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The Interaction of Host and Viral MicroRNAs with Infectious Laryngotracheitis Virus Transcripts Jack Ferguson Thesis presented for the degree of Doctor of Philosophy The University of Edinburgh 2019 Declaration I declare that this thesis is of my own composition, and that it contains no material previously submitted for the award of any other degree. The work described in this thesis has been executed by myself, all work of other authors is duly acknowledged. Jack Ferguson Division of Infection and Immunity The Roslin Institute Royal (Dick) School of Veterinary Studies University of Edinburgh Easter Bush Campus Edinburgh Midlothian EH25 9RG ii Acknowledgements Firstly, I would like to thank my supervisors Bob Dalziel and Finn Grey. They have provided help, support and guidance throughout the duration of my PhD and listened irrespective of how daft the question/comment or suggestion may have been. The project would not have been a success without their insights and knowledge. I’d also like to thank Bob’s whiteboard for the endless diagrams and scrawling’s that have helped shaped this project from the get go. Secondly, I would like to thank Inga Dry and Spring Tan as current members of the Dalziel lab for putting up with me for the last few years. Your advice, friendship and support has been invaluable to me throughout. In addition, I would like to thank Katie Nightingale and Erin Brady as previous member of the Dalziel lab for their friendship. As well as this, I would also like to thank the wider virology department at the Roslin Institute for their valued friendship and time over the last three years. In particular, I would like to thank both Marlynne Quigg-Nicol and Nikki Smith. I am also like to thank my peers and other PhD students both in the Division of Infection and Immunity and the wider Institute. They have provided support, an ear, friendship and questionably too many nights in the pub along the way. This thesis would not be complete without the acknowledgement of cake club. The club has provided a weekly dose of much needed sugar and discussions on a strange mixture of topics. I’d like to thank all members past and present but particular thanks go to emeritus president Dominique McCormick and current incumbent president Elly Gaunt. Away from the Institute, I’d like to thank the players of Dollys F.C. past and present for the laugher over the duration of my PhD. It was an honour to be able to lead the team for two seasons as captain, something that was thoroughly enjoyable. Up the Dollys! Without question, I would like to thank my family and friends back in Yorkshire and further afield. The unconditional support and patience throughout my whole education has been priceless. I would also like to thank Chloe Brown and Bruno the dog for the last 3 years of support and adventures away from the lab. iii Abstract Infectious Laryngotracheitis Virus (ILTV) is an Alphaherpesvirus of the domesticated chicken and other economically important fowl such as pheasants, peafowl and turkeys. It causes an upper respiratory disease that is clinically characterised by dyspnoea, rales and expulsion of a thick, sometimes hemorrhagic, tracheal exudate. Incidences of mortality range from 10 – 70 % whilst morbidity ranges from 50 – 100 %. The disease causes significant financial losses to the poultry industry through bird death, stunted growth and a marked decrease in egg production. Due to its economic importance, attenuated live- vaccines have been developed by serial passage of virus either in eggs or tissue culture. These have the ability to protect birds against ILTV however they do not stop latent infection which can result in reactivation of the virus termed ‘vaccinal laryngotracheitis’. The molecular biology underlying virus-host interactions for ILTV is poorly understood and there are large gaps in knowledge regarding the pathogenesis of ILTV infection. MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally regulate gene expression through targeting of specific mRNAs. Several herpesviruses have been shown to encode miRNAs that have the ability to regulate both viral and cellular gene expression which can impact virus-host interactions. Previous work in the literature has shown that ILTV encodes for 10 miRNAs with sparse data on what they may be regulating. It was hypothesised that the virus-encoded miRNAs may have an effect upon the pathogenesis of the virus by targeting both cellular and viral mRNAs. To investigate this hypothesis initially, the biochemical technique CLASH (Cross-Linking and Sequencing of Hybrids) was attempted however a lack of suitable reagents such as physiologically relevant cell lines of chicken origin made this technically challenging and this approach was halted. Instead, a bioinformatic approach was developed and split into two avenues of research. Firstly, it was hypothesised that miRNAs encoded by ILTV would target virally derived transcripts. As the virus genome is poorly annotated, transcripts for all 79 open reading frames (ORFs) were created manually using an arbitrary system of 1000 bp upstream of the ATG start site and 50 bp downstream of the designated PolyA tail. These were then fed into the online algorithm RNA Hybrid alongside sequences for all 10 virus-encoded miRNAs. Results from the bioinformatic predictions were then sorted and filtered using pre-defined conditions. This left a total of 227 predicted interactions. These were then filtered again leaving 28 novel targets that were screened in a reporter based system. Three of the predicted interactions showed a decrease in luciferase-reporter activity compared to the siRNA control (UL24, UL29 and UL46/48), however only the latter two showed statistically iv significant decreases in activity of 15 % and 20 % respectively. Mutation of the seed sequences in both UL29 and UL46/48 targets abrogated the effects of the miRNA mimic. Further work on UL29 and its interaction with ILTV-miR-I2 looked at validating this interaction by western blotting however these results were inconclusive. Investigations into the interaction between UL46/48 and ILTV-miR-I6-5p first confirmed by RT-PCR that UL46 was targeted by ILTV-miR-I6-5p. Validation of the interaction between UL46 and ILTV-miR-I6-5p by western blotting was inconclusive. Investigations into the interplay between UL46, UL48 and the ICP4 promoter were also characterised with UL46 able to negatively modulate the effects of UL48 on ICP4 promoter activity in a reporter-based system. Secondly, the same viral transcripts were then used in conjunction with high confidence chicken miRNAs as per MiRBase (Release 21, Jun 2014). The sorting and filtering of results mirrored that of the viral transcript study giving a final list of 103 predicted targets. From the list, three targets were picked that were all targeted by the cellular miRNA gga- miR-133a-3p and tested using the same reporter system. Two targets, one in UL20 and one in the coding region of ICP4 showed no statistical difference between the miRNA mimic and siRNA control. In contrast, one target, located in the 5’UTR of ICP4 and confirmed by RT- PCR to be within the expressed mRNA transcript was found to cause a 55 % reduction in luciferase activity. This effect was then abrogated upon mutation of the miRNA seed sequence. Further investigations found that this miRNA can cause an apparent reduction in virus titer and a statistically significant decrease in plaque size morphology when virus is harvested from cells transfected with the miRNA mimic and used to infect naïve cells. Moreover, a combination RT-qPCR and sequencing was used to confirm the sequence of gga-miR-133a-3p in several tissues of the chicken including the Dorsal Root Ganglia (DRG) and Harderian gland (HG). These are of importance to ILTV biology as the DRG is a site of latent infection and the HG is a secondary lymphoid organ (SLO) in the bird which monitors the upper respiratory tract, the site of lytic replication/clinical symptoms. Finally, CRISPR-Cas9 genome editing was used to delete a cluster of five miRNAs from the viral genome. Guide RNAs (sgRNAs) were designed to target the miRNA cluster and shown to efficiently direct cleavage of target DNA in an in vitro system. Following transfection/infection of cells, virus was harvested and subsequent sequencing showed that this approach was successful in creating a recombinant ILTV. This was detectable after passage of the virus through naïve cells although a pure population of recombinant virus was not obtained due to a lack of time. v Lay Summary Infectious Laryngotracheitis (ILT) is a deadly disease of chickens caused by a virus called Infectious Laryngotracheitis Virus (ILTV). The disease is financially important all over the world due to how much chicken is eaten. If birds catch this virus, they have symptoms such as shortness of breath and produce a thick, sometimes bloodied mucus that they cough up. Birds also do not grow properly and egg laying birds do not lay as many eggs as healthy animals would.