Characterization of the Human Papillomavirus 16 Oncogenes in K14HPV16 Mice: Sublineage A1 Drives Multi-Organ Carcinogenesis

Abstract

The study of human papillomavirus (HPV)-induced carcinogenesis uses multiple in vivo mouse models, one of which relies on the cytokeratin 14 gene promoter to drive the expression of all HPV early oncogenes. This study aimed to determine the HPV16 variant and sublineage present in the K14HPV16 mouse model. This information can be considered of great importance to further enhance this K14HPV16 model as an essential research tool and optimize its use for basic and translational studies. Our study evaluated HPV DNA from 17 samples isolated from 4 animals, both wild-type (n = 2) and HPV16-transgenic mice (n = 2). Total DNA was extracted from tissues and the detection of HPV16 was performed using a qPCR multiplex. HPV16-positive samples were subsequently whole-genome sequenced by next-generation sequencing techniques. The phylogenetic positioning clearly shows K14HPV16 samples clustering together in the sub-lineage A1 (NC001526.4). A comparative genome analysis of K14HPV16 samples revealed three mutations to the human papillomaviruses type 16 sublineage A1 representative strain. Knowledge of the HPV 16 variant is fundamental, and these findings will allow the rational use of this animal model to explore the role of the A1 sublineage in HPV-driven cancer.

Keywords: HPV16; K14HPV16; carcinogenesis; lineage; variant.

Figure 1

Schematic of the HPV-16R genome. The genome organization of 7906 base pairs (bp) arranged in a circular form represents the three functional regions, early, late, and long control (LCR); and a non-coding region (NCR). These are separated by two polyadenylation sites, described as early (pAE) and late (pAL). In total, the genome encodes eight open reading frames (ORFs), of which E1, E2, E4, E5, E6, and E7 are from the early region; and L1 and L2 are from the late region. The LCR, located between the E6 and L1 gene, is a regulatory region that includes the origin of replication (ori) and the p97 promoter.

Figure 2

Histological findings of WT and MUT tongue and oral cancer. Legend: Histological samples from wild-type and transgenic K14HPV16 mice stained with H&E. (A) low power view of normal squamous cell epithelium of the tongue from a wild-type mouse; (B) low power view of mild dysplastic squamous cell epithelium with enlarged hyperchromatic nuclei in the upper layers of the epithelium from a transgenic K14HPV16 mouse and (C) low power view of a focally invasive squamous cell carcinoma with thickened rete ridges and dysplastic cells with hyperchromatic nuclei, irregular basement membrane and focally disrupted associated with inflammatory cells in the stroma from a transgenic K14HPV16 mouse; the inset shows the invasive front. (D) low power view of the liver of one transgenic K14HPV16 mouse showing normal structure and cytology.

Figure 3

Phylogenetic positioning of K14HPV16 samples within the major HPV16 lineages (A–D). Legend: the phylogenetic tree was generated using the neighbor-joining method [18] with the maximum composite likelihood model [17] and depicts the genetic relationships of the obtained K14HPV16 genome consensus sequences to each representative genome. Numbers next to the branch nodes indicate the bootstrap values (1000 replicates). HPV16 lineages are shown in grey boxes next to the main tree branches, while sublineages (A1–A4, B1, B2, and D1–D3) are illustrated in black boxes.