In vivo DNA replication assay of human papillomavirus type 18

Abstract

Human papillomavirus DNA replication requires the viral trans-acting elements E1 and E2 proteins in addition to the host replication machinery. The origins of DNA replication in human papillomavirus genomes have been located in upstream regulatory region (URR) which includes recognition sites for E1 and E2 proteins. To determine significance of the core region and the relative contributions of cis-element, we studied HPV-18 URR (nt 6929 to 119) using deletion analysis and site-directed mutagenesis. Densitometric analysis of the autoradiogram showed that whole URR and serial deleted mutants (p1047, p920, p728, p641, p428, p353, p313, p243) were replicated to approximately the same levels as p210 (nt 7766 to 119), indicating that HPV-18 origin may be located within p210 (nt 7766-119). The linker substitution mutations which interfered with E1 and E2 binding activities reduced the replication activities. These observations implied that E1 and E2 binding sites were the major determinants of HPV-18 DNA replication. Mutants p210#2 which lose GRE sequence showed a slight increase of replication activity (135%). It is inferred that some indirect effects of GRE motif will be engaged in. Although replication is signicantly diminished, it is not abolished completely by mutation of the binding sites for either E1 or E2. When double mutant p210#13 was cotransfected with E1 and E2 expression vectors, it showed stronger levels of replication than those of replication by p210 cotransfected with only E1 expression vectors, suggesting that E1/E2 complex may be ewquired for binding to the half palindrome of mutant E2BSs and may initiate DNA replication.