Assembly of human papillomavirus type 16 pseudovirions in Saccharomyces cerevisiae

Studies of the encapsidation of papillomavirus (PV) DNA, and production of preparative amounts of PVs in vitro, have met with only limited success. To circumvent this problem we established a system in yeast to generate infec tious HPV-16 pseudovirions. Saccharomyces cerevisiae strain 1699 was transf ormed with a construct to allow production of HPV-16 virus-like particles ( VLPs). This strain was then transformed with a second construct (target pla smid), the same size as the HPV-16 genome and containing the HPV-16 upstrea m regulatory region (URR) and the HPV-16 E2 open reading frame, In addition , the target plasmid contained the green fluorescent protein gene to monito r delivery of the target plasmid into mammalian cells after infection. We c onclude that this system allows HPV DNA encapsidation because (1) HPV-16 VL Ps of two different types (heavy and light) were detected by CsCl gradient centrifugation, (2) DNase I-resistant DNA was detected by PCR/Southern blot analysis in fractions of CsCl gradients at a density corresponding to heav y VLPs, (3) in vitro infection of mammalian cells, including primary mouse splenocytes, with pseudovirions resulted in delivery of the reporter gene a s demonstrated by FAGS analysis for GFP expression, and (4) after injection of pseudovirions into mice, in vivo reporter gene expression was detected by confocal microscopy in sections of muscle tissue. We conclude that HPV-1 6 pseudovirions produced in yeast may be useful both for in vitro transduct ion and for gene delivery in vivo.