Propagation of infectious human papillomavirus type 16 by using an adenovirus and Cre/LoxP mechanism

doi:10.1073/pnas.0308615100

Propagation of infectious human papillomavirus type 16 by using an adenovirus and Cre/LoxP mechanism

Departments of ^*Otolaryngology, Head and Neck Surgery, ^‡Microbiology, ^§Internal Medicine, and ^¶Physiology and Biophysics and ^∥Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242; and ^**Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131

Contributed by Michael J. Welsh, December 26, 2003

Abstract

Human papillomavirus type 16 (HPV16) infection is a major risk factor for the development of squamous cell cancers of the cervix and of the head and neck. A major barrier to understanding the progression from initial infection to cancer has been the lack of in vitro models that allow infection, replication, and persistence of the viral genome as an episome in differentiated epithelial cells. To overcome this barrier, we designed an adenoviral delivery vector that contained a full HPV16 genome flanked by LoxP homologous recombination sites and a fluorescent reporter that was expressed only after the HPV genome was excised by Cre recombinase. This system delivered circular HPV16 genomes to cervical epithelial cells and well differentiated human airway epithelia. After delivery, the HPV16 genome replicated and persisted as an episome in cervical keratinocytes. These cells developed an immortalized phenotype and a dysplastic epithelial appearance. Moreover, induction of differentiation led to the expression of late genes and production of infectious HPV16 virions. This work provides a means of introducing biologically active HPV genomes into epithelial cells, which are normally difficult to transfect. These methods allow the study of HPV genome replication and gene expression in the earliest stages of HPV genome establishment, and they may provide a means to study nononcogenic HPV viral types.

Footnotes

↵ † To whom correspondence should be addressed at: University of Iowa Hospitals and Clinics, Department of Otolaryngology, Head and Neck Surgery, 200 Hawkins Drive, 21265 Pomerantz Family Pavilion, Iowa City, IA 52242. E-mail: john-h-lee{at}uiowa.edu.
Abbreviations: Ad, adenovirus; CMV, cytomegalovirus; eGFP, enhanced GFP; HPV16, human papillomavirus type 16; LCR, long control region; vge, viral genome equivalents.