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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:BackgroundVariations in human papillomavirus (HPV) E6 and E7 have been shown to be closely related to the persistence of the virus and the occurrence and development of cervical cancer. Long control region (LCR) of HPV has been shown multiple functions on regulating viral transcription. In recent years, there have been reports on E6/E7/LCR of HPV-16 and HPV-58, but there are few studies on HPV-52, especially for LCR. In this study, we focused on gene polymorphism of the HPV-52 E6/E7/LCR sequences, assessed the effects of variations on the immune recognition of viral E6 and E7 antigens, predicted the effect of LCR variations on transcription factor binding sites and provided more basic date for further study of E6/E7/LCR in Chengdu, China.MethodsLCR/E6/E7 of the HPV-52 were amplified and sequenced to do polymorphic and phylogenetic analysis. Sequences were aligned with the reference sequence by MEGA 7.0 to identify SNP. A neighbor-joining phylogenetic tree was constructed by MEGA 7.0, followed by the secondary structure prediction of the related proteins using PSIPRED 4.0. The selection pressure of E6 and E7 coding regions were estimated by Bayes empirical Bayes analysis of PAML 4.9. The HLA class-I and II binding peptides were predicted by the Immune Epitope Database server. The B cell epitopes were predicted by ABCpred server. Transcription factor binding sites in LCR were predicted by JASPAR database.Results50 SNP sites (6 in E6, 10 in E7, 34 in LCR) were found. From the most variable to the least variable, the nucleotide variations were LCR > E7 > E6. Two deletions were found between the nucleotide sites 7387-7391 (TTATG) and 7698-7700 (CTT) in all samples. A deletion was found between the nucleotide sites 7287-7288 (TG) in 97.56% (40/41) of the samples. The combinations of all the SNP sites and deletions resulted in 12 unique sequences. As shown in the neighbor-joining phylogenetic tree, except for one belonging to sub-lineage C2, others sequences clustered into sub-lineage B2. No positive selection was observed in E6 and E7. 8 non-synonymous amino acid substitutions (including E3Q and K93R in the E6, and T37I, S52D, Y59D, H61Y, D64N and L99R in the E7) were potential affecting multiple putative epitopes for both CD4+ and CD8+ T-cells and B-cells. A7168G was the most variable site (100%) and the binding sites for transcription factor VAX1 in LCR. In addition, the prediction results showed that LCR had the high probability binding sites for transcription factors SOX9, FOS, RAX, HOXA5, VAX1 and SRY.ConclusionThis study provides basic data for understanding the relation among E6/E7/LCR mutations, lineages and carcinogenesis. Furthermore, it provides an insight into the intrinsic geographical relatedness and biological differences of the HPV-52 variants, and contributes to further research on the HPV-52 therapeutic vaccine development.

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.

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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