Deterministic and Stochastic Modeling of Human Papillomavirus Gene Regulatory Network

In this thesis a novel stochastic and deterministic mathematical model of Human papillomavirus (HPV) gene regulatory network was developed. The novelty of this project is both on methodological and biological /clinical site. The former is in line with the current challenge in recent years to have a holistic view of the basics regulatory mechanisms interconnected to form a complex machinery, where complex patterns can arise, only form the interconnection of basics modules. In fact, HPV offers a case of study of great interest in molecular systems biology. It involves a number of relevant regulatory mechanisms (e.g. transcription, translation, promoter modulation, polyadenylation regulation, splicing,…) connected together to form a complex network, albeit its genome is relatively simple, thus suitable for an accurate deterministic and even stochastic modeling. HPVs cause a series of diseases of the cutaneous and mucosal epithelium, ranging from minor lesions to precancerous cervical lesions and cervical cancer, which is considered one of the most common cancer in the women worldwide. Therefore, on the biological/clinical aspect the development of a mathematical model of HPV gene expression, is of great interest in order to dispose of an in silico simulator useful to achieve a better comprehension of the complex gene regulatory network, and capable to predict different scenarios from the first stages of viral infection up to a cervical cancer condition. As far as we know, there is no model of HPV gene regulation available in literature. A new synthesis of the HPV molecular biology with especial regard to gather/infer from literature the parameters useful for designing a dynamical model, and to shed light in what is still lacking in the biological literature, was preformed. The biological knowledge was translated into a stochastic model in terms of biochemical reactions. In particular, we modeled the HPV early and late promoters that account for the transcripts and proteins evolution during the entire viral life cycle. Even the post-transcriptional and post-translational modifications were modeled in order to properly capture the complex viral regulation known from literature. As far as we know, it is the first time a stochastic model accounts for the complex post-transcriptional control, modeling the splicing and polyadenylation sites regulation, and connect this latter to the transcriptional control layer, mediated by the promoters activities, in order to explore complex patterns that can arise only from the interconnection of different control layers. The Master Equation (ME) of the system was considered in order to predict and investigate its stochastic behavior. Because of the complex system structure it wasn't possible to solve the whole ME analytically, hence numerical exact simulations were performed by means of the Gillespie's algorithm. A quasi-equilibrium approximation of the ME was developed in order to get a deterministic approximation of the model. The model structure together with the fixed parameters we have gathered/inferred from literature was able to fit a dataset consistent of the early promoter activity and to qualitatively reproduce the main dynamical behavior of two of the most important regulatory transcripts during viral late phase. Different in silico experiments were designed to opportunely explore both the capability of the stochastic model to follows the deterministic predictions, when in fast fluctuations regimen, and to discover complex stochastic patterns, that can arise through the interconnection of the transcriptional and post-transcriptional control layers. In general, both the stochastic and deterministic formulation of the model showed the capability to reproduce the HPV gene expression dynamics, during the entire viral life cycle, in good agreement with the current biological knowledge

Papilloomiviiruse transkriptsiooni ja regulaatorvalgu E2 uurimine

Väitekirja elektrooniline versioon ei sisalda publikatsioonePapilloomiviirused on viiruste perekond, mis nakatab nii inimeste kui loomade epiteelrakke ja põhjustab papilloome ehk näsakasvajaid, mis võivad teatud tingimustes areneda halvaloomulisteks kasvajateks. Inimese papilloomiviiruseid on teada üle 200 tüübi ja nad jagatakse kaheks grupiks nende koespetsiifilisuse alusel: limaskesti nakatavad papilloomiviirused ja naharakke nakatavad papilloomiviirused. Naha papilloomiviirused on siiani pälvinud vähem tähelepanu, sest nende võimalik seos nahakasvajatega on tulnud ilmsiks alles viimasel kümnendil. Meie keskendusime oma töös naha papilloomiviirusele HPV5, mida on viimasel ajal hakatud seostama nahakasvajate tekkega, aga mida on siiani ikkagi suhteliselt vähe uuritud. Meie töö tulemusena valmis HPV5 transkriptsiooni kaart, mille iseloomustamiseks me kasutasime inimese sääreluu kasvaja rakuliini U2OS. Selle rakuliini abil õnnestus meil kirjeldada 14 erinevat viiruse mRNAd. Edasises töös keskendusime viirusvalgu E2 uurimisele, mis on põhiline viiruse elutsükli regulaator. E2 valgul on lisaks täispikale valgule veel kaks isovormi, valgu C-terminaalset osa sisaldav E2C ja alternatiivse splaissinguga saadud E8/E2. Me uurisime E2 paiknemist rakus ja leidsime, et lühike E8 valgu järjestus on piisav selleks, et valk suunata raku tuuma. Meile teadaolevalt on see järjestus lühim tuuma suunav järjestus, mis on siiani teada. Tänu proteoomika arengule on siiani leitud üle 200 E2 partnervalgu, millest enamuse funktsioon viiruse elutsüklis on siiani teadmata. Meie leidsime uue E2-ga seonduva rakulise valgu, milleks on tuumamüosiin 1. See on esimene müosiini perekonda kuuluv E2 partnervalk ja katsed näitasid, et tuumamüosiin 1 mõjutab HPV5 DNA paljunemist rakus.Cutaneous papillomaviruses infect human cutaneous epithelium and in most cases these infections pass without symptoms, but in some instances, they can cause lesions and induce squamous cell carcinomas. One of the most prevalent virus types detected in skin cancer is HPV5. We tried to bring through our work more understanding about the properties of these viruses and focused our attention to HPV5 and to the first part of its life-cycle in the cells. We wanted to characterize HPV5 more thoroughly at RNA level and managed to identify HPV transcription map. We also studied papillomavirus main regulator protein E2, which influences all main viral life events. We studied E2 localization in the cell and identified short nuclear targeting signal, that sends proteins to the nucleus. We also found new E2 interaction partner nuclear myosin 1, which influences HPV5 replication

G-quadruplexes and G-quadruplex ligands: targets and tools in antiviral therapy

G-quadruplexes (G4s) are non-canonical nucleic acids secondary structures that form within guanine-rich strands of regulatory genomic regions. G4s have been extensively described in the human genome, especially in telomeres and oncogene promoters; in recent years the presence of G4s in viruses has attracted increasing interest. Indeed, G4s have been reported in several viruses, including those involved in recent epidemics, such as the Zika and Ebola viruses. Viral G4s are usually located in regulatory regions of the genome and implicated in the control of key viral processes; in some cases, they have been involved also in viral latency. In this context, G4 ligands have been developed and tested both as tools to study the complexity of G4-mediated mechanisms in the viral life cycle, and as therapeutic agents. In general, G4 ligands showed promising antiviral activity, with G4-mediated mechanisms of action both at the genome and transcript level. This review aims to provide an updated close-up of the literature on G4s in viruses. The current state of the art of G4 ligands in antiviral research is also reported, with particular focus on the structural and physicochemical requirements for optimal biological activity. The achievements and the to-dos in the field are discussed

Regulation of Human Papillomavirus Type 16 Late L1 mRNA Splicing

Human papillomaviruses (HPVs) cause almost half of the human cancers that are attributable to viruses. HPV type 16 is the most carcinogenic type among the HPVs and is detected in 50% of all cervical cancers. HPV-16 infects epithelial cells and HPV-16 gene expression is tightly linked to the differentiation stage of the infected cells. Early HPV-16 genes are expressed in basal layers of the epithelium whereas the late genes which encode highly immunogenic viral structural proteins are only expressed in the suprabasal layers. HPV-16 infections are normally cleared within 18-24 months, but HPV-16 can establish persistent infections that progress to cancer. Such HPV-infected cancer cells express early HPV-16 genes but never expressed the late genes. We speculate that inhibition of HPV-16 late gene expression is a prerequisite for viral persistence and progression to cervical cancer. HPV-16 uses alternative splicing to regulate expression of early and late genes. HPV RNA elements and cellular factors control the expression level of viral proteins by regulating alternative splicing. This project was carried out to enhance our understanding of the regulation of HPV-16 gene expression, in particular at the RNA splicing level. The goal of this thesis was to identify viral RNA elements and cellular factors that regulate the processing of HPV-16 early and late mRNA splicing. These studies may also contribute to the identification of diagnostic biomarkers for premalignant infections at risk of progressing to cervical cancer. We identified a splicing silencer that interacts with hnRNP D proteins and hnRNP A2/B1 to suppress HPV-16 late gene expression in mitotic cells, including cervical cancer cells. We also characterized a splicing enhancer that promotes HPV-16 early gene expression, thereby indirectly inhibiting late gene expression. Mutation in this enhancer reduced its binding to the ASF/SF2 splicing factor. This resulted in decreased expression of the viral oncogenes E6 and E7 and a reduced ability of HPV-16 to immortalize human epithelial cells, thereby, linking HPV-16 mRNA splicing regulation to its pathogenic prospects. We also identified the hnRNP G protein binding to this enhancer and has opposite effects to ASF/SF2 on splicing matched by antagonism in RNA binding

EPMA position paper in cancer:current overview and future perspectives

At present, a radical shift in cancer treatment is occurring in terms of predictive, preventive, and personalized medicine (PPPM). Individual patients will participate in more aspects of their healthcare. During the development of PPPM, many rapid, specific, and sensitive new methods for earlier detection of cancer will result in more efficient management of the patient and hence a better quality of life. Coordination of the various activities among different healthcare professionals in primary, secondary, and tertiary care requires well-defined competencies, implementation of training and educational programs, sharing of data, and harmonized guidelines. In this position paper, the current knowledge to understand cancer predisposition and risk factors, the cellular biology of cancer, predictive markers and treatment outcome, the improvement in technologies in screening and diagnosis, and provision of better drug development solutions are discussed in the context of a better implementation of personalized medicine. Recognition of the major risk factors for cancer initiation is the key for preventive strategies (EPMA J. 4(1):6, 2013). Of interest, cancer predisposing syndromes in particular the monogenic subtypes that lead to cancer progression are well defined and one should focus on implementation strategies to identify individuals at risk to allow preventive measures and early screening/diagnosis. Implementation of such measures is disturbed by improper use of the data, with breach of data protection as one of the risks to be heavily controlled. Population screening requires in depth cost-benefit analysis to justify healthcare costs, and the parameters screened should provide information that allow an actionable and deliverable solution, for better healthcare provision

Sensitivity analysis methods for dynamical models

Sensitivity analysis methods are widely applied to study biological systems in order to investigate the effect of parameter change on the solutions of mathematical models. In this work, conventional Parametric Sensitivity Analysis main methods were reviewed along with the novel analysis called Impulse Parametric Sensitivity Analysis presented by Perumal et al. Both analysis were then applied on the HPV early promoter regulation model presented by Giaretta et al. and their results were compare

Genome-wide methylome analysis using MethylCap-seq uncovers 4 hypermethylated markers with high sensitivity for both adeno- and squamous-cell cervical carcinoma

Background: Cytology-based screening methods for cervical adenocarcinoma (ADC) and to a lesser extent squamous-cell carcinoma (SCC) suffer from low sensitivity. DNA hypermethylation analysis in cervical scrapings may improve detection of SCC, but few methylation markers have been described for ADC. We aimed to identify novel methylation markers for the early detection of both ADC and SCC. Results: Genome-wide methylation profiling for 20 normal cervices, 6 ADC and 6 SCC using MethylCap-seq yielded 53 candidate regions hypermethylated in both ADC and SCC. Verification and independent validation of the 15 most significant regions revealed 5 markers with differential methylation between 17 normals and 13 cancers. Quantitative methylation-specific PCR on cervical cancer scrapings resulted in detection rates ranging between 80% and 92% while between 94% and 99% of control scrapings tested negative. Four markers (SLC6A5, SOX1, SOX14 and TBX20) detected ADC and SCC with similar sensitivity. In scrapings from women referred with an abnormal smear (n = 229), CIN3+ sensitivity was between 36% and 71%, while between 71% and 93% of adenocarcinoma in situ (AdCIS) were detected; and CIN0/1 specificity was between 88% and 98%. Compared to hrHPV, the combination SOX1/SOX14 showed a similar CIN3+ sensitivity (80% vs. 75%, respectively, P>0.2), while specificity improved (42% vs. 84%, respectively, P < 10(-5)). Conclusion: SOX1 and SOX14 are methylation biomarkers applicable for screening of all cervical cancer types

Evolutionary variation of papillomavirus E2 protein and E2 binding sites

Background: In an effort to identify the evolutionary changes relevant to E2 function, within and between papillomavirus genera, we evaluated the E2 binding sites (E2BS)s inside the long-control-region (LCR), and throughout the genomes. We identified E2BSs in the six largest genera of papillomaviruses: Alpha, Beta, Gamma, Delta, Lambda, and Xi-papillomaviruses (128 genomes), by comparing the sequences with a model consensus we created from known functional E2BSs (HPV16, HPV18, BPV1). We analyzed the sequence conservation and nucleotide content of the 4-nucleotide spacer within E2BSs. We determined that there is a statistically significant difference in GC content of the four-nucleotide E2BS spacer, between Alpha and Delta-papillomaviruses, as compared to each of the other groups. Additionally, we performed multiple alignments of E2 protein sequences using members of each genus in order to identify evolutionary changes within the E2 protein. Results: When a phylogenetic tree was generated from E2 amino acid sequences, it was discovered that the alpha-papillomavirus genera segregates into two distinct subgroups (α1 and α2). When these subgroups were individually analyzed, it was determined that the subgroup α1 consensus E2BS favored a spacer of AAAA, whereas subgroup α2 favored the opposite orientation of the same spacer; TTTT. This observation suggests that these conserved inverted linkers could have functional importance

Cancer as a defective network for NF-κB

In a recent review we addressed the role of the transcription factor NF-κB, in shaping the cancer microenvironment. NF-κB, which interacts with chromatin modulators by cell-specific dynamics, controls cell interactions during inflammation, and its abnormal feedback regulation is implicated in cancer. Inflammation normally reprograms cells through changes in key topological elements of chromosomal DNA. As a result, inflammation overrides cell phenotype: initially, reprogramming cell function halts processes that impede the response of a damaged tissue to the cause of the harm, and eventually, late reprogramming of cells will replenish tissue structure and restore function. Each cell type provides a distinct resource for restoration of tissue integrity, tissue function, and for replenishment of the responsiveness of the immune system. Modulators of NF-κB transcriptional activity alter key aspects of gene expression and tissue integrity. NF-κB network alterations confer transcriptional plasticity to cancer

Papilloomiviiruse E2 valkude rolli uurimine viiruse DNA replikatsioonil

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Papilloomiviirused on selgroogsete seas laialt levinud väikesed, koespetsiifilised DNA viirused, mis nakatavad naha või limaskesta epiteelkoes asuvaid keratinotsüüte. Inimese papilloomiviiruse (HPV) suurim meditsiiniline tähtsus seisneb tema võimes põhjustada mitmeid pahaloomulisi kasvajaid, sealhulgas emakakaelavähki, mis on naiste seas esinemissageduselt teisel kohal rinnavähi järel ning kõrge suremusega. HPV-d jagunevad kõrge ja madala vähitekke riskiga viirusteks. Madala riskiga viirused (HPV11, HPV6 jt) põhjustavad healoomulisi vohandeid, näiteks soolatüükaid ja kondüloome, mis tervetel inimestel kaovad immuunsüsteemi toimel aasta või paari jooksul. Pahaloomulised kasvajad võivad aja jooksul areneda rakkudest, mis on nakatunud kõrge riski HPV-dega (HPV16, HPV18 jt). Praeguseks saadaolevad HPV-vastased vaktsiinid on küll efektiivsed uue nakkuse ärahoidmisel, kuid ei paku paraku kaitset kõigi kõrge riskiga HPV-de osas, samuti on nad kulukad ning halvasti kättesaadavad arengumaades, kus leiab aset 80% emakakaelavähist tingitud surmadest. Kuigi maailmas on miljoneid HPV-ga nakatunud inimesi, ei ole tänaseks veel välja töötatud spetsiifilist viirusvastast ravi. Seetõttu on HPV-alane uurimistöö endiselt päevakorral, et paremini mõista viiruse onkogeensust ja leida uusi viise nakkuse ärahoidmiseks ning olemasolevate ravivõtete täiendamiseks. Papilloomiviiruse E2 valk osaleb viiruse DNA replikatsiooni algatamisel koos viiruse peamise replikatsioonivalguga E1. Lisaks reguleerib E2 valk viiruse varajaste geenide avaldumist ning mängib olulist rolli viiruse genoomi episomaalsel säilimisel kinnitades rakkude jagunemise käigus viiruse genoomid raku kromosoomide külge. Samas ekspresseerib papilloomiviirus lisaks täispikale E2 valgule ka sama valgu lühikesi vorme, mille ülesandeks on reguleerida täispika E2 valgu funktsioone. Eelnevalt on meie grupp näidanud, et täispikast ja transaktivatsiooni domeeni mitteomavast lühikesest E2 valgust moodustunud E2 heterodimeerid on võimelised toetama viiruse varajaste geenide transkriptsiooni ja DNA replikatsiooni. Antud töö esimeses publikatsioonis uurisin detailsemalt E2 heterodimeeri paiknemist rakus ning selle rolli viiruse DNA replikatsioonil veise papilloomiviiruse tüüp 1 (BPV1) E2 näitel. Leidsin, et BPV1 E2 heterodimeer paikneb sarnaselt täispika E2 homodimeerile rakulise kromatiini fraktsioonis, mis on kooskõlas E2 heterodimeeri võimega toetada viiruse geenide transkriptsiooni ja DNA replikatsiooni. Lisaks näitasin, et BPV1 E2 heterodimeer on võimeline siduma viiruse E1 valgu replikatsiooni alguspunktile viiruse genoomis ning toetama viiruse DNA replikatsiooni initsiatsiooni. Samas ei suutnud E2 heterodimeer säilitada viiruse genoomi jagunevates rakkudes pikema aja jooksul. Käesoleva tööga seotud järgnevas publikatsioonis uurisin HPV18 ja HPV11 E2 heterodimeeride osalust viiruse DNA replikatsioonil ning leidsin, et sarnaselt BPV1 E2 heterodimeerile toetab ka HPV E2 heterodimeer viiruse DNA replikatsiooni initsiatsiooni, kuid pole võimeline tagama viiruse genoomi pikaajalist säilimist rakus. Seega on E2 heterodimeeri võime toetada viiruse genoomi replikatsiooni initsiatsiooni konserveerunud erinevate papilloomiviiruste seas ning täispika E2 homodimeeri ja E2 heterodimeeri vaheline dünaamika on oluline reguleerimaks viiruse DNA paljundamist ja säilumist viirusega nakatunud rakkudes. HPV E2 heterodimeer oli võimeline ka maha suruma viiruse varajaste geenide avaldumist samas ulatuses kui täispika E2 homodimeer. Lisaks leidsin, et HPV18 on võimeline kodeerima transaktivatsiooni domeeni mitteomavat E8^E2 valku, mille üheks rolliks on inhibeerida HPV18 genoomi replikatsiooni, käitudes seega sarnaselt teiste papilloomiviiruste seas tuvastatud E8^E2 valkudega. Käesoleva töö kolmandaks tahuks on papilloomiviiruse DNA replikatsiooni toimumiskoha uurimine rakus. Leidsin, et HPV replikatsioonikeskuste paiknemine raku tuumas kattub osaliselt tuumsete struktuuriüksuste ND10 asetusega. Nimelt paiknesid HPV18 ja HPV11 replikatsioonivalgud E1 ja E2 kas osaliselt koos või kõrvuti ND10 struktuursete valkudega PML ja DAXX, viidates sellele, et sarnaselt teistele raku tuumas replitseeruvatele DNA viirustele (herpesviirused, adenoviirused jt) algatab ka HPV oma replikatsioonitsükli ND10 läheduses. Kuigi ND10 mängib olulist rolli rakku sisenevate viiruste replikatsiooni inhibeerimisel, on mitmed viirused leidnud viise ND10 inhibeerivast toimest vabanemiseks, sealhulgas ND10 struktuuri lõhkumise või ND10 struktuursete valkude lagundamise läbi. Üks ND10 valkudest, mis inhibeerib mitmete ND10 läheduses paljunevate viiruste replikatsiooni, on DAXX valk. Oma töös leidsin, et DAXX valk moduleerib ka HPV18 ja HPV11 varajaste geenide avaldumist ja DNA replikatsiooni. Üllatuslikult ei omanud aga DAXX valk negatiivset toimet, vaid mõjutas positiivselt HPV geeniekspressiooni ja genoomi lühiajalist replikatsiooni uuritavates rakkudes. Kokkuvõtvalt uurisin töö käigus papilloomiviiruse peamise regulaatorvalgu E2 rolli viiruse DNA replikatsioonil ning viiruse replikatsiooni toimumiskohta rakus ja sellega seotud ühe rakulise faktori, DAXX valgu, mõju viiruse replikatsioonile.Papillomaviruses are small double-stranded DNA viruses that infect the epithelial tissue of a wide variety of vertebrates. The viruses are tissue-specific and infect the keratinocytes either in the cutaneous or internal mucosal epithelium. Human papillomaviruses (HPVs) are highly prevalent pathogens of tremendous medical importance due to their association with several human cancers, including cervical cancer which is the second most common cancer in women worldwide and has a high mortality rate. HPVs are divided into low-risk and high-risk categories based on the spectrum of lesions they are associated with and the potential of these lesions to progress to cancer. Low risk HPVs (i.e. HPV11, HPV6) cause benign epithelial hyper-proliferative conditions (i.e. warts, condylomas) which in healthy individuals are frequently cleared by the immune system in less than a year or two. Persistent infections by high-risk HPVs (i.e. HPV16, HPV18) can at low frequency progress to high grade dysplasias and carcinomas. Available prophylactic HPV vaccines are effective against new infections, but have several limitations, including incomplete coverage of all high-risk HPVs, high cost and limited availability in developing countries, which account for 80% of the deaths due to cervical cancer. In addition, they cannot cure the millions of people that are already infected. There is also no virus-specific treatment currently available. Thus, there is still a need for ongoing biomedical research into HPVs and their associated diseases to lead to the development of better strategies for prevention and disease treatment, which are necessary to complement current methods of disease management. The papillomavirus life cycle is regulated by a family of proteins encoded by the E2 gene. The full-length E2 protein participates in the initiation of viral DNA replication by loading viral helicase E1 onto the viral origin of replication and in viral genome episomal maintenance by tethering viral genomes to host mitotic chromosomes during cell division. E2 also regulates transcription of viral early promoters. In addition to the full-length E2 protein, all papillomaviruses encode shorter forms of E2 which regulate the functions of the full-length E2 protein. Previously, our group has shown that E2 heterodimer formed between the full-length and truncated E2 proteins functioned as an effective transcriptional activator and was able to support papillomavirus DNA replication. In the first publication of this thesis I investigated in detail the cellular localization and the replication function of the E2 heterodimer by using the bovine papillomavirus type 1 (BPV1) E2 protein as a model. I determined that BPV1 E2 heterodimer with single transactivation domain localized similarly to the full-length E2 homodimer into cellular chromatin which is in agreement with the results that the E2 heterodimer is able to support papillomavirus DNA replication and transcription. I found that BPV1 E2 heterodimer is able to load the viral helicase E1 to the origin of replication in order to initiate viral DNA replication. Although the E2 heterodimer was able to initiate papillomavirus DNA replication, it could not maintain it for a long time. In the second publication I studied the replicative activity of HPV18 and HPV11 E2 heterodimers and found that HPV E2 heterodimer supports the initiation of viral DNA replication similarly to the BPV1 E2 heterodimer, but is not able to ensure the stable maintenance of viral genomes in dividing cells. Thus, the replication initiation function of E2 heterodimers is conserved between different types of papillomaviruses. The dynamic change in the balance between the full-length E2 homodimers and E2 heterodimers are important for the regulation of virus DNA replication and viral genome maintenance in virus-infected cells. The HPV E2 heterodimer was also able to repress expression of viral early genes to the same extent as the full-length E2 homodimer. In addition, I determined that HPV18 has the capacity to encode a truncated E2 protein, E8^E2, which serves as a negative regulator of HPV18 genome replication similarly to E8^E2 proteins of other papillomaviruses identified so far. In the third part of the thesis I studied the cellular site of papillomavirus DNA replication. I found that HPV replication compartments partially co-localize with subnuclear structures ND10 in the cell nucleus. Specifically, HPV18 and HPV11 replication proteins E2 and E1 partially co-localized or were located adjacent to ND10 structural proteins PML and DAXX. This suggested that similarly to other nuclear replicating DNA viruses, HPV also begins its replicative cycle near the ND10. The ND10 has been implicated in cellular response dedicated to counter invasion of viruses by inhibiting their replication. However, several viruses encode proteins that cause major changes in ND10 by a variety of mechanisms that either affect ND10 structural integrity or involve degradation of restrictive ND10 proteins. One of the ND10 proteins, that inhibit the replication of several viruses at ND10, is the DAXX protein. In this study, I found that the DAXX protein also modulates early gene expression and transient DNA replication of HPV18 and HPV11. Surprisingly, however, DAXX did not play a restrictive role but appeared to have a positive role in the regulation of HPV transcription and replication in our model system. In summary, I studied the role of the master regulator E2 protein in papillomavirus DNA replication, the cellular site where virus DNA replication takes place and the effect of one of the proteins associated with this site, the DAXX protein, on virus replication