Comprehensive comparison of the interaction of the E2 master regulator with its cognate target DNA sites in 73 human papillomavirus types by sequence statistics

Mucosal human papillomaviruses (HPVs) are etiological agents of oral, anal and genital cancer. Properties of high- and low-risk HPV types cannot be reduced to discrete molecular traits. The E2 protein regulates viral replication and transcription through a finely tuned interaction with four sites at the upstream regulatory region of the genome. A computational study of the E2–DNA interaction in all 73 types within the alpha papillomavirus genus, including all known mucosal types, indicates that E2 proteins have similar DNA discrimination properties. Differences in E2–DNA interaction among HPV types lie mostly in the target DNA sequence, as opposed to the amino acid sequence of the conserved DNA-binding alpha helix of E2. Sequence logos of natural and in vitro selected sites show an asymmetric pattern of conservation arising from indirect readout, and reveal evolutionary pressure for a putative methylation site. Based on DNA sequences only, we could predict differences in binding energies with a standard deviation of 0.64 kcal/mol. These energies cluster into six discrete affinity hierarchies and uncovered a fifth E2-binding site in the genome of six HPV types. Finally, certain distances between sites, affinity hierarchies and their eventual changes upon methylation, are statistically associated with high-risk types

Different papillomaviruses have different repertoires of transcription factor binding sites: convergence and divergence in the upstream regulatory region

BACKGROUND: Papillomaviruses (PVs) infect stratified squamous epithelia in warm-blooded vertebrates and have undergone a complex evolutionary process. The control of the expression of the early ORFs in PVs depends on the binding of cellular and viral transcription factors to the upstream regulatory region (URR) of the virus. It is believed that there is a core of transcription factor binding sites (TFBS) common to all PVs, with additional individual differences, although most of the available information focuses only on a handful of viruses. RESULTS: We have studied the URR of sixty-one PVs, covering twenty different hosts. We have predicted the TFBS present in the URR and analysed these results by principal component analysis and genetic algorithms. The number and nature of TFBS in the URR might be much broader than thus far described, and different PVs have different repertoires of TFBS. CONCLUSION: There are common fingerprints in the URR in PVs that infect primates, although the ancestors of these viruses diverged a long time ago. Additionally, there are obvious differences between the URR of alpha and beta PVs, despite these PVs infect similar histological cell types in the same host, i.e. human. A thorough analysis of the TFBS in the URR might provide crucial information about the differential biology of cancer-associated PVs

Impact of non-coding RNAs in genetic disease and papillomavirus lifecycle

Non-coding RNAs are vital to several diverse biological processes. Untranslated regions of mRNAs (UTRs) and variability therein can often cause differential regulation of transcripts. This regulation is often effected by microRNAs (miRNAs), small non-coding RNAs that interacts with 3’UTRs to regulate gene expression post-transcriptionally. Here we investigate the variation in the 3’UTR of SERPINA1 gene, mutations of which cause the often life-threatening Alpha-1-antitrypsin deficiency (A1AD), and effects on miRNA. We screened 50 miRNA mimics and determined their overexpression does not affect secreted alpha-1-antitrypsin (AAT). Additionally, we showed through the use of a poxvirus protein to degrade miRNAs that underexpression of miRNAs does not affect secreted AAT. Using luciferase reporters we determine that in cultured cells, there are no liver-specific trans-factors that act on the SERPINA1 3’UTR, of any length, to regulate gene expression. We sequenced the 3’UTRs from the largest cohort of of A1AD patients to date and determined that 3’UTR variation does not contribute to disease severity in patients with one form of A1AD, and that in a cell culture context, miRNAs do not regulate secreted AAT. We conclude that neither 3’UTR variation nor miRNAs affect wild-type secreted AAT. In addition to genetic disease, miRNAs regulate multiple aspects of the host-pathogen interface. Until now no widely accepted PV-encoded miRNAs have been described. We have developed miRNA Discovery by forced Genomic Expression (miDGE), a new wet bench approach to miRNA identification that screens numerous pathogen genomes in parallel. Using miDGE, we screened over 75 different PV genomes for the ability to code for miRNAs. We conclusively demonstrate a lack of PV miRNA expression in cancers associated with infections of several high risk HPVs. However, we identified five different miRNAs encoded by four different PVs (Human PVs 17, 37, 41 and Fringilla colebs (Fc) PV). We show that miRNAs from two PVs (HPV41 & FcPV) are able to regulate transcripts corresponding to the early region of the PV genome.. Combined, these findings identify the first canonical PV miRNAs and support that miRNAs of either host or viral origin are important regulators of the PV life cycle.Microbiolog

Identification of Prognostic Cancer Biomarkers through the Application of RNA-Seq Technologies and Bioinformatics

MicroRNAs (miRNAs) are short single-stranded RNAs that function as the guide sequence of the post-transcriptional regulatory process known as the RNA-induced silencing complex (RISC), which targets mRNA sequences for degradation through complementary binding to the guide miRNA. Changes in miRNA expression have been reported as correlated with numerous biological processes, including embryonic development, cellular differentiation, and disease manifestation. In the latter case, dysregulation has been observed in response to infection by human papillomavirus (HPV), which has also been established as both oncogenic in cervical cancers and oropharyngeal cancers and favorable for overall patient survival after tumor formation. The identification of dysregulated miRNAs associated with both HPV infection and cancer survival requires large datasets of high-throughput sequencing data, which were obtained through The Cancer Genome Atlas. By analyzing this public data, we have identified a series of proposed mechanisms for cancer formation and survival that is mediated through the miRNA-RISC regulatory mechanism in response to HPV infection. We have also identified a diverse set of miRNA biomarkers that have been incorporated into linear expression-based risk signatures that are prognostic for overall patient survival after tumor diagnosis in HPV-related cancers. The tools that were used to identify both miRNA biomarkers and proposed targets in public datasets, such as The Cancer Genome Atlas, have since been incorporated into an web-accessible resource, OncomiR.org, to streamline the process of biomarker identification for the cancer research community

Bioinformatics analysis of HPV associated host microRNA functions and identification of viral microRNA

Human papillomaviruses (HPVs) form a large family among double stranded DNA (dsDNA) viruses, some types of which are the major causes of cervical cancer. HPV 16 is widely distributed and the most common high-risk HPV type and approximately half of the cervical cancers are associated with HPV type 16. Of the three HPV 16 encoded oncogenes, the function of E5 in regulating viral replication and pathogenesis is less well understood than E6 and E7. The microRNAs (miRNAs) are important small noncoding RNA molecules that regulate wide range of cellular functions. Some dsDNA viruses, such as SV40 and human polyomaviruses, have functional viral miRNAs. The functional and molecular similarities among dsDNA viruses suggest that HPV could encode viral miRNAs, which have not been validated thus far. The aim of this thesis was to study the functions of the host miRNAs in HPV 16 oncogene induction and identify novel HPV encoded viral miRNAs. We utilized microarray technology to investigate the effect of E5 on host miRNAs and mRNAs expression in 0 96 hours after E5 induction in a cell line model. Among the differentially expressed cellular miRNAs, we further validated the expression of hsa-mir-146a, hsa-mir-203, and hsa-mir-324-5p and some of their target genes in a time series of 96 hours of E5 induction. Our results indicate that HPV E5 expression has an impact through complex regulatory patterns of gene expression in the host cells, and part of those genes is regulated by the E5 protein. Second, high throughput sequencing was used to identify virus-encoded miRNAs. We prepared small RNA sequencing libraries from ten HPV-associated cervical lesions, including cancer and two HPV-harboring cell lines. For more flexible analysis of the sequencing data we developed miRSeqNovel, an R based workflow for miRNA sequencing data analysis, and applied it to the sequencing data to predict putative viral miRNAs and discovered nine putative papillomavirus encoded miRNAs. Viral miRNA validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38, and one by HPV 68. The expression of two HPV 16 miRNAs was further supported by in situ hybridization, and colocalization with p16INK4A staining, a marker of cervical neoplasia. Prediction of cellular target genes of HPV 16 encoded miRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development and cancer, which were also among the functions targeted by the E5 regulated host cell mRNA and miRNAs. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene, and one in the long control region (LCR).Ihmisen papilloomavirukset (HPV) muodostavat suuren heimon kaksijuosteisen DNA-virusten (dsDNA) joukossa, ja niistä jotkin virustyypit ovat kohdunkaulasyövän pääasiallisia aiheuttajia. HPV 16 on laajalle levinnyt ja yleisin suuren riskin HPV-tyyppi, joka aiheuttaa noin puolet kohdunkaulasyövistä. Kolmesta HPV 16:n koodittamasta onkogeenistä E5:n toiminta viruksen replikaation ja patogeneesin säätelyssä tunnetaan huonommin kuin E6:n ja E7:n. Mikro-RNA:t (miRNA) ovat pieniä ei-koodittaavia RNA-molekyylejä, joilla on tärkeä merkitys solun toimintojen säätelyssä. Jotkin dsDNA-virukset, kuten SV40 ja ihmisen polyoomavirukset, koodittavat omia toiminnallisia mikro-RNA:ita. HPV:n koodittamia mRNAita ei ole aiemmin validoitu, mutta dsDNA-virusten toiminnalliset ja molekulaariset samankaltaisuudet viittaavat siihen, että myös HPV voisi koodittaa omia mikro-RNA:ita. Väitöskirjan tavoitteena oli tutkia isäntäsolun mikro-RNA:iden toimintaa HPV 16 onkogeeni-induktiossa ja löytää uusia, HPV:n koodittamia miRNA:ita. Tutkimme sirutekniikan avulla E5:n vaikutusta isännän miRNA- ja mRNA-ekspressioon solulinjamallissa 0-96 tunnin kuluessa E5:n induktiosta. Eri tavoin ekpressoituneista miRNA tutkimme tarkemmin hsa-mir-146, hsa-mir-203 ja hsa-mir-324-5p sekä muutamien näiden kohdegeenien ekspressiota 96 tunnin aikasarjana E5 induktiosta. Tulokset osoittavat HPV E5 ekspression vaikuttavan isäntäsolun geenien ilmentymiseen monimutkaisen säätelymallin välityksellä ja E5-proteiinin myös säätelevän osaa näistä geeneistä. Next generation sekvensointia käytettiin tunnistamaan virusten koodaamia miRNA:ita. Joustavampaa miRNA sekvenssidatan analysointia varten kehitimme miRSeqNovel-nimisen R-pohjaisen työkalun ja käytimme sitä ennustamaan mahdollisia virusten koodaamia miRNA:ita. Löysimme yhdeksän mahdollisesti HPV:n koodaamaa miRNA:ta, joista viisi otettiin mukaan miRNAn validointiin. Neljä viidestä HPV:n koodaamasta miRNAsta pystyttiin validoimaan qPCR:n avulla kohdunkaulan kudosnäytteistä ja solulonjoista. Näistä kaksi miRNA:ta on HPV 16 koodaamaa ja yhdet HPV38 ja HPV68 koodaamia. Kahden HPV16 koodaaman miRNA:n ekspressiota osoitettiin myös in-situ-hybridisaatiossa kolokalisoituneena värjäyksessä kohdunkaulan neoplasiasta kertovan P16INK4a kanssa. HPV16 mikro-RNA:n kohdegeenien ennusteet viittaavat miRNA:iden mahdolliseen rooliin solusyklissä, immuunijärjestelmän toiminnoissa, soluadheesiossa ja migraatiossa, yksilönkehityksessä ja syövässä. Nämä kohteet löytyvät myös E5:n säätelemien mRNA:iden ja miRNA:iden kohteista. Kaksi HPV16 miRNA:n mahdollista kohdetta löytyvät viruksen E5-geenistä, yksi kohden E1 geenistä, kaksi L1 geenistä ja yksi pitkästä kontrollialueesta (LCR)

The role of the conformational plasticity in the DNA-protein recognition mechanism: NMR study of the Human Papillomavirus E2 system

Maurizio Delfini, Bruno Samorì, Roberto Fttoruss

Human papillomaviruses and their association with cervical disease

The overall aim of the project was to establish whether human papillomaviruses (HPV) are associated with cervical intraepithelial neoplasia (CIN) and cervical carcinoma and examine the use of HPV 16 as an indicator of cervical disease. For this purpose, a Southern blot hybridization system was developed to detect HPV DNA in cervical scrape samples. However, following the description of the polymerase chain reaction for detection of nucleic acid a PCR system for detection of HPV DNA was developed. The ability of Southern blot hybridization and PCR to detect HPV DNA in cervical scrape samples was compared to determine the most suitable method for use as a diagnostic test for HPV. The PCR method was 100,000 times more sensitive than Southern blot and was more accurate in identifying women with cervical disease. The PCR system was used to analyse cervical scrape samples from two study groups for the presence of HPV 16 DNA. The first group (Study A) consisted of 200 women from a General Practice population who were expected to have normal cervical cytology.HPV 16 was present in 17% of women with no cervical abnormalities. In those women from Study A who had cervical disease (n = 22) the prevalence of HPV 16 increased with greater severity of disease from 15.4% in those with CIN 1, 40% in those with CIN 2, to 75% of those with CIN 3. The presence of HPV 16 DNA was significantly associated with CIN 2 and 3 (p = 0.009) and was therefore useful as an indicator of severe cervical disease in this population. The ability of PCR for HPV 16 to identify women with disease was compared with that of standard cytological analysis. There was no significant difference between the two methods, although a combination of screening by cytology and PCR resulted in the identification of a higher proportion of women with disease and PCR was associated with a higher false positive rate. The second group (Study B) consisted of 200 women who had been referred to the Royal Free Hospital colposcopy clinic with a smear report suggesting mild dyskaryosis. Within this group there were 54 women who were cytologically normal, 59 women who had CIN 1 or WVI and 66 women with severe cervical disease (CIN 2 or 3). The results of Study B concurred with Study A in demonstrating an increasing prevalence of HPV 16 with greater severity of disease from 53% in women with CIN 1, 64% of women with CIN 2 to 74% of patients with CIN 3. However, the prevalence of HPV 16 in the normal women in Study B was 63%, and this high value precludes the use of HPV 16 as an indicator of severe cervical disease in this population. Duplicate analysis of each cervical scrape sample from Study A and Study B allowed the reproducibility of the HPV 16 PCR system to be determined. The false positive rate was 0.1% and the false negative rate was 0.77%. The long control region (LCR) of HPV 16 was cloned from a woman without cervical disease (CO) and a woman with CIN 3 (C3). The DNA sequence of each isolate was determined and compared with the prototype HPV 16 sequence. Nucleotide variations were evident in both isolates, but LCRC3 shared less homology with the prototype sequence than LCRCO. A single nucleotide mutation occurred within the glucocorticoid responsive element of LCRC3, which disrupts the palindrome of the protein binding domain. The level of expression from the HPV 16 LCR was determined using a chloramphenicol acetyltransferase assay and found to be 5-fold lower than that of the SV40 early promoter