Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells.
We have shown that El and E2 proteins of human papillomavirus type 11 (HPV-11) were essential to support the replication of the homologous viral origin (on) in a transient replication assay, similar to reports on bovine papillomavirus type 1 (BPV-1). Unexpectedly, matched or even mixed combinations of El and E2 proteins from HPV-11 or BPV-l replicated either on in human, monkey, and rodent cell lines of epithelial or fibroblastic lineage, albeit with varied efficiencies. Either set of viral proteins was also able to initiate replication of ori-containing plasmids from many other human and animal papillomaviruses. Thus the interactions among the cis elements and trans factors of papillomaviruses are more conserved than expected from the other members of the papovavirus family, simian virus 40 and polyomavirus, for which large tumor antigen does not replicate a heterologous on in either permissive or nonpermissive cells. We infer that the stringent species and tissue specificities observed for papillomaviruses in vivo are not entirely due to direct restrictions on viral DNA replication. Rather, transcriptional control of viral gene expression must play a dominant role.The initiation of DNA replication occurs at precisely defined origins (ori) to which specific control proteins bind. Simian virus 40 (SV40) and polyomavirus have long been used as models for investigations of mammalian DNA replication. Initiation of SV40 or polyomavirus replication requires the homologous large tumor antigen, is species specific, and occurs only in a permissive cell environment (1-4). However, the uncontrolled replication of these lytic viruses does not reflect the precise regulation of cellular DNA replication, which takes place once per cell cycle in the S phase. In contrast, papillomaviruses have regulated and uncontrolled replication phases during their life cycle and therefore offer a unique opportunity to study eukaryotic DNA replication.Human papillomavirus (HPV) types trophic for mucosal epithelia, including HPV-11, are highly infectious pathogens that cause genital warts and laryngeal papillomatosis. A subset of the genital types, including HPV-16 and -18, is also associated with the development of neoplasia and progression to cancer. In subclinical or benign infections, viral DNA persists as low-copy-number plasmids in the nuclei of the undifferentiated basal stem cells. Productive replication takes place only in the more differentiated upper strata of the epithelium. Although a culture system has recently been developed in which preinfected tissues produce progeny HPV-11 virions (25), propagation of HPVs has not been achieved in vitro starting from virions or transfected DNA. In transfected or infected cells, HPV DNA either integrates into host chromosomes or is lost. Therefore it has not been possible, until now, to perform molecular studies of autonomous HPV DNA replication in cell culture.In contrast, bovine papillomavirus type 1 (BPV-1) in transformed rodent cell lines replicates extrachromosomally at a constan...
Expression of the viral polypeptides E1 and E2 is necessary and sufficient for replication of BPV in mouse C127 cells. By providing these factors from heterologous expression vectors we have identified a minimal origin fragment from BPV that contains all the sequences required in cis for replication of BPV in short term replication assays. This same sequence is also required for stable replication in the context of the entire viral genome. The identified region is highly conserved between different papillomaviruses, and is unrelated to the previously identified plasmid maintenance sequences. The minimal ori sequence contains a binding site for the viral polypeptide E1, which we identify as a sequence specific DNA binding protein, but surprisingly, an intact binding site for the viral transactivator E2 at the ori is not required. The isolated origin shows an extended host region for replication and replicates efficiently in both rodent and primate cell lines.
The DNA context of nucleotides that a protein recognizes can influence the strength of the protein-DNA interaction. Moreover, in prokaryotes, understanding the quantitative differences in binding affinities that result in part from the DNA context is often important in describing regulatory mechanisms. Nevertheless, these issues have not been a major focus yet for the investigation of protein-DNA interactions in eukaryotes. In this study, we explored the binding specificity and the range of affinities that the BPV-1 E2 transcriptional activator has for DNA. Because E2 binding sites are positioned near several different BPV-1 promoters, such quantitative information may be important to understand transcriptional regulatory mechanisms in BPV-1. Gel retardation assays and DNA footprinting were used to quantitate the affinities of the E2 binding sites in the viral genome. In the process, five sites were discovered, which, on the basis of sequence, had not been predicted previously to interact with the E2 protein. Equilibrium and kinetic studies show that the range of E2 affinities of the 17 sites varied over 300-fold. The sequence elements responsible for E2 recognition of DNA were determined by missing contact analysis of several sites and a point mutation analysis of one site. The results presented show that the affinity of an E2 binding site is to a large extent determined by the availability of specific contacts, but the data also strongly suggest that DNA structure plays an important role.
The E1 polypeptide from bovine papillomavirus binds to the origin of replication (ori) and possesses the activities attributed to initiator proteins. E1 is also the only viral protein required for replication in a cell‐free replication system. Replication in vivo, however, absolutely requires in addition the viral transcription factor E2. We demonstrate that the basis for this distinction between in vitro and in vivo requirements is the limited sequence specificity of the E1 protein. E1 and E2, which bind the ori individually with low sequence specificity, together bind with greatly increased sequence specificity. This combinatorial effect provides a function for the involvement of transcriptional activation domains in replication and suggests common mechanisms of action for transcription factors in both transcription and replication. It also provides a possible explanation for the differential specificity that is observed for auxiliary transcription factors in vivo.
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