Table 1.
Distribution of the HPV16 variants within different grades of cervical lesions.
Table 2.
Positions of nucleotide and amino acid changes within the whole genome and E1 sequences of the major HPV16 variants containing the E1-1374∧63 duplication.
Figure 1.
Phylogenic clustering of E1-1374∧63nt variant sequences with other currently available published HPV16 whole genome sequences.
The whole genome sequences of four E1-1374∧63nt variant samples (ZG01-118, ZG01-258, ZG03-145 and ZG05-249) were aligned with the available published whole genome sequences and the phylogenetic tree was created using MEGA5. The leftmost part shows the percent nucleotide sequence differences calculated using MEGA5 to determine variant lineages according to a proposed taxonomic classification [18], [19]. The top part of the figure was adapted from the transcription map of HPV16 available at http://pave.niaid.nih.gov/. Sequences at respective positions are shaded in different shades of blue according to the distance from the E1-1374∧63nt variant. Dots "." indicate no change from referent sequence while "∼" and "INS" indicate insertions. The European (or A) lineage appears to be subdivided into 3 sublineages A1–A3, based on the tree topology and sequence percent difference similarly as previously described [17]–[19]. The A1 lineage contains the reference HPV16 genome, the A2 lineage contains the E-G350 related variants, including the E1-1374∧63nt variant samples and the E-12 variant described in Bhattacharjee et al. [15]. The A3 sublineage contains only the E-G131 sample, which exhibits the most difference from other sequences. Variant "E-12" was previously shown to be more prevalent in cancer samples [15], while the variation 7450 T>C was shown to be statistically more prevalent in cancer samples [49]. Positions where samples ZG01-118 and ZG01-258 differ have been sequenced for 3 additional samples and in each case only one of 5 sequenced samples has this change. Additional whole length E1 sequencing has revealed that changes at positions 1656 and 1692 were only present in 2 of 12 samples. Thus, all those changes are unlikely to be the major cause of the association of the E1-1374∧63nt E-G350 variant with low grade cervical lesions. The major difference between E1-1374∧63nt E-G350 variant and other variants are the positions 1053 and 1374, while the only differences between the E-G350 and E-C109/G350 sequenced variants are at positions 24 and 109.
Figure 2.
Prediction of E1 protein structure.
Panel A shows 3D structure comparison between superposed monomer of the previously solved BPV1 E1 helicase domain hexamer structure (cyan; PDB ID:2GXA) [25], HPV18 E1 helicase domain structure (yellow; PDB ID:1TUE) [26], BPV1 E1 DNA binding domain structure (red; PDB ID:1F08) [27] and the predicted referent E1 model (white). The sequence at the position where the duplication occurs within the E1-1374∧63nt variants is highlighted in green, as is the amino acid 63 that is also changed in the E1-1374∧63nt variants. Panel B depicts superposed structures of the referent (white) and E1-1374∧63nt variant (magenta) E1 models. As before the referent sequence is highlighted in green and the changes specific to E1-1374∧63nt variant are highlighted in red. It can be seen that the newly added 21 amino acids (highlighted in red) partially overlap the potentially phosphorylated threonines (highlighted in green) at positions 153 and 155 and possibly influence the phosphorylation regulation of the E1 function. Panels C and D show the hexamer structure derived from the predicted E1 reference or E1-1374∧63nt variant proteins, respectively. The individual monomers are colored differently. The original referent sequence is colored in green within the white referent E1 monomer and the E1 variant magenta monomer. The sequence duplication in the E1-1374∧63nt variants is located at the junction between 2 adjacent E1 monomers within the structure and is highlighted in red on panel D. Positions of potentially phosphorylated threonines and amino acid substitution at position 63 are only highlighted on the white and magenta monomers.