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Title: Study of the structure and function of the influenza virus RNA-dependent RNA polymerase
Author: Martin, Itziar Serna
ISNI:       0000 0004 7230 0600
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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Influenza viruses are segmented, negative-sense RNA viruses responsible for influenza disease. Unlike other negative-sense RNA viruses, influenza virus genome replication occurs in the nucleus of infected cells. The virus encodes a heterotrimeric RNA-dependent RNA polymerase (FluPol) that is responsible for transcribing and replicating the viral genome. In order to transcribe, the FluPol generates capped RNA primers by a process termed 'cap-snatching'. In it, the polymerase binds host capped RNAs and cleaves them 10-13 nucleotides downstream of the cap structure. The catalytic core of FluPol is found in the PB1 (Polymerase Basic protein 1) subunit, which associates with PB2 (Polymerase Basic protein 2) and PA (Polymerase Acidic protein). PB2 and PA contain a cap binding domain and an endonuclease domain respectively, which enable cap-snatching to take place. Viral transcription and replication occur in the context of a ribonucleoprotein (RNP) complex, where an RNA segment is coated by nucleoprotein and bound by a single copy of FluPol. Regulation of transcription and replication during infection is dependent on multiple viral and host factors, including nucleoprotein, cellular DNA-dependent RNA polymerase II (Pol II) and nuclear import factors. While high-resolution structures of the FluPol heterotrimer and low-resolution structures of RNPs are available, multiple aspects of the mechanisms involved in polymerase activity and its modulation remain poorly understood. In this DPhil project, I have employed a combination of functional and structural approaches towards exploring FluPol activity. Here I present the work carried out towards optimising the methods for FluPol structural analysis, more specifically, the use of cryo-electron microscopy to explore both the structure of the heterotrimer in complex with modulators of polymerase activity as well as RNPs. I also describe the work carried out on characterisation of the requirements for transcription and replication by FluPol. This includes the role of Pol II in transcription regulation as well as those of FluPol, nucleoprotein and host factor ANP32 in controlling viral replication. Furthermore, I also present the characterisation of a panel of 24 camelid single-chain antibodies, known as nanobodies, as tools to explore polymerase activity. Within these, nanobodies which exhibited robust inhibition of all polymerase activity were found, which opens the possibility of employing these to identify sites on the polymerase that would be suitable targets for small molecule inhibitor design.
Supervisor: Fodor, Ervin ; Grimes, Jonathan Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available