ABSTRACT
The study of human papillomavirus replication has been hampered by the lack of an in vitro system which reliably supports virus replication. Recent results from the bovine papillomavirus (BPV) system indicate that the E1 and E2 proteins are the only viral gene products required for replication. By analogy with simian virus 40 large T antigen, E1 is thought to possess ATPase and helicase activity, which may play a direct role in viral DNA replication. The precise role of E2 is unclear, but it may function in part to help localize E1 to the replication origin. We have initiated a study of replication in the human papillomavirus type 11 system which, by analogy to BPV, has focused on the E1 and E2 proteins of this virus. We have expressed the full-length E1 and E2 proteins in Sf9 insect cells by using a baculovirus expression vector. Both the 80-kDa E1 protein and the 42.5-kDa E2 protein are nuclear phosphoproteins. The E1 and E2 proteins form a heteromeric complex within the insect cells, and both proteins localize to a DNA fragment which contains the viral origin of replication. In addition, we have detected an E1-associated ATPase and GTPase activity, which is likely part of an energy-generating system for the helicase activity which is predicted for this protein. The human papillomavirus type 11 E1 and E2 proteins possess the same replication-associated activities exhibited by the corresponding BPV proteins, suggesting that the replication activities of these viruses are tightly conserved.
Subject(s)
DNA Replication , Oncogene Proteins, Viral/metabolism , Papillomaviridae/metabolism , Pyrophosphatases , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Animals , Baculoviridae/genetics , Base Sequence , DNA, Viral/genetics , Humans , Macromolecular Substances , Molecular Sequence Data , Moths/cytology , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/immunology , Papillomaviridae/genetics , Phosphoproteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Plasmids/genetics , Recombinant Proteins/metabolismABSTRACT
The bovine papillomavirus upstream regulatory region represents a common element in the regulation of transcription from the five early viral promoters. We have determined the sequences required for transcription from the viral P1 promoter, which is located at the 5' end of the upstream regulatory region. In vitro transcription from P1 requires a 123-bp fragment (nucleotides 7153 to 7275; -33 to +90) consisting of an upstream TATA-like sequence as well as an unidentified protein which binds to sequences immediately downstream of the initiation site. In vivo, this promoter requires additional downstream sequences (to position +160; nucleotide 7345) for maximal activity but does not require any additional DNA sequence upstream of a putative TATA box. Four regions within the downstream sequence from +9 to +160 are protected from DNase I digestion by proteins present in a HeLa cell extract. The presence of these sites correlates with the level of P1 activity. A constitutive enhancer maps to this same region, and mutations in this enhancer have been shown to affect downstream promoters. Deletion analysis indicates that the same sequences are required by both the P1 promoter and the constitutive enhancer, suggesting that the same proteins function in both activities.
Subject(s)
Bovine papillomavirus 1/genetics , Enhancer Elements, Genetic , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Transcription, Genetic , Animals , Base Sequence , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , DNA, Viral/genetics , HeLa Cells , Humans , Molecular Sequence Data , Plasmids , Recombinant Proteins/metabolism , Restriction Mapping , Sequence Deletion , TATA BoxABSTRACT
Extrachromosomal elements that are stably maintained at a constant copy number through cell doublings are a good model system for the study of the regulation of DNA replication in higher eukaryotes. Previous studies have defined both cis and trans functions required for the regulated plasmid replication of the bovine papilloma virus in stably transformed cells. Here, a sequence known to be a cis-dominant element of the replication origin of the plasmid is shown to contain a promoter for transcription. Both in vitro and in vivo assays have been used to define this promoter and show that a sequence located just 3' to the transcriptional start site is required for activity. This DNA sequence element, which has been defined through deletions, coincides with a binding site for a cellular factor and is also required for a functional origin of replication. Possible models for how a transcription factor may play a role in the regulation of DNA replication are discussed.
Subject(s)
Bovine papillomavirus 1/genetics , DNA Replication , Papillomaviridae/genetics , Promoter Regions, Genetic , Virus Replication , Acetyltransferases/genetics , Base Sequence , Bovine papillomavirus 1/physiology , Chloramphenicol O-Acetyltransferase , Chromosome Deletion , Cycloheximide/pharmacology , Genes, Viral , RNA, Viral/analysis , Templates, Genetic , Transcription, GeneticABSTRACT
The complete DNA sequence of the human JC virus, which was found to consist of 5,130 nucleotide pairs, is presented. The amino acid sequence of six proteins could be deduced: the early, nonstructural proteins, large T and small t antigens; the late capsid proteins, VP1, VP2, and VP3; and the agnogene product encoded within the late leader sequence, called the agnoprotein in simian virus 40. The extent of homology between JC virus DNA and the genomes of simian virus 40 (69%) and BK virus (75%) confirmed the close evolutionary relationship of these three polyomaviruses. The sequences showing the greatest divergence in these viral DNAs occurred within the tandem repeats located to the late side of the replication origins.
