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Genetic variability in E6 and E7 oncogenes of human papillomavirus Type 16 from Congolese cervical cancer isolates.

ABSTRACT: BACKGROUND:The molecular epidemiological studies showed that some variants of HPV-16, distributed geographically, would present a higher risk of causing cervical cancer. This study aimed to analyze nucleotide changes of HPV-16 E6 and E7 genomic regions from infected Southwestern Congolese women. METHODS:DNA of twenty HPV-16 isolates was analyzed by amplifying the E6 and E7 genes using type-specific primers PCR and direct sequencing. The sequences obtained were aligned with the HPV-16 GenBank reference sequences. RESULTS:Thirteen (65.0%) out of 20 DNA-samples were successfully amplified. Genetic analysis revealed 18 and 4 nucleotide changes in E6 and E7 genomic regions respectively. The most frequently observed nucleotide variations were the missense C143G, G145T and C335T in E6 (100%), leading to the non-synonymous amino acid variation Q14D and H78Y. E7 genomic region was found to be highly conserved with two most common T789C and T795G (100%) silent variations. All HPV-16 variants identified belonged to the African lineage: 7 (53.8%) belonged to Af-1 lineage and 6 (46.1%) to Af-2 lineage. The missense mutation G622A (D21N) in the E7 region seems to be described for the first time in this study. CONCLUSION:This study reported for the first time the distribution of HPV-16 E6 and E7 genetic variants in infected women from southwest Congo. The findings confirmed almost ascendancy of the African lineage in our study population.

SUBMITTER: Boumba LM

PROVIDER: S-EPMC4437748 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Genetic variability in E6 and E7 oncogenes of human papillomavirus Type 16 from Congolese cervical cancer isolates.

Boumba Luc Magloire Anicet LM Assoumou Samira Zoa SZ Hilali Lahoucine L Mambou Jean Victor JV Moukassa Donatien D Ennaji Mustapha Moulay MM

Infectious agents and cancer 20150514

<h4>Background</h4>The molecular epidemiological studies showed that some variants of HPV-16, distributed geographically, would present a higher risk of causing cervical cancer. This study aimed to analyze nucleotide changes of HPV-16 E6 and E7 genomic regions from infected Southwestern Congolese women.<h4>Methods</h4>DNA of twenty HPV-16 isolates was analyzed by amplifying the E6 and E7 genes using type-specific primers PCR and direct sequencing. The sequences obtained were aligned with the HPV ...[more]

PMID: 25991921

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Project description:Patients with HPV-positive oropharyngeal cancer show better tumor response to radiation or chemotherapy than patients with HPV-negative cancer. HPV oncoprotein E6 binds and degrades a typically wild-type p53 protein product. However, HPV16 infection and p53 mutation infrequently coexist in a subset of HNSCCs. The purpose of this study was to investigate the mechanisms through which tumor biology and molecular genetic mechanisms change when two HPV-negative, p53-mutated oropharyngeal cell lines (YD8, non-disruptive p53 mutation; YD10B, disruptive p53 mutation) derived from patients with a history of heavy smoking are transfected with HPV E6 and E7 oncogenes in vitro. Transfection with HPV E6 and E7 oncogenes in YD8, reduced the abundance of proteins encoded by tumor suppressor genes, such as p-p53 and p-Rb. Cell proliferative activity was increased in the cells transfected with E6E7 compared to cells transfected with vector alone (P=0.09), whereas the invasiveness of E6E7-transfected cells was significantly reduced (P=0.02). cDNA microarray of the transfected cells with E6E7 showed significant changes in mRNA expression in several signaling pathways, including focal adhesion, JAK-STAT signaling pathway, cell cycle and p53 signaling pathway. Regarding the qPCR array for the p53 signaling pathway, the mRNA expression of STAT1 was remarkably upregulated by 6.47-fold (P<0.05); in contrast, IGF-1R was significantly downregulated by 2.40-fold in the YD8-vector compared toYD8-E6E7 (P<0.01). Finally, data collected from these two array experiments enabled us to select two genes, STAT1 and IGF-1R, for further study. In immunohistochemical study, nuclear STAT1 expression was slightly higher in HPV-positive compared to HPV-negative oropharyngeal tumors (P=0.18); however, cytoplasmic STAT1 was significantly lower in HPV-positive cases (P=0.03). IGF-1R expression levels were remarkably lower in HPV-positive compared to HPV-negative cases (P=0.01). Our data suggest that upregulated STAT1 and interferon signals by HPV16 E6 and E7 genes may play a major role in the relatively favorable prognosis for HPV-positive oropharyngeal squamous cell carcinoma cases with non-disruptive p53 mutations.

Project description:UnlabelledTranscripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41 N-terminal amino acid (aa) residues of E6 and the 38 C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy.ImportanceHPV16 is the most prevalent HPV genotype, being responsible for 60% of invasive cervical cancer cases worldwide. What makes HPV16 so potent in the development of cervical cancer remains a mystery. We discovered in this study that, besides producing two well-known oncoproteins, E6 and E7, seen in other high-risk HPVs, HPV16 produces E6^E7, a novel splice isoform of E6 and E7. E6^E7, in addition to self-interacting, binds cellular chaperone proteins, HSP90 and GRP78, and viral E6 and E7 to increase the steady-state levels and half-lives of viral oncoproteins, leading to cell proliferation. The splicing cis elements in the regulation of HPV16 E6^E7 production are highly conserved in 11 oncogenic or possibly oncogenic HPVs, and we confirmed the production of HPV18 E6^E7 in HPV18-infected cells. This study provides new insight into the mechanism of splicing, the interplay between different products of the polycistronic viral message, and the role of the host chaperones as they function.

Project description:BackgroundHuman 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.MethodsHPV 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.ResultsUsing 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.ConclusionsHPV62 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.

Project description:BackgroundHuman papillomavirus type 39 associated with genital intraepithelial neoplasia and invasive cancers, has a high prevalence in Southwest China. HPV E6, E7 are two main papillomavirus oncoproteins, closely relate to the function of HPV immortalization, cell transformation, and carcinogenesis. L1 is the major capsid protein, can reflect the replication status of the virus in cells and the progression of cervical lesions. The purpose of this study is to reveal the prevalence of HPV 39 and the genetic polymorphisms of HPV39 based on E6, E7 and L1 gene in southwest China.MethodsCell samples were collected by cervical scraped for HPV detecting and typing, and HPV39 positive samples were selected out. Important E6, E7 and L1 genes of HPV39 were sequenced and analyzed for the study of HPV39 genetic polymorphisms. Phylogenetic trees were constructed by Maximum-likelihood and Kimura 2-parameters methods in Molecular Evolutionary Genetics Analysis version 6.0. The selection pressures of E6, E7 and L1 genes were estimated by Datamonkey web server. The secondary and three-dimensional structure of HPV39 E6, E7 proteins were created by sopma server and SWISS-MODEL software.Results344 HPV39 positive samples were selected from 5718 HPV positive cell samples. Among HPV39 E6-E7 sequences, 20 single nucleotide mutations were detected, including 10 non-synonymous and 10 synonymous mutations; 26 single nucleotide mutations were detected in HPV39 L1 sequences, including 7 non-synonymous and 19 synonymous mutations respectively. 11 novel variants of HPV39 E6-E7 (5 in E6 and 6 in E7) and 14 novel variants of HPV39 L1 were identified in this study. A-branch was the most frequent HPV39 lineage in southwest China during our investigation. Selective pressure analysis showed that codon sites 26, 87, 151 in E6 and 75, 180, 222, 272, 284, 346, 356 in L1 were positively selected sites, as well as codon sites 45, 138, 309, 381 were negative selection sites in L1 gene, E7 has neither positive selection sites nor negative selection sites. A certain degree of secondary and three-dimensional structure dislocation was existed due to the non-synonymous mutations.ConclusionsAmino acid substitution affected the secondary and three-dimensional structure of HPV39, and resulting in the differences of carcinogenic potential and biological functions as well as the immune response due to the antigen epitopes difference, the antigen epitopes with stronger adaptability in Southwest will be screened out based on the above research results for the later vaccine development. And gene polymorphism of HPV39 in Southwest China may improve the effectiveness of clinical test and vaccine design, specifically for women in Southwest China.

Dataset Information

Genetic variability in E6 and E7 oncogenes of human papillomavirus Type 16 from Congolese cervical cancer isolates.

Publications

Genetic variability in E6 and E7 oncogenes of human papillomavirus Type 16 from Congolese cervical cancer isolates.

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