Our Model Systems

 

We use the organotypic (raft) culture system to study the differentiation-dependent life cycles of papillomaviruses (PVs) and to purify infectious human papillomavirus (HPV) virions.

The organotypic, or “raft” raft system was adapted for the study of papillomaviruses by McCance, Kopan, Fuchs, and Laimins [1].  It is the only in vitro system proven to consistently mimic epithelial differentiation to the extent that infectious papillomaviruses can be synthesized and purified.

Craig Meyers (the PI’s postdoc mentor) and coworkers were the first to reproduce the complete HPV life cycle in vitro using a continuous cell line, CIN-612 9E established from a low-grade cervical biopsy and persistently infected with HPV31 [2, 3].  Growth of CIN-612 9E organotypic tissues results in the reproducible production of HPV31 virions that can be purified and used for experimental infections [3-5].  Much information on high-risk HPV life cycles has come from the Laimins’ lab, the Meyers’ lab and our analyses of HPV31.

The organotypic (raft) culture system is represented by the figure above [6]. Epithelial cells persistently infected with papillomavirus DNA can be obtained from biosies or by transfecting human keratinocytes. [A], Epithelial cells containing episomal HPV genomes are seeded onto submerged type I collagen matrices containing fibroblasts. [B], When the epithelial cells reach confluence, growth media are removed and the collagen matrices are lifted onto stainless steel support grids.  [C], The epithelial cells are fed by diffusion from below the air-liquid interface; the collagen-fibroblast matrix acts as the dermal equivalent. Cell culture medium for monolayer and raft epithelium is E medium containing 5% fetal calf serum. For PKC induction, the medium is supplemented with either 10 µM 1,2- dioctanoyl-sn-glycerol (C8:0) or 16 nM 12-O- tetradecanoyl phorbol-13-acetate (TPA). C8:0 treatment is every other day, concurrent with feeding; TPA treatment is for 16-20 h, then the medium is replaced with fresh E medium containing 5% fetal calf serum.  [D], Epithelial tissues are allowed to stratify and differentiate at the air-liquid interface over a 2-week period.

 

High-yield production (HiP) of PV virions independent of viral replication was recently reported by Pyeon, Lambert, and Ahlquist [7]. 

 

The method builds upon work of Buck et al. whereby small DNAs like reporter plasmids are packaged in self-assembling L1+L2 capsids to make pseudovirions in transiently transfected mammalian cells [8].  HiP yields >100X more infectious virus per cell than organotypic cultures; another advantage of this method is the ability to create any genetic mutant viral genome, regardless of replication ability, for experimental infections.  A recent study compared infections in rabbits inoculated with either tissue-derived cottontail rabbit PV (CRPV) virions or HiP-CRPV virions[9].  They carefully established that the virion stocks were identical as assayed by susceptibility to antibody-mediated neutralization, papilloma induction, and gene expression within lesions in rabbits indicating that HiP virions are biologically indistinguishable from tissue-derived virions [9].  We use this approach to make infectious virions from HPV types 16, 18, 31, 11, and RhPV1.

 

 

 

 

 

 

 

RhPV1 as a model of HPV induced infection and disease in rhesus macaques. 

The RhPV1 genome was recovered from a metastatic tumor arising from a penile carcinoma in a rhesus monkey (Macaca mulatta) [66].  This virus resides in the alpha genus of papillomaviruses and is most genetically homologous to HPV16 [99, 101]. The RhPV1 genome has transforming abilities similar to the oncogenic HPV genomes [85, 129].  Furthermore, RhPV1 is sexually transmitted in the rhesus population [101], just as anogenital HPVs are sexually transmitted in humans.  Of thirty female monkeys having sexual contact with the index male that developed penile cancer, two females developed cervical carcinomas, eleven showed clinical signs of genital PV infection (i.e., SIL), and nine were positive for RhPV1 DNA genomes but showed no other signs of disease [101].  This equates to infection of 71% of the tested population.  Furthermore, analysis of rhesus monkeys from four geographically separate primate institutions revealed RhPV1 DNA and/or serological responses to RhPV1 infections in up to 52% of the animals tested [98].  All data are consistent with the classification of RhPV1 as an oncogenic virus.  

We hold U.S. Patent Serial No. 7,285,386: “RhPV as a model for HPV-induced cancers”.         Contact us about using this model.

We have been using the HiP system to obtain infectious virions and are performing experimental infections with our colleagues at Tulane National Primate Research Center.  Thus far we have positive indications that animal are becoming persistently infected and developing disease.  We are preparing to launch testing of potential HPV microbicides in this model.

 

References

1.  McCance, D.J., R. Kopan, E. Fuchs, and L.A. Laimins, Human papillomavirus type 16 alters human epithelial cell differentiation in vitro. Proc. Natl. Acad. Sci. U.S.A., 1988. 85: p. 7169-7173.
2.  Bedell, M.A., J.B. Hudson, T.R. Golub, M.E. Turyk, M. Hosken, G.D. Wilbanks, and L.A. Laimins, Amplification of human papillomavirus genomes in vitro is dependent on epithelial differentiation. J. Virol., 1991. 65(5): p. 2254-2260.
3.  Meyers, C., M.G. Frattini, J.B. Hudson, and L.A. Laimins, Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation. Science, 1992. 257: p. 971-973.
4. Ozbun, M.A., Human papillomavirus type 31b infection of human keratinocytes and the onset of early transcription. J. Virol., 2002. 76: p. 11291-11300.
5. Ozbun, M.A., Infectious human papillomavirus type 31b: purification and infection of an immortalized human keratinocyte cell line. J. Gen. Virol., 2002. 83: p. 2753-2763.
6.  Meyers, C., Organotypic (raft) epithelial tissue culture system for the differentiation-dependent replication of papillomavirus. Methods in Cell Sci, 1996. 18: p. 201-210.
7. Pyeon, D., P.F. Lambert, and P. Ahlquist, Production of Infectious Human Papillomavirus Independently of Viral Replication and Epithelial Cell Differentiation. Proc Natl Acad Sci USA, 2005. 102: p. 9311-9316.
8.  Buck, C.B., D.V. Pastrana, D.R. Lowy, and J.T. Schiller, Efficient intracellular assembly of papillomaviral vectors. J. Virol., 2004. 78(2): p. 751-757.
9.  Culp, T.D., N.M. Cladel, K.K. Balogh, L.R. Budgeon, A.F. Mejia, and N.D. Christensen, Papillomavirus particles assembled in 293TT cells are infectious in vivo. J. Virol., 2006. 80(22): p. 11381-11384.