Theses - Pathology
Permanent URI for this collection
Browse
Recent Submissions
Item Open Access A Deep Learning Approach to Automated Coeliac Disease DiagnosisSchreiber, BenjaminCoeliac disease (CD) is a small intestinal autoimmune disorder triggered by the consumption of gluten. Manifestations are variable and non-specific, ranging from no symptoms, through fatigue, diarrhoea, and vomiting, and, in rare cases, lymphoma and duodenal cancer. The treatment consists of a lifelong gluten-free diet, making early and accurate diagnoses of critical importance. The current gold-standard for CD diagnosis is through the procurement and assessment of a hematoxylin and eosin (H&E)-stained duodenal biopsy. However, this is an unavoidably subjective process with low interobserver concordance, resulting in as many as 8/9 affected individuals remaining undetected. There is a clear unmet need for a new gold-standard in CD diagnosis. A potential method for developing such a diagnostic tool is through digital image processing. Modern machine learning techniques, specifically deep learning, have found success in reliably automating complex image processing tasks. Additionally, biopsies can now be scanned digitally as whole-slide images (WSI), allowing biopsy analysis to be automated using digital image processing. In this thesis, I address the following question: “Can modern machine learning techniques provide a sensitive, objective and reproducible test for CD by learning from WSIs of H&E-stained duodenal biopsies?” Firstly, I describe in detail how pathologists diagnose CD, how biopsies are represented digitally, and the advantages and challenges of using digital image processing to automate CD diagnosis. Secondly, I assess the use of traditional image processing techniques for providing an automated CD diagnosis tool. I then develop and analyse tools required for a deep learning approach. I build an image processing tool that can segment the tissue in WSIs from background and artefacts, and I compare different data processing techniques to improve the generalizability of a deep learning based CD diagnosis tool. Finally, I use the tissue segmentation tool and data processing techniques discussed previously to develop a deep learning based CD diagnosis tool which focuses on accuracy, generalizability and interpretability. This thesis aims to assess whether modern machine learning can be used as a much-needed new gold-standard in CD diagnostics.Item Embargo RNA regulatory elements and translational controlLastovka, FilipRegulation of RNA translation is crucial for optimal allocation of resources and co-ordinated production of cellular machinery, which allows maintenance of physiological functions, response to environmental stimuli, and control of virulence to establish an infection. In this work, the translational landscape of *Salmonella* Typhimurium and *Listeria monocytogenes* was analysed using a ribosome profiling method adapted for examination of bacteria which yielded single-nucleotide-resolution data. The ribosome profiles in combination with parallel RNA-Seq data were used to examine genetic elements that control translation, and were found to differ between *Salmonella* and *Listeria*. In *Listeria*, we have found that a subset of efficiently translated genes had ribosomal footprints seven nucleotides upstream of the initiation position, which indicates the existence of a novel translation initiation mechanism. Moreover, the high resolution of the data allowed us to identify novel open reading frames. The bacterial ribosome profiling method was then used to examine cultured *Salmonella* as it transitions from the exponential phase towards the stationary phase, and investigated the response of *Salmonella* at four time points after application of acid stress. The project characterized changes in the expression of genes encoding complexes involved in virulence, and response to low pH. Furthermore, a ribosome profile of the *Cowpea mosaic virus*, a model plant RNA virus, was obtained during an infection of *Nicotiana benthamiana*. The results show that ribosomes pause at four sites during viral polyprotein synthesis. They have confirmed the existence of a putative upstream open reading frame and indicated the possible existence of two additional uORFs. And finally, the work focussed on a plant RNA ThermoSwitch. I established a reporter-based experiment used to test the effect of ThermoSwitch sequences in a transient expression system in *N. benthamiana*. The results mark a step towards the development of a plant thermosensitive regulatory system with potential application in molecular farming. The project has described a number of translational features and genetic elements involved in translational regulation, contributing towards our understanding of the physiology and virulence of the examined pathogens, and provides basis for experiments characterizing these features further.Item Open Access Application of polyhedrin protein microparticles for in vivo vaccine and biologics delivery: a proof-of-concept studyYakovleva, GannaDespite significant advancements in preventative and therapeutic strategies against viral infections, further improvements in global management of diseases require ongoing innovations to address long standing as well as newly emerging problems. For example, inherent vaccine instability results in strict cold-chain maintenance guidelines to preserve vaccine efficacy, which in turn increases costs. Consequently, underserved healthcare systems are often unable to afford vaccination programmes. In the case of biologic based antivirals, chemical modifications such as the conjugation of polyethylene glycol (PEG) and its derivates, are commonly used to increase drug potency. However, recent studies have reported an increase in anti-PEG immunity within the general population due to the widespread use of PEG not only in medicine but also cosmetics and foods, resulting in in the termination of several clinical trials due to adverse effects linked to high anti-PEG antibody titres. In this study, we assess the utilisation of polyhedrin protein based nanoparticles, for *in vivo* delivery of vaccines and biologics. The polyhedrin crystal phenomenon is naturally found within the life-cycle of insect viruses, whereby the encapsulation of mature viral particles by polyhedrin crystals enables the insect viruses to survive degradation in harsh environments prior to infection with a new host. Using protein tags derived from a polyhedrin expressing insect virus, we develop novel polyhedrin based Norovirus and Zika virus nanoparticle vaccines, as well as IFN-λ nanoparticles. We highlight the potential to utilise this technology in medicine. Firstly, we demonstrate the possibility to induce high titres of neutralising Norovirus antibodies. Secondly, we confirm thermostable properties of polyhedrin based nanoparticles in retaining immunogenic properties in Zika vaccine immunisation in mice. Finally, we characterise the retainment of antiviral properties in tagged IFN-λ in vitro, prior to their encapsulation. We also outline major limitations and issues faced due to the relative immaturity of this novel technology, and discuss important considerations for future improvements and adaptations.Item Embargo The Interplay of RSV and IFITs: A comprehensive analysis of human and bovine Interferon-Induced Proteins with Tetratricopeptide Repeats induction and interaction with human and bovine Respiratory Syncytial VirusVarga, MichalThe interferon-induced proteins with tetratricopeptide repeats (IFITs) are early-response antiviral proteins conserved across vertebrates. Following stimulation of cells, they are amongst the most highly induced genes, although the patterns of induction kinetics of individual IFIT proteins are species-, cell type-, tissue-, virus-, and inducer-specific. Most mammals, including Homo sapiens and Bos taurus, express four IFITs: IFIT1, IFIT2, IFIT3, and IFIT5. These proteins exert their antiviral action through mechanisms well described in human systems, involving the potentiation of innate immune signalling cascades (IFIT1, IFIT3, and IFIT5), promotion of apoptosis (IFIT2), inhibition of cell cycle progression (IFIT3), and detection of non-self single-stranded RNA (IFIT1 and IFIT5). Human IFITs were shown to be the restrictors of several RNA viruses such as parainfluenza virus 3 and influenza A virus. Most importantly, IFITs, particularly IFIT1, IFIT2, and IFIT3, globally exhibit antiviral properties against respiratory syncytial virus (RSV), evidenced by decreased viral mRNA production upon ectopic expression and an opposing effect upon IFIT gene silencing. Human and bovine RSV stand as the predominant causes of lower respiratory tract infections, posing significant health risks to young calves, children under five, the elderly, and immunocompromised individuals. RSV, an enveloped, single-stranded negative-sense RNA virus, is host-restricted *in vivo*. In infected cells, RSV forms membrane-less perinuclear cytoplasmic inclusion bodies (IBs), recognised as sites for viral RNA transcription and replication. These IBs also manipulate cellular components, either repurposing them for viral benefit (e.g., components of the eIF4F complex) or inhibiting their function (e.g., MAVS and MDA5). Our study aimed to determine if the IFITs are induced during RSV infection and thus provide support that their reported ectopic inhibition of RSV is relevant *in vivo*. Additionally, we sought to unravel the nature of this inhibition, specifically by examining IFIT interaction with RSV IBs. Lastly, we aimed to understand if this induction and subsequent inhibition are consistent between species by assessing bovine IFIT interaction with bovine RSV, as this a poorly characterised research area. Our observations revealed the induction of human IFITs by both human and bovine RSV infections. Furthermore, we established that the IFIT induction by human RSV is dependent on viral replication and functional interferon signalling. Lastly, minimal induction of bovine IFITs was observed following infection with either bovine or human RSV. Subsequently, our focus shifted to elucidating the mechanism by which RSV fitness is reduced by IFIT proteins. We assayed the interaction phenotypes of human and bovine IFITs during human and bovine RSV infection, revealing a phenotypically diverse set of interactions for each IFIT. Importantly, these interactions were consistent between species, ranging from intra-IB inclusion formation, colocalisation with the IB boundary, diffusion throughout the cytoplasm and IB structure, to exclusion from these structures. Further analysis using pseudo-inclusion bodies (pIBs), IB-like structures that spontaneously emerge after the expression of RSV nucleoprotein and phosphoprotein, and overexpressed IFIT proteins during RSV infection showed consistent interaction of IFIT1, IFIT2, and IFIT5 with both pIBs and IBs. This suggests that the anti-RSV action of IFITs is mediated via interactions with these structures, potentially hindering viral RNA transcription and replication.Item Embargo Understanding the function and structure of herpes simplex virus type 1 pUL55, a novel immune evasion proteinBoparan, NavneetHerpes simplex virus-1 (HSV-1) infects approximately 67% of the human population, causing disease ranging from asymptomatic to severe. The virus affects individuals around the world, and results in lifelong infection. This work focusses on pUL55, a HSV-1 tegument protein, which we hypothesised to be an innate immune modulator with similar functions to ICP0. ICP0 exhibits E3 ubiquitin ligase activity and is known to stimulate proteasomal degradation of several host proteins in HSV-1 infected cells. Multiple studies to date have used the *dl*1403 mutant strain of HSV-1, engineered to lack ICP0, which has demonstrated the importance of ICP0 activity in the establishment of viral infection in multiple model systems. However, a recently generated mutant strain of HSV-1 lacking ICP0 did not recapitulate many of the published characteristics of the well-established *dl*1403 strain. Comparative proteomic analysis had uncovered that the *dl*1403 mutant lacked expression of pUL55 in addition to ICP0. Accordingly, it was hypothesised that the additional loss of pUL55 may be required for some of the observed phenotypes in cells infected with the *dl*1403 mutant. The aim of this research was to identify whether pUL55 has a similar function to ICP0 and investigate the mechanism by which pUL55 can modulate the host proteome. Recombinant viruses lacking pUL55 or lacking both pUL55 and ICP0 were generated and examined for their ability to form plaques on monolayers of cells known to restrict *dl*1403 as well as their ability to degrade specific host proteins that are known to be ICP0 targets. These data demonstrated that viruses lacking either pUL55 or ICP0 individually are able to form plaques effectively on restrictive cells and degrade both PML and IFI16, known targets of ICP0-mediated degradation. Conversely, the newly generated virus lacking both pUL55 and ICP0 was significantly inhibited in its ability to form plaques on restrictive cell monolayers and was unable to degrade PML or IFI16 in a similar manner to *dl*1403. These data confirmed the hypothesis that pUL55 mediates similar host-modulatory effects as ICP0 in infected cells and loss of both is required for replication deficits previously ascribed just to ICP0 activity. Multiple immunoprecipitation-based experiments were conducted to identify and validate pUL55 interaction partners. These data uncovered two viral tegument proteins (pUL13 and pUS10) as well as a cellular ubiquitin ligase (HUWE1) as candidate binding partners. The viral proteins pUL55, pUL13, and pUS10 were found to form a tripartite complex with both pUL55 and pUS10 interacting with pUL13. Expression of both pUL13 and pUS10 was shown to be necessary for pUL55-dependent modulation of host cell functions, suggesting activity of all three viral proteins is required for a functional complex. Further data suggested the kinase activity of pUL13 was required for pUL55:pUL13:pUS10 complex activity. In addition, data suggested that HUWE1 activity is also necessary for pUL55:pUL13:pUS10 function. Recombinantly expressed pUL55 was purified at high yield and crystalised for structural studies. Using both crystal diffraction data and structural prediction data from AlphaFold2 allowed the structure of pUL55 to be solved using molecular replacement. The interaction between pUL55:pUL13 was modelled using AlphaFold2 Multimer. Point mutations predicted to disrupt the interaction from the model were incorporated expression plasmids and recombinant viruses. All tested mutations inhibited interaction between pUL55 and pUL13 and function of the pUL55:pUL13:pUS10 complex. These data have demonstrated HSV-1 expressed a previously unknown complex of three tegument proteins that modulates the host cell in an analogous way to ICP0, uncovering a novel mechanism of virus-host interactions.Item Embargo Investigating the functional roles of the viral frameshift protein 2B* in EMCV infectionNguyen, SamanthaEncephalomyocarditis virus (EMCV) is capable of infecting a wide range of mammalian hosts, causing encephalitis, diabetes, myocarditis in mice and primates as well as reproductive disorders in pigs. The severe pathogenicity is considered to be a result of the rapid cell lysis caused by EMCV. This highly efficient method of viral release also induces a severe inflammatory response, leading to disease and death. Viral propagation is further heightened by the ability of EMCV proteins to antagonise the host innate immune system, reducing the antiviral response. In 2011 the genome of EMCV was found to encode a thirteenth viral protein, 2B*, in an overlapping open reading frame within the gene for 2B. 2B* is translated via programmed ribosomal frameshifting (PRF). Unusually, the PRF is temporally regulated, increasing in efficiency from 0% at early stages of infection to 70% by 6-8 hours post-infection (hpi), thus resulting in time-dependent translation of 2B*. Truncation mutation of 2B* had previously been shown to result in a small plaque phenotype, indicative of a reduction in viral spread. However, the mechanism by which 2B* affects viral spread was left undetermined and the 2B* protein remained uncharacterised. The work presented here utilises time-course experiments to investigate the source of the small-plaque phenotype associated with loss of 2B*. Viral growth curves and cytotoxicity assays indicate that 2B* temporally regulates lytic virus release. A combination of flow cytometry, live-cell imaging and immunoblotting identified an increase in caspase-3 activation and gasdermin E (GSDME) cleavage in WT EMCV-infected samples relative to infection with 2B\*KO EMCV prior to 24 hpi. Work with caspase KO cell lines confirmed that EMCV-induced rapid cell lysis is supported by caspase-3 but that WT EMCV can also induce a caspase-3-independent lysis pathway, which 2B\*KO EMCV cannot. Together, these results show that timed production of 2B* enables temporal regulation of lytic virus release via both a caspase-3-mediated cell lysis pathway, likely to be GSDME-mediated pyroptosis, and a second caspase-3-independent pathway. The synergistic relationship between 2B* and caspase-3-mediated cell lysis means that EMCV can induce lysis independently of one of either caspase-3 or 2B* but when both are present lysis occurs more rapidly. Furthermore, interaction partners of 2B* were identified and characterised, both during infection and overexpression. A tagged virus allowed immunoprecipitation mass-spectrometry (IP-MS) of tagged-2B* and its interaction partners during infection, leading to the identification of an interaction between 2B* and every member of the 14-3-3 family of cellular proteins. As viral proteins which antagonise the innate immune response by sequestering 14-3-3 proteins had previously been identified for other virus species, this was investigated as a potential role of 2B*. Following identification of the 14-3-3 binding site of 2B* using bioinformatic prediction tools, we mutated this motif and this was shown to ablate the interaction. Further work confirmed that 2B* impedes innate immune signalling via this motif, with transcription of IFNB1 and IL6 being reduced following transient transfection. In summary, the findings of this thesis have uncovered two seemingly unrelated functions of 2B*: 1) promoting lytic virus release via caspase-3 activation in addition to a second caspase-3-independent pathway, and 2) antagonising innate immune signalling via interaction with the cellular 14-3-3 proteins. This study confirms that 2B* is a functional accessory protein and not a mere biproduct of a functionally important frameshift. Furthermore, this work contributes to the understanding of EMCV pathogenesis by uncovering novel virus-host interactions and identifying 2B* as the source of increased efficiency in EMCV-induced cell lysis relative to other picornaviruses.Item Embargo Molecular Insights of Primary Thyroid B-cell Lymphomas by Somatic Genetic and Immunogenetic ProfilingTzioni, MariaPrimary thyroid lymphomas are commonly derived from a background of Hashimoto’s thyroiditis and comprise largely of extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (EMZL) and diffuse large B-cell lymphoma (DLBCL), followed by follicular lymphoma (FL). The Du group has previously shown that thyroid EMZL has a distinct mutation profile, characterised by frequent and often concurrent deleterious mutations in *TET2* (86%), *TNFRSF14* (53%) and *CD274* (53%), which are absent or rarely seen in EMZL of other anatomic sites. Such distinct mutation profiles may be due to the influence of aetiology on the acquisition and selection of genetic changes that cooperate with antigenic stimulations during lymphomagenesis. The present thesis aimed to further elucidate the somatic genetic and immunogenetic profiles of primary thyroid lymphomas including EMZL, FL and DLBCL. In particular, the findings presented in this thesis help to improve our understanding of their pathogenesis and aid in the differential diagnosis between EMZL and FL, which could be problematic due to prominent follicular colonisation of the former. A total of 98 cases of primary thyroid lymphomas were successfully investigated, and where indicated their diagnosis was reviewed, supported by additional immunohistochemistry and fluorescent in situ hybridisation analysis for *BCL2* and *BCL6* translocation. The final diagnosis included 40 cases of EMZL (9 *BCL6* translocation positive), 22 cases of FL (5 *BCL2* translocation positive, 11 *BCL6* translocation positive, 1 both *BCL2/BCL6* translocation positive) and 36 cases of DLBCL (3 *BCL6* translocation positive). DNA from formalin-fixed paraffin-embedded (FFPE) tissue was investigated for mutations by next generation sequencing using a panel of 174 genes found to be recurrently mutated in marginal zone lymphoma, FL and DLBCL. All three lymphoma groups had considerable overlap in their mutation profiles, including frequent mutations in somatic hypermutation targeted (*TNFRSF14*, *IGLL5*) and non-targeted genes (*TET2*, *CD274*, *FAS*, *GNA13*). Subset analysis according to chromosome translocation revealed that *BCL2* translocation positive FLs were enriched for *CREBBP*, *KMT2D*, *EZH2*, but lacked *TET2*, *CD274* and *IGLL5* mutations, while *BCL2* translocation negative FL including those with *BCL6* translocations displayed few differences from EMZL and DLBCL. There were also few differences in the mutation profile between EMZL with and without *BCL6* translocation. To investigate the role of chronic BCR signalling in the pathogenesis of thyroid lymphomas, I investigated the rearranged IGH genes in a cohort of 41 thyroid lymphoma cases. I found significant overrepresentation of several *IGHV* genes including *IGHV1-46*, *IGHV3-23*, *IGHV3-49*, *IGHV4-61* and *IGHV4-34* in thyroid lymphomas. A high proportion (70%) of *IGHV3-23* rearrangements in thyroid lymphoma had a CDR3 sequence with shared amino acid motifs to those of thyroid autoantibodies seen previously in patients with Hashimoto’s thyroiditis. Additionally, 26% of EMZL cases as well as the majority of thyroid DLBCL (83%) and *BCL2* translocation positive FL cases (100%) investigated harboured newly acquired N-glycosylation sites rarely found in normal B-cell populations likely promoting BCR signalling through the binding of lectins present in the lymphoma microenvironment. We have also collected 5 cases of metachronous EMZL and FL, and 4 cases of primary thyroid lymphomas with relapse together with 1 case of composite thyroid EMZL and DLBCL. These cases provide an excellent opportunity to investigate their clonal evolution. The metachronous EMZL and FL in 4 out of the 5 cases had identical IGH::BCL2 and/or rearranged IG genes, and both common and unique mutations, suggesting that they developed independently from a common premalignant cell (CPC) population respectively. In the remaining case, the metachronous EMZL and FL were clonally unrelated. Similarly, the paired thyroid lymphoma and relapse cases showed a divergent evolution from a common CPC population in 2 cases, and a linear evolution in 2 cases (including the composite thyroid EMZL and DLBCL), 1 case did not harbour any mutations and thus its evolutionary trajectory could not be determined. These findings highlight the multi-malignant potential of IGH::BCL2-positive B-cells and clonal B cells arising in a chronic autoimmune setting. Taken together, this thesis provides several novel molecular insights into the pathogenesis of primary thyroid lymphomas and their evolution in the context of chronic autoimmune background. The common mutation and *IGHV* gene usage profile among these different thyroid lymphoma entities emphasises the role of their common aetiology (Hashimoto’s thyroiditis) and hence similar pathogenic processes in the acquisition and selection of genetic changes in the development of these lymphomas. The frequent and concurrent inactivation changes in *TET2*, *CD274* and *TNFRSF14* exclusively in thyroid lymphomas are likely the consequence of autoimmune selection associated with Hashimoto’s thyroiditis.Item Embargo A role for myotubularins in vaccinia virus egressGali Macedo, AnaVaccinia virus (VACV) generates multiple distinct forms of infectious virus. Intracellular mature virus (IMV) particles are surrounded by a single lipid membrane and are mostly released upon lysis of the infected cell. Some IMVs are wrapped by an additional double membrane to form intracellular enveloped virus (IEV), also called wrapped virus (WV). IEVs are transported on microtubules to the cell surface where the outer membrane fuses with the plasma membrane (PM) to expose a double enveloped virion outside the cell. Extracellular enveloped virus (EEV) and CEV are surrounded by two membranes and are morphologically indistinguishable, but whereas EEV are released from the cell to mediate long range virus spread, CEV are retained on the cell surface and induce the formation of actin tails to propel virions to neighbouring cells. EEV and CEV are essential for efficient virus spread within the host. This thesis concerns viral and cellular factors that influence transport of IEV particles from the site of wrapping to the cell membrane. For transport to the cell surface, IEVs rely on active microtubular transport, mediated by the cellular motor protein kinesin-I and the viral proteins E2, F12, which form a complex, and A36. While some of the players in this process are known, other aspects remain elusive, such as the mechanism by which E2/F12 are recruited to IEV, because they lack transmembrane regions. To study factors affecting IEV egress, a proteomic study was performed with A36, E2 and F12 as bait, and many potential interaction partners were identified. Amongst them, two cellular phosphoinositide (PI) phosphatases, myotubularin related protein (MTMR) 1 and su(var), enhancer of zeste, trithorax (SET)-binding factor (SBF) 1 were identified as major hits for the joint interactome of E2 and F12. This thesis has investigated the potential role of these and other cellular proteins in IEV egress. MTMR1 and SBF1 both co-precipitated with E2 and F12 only when both viral proteins were present and required the full length E2 protein. The co-purification of the catalytically active myotubularin MTMR1 with the E2/F12 complex required the inactive SBF1. Knockout (KO) of SBF1 was found to reduce virus spread, EEV titres and CEV numbers, confirming its involvement in IEV morphogenesis or egress. Interestingly, KO of MTMR1 did not cause a phenotype, likely because of functional redundancy between different myotubularins (MTMs). Indeed, when MTMR1 and MTMR2 were both deleted, a similar phenotype to that of SBF1 KO cells was observed. While a level of redundancy seems to exist between the active forms, the KO of the inactive myotubularin SBF2 in SBF1 KO cells did not further the defect in spread. The defect in spread observed in both SBF1 KO and MTMR1/2 double KO cells was dependant on the presence of F12/E2 and resulted from an accumulation of IEV at the sites of wrapping, partially recapitulating the phenotype observed in infections with viruses lacking E2 or F12. Furthermore, the contribution of myotubularins to IEV egress depended on the presence of the CC domain of SBF1 and MTMR1, that allows them to heterodimerise, their membrane recruitment domains (RID) and the phosphoinositide phosphatase activity of MTMR1. All together, these data show VACV hijacks phosphoinositide signalling through the recruitment of myotubularins to promote IEV egress and supports endosomes as a source of membranes for wrapping of IEVs. Finally, we also identified a role for PI3P regulation in IMV morphogenesis.Item Embargo Gene expression during the host-bacteria interactionWood, GeorgeThe gene expression profile of both host and pathogen can be altered dramatically upon their interaction. This response is shaped by the co-evolutionary arms race where the host seeks to detect and counter the invading pathogen, whilst the pathogen aims to evade this response and modify the host to better suit its survival and replication. Changes in gene expression during infection are thus the net result of these competing goals, the balance of which can ultimately determine the infection outcome. While the transcriptional response to bacterial infection is well studied, the analysis of gene expression is incomplete without also looking at translation. Translation regulation allows for a more rapid and dynamic response than transcriptional regulation and it is well characterized that translation is highly modulated under stress, including in infection. Where there are many studies on the translational response to viral infection, the translational response to bacterial pathogens is understudied. This thesis centres on the changes in host macrophage transcription and translation upon infection with intracellular pathogenic bacteria, with the aim to uncouple the contribution of various bacterial features or intracellular life cycle stages from the more general response to the bacteria. For this, I utilise the Gram-negative bacteria Salmonella Typhimurium and the Gram-positive bacteria Listeria monocytogenes. As such, I can identify both responses in common and unique to the two very different intra-cellular bacterial pathogens. In Salmonella infection, detection of bacterial LPS by TLR4 is critical to mount a rapid immune response. TLR4 activation has been well studied in isolation as a driver in increasing transcription and translation of immune response genes. However, stimulation of TLR4 by purified LPS does not recapitulate how it will be activated in an infection and excludes potential competing or synergising pathways that are activated by invading bacteria. As such, using host TLR4 mutants and bacterial invasion mutants, I am able to uncouple the TLR4-specific changes in host transcription and translation in Gram-negative bacterial infection. Unlike Salmonella, after invading a host cell Listeria do not remain in a vesicle. Rather, they lyse the endosome and enter the host cytoplasm. As such, the machinery that detects the infecting Listeria differs depending on the stage in the Listeria intracellular lifecycle. Therefore, by utilising mutants that stall Listeria at various lifecycle stages, I can tie specific transcriptional and translational responses to a specific lifecycle stage. From this not only do I gain insight into the temporal dynamics of gene expression in response to Listeria infection, but I can also link it to the specific modes of Listeria detection.Item Embargo Decoding aberrant RNA Synthesis by the Influenza A VirusPitre, EmmanuelleInfluenza A viruses (IAV) are negative-strand segmented RNA viruses and are responsible for causing severe respiratory diseases and fatalities in humans. IAV encodes an RNA-dependent RNA polymerase (FluPolA), which is responsible for transcribing and replicating each segment of the viral genome. In addition to the full- length vRNAs and cRNAs, the FluPolA synthesizes aberrant RNA products, such as defective interfering RNAs (diRNAs) and mini viral RNAs (mvRNAs). The innate immune system serves as the body's primary defence against viruses. Within this intricate defence network, RIG-I is responsible for detecting viral RNAs in the host cell, initiating a signalling cascade that ultimately triggers the expression of interferons and innate immunity. Adding to this complexity, IFI16, another essential cellular factor, is recognized for its ability to enhance the binding of specific full-length viral segments to RIG-I and increase RIG-I signalling. In this study, I set out to understand what factors determine which IAV RNAs are RIG-I agonists. Additionally, I sought to determine whether IFI16 demonstrates analogous behaviour when engaging with mvRNAs as it does with full-length viral segments. Finally, I aimed to improve our knowledge regarding the factors that govern transcription and replication processes. Here, I present an RNA structure-dependent (t-loop) innate immune activation mechanism that potentially underlies how mvRNAs induce RIG-I-dependent signalling. T-loop-containing mvRNAs are poorly replicated and strong t-loops are even capable of FluPolA stalling. Further experiments show that FluPolA stalling does not result in release of the mvRNA templates or nascent RNAs, but instead I observe that the stalled mvRNA- FluPolA complex itself activates innate immune signalling in a manner which seems to be dependent on the helicase activity of RIG-I. I also observe that, in contrast to mvRNAs, the full-length IAV segments can form interactions with IFI16 in a length-dependent manner. It also appears that IFI16 competes with RIG-I for binding to the NA RNA segment. Additionally, mvRNA transcription can lead to the production of an additional aberrant product; a cRNA with a cap1 structure (ccRNA), which appears to interact with RIG-I. This aberrant RNA is the result of a defect during polyadenylation. Finally, this study provides evidence that both the PB1 C-terminal residues and priming loop residues play a critical role at every stage of IAV transcription initiation. Moreover, these PB1 C-terminal residues also exert a significant influence over the replication process. Overall, my research contributes to a better understanding of the mechanisms that underlie the innate immune response to IAV and sheds light on the roles of different viral components in the transcription and replication of the viral genome.Item Embargo Role of RNA methyltransferases in lung cancerKlimontova, MariiaIn recent years, RNA-modifying enzymes have gained significant attention because their activities are relevant to cancer biology, and they are therefore potential targets for novel therapeutic intervention. Here, I target validated two RNA methyltransferases. I performed a late-stage study of METTL3, and I initiated and developed an early-stage analysis of THUMPD3. Advanced validation of METTL3, a well characterised m⁶A RNA methyltransferase, was performed based on the availability of a specific small molecule inhibitor, METTL3i. I explored whether differences in sensitivities of lung cancer cell lines to METTL3i might be due to METTL3 binding specific gene promoters in a cell-dependent manner. My findings indicate that this is not the case. I also performed a synthetic lethality screen to identify epigenetic-related inhibitors that sensitise a METTL3i-resistant lung cancer cell line to the inhibitor. This identified potential pathways that could be targeted to overcome resistance to METTL3i-based therapy. Independently of the above, an in-house bioinformatic analysis indicated a potential human RNA methyltransferase, THUMPD3, was linked to lung cancer biology. Furthermore, early in my project, THUMPD3 was reported to catalyse m²G in tRNAs. However, whether THUMPD3 also methylates non-tRNA substrates was not addressed. My project sought to address whether other RNAs are indeed m²G modified by THUMPD3 and to further investigate and validate the role of the enzyme in lung cancer biology. Consistent with our bioinformatic data, depletion of THUMPD3 from lung cancer cells induced several notable effects; it negatively impacted cellular proliferation, and migration. It also induced apoptosis and disturbances in RNA splicing. Importantly, exogenous expression of THUMPD3 in normal lung fibroblasts stimulated their proliferation rate. Furthermore, transcriptome analysis indicated that changes in expression of proteins on the cell surface and the extra cellular matrix (ECM) may occur. To identify potential RNA substrates containing THUMPD3-dependent m²G, I leveraged a newly invented method, PhOxi-seq, which is based upon blue light-induced photo-oxidation of certain modified nucleotides in RNA, including m²G. Initially, I further developed and optimised PhOxi-seq on tRNA, leading to the discovery of THUMPD3-dependent alterations in m²G6 tRNA modification. Subsequently, I broadened the scope of my screen by including purified human rRNA-depleted RNA from lung cancer cells as substrate in the photo- oxidation reaction. This approach unveiled a cohort of RNAs, mainly mRNAs, several of which are implicated in the ECM. This links in well with my phenotypic analysis on the same cells. Notably, this is the first time that m²G has been reported in human mRNAs, as well as other polyA+ RNAs. The implications of these findings are discussed. In summary, my work extends the application of PhOxi-seq to identifying THUMPD3- dependent m²G sites (and potentially other oxidisable modifications induced by different enzymes) in various RNA types. My works also identifies a potential oncogenic role for THUMPD3 in lung cells. Thus, my findings highlight THUMPD3 as a potential therapeutic target, especially in a lung cancer setting.Item Open Access Using Tumour Evolution to Understand the Epigenetic and Transcriptional Adaptations of Cancer to Host ImmunityBaird, TarrionImmunoediting describes the process in tumour development whereby tumours evolve to avoid the immune system. The relevance of immunoediting in carcinogenesis, metastasis and immunotherapy resistance makes understanding the mechanisms of immunoediting essential in informing cancer therapy and prevention. Previous work on immunoediting has largely focussed on neoantigen loss, but this thesis aimed to expand the immunoediting paradigm to incorporate transcriptomic and epigenetic changes in tumour cells. This thesis describes the epigenetic and transcriptomic characterisation of carcinogen-induced immunoedited and unedited tumour lines after growth *in vitro*. The transcriptome and immune infiltrate of immunoedited tumours undergoing progressive growth, or unedited tumours undergoing immune-mediated regression in wild-type mice was investigated. The results suggested that expression of T cell inhibitory molecules, a reduction in inflammation, and reduced expression of L1 transposable elements may be involved in immune escape in immunoedited tumours. An over-expression screen was performed to attempt to identify key genes involved in immunoedited tumour escape *in vivo*. Finally, a collection of bioinformatic tools were developed to perform process-based analysis of The Cancer Genome Atlas. These tools can suggest hypotheses of gene function and regulatory mechanisms and explore the pathological significance of gene expression signatures in human cancers. These tools are publicly available as an online web-tool. Both the identification of potential mechanisms of immunoediting and the bioinformatics tools developed in this thesis can support and inform further research.Item Embargo Characterisation of the Mechanism of Norovirus VPg-NucleotidylylationPinckert, MalteNoroviruses, which belong to the *Caliciviridae* family, have a substantial yearly impact on the global economy of about $60 billion and cause about 200,000 deaths per annum in children under the age of 5. Despite causing most cases of viral gastroenteritis, noroviruses remain poorly characterised compared to other viruses. Infectious norovirus RNA is covalently linked to VPg (viral protein genome-linked) at the 5’ end of the genome through VPg-mediated RNA priming of viral RNA synthesis. While poorly understood mechanistically, this process is reliant on the addition of a nucleotide to a highly conserved tyrosine residue within VPg - this process is referred to as VPg nucleotidylylation and is catalysed by the viral polymerase (NS7). Using murine norovirus (MNV) as the primary model, this project sought to better understand this key stage of the norovirus life cycle, providing insight into a previously ill-characterised crucial stage of the viral life cycle and may help to identify key points which could be therapeutically targeted. Mass spectrometric analysis of the 5’ end of infectious norovirus RNA purified from infected cells, confirmed that VPg is genome-linked via a GDP moiety at a highly conserved tyrosine residue. These results however are contradicted by the state of the art on calicivirus VPg nucleotidylylation and has, to date, not been replicated *in vitro*. Therefore, developing a specific assay to measure *in vitro* VPg nucleotidylylation producing physiologically relevant readouts laid at the core of this project. Subsequently, this, for the first time, brought previous *in vivo*, genomic and *in vitro* observations of VPg nucleotidylylation in agreement and enabled me to study this reaction. I identified negative sense subgenomic RNA (-sgRNA), magnesium ions (Mg2+) and the viral protease (NS6) as essential components that facilitate specific and physiologically relevant VPg nucleotidylylation. Further, I showed precursor forms of NS6, NS56 and NS67, to stimulate the reaction more efficiently than mature NS6. Mutations within potential RNA binding regions of NS6 affected VPg nucleotidylylation *in vitro*, suggesting a potential role in the reaction for it as stabilizing the nucleotidylylation complex, through binding the RNA template. I identified a region within the -sgRNA of MNV, spanning region in the 3’ end of open reading frame 3 (ORF3) and the untranslated region (UTR), to be both sufficient and necessary for stimulating VPg nucleotidylylation. Through subsequent serial truncations, internal deletions and point mutations, I was then able to refine this understanding. My findings indicate VPg nucleotidylylation to be reliant on three distinct structural elements within this region, one of them a small, predicted stem-loop in the UTR. Utilising a panel of MNV mutants within the 3’ region of the genome, previously found to affect virus viability, I observed a clear correlation between virus viability and the negative sense 3’ region’s ability to enhance VPg nucleotidylylation. Lastly, I demonstrated the ORF3 UTR region of HuNoV RNA to be able to template MNV VPg nucleotidylylation, suggesting template and positional conservation between MNV and HuNoV. In summary, this work provides new key insights into VPg nucleotidylylation - a previously poorly characterised, but vital, process of norovirus replication and may pave the way for further exploration of this process for therapeutic targeting.Item Open Access The role of RNA structures in the evolution of respiratory RNA virus genomes.Rigby, Charlotte; Rigby, Charlotte [0009-0004-3533-6679]RNA secondary structures in RNA viral genomes play important roles in a range of functions, including packaging, protection from cellular nucleases, and regulating viral replication. Furthermore, RNA structures have been shown to be immunostimulatory. In the case of Influenza A virus (IAV), during infection aberrant RNAs are produced. One of these are mini-viral RNAs (mvRNAs) which are very potent RIG-I stimulators. However, our understanding of the mechanisms that underlie the role of RNA structures in the above processes and how RNA structures evolve is still limited. In my thesis I first investigate whether mvRNAs are produced in human patients. Clinical samples were obtained from three influenza seasons and presence of mvRNAs were analysed via RT-PCR. Due to both technical and sample issues, no conclusion was reached. I next examined how RNA structures, including those capable of inducing mvRNA synthesis and increasing their immunostimulatory potential, are changing during pandemic IAV H3N2 evolution. Using in silico analyses, I observed a decline in RNA structure stability in the polymerase segments over time. Control analyses showed that this change was independent to amino acid mutation and reduced in human-adapted viruses, but present in emerging H1N1 IAV. Using transfections, I subsequently identified that amino acid changes in the IAV RNA polymerase make the enzyme more sensitive to RNA structures, suggesting that the loss in RNA structure stability and change in enzyme activity are linked. To determine if the loss of RNA structure was present in other emerging viruses, I analysed sequences of SARS-CoV-2 and found a similar decline in thermodynamic stability over time. Having identified a pattern of RNA structure loss in both IAV and SARS-CoV-2 I repeated the analysis on Ebola Zaire and Lassa virus to determine if similar patterns are observed in zoonotic viruses. I found this not to be the case. Based on these observations, I propose a novel model for emerging and pandemic RNA virus adaptation and hypothesise that immune pressure may select for viruses with improved replication efficiency, leading to RNA structure loss and a reduction in detection by immune receptors over time.Item Open Access TRIM5α is a poxvirus restriction factor: viral evasion strategies and a pathway to antiviralsZhao, Yiqi; Zhao, Yiqi [0000-0003-4348-0588]This thesis describes cellular protein TRIM5α as a restriction factor for vaccinia virus (VACV) and two VACV countermeasures. This study followed up a proteomic analysis showing that VACV infection of TERT-immortalised human foetal foreskin fibroblasts (HFFF-TERTs) induced TRIM5 downregulation. This observation was validated in HFFF-TERT and HeLa cells, and TRIM5 degradation was shown to be proteasome-dependent. By using VACV mutants lacking blocks of genes near either genomic terminus, and thereafter mutants lacking individual genes, the gene encoding the viral protein that induces TRIM5α degradation was mapped to gene *C6L*. Furthermore, the inducible expression of C6 degraded endogenous TRIM5α outwith infection, showing that TRIM5α degradation does not require other viral proteins. Subsequent mechanistic studies showed that C6 interacts directly with the RING domain of TRIM5α. Finally, highly conserved orthologues of C6 in other orthopoxviruses such as cowpox virus (CPXV), rabbitpox virus (RPXV), camelpox virus (CMLV), elephantpox virus, monkeypox virus (MPXV) clades I and II and variola virus (VARV), were shown to co-precipitate and degrade TRIM5α outwith infection. A hypothesis to explain VACV-mediated TRIM5α degradation was that TRIM5α is an anti-VACV restriction factor, and therefore is removed by VACV to facilitate infection. To test this, cell lines lacking or over-expressing TRIM5 were generated. Whereas VACV replication and spread were enhanced in TRIM5-/- cell lines, over-expression of TRIM5α had the opposite phenotype. Reintroduction of wildtype (WT) TRIM5α into TRIM5-/- cells rescued TRIM5α-mediated restriction of VACV, but TRIM5α mutants that lacked E3 ubiquitin ligase activity, the ability to oligomerise or the C-terminal PRY/SPRY domain did not. Notably, during VACV infection, TRIM5α co-localised with virus replication factories, whereas in uninfected cells, TRIM5α was diffuse throughout the cytoplasm. In addition to C6-mediated degradation of TRIM5α, this thesis reports that VACV exploits the cellular protein, cyclophilin A (CypA), as an additional measure to counteract TRIM5α-mediated restriction. This work was based on prior observations that CypA is utilised by HIV-1 to evade restriction by human TRIM5α, CypA is incorporated into both VACV and HIV-1 virions and the inhibitor of CypA, cyclosporine A (CsA), has anti-viral activity. Whilst VACV replication and spread were reduced in CypA-/- cells, this phenotype was lost when TRIM5 was also knocked out, showing that CypA is proviral in a TRIM5-dependent manner. Furthermore, CsA restricted virus plaque size in WT but not in TRIM5-/- cells, showing that the antiviral activity of CsA is also TRIM5-dependent. To determine whether the peptidyl prolyl isomerase activity of CypA is required for its proviral function, two catalytic defective mutants were re-introduced into CypA-/- cells. Whilst WT CypA rescued virus replication and spread, the mutants did not, showing that CypA enzymatic activity is needed to antagonise TRIM5-mediated restriction. Next, the binding partner(s) of TRIM5α and CypA was identified by proteomic analysis of proteins that co-precipitated with TRIM5α and CypA during VACV infection. Of the several VACV structural proteins that co-precipitated with TRIM5α, only L3 was also precipitated by CypA, and this co-precipitation was lost in the presence of CsA. These interactions were validated during VACV infection and following ectopic expression of L3, and shown to be direct. The interaction between TRIM5α and L3 was mapped to the C-terminal domains of TRIM5α. During infection in the presence of L3, TRIM5α co-localised to virus factories, whereas when L3 expression was repressed, TRIM5α was elsewhere in the cell. L3 was shown to dimerise and dimerisation was enhanced by TRIM5α and this required its E3 ubiquitin ligase activity, and was antagonised by CypA. Furthermore, TRIM5α and L3 were shown to activate NF-κB synergistically, and this was antagonised by CypA but not a catalytically defective mutant. Like C6, L3 is highly conserved amongst orthopoxviruses and the L3 from MPXV and VARV co-precipitated with both TRIM5α and CypA, and the latter interaction was disrupted by CsA. Given the 2022 MPXV epidemic and the emergence of tecovirimat-resistant MPXV, the possibility of repurposing CsA as an anti-poxviral drug was tested. Since CsA is an immunosuppressant, two non-immunosuppressive derivatives, NIM811 and alisporivir, were also tested. All three drugs reduced VACV and MPXV replication and spread and disrupted the interaction between VACV and MPXV L3 and CypA. Therefore, NIM811 and alisporivir have anti-poxviral activity and potential clinical applications.Item Embargo Algorithm development for RNA structure prediction in RNA virusesTumescheit, CharlotteRNA structures play many different roles in the life cycles of RNA viruses, including in directing translational control, genome replication, subgenomic RNA synthesis, and encapsidation. However, predicting these structures can be challenging, especially when it comes to pseudoknots, long-range interactions, and mutually exclusive interactions or switches. Furthermore, separating biologically relevant structures from the vast number of interactions that could theoretically form can be problematic. Since the sequences of RNA viruses evolve rapidly and, for many species, there are a large number of sequenced isolates, comparative genomics can be used to predict structures that are conserved and therefore more likely to be biologically relevant. Here, a new program is presented that aims to fill the gaps among existing RNA structure prediction programs by looking for all possibly functionally relevant short-range and long-range interactions that are conserved within a multiple sequence alignment, thereby allowing for the detection of pseudoknots and mutually exclusive structures. An offset parameter makes it possible to find interactions that do not align perfectly, thereby allowing for the possibility to look at more divergent sequences. This can further improve the separation of functional structures from random structures. Furthermore, a phylogenetic weighting scheme aims to balance alignments that may mix an abundance of closely related sequences with a few distantly related sequences, besides mitigating potential problems introduced by sequencing errors. The free energy is taken into account to reduce the number of false positives. The program to predict potential secondary structures in RNA viruses is embedded in a pipeline that, based on a reference sequence, obtains related sequences, builds a sequence alignment and phylogenetic tree, and creates all the necessary files to perform the analysis. The pipeline is then applied to a selection of viruses. Also, a novel tool is presented for improving the quality of multiple sequence alignments by trimming poorly aligned regions, and for visualising alignments and alignment processing steps. The structure prediction pipeline presented identifies known functionally important structures and suggests new structures for potential experimental follow up. Different options and adjustable parameters allow for individual workflows. By filling an underrepresented niche, the software will hopefully help guide future molecular understanding of RNA viruses.Item Restricted Item Open Access Role of the Innate Immune Response to DNA During Therapy with Oncolytic VirusesWagner, EmmaOncolytic viruses (OVs) are viruses which selectively replicate within and directly lyse cancer cells whilst sparing host tissue. This selectivity not only helps their safety profiles and minimise toxicities but also allows OVs to potentially increase their therapeutic dose over time as they replicate and spread throughout tumours. Whilst tumour lysis helps reduce tumour burden, a secondary purpose of OVs as immunotherapies is to stimulate the host's immune system culminating in a systemic anti-tumour response. To do so relies on OVs ability to trigger to two distinct forms of immunity simultaneously: anti-tumour and anti-viral. OV-induced anti-viral immunity typically refers to release of virions, cytokines and damage- or pathogen-associated molecular patterns (DAMPs and PAMPs) to the tumour microenvironment (TME) upon tumour lysis. Doing so helps to recruit innate and adaptive immune cells to the TME, helping to overcome its naturally immunosuppressive state and stimulate the generation, recruitment and activation of tumour antigen-specific T cell responses. In theory, this combination of anti-viral and anti-tumour immunities has the potential to build to a systemic anti-tumour T cell response that targets distant, secondary tumour sites. In reality, although effective in reducing local tumour burden in the short-term, OVs struggle to produce sustainable anti-tumour responses in the long-term as monotherapies. Recent research into improving OV efficacy has focused on enhancing anti- tumour immunity by combining OVs with other immunotherapies such as immune checkpoint inhibitors (ICI). However, to ultimately achieve a sustainable systemic anti-tumour response against future tumour relapse, a greater understanding of OV-induced anti-viral immunity is needed. In particular, little is known about cancer cell’s intrinsic ability to sense and respond to OV infection and how this may impact OV efficacy. The purpose of this study was to gain a mechanistic understanding of how tumour cells may sense infection by a vaccinia (VACV)-derived OV, superior killing virus (SKV), through cytoplasmic DNA sensing pathways and how manipulation of this pathway may impact OV efficacy. Here, I screened several syngeneic murine cancer cell lines for their expression of cytoplasmic DNA sensing proteins and susceptibility to OV infection. After establishing the BRAF-mutant melanoma cell line 4434 as our model system, I found that deletion of cytoplasmic DNA sensor cyclic GMP-AMP Synthase (cGAS) resulted in loss of STING-dependent signalling in response to DNA stimulation and SKV infection whilst deletion of DNA-dependent protein kinase (DNA-PK) complex protein Ku80 did not. Despite these differences in STING-dependent signalling, loss of either cGAS or DNA-PK resulted in loss of pro-inflammatory signalling and significantly increased SKV replication *in vitro*. This was also true *in vivo*, where cGAS-/- tumours had significantly increased SKV load in comparison to WT tumours. Interestingly, deleting cGAS also had an impact on tumour growth and immune cell infiltration to the TME prior to SKV treatment. Altogether, this work highlights the importance of investigating the underlying activity of cytoplasmic DNA sensing pathways in tumours prior to OV therapy. In particular, these results indicate that VACV-derived OV treatment efficacy of tumours expressing cGAS and DNA-PK may be enhanced by incorporating cGAS and DNA-PK inhibitors to their design.Item Embargo Comparative analysis of African and Asian/American Zika virus strains at the transcriptional and translational levelLefèvre, CharlotteZika virus (ZIKV) is an emerging mosquito-borne flavivirus. Initially, ZIKV was considered of low importance as infection was usually asymptomatic or caused mild illness. However, the emergence of the Asian/American lineage, in contrast to the African lineage, was considered a public health threat. The Asian/American lineage has been associated with neurological complications such as Guillain-Barré syndrome in adults and congenital ZIKV syndrome (e.g., microcephaly) in newborns. Currently, it is not understood why this newly emerged Asian/American ZIKV results in a more severe disease than the African ZIKV. This project focused on understanding the molecular mechanisms underlying the differences in virulence and pathogenicity between these two ZIKV lineages in human brain cells. In order to identify the molecular determinants that influence viral replication and subsequent ZIKV virulence, the differences between an African and an American ZIKV strain were studied at different levels. Firstly, RNA-Seq and ribosome profiling (Ribo-Seq) was performed on astrocytoma-glioblastoma cells (U251 cells) infected with an African and an American ZIKV strain to determine the transcriptional and translational profile of each virus. Two previously overlooked upstream open reading frames (uORFs) in the 5′ untranslated region (UTR) of the American ZIKV genome were identified, initiating at non-canonical CUG and UUG codons; and a single uORF in the 5′UTR of the African ZIKV, initiating at a CUG start codon. The uORFs in the American ZIKV are referred to as ‘uORF1’ and ‘uORF2’, and the one in the African ZIKV as the ‘African uORF’. Secondly, considering their overlap with the main ORF, the viral polyprotein, it was hypothesised that these uORFs could play a role in the translational regulation of the main ORF. To investigate this, luciferase reporter assays were used, which showed that when the ZIKV 5′UTR harboured an African uORF or when uORF1 was not expressed, the main ORF was better translated compared to the wild-type, in contrast to the knock-out of uORF2. Thirdly, the presence of these uORFs was shown to modulate virus growth and replication by using mutant viruses that modulate the expression of the different uORFs and comparing these to the wild-type American ZIKV strain by performing viral growth curves and competition assays. Interestingly, the uORF2 knock-out virus showed a similar infectivity as the wild-type American virus, whereas the uORF1 knock-out virus and the African-like virus reached higher fitness than the wild-type virus. Additionally, transient overexpression of the uORF1 peptide and its subcellular localisation within the cytoskeletal fraction suggests a contribution to the dysregulation of the cytoskeleton, which has been associated with neurodegenerative diseases. Finally, a differential gene expression analysis was performed in which cellular genes that were differentially transcribed by using the RNA-Seq datasets, but also the genes that had a different translational efficiency by using the Ribo-Seq datasets in response to infection with different ZIKV strains were thoroughly analysed. Infection of U251 cells with an African ZIKV strain led to an early upregulation of innate immune response genes, but infection with an American ZIKV strain induced the expression of a broader range of genes related to the antiviral response. With this first analysis of neurotropic flavivirus gene expression using ribosome profiling, the functional characterisation of novel uORFs and the comparative analysis of differentially up- or down-regulated cellular genes, novel insights were provided into important differences between the African and the Asian/American ZIKV lineages, two viruses that result in different pathologies.