Genetic variability in E6, E7 and L1 genes of Human Papillomavirus 62 and its prevalence in Mexico

Abstract

Background: Human papillomavirus (HPV) is the main etiological agent of cervical cancer, the third most common cancer among women globally and the second most frequent in Mexico. Persistent infection with high-risk HPV genotypes is associated with premalignant lesions and cervical cancer development. HPVs considered as low risk or not yet classified, are often found in coinfection with different HPV genotypes. Indeed, HPV62 is one of the most prevalent HPV detected in some countries, but there is limited information about its prevalence in other regions and there are no HPV62 variants currently described. The aim of this study was to determine the prevalence of HPV62 in cervical samples from Mexican women and to identify mutations in the L1, E6 and E7 genes, which have never been reported in our population.

Methods: HPV screening was performed by Cobas HPV Test in women who attended prevention health programs and dysplasia clinics. All HPV positive samples (n = 491) and 87 additional cervical cancer samples were then genotyped with Linear Array HPV Genotyping test. Some samples were selected to corroborate genotyping by Next-Generation sequencing. On the other hand, nucleotide changes in L1, E6 and E7 genes were determined using PCR, Sanger sequencing and analysis with the CLC-MainWorkbench 7.6.1 software. L1 protein structure was predicted with the I-TASSER server.

Results: Using Linear Array, HPV62 prevalence was 7.6% in general population, 8% in Cervical Intraepithelial Neoplasia grade 1 (CIN1) samples and 4.6% in cervical samples. The presence of HPV62 was confirmed with Next-Generation sequencing. Regarding L1 gene, novel sequence variations were detected, but they did not alter the tertiary structure of the protein. Moreover, several nucleotide substitutions were found in E6 and E7 genes compared to reference HPV62 genomic sequence. Specifically, three non-synonymous sequence variations were detected, two in E6 and one in E7.

Conclusions: HPV62 is a frequent HPV genotype found mainly in general population and in women with CIN1, and in 90.5% of the cases it was found in coinfection with other HPVs. Novel nucleotide changes in its L1, E6 and E7 genes were detected, some of them lead to changes in the protein sequence.

Keywords: Cervical cancer; E6; E7; HPV62; L1.

Fig. 1

Frequency of HPV genotypes found in coinfection with HPV62. The graphic depicts the number of samples in which each HPV genotype was found together with HPV62

Fig. 2

Sequence and structure alignment of HPV62 L1 protein. a Protein sequence alignment of the AY395706 NCBI sequence (HPV62-L1 REF) with that obtained from cervical sample P9 (HPV62-L1 P9); dots indicate matching residues, amino acid changes are darkened. b Structure alignment of both 503 amino acid complete proteins (HPV62-L1 REF in red and HPV62-L1 P9 in grey) and location of the four detected mutations shown as yellow dots. c Magnification of the structure alignment region containing the mutated amino acids threonine (p.T84A), alanine (p.A88G), lysine (p.K380R); and d) alanine (p. A497T)

Fig. 3

Protein alignment of HPV62-E6 and HPV62-E7. a Alignment of the HPV62-E6 reference protein sequence from AY395706 genome (HPV62-E6 REF) relative to the HPV62-E6 from 13 cervical samples (HPV62-E6 P1-P13). Amino acid changes p.R53W and p.K135C are shown. b Alignment of the HPV62-E7 reference protein sequence (HPV62-E7 REF) relative to the HPV62-E7 from the same 13 cervical samples (HPV62-E7 P1-P13). Amino acid change p.A42G is shown. Dots indicate matching residues and dashes indicate that no information is available for the corresponding region. Arrows point to cysteines that form the zinc binding domains

Fig. 4

Phylogenetic tree showing reference HPVs from alpha-3 species and 13 HPV62 Mexican sequences based on E6/E7 genes. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model [37]. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 18 nucleotide sequences. Evolutionary analyses were conducted in MEGA7 [38]. The GenBank accession number of each HPV genotype reference from alpha-3 species is included in parentheses