Fluorescently Labeled Human Papillomavirus Pseudovirions for Use in Virus Entry Experiments

Human papillomaviruses (HPV) infect skin or mucosal epidermis. The simplistic capsid consists of a major capsid protein L1, a minor capsid protein L2, and a double-stranded circular DNA of about 8 kB in size. The development of HPV-based vectors [i.e., pseudovirions (PsV)] as tools to study the initial infection has facilitated our understanding of HPV entry. The covalent coupling of fluorescent molecules to these PsV allows following the viruses en route to the nucleus, i.e., the site of replication. In the first section, we describe a facile method to covalently label HPV PsV that retain their infectivity. In this method, fluorophores coupled to a reactive succinimidyl ester are covalently attached to amine residues in the virion in a one-step chemical reaction. In the second section of this unit, several assays are outlined that use the fluorescently labeled virions for entry studies in live and fixed cells.