INFECTIOUS HUMAN-PAPILLOMAVIRUS TYPE-18 PSEUDOVIRIONS

Human papillomavirus type 18 (HPV18) capsid proteins L1 and L2, synthe sised in mammalian cells using recombinant vaccinia viral expression v ectors, are transported to the nucleus and assembled into virus-like p articles. When 293T cells, which express SV40 T antigen, were transfec ted with plasmid DNAs containing an SV40 origin of replication then in fected with vaccinia viral vectors encoding L1 and L2, plasmid DNA was encapsidated into the particles. The DNAs ranged in size from 5.4 to 7.9 kb. By encapsidating plasmids containing either the beta-galactosi dase gene or the puromycin-resistance gene, the pseudovirions were sho wn to be infectious in that they could transfer beta-galactosidase act ivity or confer resistance to puromycin to a number of cell types, ind icating that the uptake and decapsidation of HPV particles are not the main determinants of cell type specificity of HPV. Episomal HPV16 DNA in a cervical keratinocyte line could also be encapsidated. Further i nvestigation showed that DNA encapsidation is independent of HPV DNA s equences and of T antigen-mediated plasmid DNA replication. Instead, t he minor capsid protein, L2, was found to be attached to plasmid mini- chromosomes extracted from these cells, suggesting a role for L2 in en capsidation. Consistent with this, the L1 protein alone was unable to encapsidate DNA, although it was able to form virus-like particles. Th e results suggest that intracellular episomal DNAs of suitable size ca n be encapsidated by the HPV18 L1 and L2 proteins without the need of any HPV packaging signal, and reintroduced into cells. (C) 1998 Academ ic Press.