Molecular cloning and functional analysis of human Papillomavirus type 59 DNA

Abstract

The complete nucleotide sequence and genomic organization of human papillomavirus type 59 DNA isolated from vulvar intraepithelial neoplasia was determined. It consists of 7896 nucleotides. A comparative analysis of HPV59 with other sequenced HPVs revealed that genetic features of HPV59 are similar to those of other mucosal HPVs. This virus is most closely related to both HPV18 and 39 (60% identical nucleotide). Using distance matrix phylogenetic analysis, we compared the full L1 ORF of HPV59, 45 other HPV types, 2 subtypes, and 9 animal papillomaviruses. The resulting trees include virtually identical major branches to previous studies; however, they distinctly show subgrouping of all "high risk" HPVs, including HPV 59, into two minor branches. Phylogenetic analysis of the L2 ORF, a putative determinant of tissue specificity, was also performed, revealing identical "high risk" minor branches. Furthermore, the L2 motif thr-thr-pro-ala-val/ile-leu/ile-asp/asn-val/lie, an extension of a previously reported mucosal motif, is highly conserved and found exclusively in all HPV types which have been reported in mucosal lesions. cis-regulatory element of the HPV 59 URR are identified by using CAT (chloramphenicol acetyl transferase) assay of deletion mutants and EMSA (electrophoresis mobility shift assay) of cis-elements for cellular transcribing factor, which are capable of independent enhancing activity. these results suggested that the structural organization of HPV59 E6 promoter is subdivided into three parts : distal (nt 7149 to 7493), central region (nt 7493 to 7742), and proximal region (nt 7742 to 48). In these subregions, the 249 bp (nt 7493 to 7742) of the central region plays an important role as the enhancer element for the maximal transcription of the E6 promoter. It also found that the HPV59 E2 transactivator can repress the transcription of the E6 promoter via inhibiting the formation of initiation complex. When the cellular factor binding to the HPV59 U...