HPV16 E7 Genetic Conservation Is Critical to Carcinogenesis

Abstract

Although most cervical human papillomavirus type 16 (HPV16) infections become undetectable within 1-2 years, persistent HPV16 causes half of all cervical cancers. We used a novel HPV whole-genome sequencing technique to evaluate an exceptionally large collection of 5,570 HPV16-infected case-control samples to determine whether viral genetic variation influences risk of cervical precancer and cancer. We observed thousands of unique HPV16 genomes; very few women shared the identical HPV16 sequence, which should stimulate a careful re-evaluation of the clinical implications of HPV mutation rates, transmission, clearance, and persistence. In case-control analyses, HPV16 in the controls had significantly more amino acid changing variants throughout the genome. Strikingly, E7 was devoid of variants in precancers/cancers compared to higher levels in the controls; we confirmed this in cancers from around the world. Strict conservation of the 98 amino acids of E7, which disrupts Rb function, is critical for HPV16 carcinogenesis, presenting a highly specific target for etiologic and therapeutic research.

Keywords: E7 gene; HPV epidemiology; HPV genomics; HPV16; cervical carcinogenesis.

Figure 1. Individual SNP Associations with CIN3⁺ Compared to the Controls within the A1/A2 Lineages in Women in the PaP Cohort

Logistic regression models were used to obtain the odds ratio and 95% confidence intervals for CIN3⁺ risk for eachSNP minor alleleusing the controls asthe referent group. The x axis indicates the viral genome nucleotide position; The y axis indicates the p-value. The vertical arrows indicate the most significant SNPs identified. SNPs are colored by gene region in the legend. URR, upstream regulatory region; E6, early gene 6; E7, early gene 7; E1, early gene 1; E2, early gene 2; E4, early gene 4; E5, early gene 5; L2, late gene 2; L1, late gene 1; NC, non-coding region. See also Table S2.

Figure 2. Rare Non-silent Variant Distributions across the Viral Genomes of A1/A2 Lineages for the CIN3⁺ Cases and Controls in the PaP Cohort

Each viral gene region is represented by a wedge in the plots and the size of the wedge corresponds to the nucleotide size of the region. The nucleotide positions of each viral gene region are shown in the center. The two inner rings illustrate the genome positions of the rare variants within each of the viral gene regions for cases in red and controls in blue. Each vertical line represents a variant and the height of the line corresponds to how many individuals had this variant. The total variant counts for each viral region are given in Table 4. The outermost ring illustrates the cumulative rare variant counts by viral gene region. The density of the variants and the variant increase across each region are represented by the slope of the line for the cases and controls. URR, upstream regulatory region; E6, early gene 6; E7, early gene 7; E1, early gene 1; E2, early gene 2; E4, early gene 4; E5, early gene 5; L2, late gene 2; L1, late gene 1. See also Figure S1 and Tables S3 and S4–S6.

Figure 3. HPV16 Rare Nonsynonymous and Nonsense Nucleotide Variants Observed in the E7 ORF

Women in the PAP Cohort with HPV16 A1/A2 lineage infections were included in this analysis. Rare nonsynomous and nonsensense variants are shown as black and red sticks, respectively. Controls are shown as blue lollipops and CIN3+ cases as either red or grey lollipops corresponding to CIN3 or AIS, respectively. Amino acid changes are indicated with each lollipop, and number of lollipop circles indicates the number of individuals with that variant. The domains of E7 are colored, see legend, and the stars indicate changes consistent with an APOBEC3-induced change. CR1, conserved region 1; CR2, conserved region 2; RB1, retinoblastoma (Rb) protein interacting region. CIN3, cervical intraepithelial neoplasia grade 3; AIS, adenocarcinoma in situ. See also Figure S2.