Genetic analysis of human papilloma virus 16 E6/E7 variants obtained from cervical cancer cases in Chhattisgarh, a central state of India

Abstract

Human papilloma virus genotype 16 (HPV-16), a predominant etiological cause of cervical cancer (CC) vary in inflicting oncogenicity according to their geographical distribution and mutational changes. With no published data from central India, the present study aimed to genetically analyze HPV-16 E6/E7 variant obtained from CC women of Chhattisgarh. In twenty one CC patients, PCR amplified E6/E7 genes were decoded by DNA sequencing to study phylogenetic relatedness, mutational changes and their in-silico effect on protein structure. E6 analysis revealed nineteen sequences exhibited intratypic variation. L83V mutation was observed in 76.2% sequences followed by S71C seen in 28.6% sequences. Mutations of E41G, A46G, F47V, R77S, L99V and Q107K were observed in three sequences each. C140 Stop codon mutation has caused early truncation of E6 in three sequences to produce the conformational structural change. In contrast, E7 was relatively more conserved showing D4E (4.7%), G88R (23.8%), I93T (9.5%) and C94S (9.5%) mutations. Other than L83V and S71C, E6 and E7 mutations were reported for the first time from India. E6/E7 nonsynonmous mutations have a spectrum of biological effect in progression of CC. Phylogenetic analysis revealed ten sequence belonged to Asian while eleven to European sublineage to show CC cases in Chhattisgarh are a mix of Asian and European lineage. Asian sequences showing higher frequency of L83V mutations and exclusive presence of S71C and C140 Stop codon mutations may be linked with higher oncogenicity. Various E6/E7 mutational data may prove useful for development of better diagnostic and vaccine for the region of Chhattisgarh.

Keywords: Cervical cancer; E6/E7 oncoprotein; Human papilloma virus genotype 16; Nonsynomous mutations.

Fig. 1

Schematic flow chart to explain inclusion, exclusion and total number of cases processed in the present study

Fig. 2

Mutational changes in E6 leading to change of various AA. Number shows the AA positions and all single capital letter indicate AA. A, Alanine; D, Aspartic acid; C, Cysteine; E, Glutamic acid; F, phenylalanine; G, Glycine; K, Lysine; L, Leucine; Q, Glutamine; R, Arginine; S, Serine; V, Valine

Fig. 3

Phylogenetic analysis of 21 HPV E6 ORF sequences in comparison of various lineage and sublineage reference E6 gene sequences using the Neighbor-Joining algorithm in MEGA software (version 7.0). Bootstrap analysis of 1000 replicates were performed on each tree to determine the confidence. All twenty one sequences were marked in dark circle. Among, the 18 reference sequences NC 001526, U89348 and K02718 represented reference sequences of HPV-16. The rest of the sequences represents various lineage (A/B/C/D) and sublineage reference sequences namely MH921951 and LC368988 (A1), HQ537752 and AF536179 (A2), HQ537758 and HQ644236 (A3), AF534061 and LC456628(A4), AF536180(B1), HQ644298(B2), AF472509(C), HQ644257(D1), AY68579(D2), AF4022678(D3), AF472508 (Af-1)

Fig. 4

Phylogenetic analysis of 21 HPV E7 ORF sequences and 18 reference representative E7 gene sequences of various lineage and sublineage conducted using the Neighbor-Joining algorithm in MEGA software (version 7.0). Bootstrap analysis of 1000 replicates was performed on each tree to determine the confidence. All twenty one sequences were marked in dark circle. The reference and various lineage and sublineage sequences are similar as described with their accession number in the legend of Fig. 3

Fig. 5

3D Models of E6 proteins (Wild type, Truncated and Variant-958) of HPV-16. A E6 wild type protein, Yellow color indicates the chain of 12 AA, which was absent in the truncated protein. Last AA residue of wild type E6 may interact to the AA residues (76, 79 and 80). B E6 truncated protein in which last 12 AA were missing due to the early truncation. This truncation inhibiting the interaction with N-terminal domain. C E6-958 Variant protein in which AA changes due to point mutations could leads to conformational changes

Fig. 6

3D mode A shows E7 wild type protein whereas B represent E7-143 variant showing some AA variation at the protein tail and may affect its conformations