Defective interfering particles of parainfluenza virus subtype 5 and interferon induction

The innate immune response is the first line of defence against virus infection. Cells contain a diverse array of pathogen recognition receptors (PRRs) that are able to recognise multiple pathogen associated molecular patterns (PAMPS) that present themselves during virus infection. The RIG-I (Retinoic acid inducible–gene-I) and MDA5 (melanoma differentiationassociated gene 5) PRRs detect specific viral RNA ligands and subsequently induce the expression of the cytokine Interferon-β (IFN-β). IFN-β is secreted, acting on the infected cell and neighbouring uninfected cells to generate an antiviral state that is hostile to virus transcription, replication and dissemination, whilst also orchestrating adaptive immune responses. Given IFN-βs crucial cellular antiviral role, understanding its induction is of great importance to developing future antiviral drugs and vaccine strategies. Using A549 reporter cells in which GFP expression is under the control of the IFN-β promoter, we show that there is a heterocellular response to parainfluenza virus 5 (PIV5) and infection with other negative sense RNA viruses. Only a limited number of infected cells are responsible for IFN-β induction. Using PIV5 as a model, this thesis addresses the nature of the PAMPs that are responsible for inducing IFN-β following PIV5 infection. The previous work has shown that PIV5 Defective Interfering particle (DI) rich virus preparations acted as a better inducer of IFN-β compared to DI poor stocks. DIs are incomplete virus genomes produced during wild-type virus replication as a result of errors in the viral polymerase. To investigate this further, A549 Naïve, MDA5/RIG-I/LGP2 Knock down reporter cells were infected with PIV5 W3 at a low MOI to examine the inverse correlation of NP and GFP of DIs generated during virus replication and not from the initial infection. GFP+ve cells were cell sorted, and using QPCR it was found that cells that have the IFN-β promoter activated contain large amounts of DIs relative to GFP-ve cells. This data supports the Randall group’s findings that DIs generated during errors of wild-type replication by the viral RNA polymerase are the primary PAMPs that induce of IFN-β, as opposed to PAMPs being generated during normal wild-type virus replication. 1. Candidate’s declarations: I, John A L Short hereby certify that this thesis, which is approximately 45,000 words in length, has been written by me, and that it is the record of work carried out by me, or principally by myself in collaboration with others as acknowledged, and that it has not been submitted in any previous application for a higher degree. I was admitted as a research student in Oct, 2009 and as a candidate for the degree of Doctor of Philosophy (PhD) in Molecular Virology; in Oct, 2014; the higher study for which this is a record was carried out in the University of St Andrews between 2009 and 2014. Date . Signature of candidate . 2. Supervisor’s declaration: I hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriate for the degree of PhD in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree. Date . Signature of supervisor . Prof. R. E. Randall 3. Permission for publication: In submitting this thesis to the University of St Andrews I understand that I am giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby. I also understand that the title and the abstract will be published, and that a copy of the work may be made and supplied to any bona fide library or research worker, that my thesis will be electronically accessible for personal or research use unless exempt by award of an embargo as requested below, and that the library has the right to migrate my thesis into new electronic forms as required to ensure continued access to the thesis. I have obtained any thirdparty copyright permissions that may be required in order to allow such access and migration, or have requested the appropriate embargo. The following is an agreed request by candidate and supervisor regarding the publication of this thesis: PRINTED COPY A) No embargo on print copy ELECTRONIC COPY A) No embargo on electronic copy