Analysis of relative binding affinity of E7-pRB of human papillomavirus 16clinical variants using the yeast two-hybrid system

A number of genotypes of the human papillomaviruses (HPV) are associated wi th malignancies of the uterine cervix. Sequencing work has revealed the exi stence of intratype HPV variants with minor differences in the nucleotide s equence. More recent data suggest the possibility that some of the variants may have different modes of clinical manifestation. In this study, sequenc es of the E6 and E7 oncogenes of 17 HPV16 isolates derived from PAP smear s amples of Taiwanese patients were analyzed. A number of E6 and E7 novel var iants were found. Particularly, a prevalent (64.7%) E6 polymorphic site A44 2C with an E113D amino acid substitution seems specific to Taiwanese patien ts. In E7, two novel but silent polymorphic sites G663A (41.2%) and T846C ( 88.2%) were also prevalent in the samples analyzed. The yeast two-hybrid sy stem was adopted for rapid assessment of relative E7-pRb binding affinity i n the variants. The relative binding affinities of the E7 proteins of diffe rent HPV types to pRB were in close agreement with previous biochemical dat a. A T663G/C24W polymorphic change in E7 correlated with a decrease in E7-p Rb relative binding affinity the significance of which remains to be clarif ied. This semi-quantitative biochemical and genetic approach may be useful as a first step in the development of clinical protocols for the screening and identification of important HPV variants for clinical interpretation an d for further functional analysis by transfection or other bioassays. (C) 2 000 Wiley-Liss, Inc.