CIS-ACTING COMPONENTS OF HUMAN PAPILLOMAVIRUS (HPV) DNA-REPLICATION -LINKER SUBSTITUTION ANALYSIS OF THE HPV TYPE-11 ORIGIN

Papillomavirus DNA replication requires the viral trans-acting factors E1 and E2 in addition to the host cell's general replication machiner y. The origins of DNA replication in bovine and human papillomavirus g enomes have been localized to a specific part of the upstream regulato ry region (URR) which includes recognition sites for E1 and E2 protein s. To fine map cis-acting elements influeucing human papillomavirus ty pe 11 (HPV-11) DNA replication and to determine the relative contribut ions of such sites, we engineered consecutive linker substitution muta tions across a region of 158 bp in the HPV-11 origin and tested mutant origins for replication function in a cell-based transient replicatio n assay. Our results both confirm and extend the findings of others. E 2 binding sites are the major cis components of HPV-11 DNA replication , and there is evidence for synergy between these sites. Differential capacity of the three E2 binding sites within the origin to affect rep lication map be attributed, at least in part, to context. At least one E2 binding site is essential for replication. The imperfect AT-rich p alindrome of the E1 helicase binding site is not essential since repli cation occurs even in the absence of this sequence. However, replicati on is enhanced by the presence of the palindromic sequence in the HPV- 11 origin. Sequence components adjacent to the E1 and E2 binding sites , comprising AT-rich and purine-rich elements and the consensus TATA b ox sequence, probably contribute to the overall efficiency of replicat ion, though they are nonessential. None of the other cis elements of t he HPV-II origin region analyzed seems to influence replication signif icantly in the system described. The HPV-11 origin of DNA replication therefore differs from those of the other papovaviruses, simian virus 40 and polyomavirus, inasmuch as an intact helicase binding site and a djacent AT-rich components, while influential, are not absolutely esse ntial.