Only persistent HPV infections lead to the development of cancer. Thus, understanding the virus-host interplay that influences the establishment of viral infection has important implications for HPV biology and human cancers. The ability of papillomaviruses to establish in cells requires the strict temporal regulation of viral gene expression in sync with cellular differentiation. This control primarily happens at the level of RNA splicing and polyadenylation. However, the details of how this spatio-temporal regulation is achieved still need to be fully understood. Until recently, it has been challenging to study the early events of the HPV lifecycle following infection. We used a single-cell genomics approach to identify cellular factors involved in viral infection and establishment. We identify protein arginine N-methyltransferase 1 (PRMT1) as an important factor in viral infection of primary human cervical cells. PRMT1 is the main cellular enzyme responsible for asymmetric dimethylation of cellular proteins. PRMT1 is an enzyme responsible for catalyzing the methylation of arginine residues on various proteins, which influences processes such as RNA processing, transcriptional regulation, and signal transduction. In this study, we show that HPV18 infection leads to increased PRMT1 levels across the viral lifecycle. PRMT1 is critical for the establishment of a persistent infection in primary cells. Mechanistically, PRMT1 inhibition leads to a highly dysregulated viral splicing pattern. Specifically, reduced PRMT1 activity leads to intron retention and a change in the E6 and E7 expression ratio. In the absence of PRMT1, viral transcripts are destabilized and subject to degradation via the nonsense-mediated decay (NMD) pathway. These findings highlight PRMT1 as a critical regulator of the HPV18 lifecycle, particularly in RNA processing, and position it as a potential therapeutic target for persistent HPV18 infections.

The aim of this study was to amplify the ancient DNA from a collection of 63 Romano-British neonatal skeletons, in order to determine their sex. The DNA testing strategy has been designed to overcome the issues associated with ancient DNA investigations, including degraded template DNA, inhibition of PCR, and modern contaminants. Ancient DNA has been extracted using a silica based method using the GENECLEAN Kit for Ancient DNA. Three different PCR-Cleanup kits were investigated to determine their effectiveness in the removal of PCR inhibitors. The PowerClean kit (MoBio Laboratories) proved the most effective, and was used throughout. Two different sets of primers were used to provide amplicons from the Amelogenin gene. The Amel-A and Amel-C primers used by Arnay-de-la-Rosa (2007) proved to be ineffective, with multiple problems with non-specific binding and lack of amplification of the target region. However, the amelogenin primers from the AmpFISTR Identifiler kit proved more effective and were chosen for use in testing the ancient material. Modern DNA is used through the study to compare to the ancient material, in order to allow for pre-testing of the techniques and optimisation of the PCR without the loss of precious material. Modern DNA was also artificially degraded in order to determine whether the PCR technique can amplify fractured DNA. In this experimentation, the Identifiler primer PCR successfully amplified modern DNA that had been degraded for 15 minutes in an ultrasonic bath, to a length of approximately 100-500bp in size. Measurements of the skeletons have also been taken in order to determine the exact age of the neonates at time of death, to identify potential patterns in the deaths of the individuals, and to identify a potential link between the age of the individual at time of death and the probability of survival of the DNA within their remains. Of the remains, 72% of the individuals were of an age of at least 38 prenatal weeks, indicating a full term child considered as an infant death. 8% of individuals provided an age range above 24 weeks, and were potentially stillbirths rather than infant deaths, while 20% of the remains showed sufficient damage to prevent complete age ranges that utilise both the length and width of the skeletal material from being calculated and therefore may have been miscarriages of younger foetuses or the fragmented remains of older infant remains. Following the DNA analysis of the remains, three of the individuals provided amplicons of the target region and therefore their sex could be determined. All three of these individuals provided an XY male genotype. All other remains tested gave negative results. Many of the results showed severe problems with PCR inhibition even after the use of the PowerClean PCR Cleanup kit. Therefore, the conclusions that can be drawn are limited. The potential for contamination to be the cause of these three results is discussed, but the low sample size of positive results makes it difficult to determine the source of the amplified DNA. Further work is suggested, including more rigorous inhibitor removal methods and qPCR to provide a larger sample size of positive neonatal DNA results, allowing for the validity of the results to be assessed more thoroughly. Inhibition and DNA degradation have proved to be the largest challenges faced in this research, which is consistent with other research that utilises ancient DNA. Further analysis of soil samples from the excavation and of the remains themselves is suggested, to determine the type and quantity of inhibitory substances present. Biochemical assessment to determine the level of preservation of the microstructure of the bone, and the use of cloning and sequencing to identify DNA damage and rule out contaminants is suggested.

................................................................................................................................... i ACKNOWLEDGEMENTS ......................................................................................................... ii DEDICATION.............................................................................................................................. iv TABLE OF CONTENTS ............................................................................................................. v LIST OF TABLES ....................................................................................................................... ix LIST OF FIGURES ...................................................................................................................... x CHAPTER 1 – INTRODUCTION .............................................................................................. 1 1.1 – Literature Review ............................................................................................................. 1 1.2 – Research Rationale, Question, Hypothesis, and Objectives ......................................... 4 1.3 – Original Scientific Contributions .................................................................................... 6 CHAPTER 2 – VIRAL-HOST INTEGRATION DUE TO SUB-LINEAGE .......................... 8 2.1 – Abstract ............................................................................................................................. 9 2.1.1 – Background .................................................................................................................. 9 2.1.2 – Results .......................................................................................................................... 9 2.1.3 – Conclusions ................................................................................................................. 9 2.2 – Background ..................................................................................................................... 10 2.3 – Results and Discussion ................................................................................................... 13 2.3.1 – Viral integration in the HPV16 AAE6 but not EPE6 epithelium ............................... 13 2.3.2 – The HPV16 AAE6 epithelium has a unique transcriptional profile .......................... 16 2.3.3 – Nature of viral-human fusion transcripts detected in HPV16 AAE6 epithelium ....... 20 2.3.4 – The HPV16 AAE6 epithelium reveals a signature of chromosomal instability conducive to host genome integration ................................................................................... 23 2.3.5 – HPV16 AAE6 epithelium exhibits a proliferating phenotype as a consequence of viral integration into the host genome ........................................................................................... 25 2.4 – Conclusions ..................................................................................................................... 31 2.5 – Methods ........................................................................................................................... 32 2.5.1 – Cell lines .................................................................................................................... 32 2.5.2 – Detection of integrated papillomavirus sequences by DNA-Seq: Capt-HPV............ 32 2.5.3 – RNA-Seq library preparation and sequencing .......................................................... 32 2.5.4 – Viral variant read alignment, mapping, and coverage plotting ................................ 33