Amino acid substitution patterns in the E6 and E7 Proteins of HPV type 16: Phylogeography and Evolution

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Abstract

The E6 and E7 proteins of human papillomavirus (HPV) play a key role in the oncogenesis of papillomavirus infection. Data on the variability of these proteins are limited, and the factors affecting their variability are poorly understood. We analyzed the variability of the currently known sequences of HPV type 16 (HPV16) E6 and E7 proteins, taking into account their geographic origin and year of sample collection, as well as the direction of their evolution in major geographic regions of the world. All sequences belonging to HPV16 genome fragments encoding E6 and E7 oncoproteins were downloaded from the NCBI GenBank database on October 6, 2022. Samples were filtered according to the following parameters: the sequence includes at least one of the two whole open reading frames, the collection date and the country of origin are known. A total of 3,651 full-genome nucleotide sequences encoding the E6 protein and 4,578 full-genome nucleotide sequences encoding the E7 protein were sampled. The nucleotide sequences obtained after sampling and alignment were converted to amino acid sequences and analyzed using MEGA11, R, RStudio, Jmodeltest 2.1.20, BEAST v1.10.4, Fastcov, and Biostrings software. The highest variability in E6 protein structure was recorded at positions 17, 21, 32, 85, and 90, while in E7, positions 28, 29, 51, and 77 were the most variable. The samples were divided geographically into 5 heterogeneous groups: African, European, American, Southwest and South Asia and Southeast Asia. Unique amino acid substitutions (AA-substitutions) in the E6/E7 proteins of HPV16, presumably characteristic of certain ethnic groups, were identified for a number of countries. They are mainly localized in sites of known B- and T-cell epitopes and relatively rarely in structural and functional domains. The revealed differences in AA-substitutions in different ethnic groups and their colocalization with clusters of B- and T-cell epitopes suggest their possible relationship with the geographical distribution of alleles and haplotypes of the major histocompatibility complex (HLA). This may lead to the recognition of a different set of B- and T-cell epitopes of the virus, resulting in regional differences in the direction of epitope drift. Phylogenetic analysis of the nucleotide sequences encoding the E6 protein of HPV16 revealed a common ancestor, confirmed regional clustering of the E6 protein gene sequences by the set of the most common AA-substitutions, and identified cases of reversion of individual AA-substitutions when the virus distribution region changed. For the E7 protein, a similar analysis was not possible due to high sequence homology. Covariance analysis of the pooled sample revealed that there was no relationship between amino acid residues in the E6 protein, in the E7 protein, and between E6 and E7. Data obtained are important for the development of therapeutic vaccines against HPV of high carcinogenic risk.

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About the authors

E. E. Zelenova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; Blokhin National Medical Research Center of Oncology

Author for correspondence.
Email: zelenovayeye@gmail.com
Russian Federation, Moscow, 119991; Moscow, 115478

A. A. Karlsen

Peoples’ Friendship University of Russia; Mechnikov Research Institute of Vaccines and Sera

Email: zelenovayeye@gmail.com
Russian Federation, Moscow, 117198; Moscow, 105064

D. V. Avdoshina

Chumakov Federal Scientific Center for Research and Development of Immunobiological Drugs, Russian Academy of Sciences

Email: zelenovayeye@gmail.com
Russian Federation, Moscow, 117218

K. K. Kyuregyan

Peoples’ Friendship University of Russia; Mechnikov Research Institute of Vaccines and Sera

Email: zelenovayeye@gmail.com
Russian Federation, Moscow, 117198; Moscow, 105064

M. G. Belikova

Peoples’ Friendship University of Russia; Chumakov Federal Scientific Center for Research and Development of Immunobiological Drugs, Russian Academy of Sciences; National Research Center for Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of the Russian Federation

Email: zelenovayeye@gmail.com
Russian Federation, Moscow, 117198; Moscow, 117218; Moscow, 123098

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Supplementary files

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2. Appendix Table S1
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3. Fig. 1. Distribution of selected HPV16 E6 and E7 protein gene sequences by geographic region: Europe (gray frame), Africa (orange frame), America (purple frame), Southeast Asia (green frame), South and Southwest Asia (red frame). N is the number of E6 and E7 sequences deposited in the GenBank database, indicating the representation of individual countries in each region.

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4. Fig. 2. Variability of HPV16 oncoproteins E6 and E7. Amino acid substitutions occurring in >1% of E6 (a) and E7 (b) sequences; distribution of samples by the number of AA substitutions in enriched samples of E6 protein (284 samples out of 356 sequences) (c) and E7 protein (92 samples out of 132 sequences) (d). Enriched samples excluded sequences with an identity exceeding 99.8%. Color coding of the number of AA substitutions is common for panels (c) and (d).

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5. Fig. 3. Heat maps of AA substitution occurrence in five major geographic regions of the world. Frequency distribution map: pattern of AA substitutions in E6 and E7 proteins, color scale on the right reflects the frequency of substitution for the region; cells with frequency >20% are circled (a). Significance map of AA substitution in E6 and E7 proteins for the region in pairwise comparison with the frequency of occurrence of this substitution in the general population. Color scale on the right reflects the significance value, p. Comparison was performed using Fisher's test (b). Heat maps were made using R language and visualized via Rstudio. Mark “x” means the absence of AA substitution in the region, therefore comparison was not performed.

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6. Fig. 4. Distribution of AK substitutions occurring in >10% of sample sequences across five geographic regions, including AK substitutions in individual countries. Purple block – USA, Central and South American countries; blue block – European countries; orange block – African countries; red block – Southwest and South Asian countries; green block – Southeast Asian countries.

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7. Fig. 5. Covariance analysis of amino acid substitutions of the E6 protein for the American region (AM) samples. The analysis was performed using the second bioinformatics method using the Biostrings package with the MI calculator (http://www.yanlilab.github.io/fastcov). The circles indicate the positions of amino acid residues, the lines between them indicate the linkage of two amino acid substitutions, the probability of linkage between which is p < 0.05 (MI).

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8. Fig. 6. Phylogenetic tree using the Bayesian approach with a time scale for the E6 gene sequences, fragment length is 477 bp (coordinates according to the reference sample NC_001526.4:7125–7601). For each sequence, the GenBank number, country and year of isolation are indicated. The nodes of the tree show the amino acid residues predicted for the hypothetical ancestor of each phylogroup. Designations: EU – countries of Europe, AF – countries of Africa, AM – countries of America, AS1 – countries of Southwest and South Asia, AS2 – countries of Southeast Asia.

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9. Fig. 6. Continuation.

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10. Fig. 6. Continuation.

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11. Fig. 6. Continuation.

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12. Fig. 6. End.

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13. Fig. 7. Fragments of the epitope map of the E6 oncoprotein. Fragments 17‒32 (a), 65‒90 (b), 119‒123 (c) of the amino acid sequence are shown. The sample included sequences with an identity exceeding 99.8%. The identification numbers (ID) of the epitopes in the IEDB database (http://www.tools.iedb.org) are shown in yellow, the sequence of the mapped epitope is shown in light yellow, and the numbers inside the block are the positions of the first and last amino acid residues. The frequency of occurrence of AA substitutions in the variable position of the E6 protein as part of the epitope cluster is indicated in the frame at the bottom of each block. The numbers at the bottom are the total number of epitopes affected by the AA substitutions in the indicated positions.

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14. Fig. 8. Epitope map of the E7 oncoprotein. Fragments 28‒51 (a) and 52‒99 (b) of the amino acid sequence are shown. The sample included sequences with an identity exceeding 99.8%. The identification numbers (ID) of the epitopes in the IEDB database are shown in yellow, the sequence of the mapped epitope is shown in light yellow, and the numbers inside the block are the positions of the first and last amino acid residues. The frequency of occurrence of amino acid substitutions in the variable position of the E7 protein as part of the epitope cluster is indicated in the frame at the bottom of each block. The numbers at the bottom are the total number of epitopes affected by the amino acid substitutions in the indicated positions.

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