Tumourigenesis Driven by the Human Papillomavirus Type 16 Asian-American E6 Variant in a Three-Dimensional Keratinocyte Model

Abstract

Infection with a transforming human papillomavirus (HPV) such as type 16 (of species Alphapapillomavirus 9) causes ano-genital and oral tumours via viral persistence in human squamous cell epithelia. Epidemiological studies showed that the naturally occurring HPV16 Asian-American (AA) variant (sublineage D2/D3) is found more often than the European Prototype (EP) (sublineage A1) in high-grade cervical neoplasia and tumours compared to non-cancer controls. Just three amino acid changes within the early gene, E6, of HPV16 AA have been linked to this augmented tumourigenicity. The AAE6 variant's greater immortalizing and transforming potential over EPE6 has recently been confirmed in retrovirally-transduced keratinocytes expressing the E6 gene only. However, the tumourigenic role of the full-length viral genome of HPV16 has not yet been addressed with regard to these E6 variants. To investigate this process in the context of these two HPV16 E6 genotypes, an organotypic tissue culture model was used to simulate the HPV infectious life cycle. The AAE6 variant demonstrated an enhanced ability over EPE6 to drive the viral life cycle toward tumourigenesis, as evidenced phenotypically—by a more severe grade of epithelial dysplasia with higher proliferation and deregulated differentiation, and molecularly—by high viral oncogene E6 and E7 expression, but lack of productive viral life cycle markers. In contrast, EPE6 had low E6 and E7 but high E1∧E4 expression, indicative of a productive life cycle. We suggest increased viral integration into the host genome for AAE6 as one possible mechanism for these observed differences from EPE6. Additionally, we found downstream effects on immortalization and host innate immune evasion. This study highlights how minor genomic variations in transforming viruses can have a significant affect on their tumourigenic ability.

Relationship between E6-E7 lineage sequences, viral loads, and integration of HPV16 in women with atypical squamous cells of undetermined significance (ASCUS) pap smears

“…This result suggests that non-European HPV16 variants are more oncogenic than European variants, and is in agreement with the findings of previous studies [5,44,59,60] demonstrating that HPV16 non-European viral variants were significantly more likely than European variants to cause persistence and a CIN3+ evolution [8,14,16,61] inducing proliferation phenotype in an organotypic epithelial model [45 ,46], probably due to 1) modification of the immunogenic properties of the virus [46,62,63], 2) stimulation of cell migration and cell invasion [64] and 3) viral DNA integration into the host genome [45,46].…”

“…Additional factors are likely to increase the probability of this progression occurring. For example, the percentage of HPV16 DNA integration, as a marker for high-grade cervical lesions, was demonstrated in some studies [23], while the increased frequency of non-European HPV16 variants in invasive lesions was described by Tornesello et al [43], suggesting greater oncogenicity for the nonEuropean HPV16 variants [44][45][46].…”

Functional variants of human papillomavirus type 16 demonstrate host genome integration and transcriptional alterations corresponding to their unique cancer epidemiology

“…To decipher the fundamental biology of HPVs and their tumourigenic features in a model system, the organotypic 3D infection model (raft culture) has the advantage of allowing reproducible and simultaneous epithelial differentiation and hence the occurrence of an active viral life cycle ([31]; Fig. 1).…”

Epithelial stratification shapes infection dynamics

“…Organotypic culture growing techniques used here have already been described in detail elsewhere [65,66]. Original experiments were performed to obtain time series data with sufficient replicates for model fitting.…”