Investigation of the Cydia pomonella granulovirus capsid and capsid associated proteins

Abstract

Baculoviruses are a family of viruses that infect insect larvae. The baculovirus lifecycle produces two distinct infectious particles: occlusion derived virions (ODV), and budded virions (BV). Late in the infection cycle, the ODV is encapsulated in a crystalline matrix to form an occlusion body. Each virion is composed a lipid membrane embedded with different sets of membrane proteins, and contains a conserved cylindrical protein shell (the capsid) that packages a circular dsDNA genome. Baculoviruses have been used to express protein for 30 years, and to control insect pest species for over 100 years. In spite of this, little is known about the structure of the capsid or its associated proteins. The structure of capsid of the baculovirus that infects the coddling moth, the Cydia pomonella Granulovirus (CpGV), is the subject of this thesis. To improve the understanding of the structural biology and assembly of the capsid, the major capsid protein, Vp39, and an associated protein kinase, PK-1, were expressed for biochemical characterisation. The capsid was also purified from occlusion body samples for visualisation using cryo-electron microscopy. Vp39 could be expressed as a soluble fusion protein and formed helical assemblies when cleaved in stabilising additives, which were characterised by electron microscopy. These helical assemblies allowed several aspects of Vp39 assembly to be revealed. The C-terminal of Vp39 was required for self-assembly, and Vp39 formed a disulphidelinked oligomer. Two surface exposed cysteine residues that likely form this disulphide bond were identified by tandem mass spectrometry. Sequence analysis of the PK-1 from two genera of baculovirus–granuloviruses (GV) and nucleopolyhedroviruses (NPV)–revealed that GV PK-1 lacks a highly conserved protein kinase motif. CpGV PK-1 was shown to be active despite lacking these important functional residues, and mass spectrometry was used to characterise substrate peptides after phosphorylation. A 2.22 Å structure was determined using X-ray crystallography, which revealed that PK-1 forms a unique dimer that locks the enzyme into the typical kinase active conformation.