Assembly of human papillomavirus type 16 pseudovirions in the yeast strain Saccharomyces cerevisiae.

摘要

Studies on encapsidation of Papillomavirus DNA, and production of preparative amounts of papillomaviruses in vitro have met with only limited success. To circumvent this problem I established a system in yeast to generate infectious human papillomavirus type 16 pseudovirions. Saccharomyces cerevisiae strain 1699 was transformed with a construct to allow production of human papillomavirus type 16 virus-like particles. This strain was then transformed with a second construct (target plasmid) having the size of the human papillomavirus type 16 genome, the human papillomavirus type 16 upstream regulatory region and the human papillomavirus type 16 E2 open reading frame. In addition, the target plasmid contained the Green Fluorescent protein gene to monitor delivery of the target plasmid into mammalian cells after infection.;I conclude that the above system allows DNA encapsidation because (a) human papillomavirus type 16 virus-like-particles of two different types (heavy and light) were detected by CsCl gradient centrifugation, (b) DNase I resistant DNA was detected by Polymerase chain reaction/Southern blot analysis in fractions of Cesium Chloride gradients at a density corresponding to heavy virus-like-particles, (c) in vitro transduction of mammalian cells, including primary mouse splenocytes, with pseudovirions resulted in delivery of the reporter gene as demonstrated by flow cytometric analysis for GFP expression, (d) after injection of pseudovirions into mice, in vivo reporter gene expression was detected by confocal microscopy in sections of muscle tissue.