Generation and applications of HPV pseudovirions using vaccinia virus.
This chapter outlines the generation and application of human papillomavirus type 33 (HPV 33) pseudovirions. These pseudovirions are structurally indistinguishable from native virions and are therefore valuable tools for the study of papillomavirus/cell interactions. The method describes (1) the construction of vaccinia viruses recombinant for the major and minor HPV capsid proteins, L1 and L2, respectively, (2) the transfection of Cos7 cells with a marker plasmid replicating to high copy numbers, (3) the expression of L1 and L2 using the vaccinia virus expression system, (4) the extraction, purification, and analysis of HPV-33 pseudovirions, (5) pseudoinfection assays, (6) pre- and post-attachment neutralization of pseudovirions, and (7) the use of inhibitors for study of binding and uptake of pseudovirions. The methods described have been successfully adopted for HPV 16 and 18 and may thus be applied for other HPV types, too.
Sapp
Martin
M
Institute for Medical Microbiology and Hygiene, University of Mainz, Germany.
Selinka
Hans-Christoph
HC
D000818:Animals; D019556:COS Cells; D002213:Capsid / Q000502:physiology; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D018014:Gene Transfer Techniques; D006801:Humans; D008854:Microscopy, Electron; D027383:Papillomaviridae / Q000235:genetics* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D015183:Restriction Mapping / Q000379:methods; D014616:Vaccinia virus / Q000235:genetics* / Q000302:isolation & purification; D014771:Virion / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure