[Kakugo Viruses]

By

ACKNOWLEDGEMENT

I would take this opportunity to thank my research supervisor, family and friends for their support and guidance without which this research would not have been possible

DECLARATION

I, [type your full first names and surname here], declare that the contents of this dissertation/thesis represent my own unaided work, and that the dissertation/thesis has not previously been submitted for academic examination towards any qualification. Furthermore, it represents my own opinions and not necessarily those of the University

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ABSTRACT

Kakugo viruses (KV) are picorna-like viruses and are grouped in genus Inflaviruswhich have not yet been placed within a virus family. Kakugo viruses have been isolated from the brains of European worker honeybee, Apismellifera L. and are proven to be responsible for their aggressive behaviour towards giant hornet, Vespa mandarinia japonica. Kakugo viruses have positive sense ssRNA genome that is monocistronic. The full length of KV RNA consists of 10,152 nucleotides, with a large single open reading frame (ORF) which encodes a polyprotein of 2,893 amino acids residues. The KV genome contains a 5' untranslated region (UTR) of 1156 nucleotides within which an internal ribosome entry site (IRES) is present. IRES are specific nucleotide sequences found within the 5' UTR and are involved in translation initiation. Translation in eukaryotes occurs via 5' 7methyl guanosin (mG) cap-dependent process where the ribosome binds to 5' cap with the help of eukaryotic initiation factors and scans the UTR until it reaches the start codon. However, in picornaviruses and picorna-like viruses the genome lacks the 5' cap but has a genome linked viral protein (VPg) at 5' end which acts as a primer in RNA replication but is not involved in translation as it gets cleaved off during infection; therefore, translation initiation occurs via cap-independent mechanism and involves the IRES. Our work focuses on finding the minimal functional sequence of KV IRES. Former PhD student had demonstrated that deletion of 500 nucleotides from 5' end of KV UTR had no effect on IRES activity. Further deletions of 600, 700, 800, 900, 950, 1000, 1050, 1075, 1101 nucleotides from the 5' end of KV UTR have been done to form 5' KV inserts of different sizes. These KV inserts were cloned into the dicistronicpGEM-CAT/LUC vector to form pGEM-CAT/IRES/LUC dicistronic constructs. KV inserts were assayed for IRES activity by transfecting Spodopterafrugiperda21 (Sf21) insect cells and measuring its activity by chloramphenicol acetyltransferase (CAT) assay and luciferase (LUC) assay. Their IRES activity will also be compared in mammalian cell i.e. rabbit reticulocyte lysate (RRL) system and plant cell i.e. wheat germ extract system. IRES activity will further be compared with other insect and mammalian picornaviruses i.e. Rhopalosiphumpadi virus and Encephalomyocarditis virus respectively. Bead binding assay was carried out for the minimal functional sequence i.e., KV 1050 to check for the proteins that binds to it. Finding the minimal functional sequence of KV IRES increases its potential for being used in insect protein expression systems where two proteins can be synthesized simultaneously in a cell.

TABLE OF CONTENTS

ACKNOWLEDGEMENTII

DECLARATIONIII

ABSTRACTIV

CHAPTER 1: INTRODUCTION1

Background of the ...