Research Article: Inactivation of p53 Rescues the Maintenance of High Risk HPV DNA Genomes Deficient in Expression of E6

Date Published: October 24, 2013

Publisher: Public Library of Science

Author(s): Laurel D. Lorenz, Jessenia Rivera Cardona, Paul F. Lambert, Ann Roman.


The human papillomavirus DNA genome undergoes three distinct stages of replication: establishment, maintenance and amplification. We show that the HPV16 E6 protein is required for the maintenance of the HPV16 DNA genome as an extrachromosomal, nuclear plasmid in its natural host cell, the human keratinocyte. Based upon mutational analyses, inactivation of p53 by E6, but not necessarily E6-mediated degradation of p53, was found to correlate with the ability of E6 to support maintenance of the HPV16 genome as a nuclear plasmid. Inactivation of p53 with dominant negative p53 rescued the ability of HPV16 E6STOP and E6SAT mutant genomes to replicate as extrachromosomal genomes, though not to the same degree as observed for the HPV16 E6 wild-type (WT) genome. Inactivation of p53 also rescued the ability of HPV18 and HPV31 E6-deficient genomes to be maintained at copy numbers comparable to that of HPV18 and HPV31 E6WT genomes at early passages, though upon further passaging copy numbers for the HPV18 and 31 E6-deficient genomes lessened compared to that of the WT genomes. We conclude that inactivation of p53 is necessary for maintenance of HPV16 and for HPV18 and 31 to replicate at WT copy number, but that additional functions of E6 independent of inactivating p53 must also contribute to the maintenance of these genomes. Together these results suggest that re-activation of p53 may be a possible means for eradicating extrachromosomal HPV16, 18 or 31 genomes in the context of persistent infections.

Partial Text

Human papilloma viruses (HPVs) are small, non-enveloped icosahedral viruses that infect epithelial linings of the body and are the causative agents of warts. Infection is thought to arise when a virus particle enters a dividing basal epithelial cell, accessed through a wound in the epithelia, wherein its viral genome is delivered to the nucleus and viral genes begin to be expressed [1]. The papillomavirus life cycle is intricately tied to the differentiation of the stratified squamous epithelia that they infect, with progeny virus exclusively generated in the suprabasal compartment [2]–[5]. HPVs are classified as cutaneous or mucosotropic depending upon the type of epithelia they infect. A subset of the mucosotropic HPVs, the so-called high risk HPVs, including HPV genotypes 16, 18 and 31, are associated with approximately 5% of human cancers including the vast majority of cervical cancers as well as other anogenital cancers and a growing fraction of head and neck cancers [6]–[10]. An important requirement for the onset of HPV-associated cancers is persistent infection by these high risk HPVs [11]. Prophylactic HPV vaccines hold great promise in preventing new infections but do not eliminate pre-existing infections [12]. Developing the means to eliminate persistent high risk HPV infections would be of great value in reducing the risk of cancer among patients already infected with high risk HPVs.

Our studies demonstrate that HPV16 E6 is required for maintenance of the HPV16 genome as an extrachromosomal nuclear plasmid and that inactivation of p53 by dominant negative p53 (p53DD) is sufficient to support maintenance of the HPV16 genome in the absence of E6 (Figure 5). Inactivation of p53 was also necessary to support maintenance of HPV16 since the HPV16 E6SAT mutant genome, which is deficient for inactivating p53, was maintained only in NIKS transduced with p53DD (Table 2). Furthermore the ability of two other mutant genomes HPV16 E6I128T and HPV16 E6Δ146–151 to inactivate p53 (as shown by their ability to attenuate a p53-dependent G1/S growth arrest) correlated with the ability of these genomes to be maintained extrachromosomally (Figures 1B, 3C and 3D).




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