Introduction
Human papillomaviruses (HPVs) are associated with a variety of epithelial lesions, including benign genital warts and cervical intraepithelial neoplasia [
1]. To date, more than 250 HPV types have been identified and each of these genotypes are associated with infection at particular anatomical sites. HPV6 may be the most prevalent low risk alpha-papillomavirus type and is commonly associated with genital warts [
2]. For example, anogenital warts are primarily caused by HPV6 (family
Papillomaviridae, genus
Alphapapillomavirus, species 10) [
3], which brings a significant burden to both the healthcare system and patients. Similarly, one third of Dutch primary school children have cutaneous warts, of which approximately 20% seek medical treatment each year [
4]. Generally speaking, these infections are classified as “not carcinogenic” or “low risk”, they often attract negative attention, and thereby cause significant psychological distress [
5]. However, some HPV6 variants are classified as “carcinogenic”, because they cause infections that lead to potentially fatal conditions, such as tonsillar, and malignant laryngeal carcinoma and/or malignant laryngeal papilloma [
6‐
9].
To date, extensive research has been conducted to investigate sequence variation among carcinogenic HPV types; nevertheless, only limited data is available regarding HPV6 variants, despite their significant impact on human health. Structurally, the HPV is a double-stranded, circular DNA virus that encodes E1, E2, E4, E5, E6, E7, L1 and L2 proteins [
10]. HPVs infect cells via the basal layer of the stratified epithelium, and viral gene expression is closely linked to the endogenous differentiation program of the host cells [
11]. Of the HPV-encoded proteins, E6 and E7 have been shown to be the most important pathogenic HPV proteins. They have been previously shown to function as oncoproteins that critically regulate HPV-induced tumorigenesis [
12]. Furthermore, they have also been demonstrated to be essential to maintain the extrachromosomal forms of HPV in undifferentiated basal cells [
13].
Genetic variability analyses have proven essential to facilitate an improved understanding of the evolution of the papillomavirus. A number of carcinogenic variants have been identified in HPV variants isolated from populations in Southwest China; however, only limited research has been conducted to identify low risk HPV variants. Thus, the present study aimed to analyze E6 and E7 sequence variability among HPV6 isolated from cervical papilloma samples collected from patients in Southwest China. Phylogenetic analyses were conducted to compare the identified nucleotide sequences with those previously described in other ethnic populations. In addition, the secondary structure of the identified sequences were predicted to assess the probably impact of the low risk variants on overall viral function. The results of the study could provide important data for the research on HPV6 prevention, diagnostic, therapeutic and even the design of therapeutic vaccines based on proteins E6 and E7 in Southwest China.
Discussion
E6 and E7 are essential HPV E-gene products, they target P53 and retinoblastoma (Rb) tumuor-suppressor proteins, respectively. The degradation of PRb can initiate abnormal cell replication; the inhibition of p53 can cause abnormal replication cell to lose control. The HPV genome can only be replicated along with the replication of the host genome. Therefore, E6 and E7 are especially important in HPV lifecycle. The present study sequenced the E6 and E7 ORFs in 143 HPV6 isolates from patients in Southwest China, to assess the HPV6 genetic diversity and evolution characteristics within this population, and help further identify specific HPV6 variants.
HPV6 was the second prevalent HPV type and the most prevalent low risk HPV type in southwest China, indicating that HPV6 is highly adaptable to the environment in southwest China compare with most HPV types that are not so prevalent.
Seven
E6 sequence variants were identified, two of which induced corresponding
E6 amino acid changes. One of these, H50Q (His to Gln), was identified in 30.08% of the analyzed HPV6 sequences, the amino acid at position 50 of prototype HPV6 E6 may be located centrally between the two internal Zn-binding motifs, which is important for the E6 protein stability [
22]. These variants have not been discovered previously in Southwest China, and their functional impact requires further analysis. Overall, the HPV6
E6 sequence was shown to be highly conserved within the analyzed patient samples, which exhibited only two main genotypes, HPV6E601 and HPV6E603, that represented 67.13% (96/143) and 29.37% (42/143) of the samples, respectively. Likewise, five
E7 variants were identified, four of which exhibited a corresponding
E7 amino-acid change. The most common
E7 nucleotide mutations were T155A and C294A. Notably, T155A mutation affected 34.27% (49/143) patients, and caused a F52Y (Phe to Tyr) amino acid conversion that was predicted impact the protein’s secondary structure. Furthermore, the
E7 sequence also was proved to be highly conserved within the patient specimens, comprising only two main genotypes HPV6E702 and HPV6E703 that were exhibited by 31.47% (45/143) and 65.73% (94/143) of the analyzed patients, respectively, and the sequence of HPV6E703 is in conformity with the reference (X00203). The fact that both sequences were so highly conserved supports the hypothesis that they play vital roles in HPV6 structure and function [
23,
24], consistent with previous studies by Dartmann [
25]. Thus, they are likely also promising targets for HPV6 primer design and diagnostic detection. Compared to deadly cancerogenic high risk HPV, low risk HPV is able to get along better with the host; low risk HPV is more likely to be ignored in clinical treatment and prevention. HPV6
E6 and
E7 were much more conserved than main high risk HPV types (like 16, 33, 53 and 58), may indicating low risk HPV has lower evolutionary pressure in gene level [
20,
21,
26].
Structurally, some of the detected mutations affected amino acids at critical positions related to known biological functions. In E6, one (I97) and three (D14Y, H50Q, and I74) mutations were found to affect the β-strand and α-helix-encoding regions, respectively, that are critical for structural stability. In E7, only one mutation (F52Y) was detected may affect the β-strand-encoding region. The nucleotides as positions 58, 61, 91, and 94 in the E7 protein sequence have been previously shown to act as zinc binding sites [
27], but no mutations were identified by the present study to occur in these positions, nor in the consensus LXCXE RB1-binding site (positions 22–26), or zinc-binding motif.
Notably, the present study is first to conduct an analysis to assess whether the
E6 and
E7 sequences are subject to positive selection in Southwest China. The main characteristic of positive selection is that it causes an unusually rapid rise in allele frequency, thereby enabling a species to adapt rapidly to environmental changes [
28]. The selective pressure analysis showed that all the sites that evolved under positive selection were common non-synonymous mutations, indicating that the positively selected variations beneficial for HPV6 to accommodate their environments are wide-spread. Moreover, remarkably, the positive sites R4E, E34K, and F52Y were observed in HPV6
E7, they may have evolutionary significance in making HPV6 adaptive to their environments.
Previous studies revealed the existence of two variant lineages (lineage A, lineage B) and five variant sub-lineages (sub-lineage B1, B2, B3, B4, B5) [
29] among HPV6 variants. The conducted phylogenetic analyses showed that 97.56% of the
E6 and
E7 sequences within the patient specimens belonged to sub-lineage A1 (Reference, HPV6b) and sub-lineage B1 (HE599232, LP243) [
29]. Lineage A has been shown to predominate in Asia; in contrast, lineage B is distributed globally [
30,
31], and consists mainly of variants from sub-lineage B1. Given its global distribution, lineage B1 may represent the oldest HPV6 sub-lineage, and likely disseminated during early human evolution. This may have enabled it to migrate to different regions of the world prior to the emergence of other HPV6 sub-lineages [
29], and for
E7, a new sub-lineage was found, however, only three samples were detected, and therefore, it requires further study.
Certain strengths of this study were that all clinical samples were collected from the Southwest region of China which had a strong regional representation and several new mutations were discovered, which will provide real and valid data for development of therapeutic vaccines for affected people in the Southwest China. Thus, the results of the present study significantly expand the current knowledge of HPV6 genetic diversity in Southwest China, and also provide a valuable resource for future studies of HPV6 epidemiology, evolution, function, pathogenesis, and use as a therapeutic target. However, the cellular level research is required, and a series of cellular experiments about the mutations should be designed for future studies.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.