Regulation of viral and host genomes by high risk human papillomavirus E2 protein in association with cellular factors

Human papillomaviruses (HPVs) are the causative agent in cervical cancer and have been implicated in a rising number of head and neck cancers in both men and women. There are two licensed prophylactic vaccines, both target HPV 16 and 18, the two most common, oncogenic types. However, there are no antiviral drugs for the treatment of HPV infection and disease. Papillomaviruses encode two DNA binding proteins, E1 and E2, which interact with host cell proteins to induce replication. Two essential cellular factors for viral transcription and replication are TopBP1 and Brd4. TopBP1 is a known interactor of HPV16 E2, and is essential for the initiation of DNA replication in eukaryotic cells. Previous studies with E2 mutants have shown that Brd4 is essential for transactivation properties of E2. Brd4 has also been implicated in the regulation of E1-E2 mediated viral DNA replication. However, the role of E2 in regulating the host genome has been less well studied. As attempts at developing a therapeutic vaccine have failed, and current small molecule inhibitors which block the interaction of replication factors, E1 and E2, are not effective across all HPV types due to slight differences in E1-E2 interactions, Brd4 and TopBP1 may present themselves as pan-type specific targets. Blocking the interaction of the host proteins Brd4 and TopBP1 with viral E2 is a viable target for HPV related cancers. This thesis set out to understand how E2 interacts with TopBP1 and Brd4 to regulate the cellular genome as well as the viral genome, to better understand how to terminate the viral life cycle. Two E2 mutants, E2-TopBP1 and E2-Brd4, which fail to bind TopBP1 and Brd4 respectively, were made to address this question. Functional assays with E2 wildtype and the two E2 mutants have enhanced our understanding of viral replication. TopBP1 and Brd4 are present at the viral origin of replication in an E1-E2 dependent manner, and optimal initiation of DNA replication is dependent on the interaction with E2. TopBP1 and Brd4 locate into E1-E2 foci and shRNA targeting these proteins destroys these replication foci. However, this has no effect on E1-E2 mediated levels of DNA replication. The results suggest a role for TopBP1 and Brd4 in the initiation of HPV16 E1-E2 DNA replication but not continuing replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. To address the question of how E2 regulates the host, we stably expressed HPV16 and 18E2 wildtype, E2-TopBP1 and E2-Brd4, in U2OS cells, a p53 positive cell line that tolerates E2 expression. These cells were used in human exon array analysis, to determine which host cellular genes E2 regulates. We determined that HPV16 and 18E2 can regulate cellular genes and a failure to bind either TopBP1 or Brd4 increases the number of cellular genes altered. Overall the results suggest that the levels of TopBP1 and Brd4, which can interact, regulate E2 function and therefore could regulate viral infection

Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation

Human papillomaviruses recruit a host of DNA damage response factors to their viral genome to facilitate homologous recombination replication in association with the viral replication factors E1 and E2. We previously demonstrated that SIRT1 deacetylation of WRN promotes recruitment of WRN to E1-E2 replicating DNA and that WRN regulates both the levels and fidelity of E1-E2 replication. The deacetylation of WRN by SIRT1 results in an active protein able to complex with replicating DNA, but a protein that is less stable. Here, we demonstrate an inverse correlation between SIRT1 and WRN in CIN cervical lesions compared to normal control tissue, supporting our model of SIRT1 deacetylation destabilizing WRN protein. We CRISPR/Cas9 edited N/Tert-1 and N/Tert-1+HPV16 cells to knock out WRN protein expression and subjected the cells to organotypic raft cultures. In N/Tert-1 cells without WRN expression, there was enhanced basal cell proliferation, DNA damage, and thickening of the differentiated epithelium. In N/Tert-1+HPV16 cells, there was enhanced basal cell proliferation, increased DNA damage throughout the epithelium, and increased viral DNA replication. Overall, the results demonstrate that the expression of WRN is required to control the proliferation of N/Tert-1 cells and controls the HPV16 life cycle in these cells. This complements our previous data demonstrating that WRN controls the levels and fidelity of HPV16 E1-E2 DNA replication. The results describe a new role for WRN, a tumor suppressor, in controlling keratinocyte differentiation and the HPV16 life cycle

RNASeq analysis of differentiated keratinocytes reveals a massive response to late events during human papillomavirus type 16 infection, including loss of epithelial barrier function.

The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalised, keratinocytes (NIKS), and NIKS stably transfected with HPV16 episomal genomes (NIKS16), were compared using RNASeq. HPV16 infection altered expression of 2862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNASeq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (>log2 = 1.8, 3.5-fold change) 670 genes were downregulated and 296 genes were up-regulated. HPV down-regulated many genes involved in epithelial barrier function that involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant, CCL20, and proinflammatory cytokines, IL1A and IL1B. However, IRF1, IFNκ and viral restriction factors (IFIT1, 2, 3, 5, OASL, CD74, RTP4) were up-regulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions and cell adhesion. qPCR and western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress.IMPORTANCE Human papillomavirus (HPV) genome amplification and capsid formation takes place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNASeq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3000 genes were differentially expressed in keratinocyte due to HPV16 infection. Strikingly, the epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted. These data provide new insights into virus-host interaction crucial for production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle

Novel properties of the phytocannabinoids and their receptors

I have investigated the pharmacological properties of cannabichromene (CBC), cannabigerol (CBG) and Δ8-tetrahydrocannabivarin (Δ8-THCV).  A concentrations in the micromolar range, CBG binds to the cannabinoid CB1 and CB2 receptors and acts as a CB1 receptor antagonist.  In the functional [35S]GTPγS binding assay performed with mouse brain membranes, CBG also behaves as a potent partial agonist at concentrations less than 100 nM. CBG is an α2-adrenoceptor agonist, and behaves as a competitive 5-HT1A antagonist at concentrations ranging from 300 nM to 10 μM. Further experiments were conducted to explore interactions between established 5-HT1A ligands and the CB1 receptor.  In mouse brain membranes, although not in hCB1 transfected cells, the 5-HT1A receptor agonist, 8-OH-DPAT, has the ability both to displace [3H]CP55940 from specific binding sites and to modulate the rate of [3H]CP55940 dissociation from these sites.  These results may reflect the presence of CB1-5-HT1A dimers in the brain. Δ9-THCV has been reported to be a potent CB2 receptor antagonist with a KB value lower than its binding affinity for CB2.  Experiments were performed in the cAMP assay to establish if Δ8-THCV did in fact target the CB2 receptor.  Antagonism of Δ8-THCV was attempted by using a selection of compounds but unfortunately these either behaved as inverse agonists in the assay or as partial agonists.  Δ8-THCV induced agonism was Pertussis toxin sensitive.  In untransfected CHO cells, Δ8-THCV did not stimulate or inhibit forskolin induced stimulation of cAMP, hence the effects of Δ8-THCV are most likely CB2 dependent. As CBG was found to behave as a potent α2-adrenoceptor agonist and 5-HT1A receptor antagonist, it may be useful clinically, for example for the treatment of depression.  Interactions between the 5-HT1A and CB1 receptors need to be explored more fully, not least because it might be possible to exploit such interactions in the clinic.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Failure to interact with Brd4 alters the ability of HPV16 E2 to regulate host genome expression and cellular movement

The E2 protein of the carcinogen human papillomavirus 16 (HPV16) regulates replication and transcription of the viral genome in association with viral and cellular proteins. Our previous work demonstrated that E2 can regulate transcription from the host genome. E2 can activate transcription from adjacent promoters when located upstream using E2 DNA binding sequences and this activation is dependent upon the cellular protein Brd4; this report demonstrates that a Brd4 binding E2 mutant alters host genome expression differently from wild type E2. Of particular note is that highly down regulated genes are mostly not affected by failure to interact with Brd4 suggesting that the E2-Brd4 interaction is more responsible for the transcriptional activation of host genes rather than repression. Therefore failure to interact efficiently with Brd4, or altered levels of Brd4, would alter the ability of E2 to regulate the host genome and could contribute to determining the outcome of infection

Robotereinsatz in Intergasatmosphaere bis 110 bar

SIGLECopy held by FIZ Karlsruhe; available from UB/TIB Hannover / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

Regulation of human genome expression and RNA splicing by human papillomavirus 16 E2 protein

Human papillomavirus 16 (HPV16) is causative in human cancer. The E2 protein regulates transcription from and replication of the viral genome; the role of E2 in regulating the host genome has been less well studied. We have expressed HPV16 E2 (E2) stably in U2OS cells; these cells tolerate E2 expression well and gene expression analysis identified 74 genes showing differential expression specific to E2. Analysis of published gene expression data sets during cervical cancer progression identified 20 of the genes as being altered in a similar direction as the E2 specific genes. In addition, E2 altered the splicing of many genes implicated in cancer and cell motility. The E2 expressing cells showed no alteration in cell growth but were altered in cell motility, consistent with the E2 induced altered splicing predicted to affect this cellular function. The results present a model system for investigating E2 regulation of the host genome