G L Bream

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.

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.

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.

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.