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J. Virol., 06 1995, 3789-3798, Vol 69, No. 6
Copyright © 1995, American Society for Microbiology

E1 recognition sequences in the bovine papillomavirus type 1 origin of DNA replication: interaction between half sites of the inverted repeats

R Mendoza, L Gandhi and MR Botchan
Department of Molecular and Cell Biology, University of California, Berkeley 94720-3204, USA.

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 efficiency, stable plasmid copy number in cell lines established from pooled foci, and transient replication of full-length viral genomes. For replication reporter constructs where E1 and E2 are supplied in trans by the respective expression vectors, distance between the half sites seems to play a major role, yet the phasing relationships are measurable. DNase I protection studies showed that E1 bound very poorly to the construct containing a 5-bp linker, and binding was close to the wild-type level for the 10-bp insertion, consistent with a requirement for a phasing function between half sites with a modulus of 10 bp. Binding to the 15- and 20-bp insertion mutants was weak, but only for the 20-bp insertions was protection over both halves of the palindrome measurable. As it had been previously reported that the 18-bp palindrome contains sufficient nucleotide sequence information for E1 binding, we speculate that a minimal E1 recognition motif is presented in each half site. A comparison between this sequence and that of an upstream region that also binds E1 (the E2RE1 region) revealed a common pentanucleotide motif of APyAAPy. Mutants with substitutions of the ATAAT elements within E2RE1 failed to bind E1 protein. We present models for how repeats of the pentanucleotide sequence may coordinate E1 binding at the dyad symmetry axis of the origin and compare the DNA sequence organization of BPV-1 with those of the simian virus 40 and polyomaviruses at their origins of DNA replication.

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