Orientation-dependent toxic effect of human papillomavirus type 33 long control region DNA in Escherichia coli cells.
ABSTRACT: The functional analysis of human papillomavirus (HPV) sequence variation requires the molecular cloning of different genomic regions of virus variants. In this study, we report an unexpected difficulty experienced when trying to clone HPV33 long control region (LCR) variants in Escherichia coli. Standard cloning strategies proved to be inappropriate to clone HPV33 LCR variants in the forward orientation into a eukaryotic reporter vector (pGL2-Basic). However, by slight modification of culture conditions (incubation at 25 °C instead of 37 °C), constructs containing the HPV33 LCR variants in the forward orientation were obtained. Transformation experiments performed with different HPV33 LCR constructs indicated that there is a sequence element in the 5' LCR of HPV33 causing temperature-dependent toxic effect in E. coli. Sequence analysis revealed the presence of an open reading frame (ORF) in the 5' part of HPV33 LCR potentially encoding a 116-amino acid polypeptide. Protein structure prediction suggested that this putative protein might have a structural similarity to transmembrane proteins. Even a low-level expression of this protein may cause significant toxicity in the host bacteria. In silico analysis of the LCR of HPV33 and some other HPV types belonging to the species Alphapapillomavirus 9 (HPV31, 35 and 58) seemed to support the assumption that the ORFs found in the 5' LCR of these HPVs are protein-coding sequences. Further studies should be performed to prove that these putative proteins are really expressed in the infected host cells and to identify their function.
Project description:The human papillomavirus (HPV) Long Control Region (LCR) encompasses the early promoter (EP) that drives transcription of the E6 and E7 oncogenes in keratinocytes and HPV-associated cancers. In this study, the transcriptional activities of the HPV33 EP from the prototype LCR and from eight variants representative of the worldwide diversity of the virus were examined in primary human keratinocytes (PHK) and in the HeLa cervical carcinoma cell line by luciferase reporter-gene assays. Remarkably, the two variations with the greatest effect on the EP in PHK were C7732G and a 79-bp deletion that were associated with high-grade cervical lesions and persistent infections, respectively, in epidemiological studies. In contrast, the three variations most active in HeLa cells were C7537A, A7874C and A7879G. A7874C, which lies within an E2-binding sequence, is also shown to increase the activity and binding of E2 at this site. Collectively, these results indicate that naturally-occurring variations affect the HPV33 EP differentially in PHK than in cancer cells and, furthermore, that they can also alter its regulation by E2. These findings provide a molecular basis for rationalizing the results of previous epidemiological studies and for understanding the contribution of LCR polymorphisms to the oncogenicity and persistence of HPV33 infections.
Project description:BACKGROUND:Human papillomavirus 16 (HPV16) species group (alpha-9) of the Alphapapillomavirus genus contains HPV16, HPV31, HPV33, HPV35, HPV52, HPV58 and HPV67. These HPVs account for 75% of invasive cervical cancers worldwide. Viral variants of these HPVs differ in evolutionary history and pathogenicity. Moreover, a comprehensive nomenclature system for HPV variants is lacking, limiting comparisons between studies. METHODS:DNA from cervical samples previously characterized for HPV type were obtained from multiple geographic regions to screen for novel variants. The complete 8 kb genomes of 120 variants representing the major and minor lineages of the HPV16-related alpha-9 HPV types were sequenced to capture maximum viral heterogeneity. Viral evolution was characterized by constructing phylogenic trees based on complete genomes using multiple algorithms. Maximal and viral region specific divergence was calculated by global and pairwise alignments. Variant lineages were classified and named using an alphanumeric system; the prototype genome was assigned to the A lineage for all types. RESULTS:The range of genome-genome sequence heterogeneity varied from 0.6% for HPV35 to 2.2% for HPV52 and included 1.4% for HPV31, 1.1% for HPV33, 1.7% for HPV58 and 1.1% for HPV67. Nucleotide differences of approximately 1.0% - 10.0% and 0.5%-1.0% of the complete genomes were used to define variant lineages and sublineages, respectively. Each gene/region differs in sequence diversity, from most variable to least variable: noncoding region 1 (NCR1) /noncoding region 2 (NCR2) >upstream regulatory region (URR)> E6/E7 > E2/L2 > E1/L1. CONCLUSIONS:These data define maximum viral genomic heterogeneity of HPV16-related alpha-9 HPV variants. The proposed nomenclature system facilitates the comparison of variants across epidemiological studies. Sequence diversity and phylogenies of this clinically important group of HPVs provides the basis for further studies of discrete viral evolution, epidemiology, pathogenesis and preventative/therapeutic interventions.
Project description:The Long Control Region (LCR) of the human papillomavirus (HPV) genome encompasses the early promoter (EP) that drives expression of the viral oncogenes in infected cells and HPV-associated cancers. Here, we report on a natural variant of HPV33 that displays higher EP activity than the prototype in transfected C33A and HeLa cervical carcinoma cells, and in the osteosarcoma U2OS cell line which supports replication of HPV episomes. This increased promoter activity was ascribed to a single nucleotide variation in the LCR, T7791C, in a putative binding site for the transcription factor C/EBP?. T7791C abrogated binding of recombinant C/EBP? to this site in vitro and stimulated the EP in vivo, suggesting that it abrogates a negatively-acting regulatory element. A second C/EBP? binding site was identified in vitro that activated the EP in vivo and whose function and location in the epithelial-specific enhancer is shown to be conserved in the highly prevalent HPV18. These results suggest that C/EBP? is both an activator and a repressor of the HPV33 EP, acting via two distinct binding sites. Prediction of C/EBP? sites in the LCR of 186 HPV types suggests that C/EBP? regulation of the EP is common among high-risk viruses from the ? genus.
Project description:Human papillomavirus (HPV) 33, a member of the HPV16-related alpha-9 species group, is found in approximately 5% of cervical cancers worldwide. The current study aimed to characterize the genetic diversity of HPV33 and to explore the association of HPV33 variants with the risk for cervical cancer. Taking advantage of the International Agency for Research on Cancer biobank, we sequenced the entire E6 and E7 open reading frames of 213 HPV33-positive cervical samples from 30 countries. We identified 28 HPV33 variants that formed 5 phylogenetic groups: the previously identified A1, A2, and B (sub)lineages and the novel A3 and C (sub)lineages. The A1 sublineage was strongly over-represented in cervical cases compared to controls in both Africa and Europe. In conclusion, we provide a classification system for HPV33 variants based on the sequence of E6 and E7 and suggest that the association of HPV33 with cervical cancer may differ by variant (sub)lineage.
Project description:HPV-16 long control region (LCR) has been shown to be the most variable region of the HPV-16 genome and may play important roles in viral persistence and the development of cervical cancer. This study aimed to assess the risk of HPV-16 LCR variants for cervical cancer in women of Southwest China. 2146 cervical scrapings of volunteer outpatients and 74 cervical cancer tissues were screened.14 entire HPV-16 LCRs from asymptomatic carriers and 34 entire HPV-16 LCRs from cervical cancer patients were successfully amplified and sequenced to align to others described. 58 different point mutations were detected in 54 nucleotide sites of HPV-16 LCR. G7193T and G7521A variants, accounting for 100% of the infections, were predicted to locate at the binding site for FOXA1 and SOX9, respectively. A7730C variant which showed a high mutation frequency in cervical cancer was predicted to be a binding site for the cellular transcription factor PHOX2A. In addition, phylogenetic analysis displayed a high prevalence of A lineage in HPV-16 LCR in this Southwest China population. This study may help understanding of the intrinsic geographical relatedness and the correlations between LCR mutations and the development of carcinogenic lesions in Southwest China population. And it provides useful data for the further study of the biological function of HPV-16 LCR variants.
Project description:A significant proportion of recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6). The long control region (LCR) contains cis-elements for regulation of transcription. Our aim was to characterize LCR HPV-6 variants in RRP cases, compare promoter activity of these isolates and search for cellular transcription factors (TFs) that could explain the differences observed. The complete LCR from 13 RRP was analyzed. Transcriptional activity of 5 variants was compared using luciferase assays. Differences in putative TFs binding sites among variants were revealed using the TRANSFAC database. Chromatin immunoprecipation (CHIP) and luciferase assays were used to evaluate TF binding and impact upon transcription, respectively. Juvenile-onset RRP cases harbored exclusively HPV-6vc related variants, whereas among adult-onset cases HPV-6a variants were more prevalent. The HPV-6vc reference was more transcriptionally active than the HPV-6a reference. Active FOXA1, ELF1 and GATA1 binding sites overlap variable nucleotide positions among isolates and influenced LCR activity. Furthermore, our results support a crucial role for ELF1 on transcriptional downregulation. We identified TFs implicated in the regulation of HPV-6 early gene expression. Many of these factors are mutated in cancer or are putative cancer biomarkers, and must be further studied.
Project description:High-risk human papillomaviruses (HPVs), particularly HPV types 16 and 18 (HPV-16 and HPV-18, respectively), play a cardinal role in the etiology of cervical cancer. The most prevalent type, HPV-16, shows intratypic sequence variants that are known to differ in oncogenic potential and geographic distribution. This study was designed to analyze sequence variations in E6, E7, and L1 genes and the LCR (for long control region) of HPV-16 in cervical cancer patients to identify the most prevalent and novel HPV-16 variants and to correlate them with the severity of the disease. Cervical biopsies from 60 HPV-16-positive cancer cases were analyzed by PCR and DNA sequencing. The most frequently observed variations were T350G (100%) in E6, T789C (87.5%) in E7, A6695C (54.5%) in L1, and G7521A (91.1%) in the LCR. In addition, only one novel variant (T527A) in E6 and four new variants each in L1 (A6667C, A6691G, C6906T, and A6924C) and in the LCR (C13T, A7636C, C7678T, and G7799A) were identified. While E7 was found to be highly conserved, the variant 350G of E6 was the most prevalent in all of the histopathological grades. The majority of LCR variants were found at the YY1 transcription factor binding sites. Interestingly, a complete absence of the Asian lineage and a high prevalence of European lineages in E6, E7, L1, and the LCR (85, 86.7, 67.7, and 63.3%, respectively) indicate a possible epidemiological linkage between Europe and India with regard to the dissemination of HPV-16 infections in India.
Project description:<h4>Objectives</h4>Human papillomavirus (HPV) causes tumors primarily Cervical cancer. Recently, inconsistent reports came up in Breast cancer (BC) too. In India, despite treatment 70,218 BC patients die each year. So, we explored the association of HPV, if any, with BC prognosis in Indian pre-therapeutic (PT) and Neo-adjuvant chemotherapy (NACT) patients with subsequent analysis of HPV profile.<h4>Methods</h4>HPV prevalence was checked and analysis of physical status, copy number, genome variation, promoter methylation and expression (mRNA and protein) of the prevalent subtype was done.<h4>Results</h4>High prevalence of HPV was observed in both PT (64.0%) and NACT (71.0%) cases with significant association with younger (20-45 yrs) PT patients. Interestingly, HPV infection was significantly increased from adjacent normal breast (9.5%, 2/21), fibro adenomas (30%, 3/10) to tumors (64.8%, 203/313) samples. In both PT and NACT cases, HPV16 was the most prevalent subtype (69.0%) followed by HPV18 and HPV33. Survival analysis illustrated hrHPV infected PT patients had worst prognosis. So, detailed analysis of HPV16 profile was done which showed Europian-G350 as the most frequent HPV16 variant along with high rate of integration. Moreover, low copy number and hyper-methylation of P97 early promoter were concordant with low HPV16 E6 and E7 mRNA and protein expression. Notably, four novel variations (KT020838, KT020840, KT020841 and KT020839) in the LCR region and two (KT020836 and KT020837) in the E6 region were identified for the first time along with two novel E6^E7*I (KU199314) and E6^E7*II (KU199315) fusion transcript variants.<h4>Conclusion</h4>Thus, significant association of hrHPV with prognosis of Indian BC patients led to additional investigation of HPV16 profile. Outcomes indicated a plausible role of HPV in Indian BC patients.
Project description:To map comprehensively the methylation status of the CpG sites within the HPV16 long control region (LCR) in HPV-positive cancer cells, and to explore further the effects of methylation status of HPV16 LCR on cell bioactivity and E6 and E7 expression. In addition, to analyze the methylation status of the LCR in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) patients.Methylation patterns of HPV16 LCR in UM-SCC47, CaSki, and SiHa cells and HPV16-positiive OPSCC specimens were detected by bisulfite-sequencing PCR and TA cloning. For cells treated with 5-aza-2'-deoxycytidine and E6 and E7 knockdown, MTS and trypan blue staining, annexin-V and 7-AAD staining, and prodidium iodide were used to evaluate cell growth and cell proliferation, cell apoptosis, and cell cycle arrest, respectively. E6 and E7 mRNA and protein expression were analyzed by quantitative real-time PCR and immunocytochemistry, respectively.Hypermethylation status of the LCR in UM-SCC47 (79.8%) and CaSki cells (90.0%) and unmethylation status of the LCR in SiHa cells (0%) were observed. Upon demethylation, the cells with different methylation levels responded differently during growth, apoptosis, and cell cycle arrest, as well as in terms of their E6 and E7 expression. In HPV16-positive OPSCC patients, the methylation rates were 9.5% in the entire LCR region, 13.9% in the 5'-LCR, 6.0% in the E6 enhancer, and 9.5% in the p97 promoter, and hypermethylation of p97 promoter was found in a subset of cases (20.0%, 2/10).Our study revealed two different methylation levels of the LCR in HPV16-positive cancer cells and OPSCC patients, which may represent different carcinogenesis mechanisms of HPV-positive cancers cells. Demethylating the meCpGs in HPV16 LCR might be a potential target for a subgroup of HPV16-positive patients with head and neck squamous cell carcinoma.
Project description:BACKGROUD:Variations in HPV LCR/E6/E7 have been shown to be associated with the viral persistence and cervical cancer development. So far, there are few reports about the polymorphisms of the HPV-58 LCR/E6/E7 sequences in Southwest China. This study aims to characterize the gene polymorphisms of the HPV-58 LCR/E6/E7 sequences in women of Southwest China, and assess the effects of variations on the immune recognition of viral E6 and E7 antigens. METHODS:Twelve LCR/E6/E7 of the HPV-58 isolates were amplified and sequenced. A neighbor-joining phylogenetic tree was constructed by MEGA 7.0, followed by the secondary structure prediction of the related proteins using PSIPRED v3.3. The selection pressure acting on the HPV-58 E6 and E7 coding regions was estimated by Bayes empirical Bayes analysis of PAML 4.8. Meanwhile, the MHC class-I and II binding peptides were predicted by the ProPred-I server and ProPred server. The transcription factor binding sites in the HPV-58 LCR were analyzed using the JASPAR database. RESULTS:Twenty nine SNPs (20 in the LCR, 3 in the E6, 6 in the E7) were identified at 27 nucleotide sites across the HPV-58 LCR/E6/E7. From the most variable to the least variable, the nucleotide variations were LCR > E7 > E6. The combinations of all the SNPs resulted in 11 unique sequences, which were clustered into the A lineage (7 belong to A1, 2 belong to A2, and 2 belong to A3). An insertion (TGTCAGTTTCCT) was found between the nucleotide sites 7280 and 7281 in 2 variants, and a deletion (TTTAT) was found between 7429 and 7433 in 1 variant. The most common non-synonymous substitution V77A in the E7 was observed in the sequences encoding the α-helix. 63G in the E7 was determined to be the only one positively selected site in the HPV-58 E6/E7 sequences. Six non-synonymous amino acid substitutions (including S71F and K93 N in the E6, and T20I, G41R, G63S/D, and V77A in the E7) were affecting multiple putative epitopes for both CD4+ and CD8+ T-cells. In the LCR, C7265G and C7266T were the most variable sites and were the potential binding sites for the transcription factor SOX10. CONCLUSION:These results provide an insight into the intrinsic geographical relatedness and biological differences of the HPV-58 variants, and contribute to further research on the HPV-58 epidemiology, carcinogenesis, and therapeutic vaccine development.