The Human Papillomavirus Type 16 E6 Gene Alone Is Sufficient To Induce Carcinomas in Transgenic Animals

ABSTRACT High-risk human papillomaviruses (HPVs) are the causative agents of certain human cancers. HPV type 16 (HPV16) is the papillomavirus most frequently associated with cervical cancer in women. The E6 and E7 genes of HPV are expressed in cells derived from these cancers and can transform cells in tissue culture. Animal experiments have demonstrated that E6 and E7 together cause tumors. We showed previously that E6 and E7 together or E7 alone could induce skin tumors in mice when these genes were expressed in the basal epithelia of the skin. In this study, we investigated the role that the E6 gene plays in carcinogenesis. We generated K14E6 transgenic mice, in which the HPV16 E6 gene was directed in its expression by the human keratin 14 promoter (hK14) to the basal layer of the epidermis. We found that E6 induced cellular hyperproliferation and epidermal hyperplasia and caused skin tumors in adult mice. Interestingly, the tumors derived from E6 were mostly malignant, as opposed to the tumors from E7 mice, which were mostly benign. This result leads us to hypothesize that E6 may contribute differently than E7 to HPV-associated carcinogenesis; whereas E7 primarily contributes to the early stages of carcinogenesis that lead to the formation of benign tumors, E6 primarily contributes to the late stages of carcinogenesis that lead to malignancy.

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Disruption of repressive p130–DREAM complexes by human papillomavirus 16 E6/E7 oncoproteins is required for cell-cycle progression in cervical cancer cells

Journal of General Virology ◽

10.1099/vir.0.035352-0 ◽

2011 ◽

Vol 92 (11) ◽

pp. 2620-2627 ◽

Cited By ~ 28

Author(s):

Nurshamimi Nor Rashid ◽

Rohana Yusof ◽

Roger J. Watson

Keyword(s):

Cell Cycle ◽

Cervical Cancer ◽

Cancer Cells ◽

Cell Cycle Progression ◽

Human Papillomaviruses ◽

G1 Arrest ◽

Cycle Progression ◽

Hpv16 E6 ◽

Cervical Cancer Cells ◽

E6 And E7

Human papillomaviruses (HPVs) with tropism for mucosal epithelia are the major aetiological factors in cervical cancer. Most cancers are associated with so-called high-risk HPV types, in particular HPV16, and constitutive expression of the HPV16 E6 and E7 oncoproteins is critical for malignant transformation in infected keratinocytes. E6 and E7 bind to and inactivate the cellular tumour suppressors p53 and Rb, respectively, thus delaying differentiation and inducing proliferation in suprabasal keratinocytes to enable HPV replication. One member of the Rb family, p130, appears to be a particularly important target for E7 in promoting S-phase entry. Recent evidence indicates that p130 regulates cell-cycle progression as part of a large protein complex termed DREAM. The composition of DREAM is cell cycle-regulated, associating with E2F4 and p130 in G0/G1 and with the B-myb transcription factor in S/G2. In this study, we addressed whether p130–DREAM is disrupted in HPV16-transformed cervical cancer cells and whether this is a critical function for E6/E7. We found that p130–DREAM was greatly diminished in HPV16-transformed cervical carcinoma cells (CaSki and SiHa) compared with control cell lines; however, when E6/E7 expression was targeted by specific small hairpin RNAs, p130–DREAM was reformed and the cell cycle was arrested. We further demonstrated that the profound G1 arrest in E7-depleted CaSki cells was dependent on p130–DREAM reformation by also targeting the expression of the DREAM component Lin-54 and p130. The results show that continued HPV16 E6/E7 expression is necessary in cervical cancer cells to prevent cell-cycle arrest by a repressive p130–DREAM complex.

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Molecular and evolutionary analysis of HPV16 E6 and E7 genes in Greek women

Journal of Medical Microbiology ◽

10.1099/jmm.0.055491-0 ◽

2013 ◽

Vol 62 (11) ◽

pp. 1688-1696 ◽

Cited By ~ 6

Author(s):

D. Tsakogiannis ◽

A. Papadopoulou ◽

G. Kontostathi ◽

I. G. A. Ruether ◽

Z. Kyriakopoulou ◽

...

Keyword(s):

Viral Evolution ◽

Greek Population ◽

Low Grade ◽

Evolutionary Analysis ◽

Amino Acid Residues ◽

Hpv16 E6 ◽

E6 Gene ◽

E6 And E7 ◽

Cervical Cancer Development ◽

The Individual

Human papillomavirus type 16 (HPV16) non-European variants have been associated with persistent infection and cervical cancer development, while the L83V variant of the E6 gene has been correlated with the progression of cervical malignancy. The present study investigated the presence of the HPV16 L83V variant in Greek women. Molecular evolutionary analysis of the HPV16 E6 and E7 oncogenes was conducted in order to estimate the evolution of the HPV16 genome in the Greek population. The E6 L83V variant was found in 78.2 % of high- and 64.28 % of low-grade specimens. Moreover, the prototype and E6 L83V variants were both prevalent in high- and low-grade malignancies in Greek women. Selective pressure analysis of the individual amino acid residues of HPV16 sequences from the Greek population indicates that codon 83 of the E6 protein, as well as codon 85 of the E7 protein, are undergoing positive selection. Novel sequence variations were recorded within the E6 and E7 genes in cervical samples, characterized as (T350G) European variants. However, no signal of intratypic recombination event was identified within the E6–E7 region. Molecular and evolutionary analyses of HPV16 genomes from distinct geographical locations might provide valuable information about viral evolution and oncogenecity.

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Structure of High-Risk Papillomavirus 31 E6 Oncogenic Protein and Characterization of E6/E6AP/p53 Complex Formation

Journal of Virology ◽

10.1128/jvi.00730-20 ◽

2020 ◽

Vol 95 (2) ◽

pp. e00730-20

Author(s):

Marcel Chris Conrady ◽

Irina Suarez ◽

Gergö Gogl ◽

Desiree Isabella Frecot ◽

Anna Bonhoure ◽

...

Keyword(s):

High Risk ◽

Complex Formation ◽

Ternary Complex ◽

Human Papillomaviruses ◽

High Sequence Identity ◽

Hpv16 E6 ◽

Cellular Targets ◽

Ternary Complex Formation ◽

Lxxll Motif ◽

E6 And E7

ABSTRACTThe degradation of p53 is a hallmark of high-risk human papillomaviruses (HPVs) of the alpha genus and HPV-related carcinogenicity. The oncoprotein E6 forms a ternary complex with the E3 ubiquitin ligase E6-associated protein (E6AP) and tumor suppressor protein p53 targeting p53 for ubiquitination. The extent of p53 degradation by different E6 proteins varies greatly, even for the closely related HPV16 and HPV31. HPV16 E6 and HPV31 E6 display high sequence identity (∼67%). We report here, for the first time, the structure of HPV31 E6 bound to the LxxLL motif of E6AP. HPV16 E6 and HPV31 E6 are structurally very similar, in agreement with the high sequence conservation. Both E6 proteins bind E6AP and degrade p53. However, the binding affinities of 31 E6 to the LxxLL motif of E6AP and p53, respectively, are reduced 2-fold and 5.4-fold compared to 16 E6. The affinity of E6-E6AP-p53 ternary complex formation parallels the efficacy of the subsequent reaction, namely, degradation of p53. Therefore, closely related E6 proteins addressing the same cellular targets may still diverge in their binding efficiencies, possibly explaining their different phenotypic or pathological impacts.IMPORTANCE Variations of carcinogenicity of human papillomaviruses are related to variations of the E6 and E7 interactome. While different HPV species and genera are known to target distinct host proteins, the fine differences between E6 and E7 of closely related HPVs, supposed to target the same cellular protein pools, remain to be addressed. We compare the oncogenic E6 proteins of the closely related high-risk HPV31 and HPV16 with regard to their structure and their efficiency of ternary complex formation with their cellular targets p53 and E6AP, which results in p53 degradation. We solved the crystal structure of 31 E6 bound to the E6AP LxxLL motif. HPV16 E6 and 31 E6 structures are highly similar, but a few sequence variations lead to different protein contacts within the ternary complex and, as quantified here, an overall lower binding affinity of 31 E6 than 16 E6. These results align with the observed lower p53 degradation potential of 31 E6.

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Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins

mBio ◽

10.1128/mbio.02170-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 30

Author(s):

Mallory E. Harden ◽

Nripesh Prasad ◽

Anthony Griffiths ◽

Karl Munger

Keyword(s):

Gene Expression ◽

High Risk ◽

Human Papillomaviruses ◽

Cancer Development ◽

Cellular Gene ◽

Hpv16 E6 ◽

Hpv E6 ◽

E6 And E7 ◽

E7 Proteins ◽

Almost All

ABSTRACT The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation. IMPORTANCE High-risk human papillomaviruses (HPVs) are the causative agents of almost all cervical cancers and many other cancers, including anal, vaginal, vulvar, penile, and oropharyngeal cancers. Despite the availability of efficacious HPV vaccines, it is critical to determine how HPVs cause cancer, as many people remain unvaccinated and the vaccine does not prevent cancer development in individuals who are already infected. Two HPV proteins, E6 and E7, are the major drivers of cancer development, and much remains to be learned about how the expression of these viral proteins reprograms infected cells, ultimately resulting in cancer development. Small, noncoding human RNAs, termed microRNAs (miRs), regulate gene expression and have been implicated in almost all human cancers, including HPV-associated cancers. Our study provides a comprehensive analysis of how E6 and E7 alter the expression of human miRs and how this potentially impacts cellular gene expression, which may contribute to cancer development.

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Development and validation of a multiplex qPCR assay for detection and relative quantification of HPV16 and HPV18 E6 and E7 oncogenes

Scientific Reports ◽

10.1038/s41598-021-83489-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alexia Bordigoni ◽

Anne Motte ◽

Hervé Tissot-Dupont ◽

Philippe Colson ◽

Christelle Desnues

Keyword(s):

Limit Of Detection ◽

Quantitative Polymerase Chain Reaction ◽

Human Papillomaviruses ◽

Molecular Techniques ◽

Clinical Samples ◽

Gene Encoding ◽

Hpv Dna ◽

E6 And E7 ◽

High Risk Hpv ◽

Development And Validation

AbstractHuman papillomaviruses (HPV) play a key role in promoting human anogenital cancers. Current high-risk HPV screening or diagnosis tests involve cytological or molecular techniques mostly based on qualitative HPV DNA detection. Here, we describe the development of a rapid quantitative polymerase chain reaction (qPCR) detection test of HPV16 and HPV18 oncogenes (E6 and E7) normalized on human gene encoding GAPDH. Optimized qPCR parameters were defined, and analytical specificities were validated. The limit of detection was 101 for all genes tested. Assay performances were evaluated on clinical samples (n = 96). Concordance between the Xpert HPV assay and the triplex assay developed here was 93.44% for HPV16 and 73.58% for HPV18. HPV co-infections were detected in 15 samples. The systems developed in the present study can be used in complement to traditional HPV tests for specifically validating the presence of HPV16 and/or HPV18. It can also be used for the follow-up of patients with confirmed infection and at risk of developing lesions, through the quantification of E6 and E7 oncogene expression (mRNA) normalized on the GAPDH expression levels.

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Intrabodies targeting human papillomavirus 16 E6 and E7 oncoproteins for therapy of established HPV-associated tumors

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01841-w ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Francesca Paolini ◽

Carla Amici ◽

Mariantonia Carosi ◽

Claudia Bonomo ◽

Paola Di Bonito ◽

...

Keyword(s):

Human Papillomavirus ◽

Antitumor Activity ◽

Tumor Growth ◽

Tumor Progression ◽

Cellular Transformation ◽

Neoplastic Progression ◽

Single Chain ◽

Hpv16 E6 ◽

Associated Lesions ◽

E6 And E7

Abstract Background The oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with cellular homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7 proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy against tumor progression of HPV16-positive cells. Methods Envisioning clinical application of the best characterized anti-HPV16 E6 and –HPV16 E7 scFvs, we verified their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum targeting sequence were delivered by injection followed by electroporation to three different preclinical models using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model, the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E and caspase-3 staining of tumor section were performed. Results We showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells towards the apoptotic pathway. Conclusion Based on our study, two scFvs have been identified that could represent a safe and effective treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed for human treatment, to deliver scFvs to tumors was confirmed.

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Nucleosome Positioning on Episomal Human Papillomavirus DNA in Cultured Cells

Pathogens ◽

10.3390/pathogens10060772 ◽

2021 ◽

Vol 10 (6) ◽

pp. 772

Author(s):

Isao Murakami ◽

Takashi Iwata ◽

Tohru Morisada ◽

Kyoko Tanaka ◽

Daisuke Aoki

Keyword(s):

Life Cycle ◽

Cultured Cells ◽

Basal Layer ◽

Nucleosome Positioning ◽

Viral Life Cycle ◽

Human Papillomaviruses ◽

Host Cells ◽

Micrococcal Nuclease ◽

Hpv Dna ◽

Dna Replication And Repair

Several human papillomaviruses (HPV) are associated with the development of cervical carcinoma. HPV DNA synthesis is increased during the differentiation of infected host keratinocytes as they migrate from the basal layer of the epithelium to the spinous layer, but the molecular mechanism is unclear. Nucleosome positioning affects various cellular processes such as DNA replication and repair by permitting the access of transcription factors to promoters to initiate transcription. In this study, nucleosome positioning on virus chromatin was investigated in normal immortalized keratinocytes (NIKS) stably transfected with HPV16 or HPV18 genomes to determine if there is an association with the viral life cycle. Micrococcal nuclease-treated DNA analyzed by Southern blotting using probes against HPV16 and HPV18 and quantified by nucleosome scanning analysis using real-time PCR revealed mononucleosomal-sized fragments of 140–200 base pairs that varied in their location within the viral genome according to whether the cells were undergoing proliferation or differentiation. Notably, changes in the regions around nucleotide 110 in proliferating and differentiating host cells were common to HPV16 and HPV18. Our findings suggest that changes in nucleosome positions on viral DNA during host cell differentiation is an important regulatory event in the viral life cycle.

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Association of cervical carcinogenesis risk with HPV16 E6 and E7 variants in the Taizhou area, China

BMC Cancer ◽

10.1186/s12885-021-08531-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Mei-Zhen Dai ◽

Yi Qiu ◽

Xing-Hong Di ◽

Wei-Wu Shi ◽

Hui-Hui Xu

Keyword(s):

Cervical Cancer ◽

Cervical Carcinogenesis ◽

Chi Square ◽

Major Health Problem ◽

Hpv16 E6 ◽

Exact Test ◽

Chi Square Test ◽

E6 And E7

Abstract Background Human papillomavirus (HPV) type 16 accounts for a larger share of cervical cancer and has been a major health problem worldwide for decades. The progression of initial infection to cervical cancer has been linked to viral sequence properties; however, the role of HPV16 variants in the risk of cervical carcinogenesis, especially with longitudinal follow-up, is not fully understood in China. Methods We aimed to investigate the genetic variability of HPV16 E6 and E7 oncogenes in isolates from cervical exfoliated cells. Between December 2012 and December 2014, a total of 310 single HPV16-positive samples were selected from women living in the Taizhou area, China. Sequences of all E6 and E7 oncogenes were analysed by PCR-sequencing assay. Detailed sequence comparison, genetic heterogeneity analyses and maximum-likelihood phylogenetic tree construction were performed with BioEdit Sequence Alignment Editor and MEGA X software. Data for cytology tests and histological diagnoses were obtained from our Taizhou Area Study with longitudinal follow-up for at least 5 years. The relationship between HPV16 variants and cervical carcinogenesis risk was analysed by the chi-square test or Fisher’s exact test. Results In this study, we obtained 64 distinct variation patterns with the accession GenBank numbers MT681266-MT681329. Phylogenetic analysis revealed that 98.3% of HPV16 variants belong to lineage A, in which the A4 (Asian) sublineage was dominant (64.8%), followed by A2 (12.1%), A1 (11.4%), and A3 (10.0%). The A4 (Asian) sublineage had a higher risk of CIN2+ than the A1–3 (European) sublineages (OR = 2.69, 95% CI = 1.04–6.97, P < 0.05). Furthermore, nucleotide variation in HPV16 E6 T178G is associated with the development of cervical cancer. Conclusion These data could provide novel insights into the role of HPV16 variants in cervical carcinogenesis risk in China.

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Phylogenetic analysis and predicted functional effect of protein mutations of E6 and E7 HPV16 strains isolated in Indonesia

Medical Journal of Indonesia ◽

10.13181/mji.v24i4.1197 ◽

2015 ◽

Vol 24 (4) ◽

pp. 197-205

Author(s):

Dwi Wulandari ◽

Lisnawati Rachmadi ◽

Tjahjani M. Sudiro

Keyword(s):

Amino Acids ◽

Phylogenetic Analysis ◽

Amino Acid ◽

Asian American ◽

Protein Function ◽

Single Amino Acid ◽

Hpv16 E6 ◽

E6 And E7 ◽

Neutral Mutations

Background: E6 and E7 are oncoproteins of HPV16. Natural amino acid variation in HPV16 E6 can alter its carcinogenic potential. The aim of this study was to analyze phylogenetically E6 and E7 genes and proteins of HPV16 from Indonesia and predict the effects of single amino acid substitution on protein function. This analysis could be used to reduce time, effort, and research cost as initial screening in selection of protein or isolates to be tested in vitro or in vivo.Methods: In this study, E6 and E7 gene sequences were obtained from 12 samples of Indonesian isolates, which were compared with HPV16R (prototype) and 6 standard isolates in the category of European (E), Asian (As), Asian-American (AA), African-1 (Af-1), African-2 (Af-2), and North American (NA) branch from Genbank. Bioedit v.7.0.0 was used to analyze the composition and substitution of single amino acids. Phylogenetic analysis of E6 and E7 genes and proteins was performed using Clustal X (1.81) and NJPLOT softwares. Effects of single amino acid substitutions on protein function of E6 and E7 were analysed by SNAP.Results: Java variants and isolate ui66* belonged to European branch, while the others belonged to Asian and African branches. Twelve changes of amino acids were found in E6 and one in E7 proteins. SNAP analysis showed two non neutral mutations, i.e. R10I and C63G in E6 proteins. R10I mutations were found in Af-2 genotype (AF472509) and Indonesian isolates (Af2*), while C63G mutation was found only in Af2*.Conclusion: E6 proteins of HPV16 variants were more variable than E7. SNAP analysis showed that only E6 protein of African-2 branch had functional differences compared to HPV16R.

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