragment (nucleotides 4921 to 4990) which contains at least two binding sites for cellular proteins. One of them is the murine protein termed CDEBP, which recognizes the octameric motif ATCACGTG, identical to the yeast CDEI element. Either deletions affecting this CDEI box or a point mutation which impairs binding of CDEBP markedly decreased the extent of viral DN...
Transient replication of bovine papilloma virus type 1 plasmids: cis and trans requirements
Proceedings of the National Academy of Sciences, 1986
A transient assay has been used to study bovine papilloma virus type 1 (BPV-1) replication. We show that BPV-1 early replication occurs faster than cellular DNA synthesis. Initial replication events are dependent on a gene product(s), encoded by the BPV-1 E1 open reading frame. Mutational analysis of the viral upstream regulatory region shows the requirement of two domains in cis for replication. Domain one, located outside of the viral 69% transforming fragment, is an enhancer-like activity and can be replaced by other known viral enhancers. Domain two lies within sequences previously defined as plasmid maintenance sequence 1. The apparent requirement for a proximal enhancer function for replication may explain why certain BPV-1 constructions, when linked to bacterial plasmid sequences, can be maintained extrachromosomally while others cannot.
Journal of Biological Chemistry, 1994
W e have established the first homologous cell-free DNA replication system for a papillomavirus. The replication of the human papillomavirus type 11 (HPV-11) origin was achieved by using human 293 cell extracts supplemented with the HPV-11 E l and E2 proteins purified from insect cells infected with recombinant baculoviruses. Efficient replication depends on the HPV-11 origin, the HPV-11 E l and E2 proteins, as well as human DNA polymerase a, 6, replication protein A, topoisomerase I, and topoisomerase 11. High concentrations of E l protein also promoted a low level of origin-independent replication which was suppressed by the addition of the E2 protein, whereas E2 protein stimulated origin-dependent replication. W e also show that an intact E2 protein binding site was absolutely necessary for origin activity, as a strong HPV-11 origin was rendered inactive when one half-site of each of the three E2 binding sites was mutated. In contrast, there was only a relatively small reduction in this mutant origin activity when the cell extracts were supplemented with the bovine papillomavirus type 1 (BPV-1) proteins. These results suggest that the HPV-11 E2 protein plays a primary role in HPV origin recognition. Furthermore, unlike transient replication in which HPV-11 and BPV-1 viral proteins promote efficient replication of homologous and heterologous origins, efficient cell-free replication took place only with the homologous combinations. Small DNA viruses have long been used as models for higher eukaryotic DNA replication. SV40 is one of the most thoroughly studied DNA viruses, and the functions of both viral and host transacting factors and enzymes have been identified (Refs. 1 and 2; for reviews, see Refs. 3 and 4). The unchecked replication of these viruses, however, does not reflect the regulated host DNA replication that occurs once per cell cycle. Human papillomaviruses (HPVs),' which infect mucosal or cutaneous epithelium and cause hyperproliferation, have two distinct modes of DNA replication. In the basal stem cells and the parabasal transit amplifying cells of squamous epithelia or stratified epithelial raft cultures, viral DNA is maintained as low copy number extrachromosomal plasmids. Only in upper layer keratino-Grant CA36200. The costs of publication of this article were defrayed in
Proceedings of the National Academy of Sciences, 1994
Chemical and enzymatic probing techniques were used to examine the interaction of the bovine papillomavirus type 1 El and E2 proteins with the viral origin of replication (on). El was found to generate significant distortions to the structure of on, as assayed by KMnO4 oxidation of DNA. The primary site of oni distortion was located within and adjacent to the AT-element of the core replicator sequence, although a number of minor structural transitions were also detected. The induction of these structural changes required ATP and appeared to require ATP hydrolysis. E2 was found to decrease the amount of El required for on distortion but did not significantly alter the pattern of structural distortion. In contrast, the presence of E2 resulted in a biphasic mechanism for El binding to oM, as assayed by nuclease protection. Under these conditions, El bound preferentially to the dyad symmetry region containing the conserved Hpa I site. Higher levels of El were required for binding to the adjacent oni AT-rich region. Thus, these data suggest that E2 can order the stepwise binding of El to on.
Journal of Virology, 1986
A bovine papillomavirus (BPV) type 1-encoded function (M) which is a negative regulator of viral plasmid replication has been described elsewhere (Berg et al. Cell, in press; Roberts and Weintraub, Cell, in press). We report here that expression of M, which is a repressor of transient BPV replication and is not required as a positive factor in these assays, is required for the establishment of the viral genome as a stable nuclear plasmid. This function is encoded in part by the 5' portion of the BPV E1 open reading frame, whereas the 3' part of this open reading frame encodes a positive replication function (R). The R function is required for early replication events. We used transient replication assays to define the phenotypes of mutants in both the R and M genes and complementation tests to show that R and M define two separate genes. We showed that R- and M- mutants could also complement each other in stable assays. In cotransfection experiments, M- mutants had a lethal ...
Journal of virology
The E1 protein encoded by bovine papillomavirus type 1 (BPV-1) is required for viral DNA replication, and it binds site specifically to an A/T-rich palindromic sequence within the viral origin of replication. The protein is targeted to this site through cooperative interactions and binding with the virus-encoded E2 protein. To explore the nature of the E1 binding site, we inserted a series of homologous DNA linkers at the center of dyad symmetry within the E1 recognition palindrome. The effects of these modifications indicated that the E1 recognition palindrome can be separated into functional half sites. The series of insertions manifest a phasing relationship with respect to the wild-type BPV-1 genome in that greater biological activity was measured when full integral turns of the DNA helix separated the palindrome than when the separations were half-turns. This phasing pattern of activity was observed to occur in a variety of biological phenotypes, including transformation effici...
Contribution of bovine papillomavirus type 1 E1 protein residue 48 to replication function
Journal of General Virology, 2000
The E1 protein of bovine papillomavirus type 1 (BPV-1) is the origin recognition protein and is essential for the initiation of viral DNA replication. We reported previously that there is a conserved motif between residues 25 and 60 of all papillomavirus E1 proteins that resembles a casein kinase II (CKII) phosphorylation site. The corresponding serine in BPV-1, serine-48, is an efficient substrate for CKII in vitro. To examine the functional role of this potential phosphorylation site, three amino acid substitutions were constructed at serine-48. Conversion of serine-48 to a glycine (S48G) resulted in a BPV-1 genome that was unable to replicate and had reduced transformation capacity. The S48G E1 protein also failed to support replication of a BPV-1 origin-containing plasmid when expressed from a heterologous vector rather than the viral genome, indicating a direct replication defect. In contrast, conversion of serine-48 to acidic residues (S48D or S48E), which mimic the charge and...