Herpesvirus virions consist of three layers: nucleocapsid, tegument, and envelope. The innermost layer, the nucleocapsid, initially assembles as an immature procapsid precursor built around viral scaffold proteins. The event that initiates procapsid maturation is unknown but it is dependent upon activation of the internal protease. Scaffold cleavage triggers angularization, or transformation into a polyhedral form, of the shell and its decoration with the accessory capsid-surface proteins – the capsid vertex specific component and the small capsid protein. In both the procapsid and mature angularized capsid, the apical region of the major capsid protein is surface exposed. I investigated whether the major capsid protein apical region contributes to intracellular transport dynamics following entry into primary sensory neurons and also tested the hypothesis that conserved negatively-charged amino acids in the apical region contribute to acquisition of the small capsid protein. To my surprise neither hypothesis proved true. Instead, mutation of glutamic acid residues in the apical region delayed viral propagation and induced focal capsid accumulations in nuclei. ', 'This dissertation reports on the fortuitous discovery that a region of the major capsid protein that is exposed on the outer surface of the capsid also contributes to capsid maturation. Examination of capsid morphogenesis based on epitope unmasking, capsid composition, and ultrastructural analysis indicated that the glutamic acid mutant focal accumulations consisted of procapsids. The results demonstrate that, in addition to established events that occur inside the capsid, the exterior capsid shell promotes capsid morphogenesis and maturation of the megastructure. ', 'In addition, this dissertation presents a modified fluorescent fusion protein design that produced the first described procapsid-specific tag. In this design, a fluorescent protein open reading frame is fused to the amino-terminus of the protease and includes the protease cleavage site immediately following the fluorescent protein’s sequence. Consequently, the fluorescent protein is cleaved from the protease upon its activation during capsid maturation and the fluorescent signal is lost from the megastructure. This procapsid-specific tag not only allows for the identification of procapsids in living cells by a single fluorescent marker but also might serve as a fluorescent readout of protease activity.

Last modified

• 10/28/2019

Creator

• Laura Lee Ruhge

DOI

• https://doi.org/10.21985/n2-x68q-7w91

Subject

• Driskill Graduate Training Program in Life Sciences

Keyword

• maturation
• capsid
• microscopy
• procapsid
• herpesvirus
• major capsid protein

Date created

• 2018-01-01

Resource type

• Dissertation

Rights statement

• In Copyright