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

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.H uman papillomavirus type 16 (HPV16) is a small doublestranded DNA virus that infects mucosal epithelia (1). It is associated with cervical cancer (2, 3) and respiratory tract papillomatosis, and it is thought to contribute to the oncogenesis of head and neck squamous cell cancers. Of such cancers arising from the tonsil, 60% contain HPV16 (4, 5).Current understanding of HPV pathogenesis related to cellular and viral gene interactions has come from studies using HPV infected keratinocytes obtained by one of two means. One method uses immortalized cell lines derived from in vivo lesions infected with HPV types 11, 16, 31, or 59 (6-11). A second method uses the transfection of full or partial HPV genomes, including 11, 16, 18, 31, and 45, as well as a chimeric HPV18͞16, into low-passage human keratinocytes (12-17). After prolonged passaging of cells transfected with complete oncogenic HPV genomes, isolated immortalized cell lines containing episomal HPV DNA could be obtained. When these HPV-containing immortalized cells were grown under differentiating conditions, they produced infectious particles (6,8,(12)(13)(14).However, genome transfection methods used to produce infectious HPV particles have limitations. Even under optimal conditions, very few immortalized clones emerge, typically after weeks in culture. In addition, because of the requirement of selection and the relative paucity of HPV-containing cells in these models, it has not been possible to study early events in the viral lifecycle, such as HPV genome establishment and virus-cell interactions before cellular immortalization. Moreover, because cellular immortalizati...

Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis

“…To study the trafficking of HPV virions, we used efficient generation of pseudoviral particles, which are considered indistinguishable from native virions but encapsidate a reporter plasmid (pseudogenome) (4,37,38). We also generated quasivirions in 293TT cells that encapsidate the HPV16 genome (39). For most experiments, we used particles containing a pseudogenome labeled with 5-ethynyl-2′-deoxyuridine (EdU), a nucleotide analog that can be detected by immunofluorescent staining using Click-iT chemistry (40).…”

Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes

“…In so doing, we overcame the inefficient amplification of the wild-type viral DNA in immortalized cells, a limitation on HPV genetic analyses. Lee et al (2004) similarly used Cre-loxP recombination to generate an HPV-16 genomic plasmid in PHKs from chimeric adenoviruses, but the immortalized cells had a very low yield of virus. Construction of such a virus is time-consuming.…”

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

“…Second we established a conceptual framework for quantitative determination of the rate of viral entry into cells by using quantum dot labeled human pseudo-papillomavirus (HPV) particles. Human papillomavirus have been identified as mediators of a number of benign and malignant cancers of the skin and mucosa (19,20).…”