Structural analysis of capsid maturation and uncoating in Picornaviridae
Open Access
- Author:
- Shingler, Kristin Leigh
- Graduate Program:
- Microbiology and Immunology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 23, 2015
- Committee Members:
- Susan Hafenstein, Dissertation Advisor/Co-Advisor
Aron Eliot Lukacher, Committee Chair/Co-Chair
Rebecca C Craven, Committee Member
Neil David Christensen, Committee Member
Clare E Sample, Committee Member
Katsuhiko Murakami, Committee Member - Keywords:
- picornavirus
cryo-EM
structure
virology
cryo-electron microscopy
A-particle - Abstract:
- Cryo-electron microscopy (cryo-EM) and single particle reconstruction analysis routinely produces high-resolution electron density maps for the study of virus capsids, both alone and in combination with binding partners. Structural virologists have historically taken advantage of the icosahedral symmetry of many viral capsids to enhance the resolution of cryo-EM reconstructions. Recent technological advances in the field have lifted this requirement for achieving high-resolution, making high-resolution reconstruction of asymmetric virus capsids possible. The work presented here parallels the advances made in the field of cryo-EM to explain conformational changes that occur in capsids of the viral order Picornavirales. The order Picornavirales contains the Picornaviridae and Iflaviridae families that infect mammals and insects, respectively. Several picornaviruses are prominent human pathogens including enterovirus 71 (EV71), poliovirus (PV), and coxsackievirus B3 (CVB3). The iflaviruses contain numerous agriculturally important pathogens, such as the honey bee virus, deformed wing virus (DWV). Structural studies of the several capsid forms used to complete the viral life cycle of these microbes have been completed. The lifecycle of the picornavirus capsid is well defined. There are five distinct capsid forms that include procapsid, provirion, infectious capsid, altered particle (A-particle), and empty capsid. Structures are available for all of these forms except the provirion, which is a short-lived intermediate. The procapsid and A-particle structures are of particular interest, as the function of the procapsid is uknown and the exact structural rearrangements needed to form A-particle have yet to be identified. Here we present structures that represent the procapsid, A-particle, and empty capsid forms to answer a variety of biological questions. The EV71 A-particle and empty capsid cryo-EM structures were formed by applying heat to the infectious capsid. The A-particle structure displayed prominent density pillars that connected the particle interior to the viral RNA. Pseudoatomic modeling and density difference map calculations were used to determine that the density pillars were composed of a portion of the N-terminus of VP1. We propose that this interaction tethers the genome to the capsid until a secondary signal for genome release is sensed by the capsid. The predicted surface charges of the A-particle and empty capsid structures also suggest that the RNA is released from an opening at the viral two-fold axis. The EV71 procapsid is expanded compared to the infectious virus. This expansion causes the two capsid forms to have different antigenic properties. The mechanism of neutralization of a monoclonal antibody (MAb 22A12) generated against a highly immunogenic peptide in the EV71 VP1 protein was investigated by cryo-EM image reconstruction. MAb 22A12 binds more readily to the procapsid than the infectious capsid. A 3-D reconstruction of FAb 22A12 in complex with EV71 procapsid reveals that the epitope is near the canyon at a loop that is flexible in the procapsid, but gains ordered structure in the infectious capsid (VP1 GH loop). The suspected mechanism of neutralization is to occlude receptor binding and to cause immune aggregates by capsid cross-linking. This study presents the first evidence for a function of a picornavirus procapsid, suggesting that it is capable of acting as an immune decoy to enhance the infectivity of infectious capsids. The CVB3 A-particle formed by focused receptor engagement using receptor embedded nanodiscs provides the first high-resolution asymmetric reconstruction of a picornavirus entry intermediate. The work presents a new definition for the picornavirus A-particle. The capsid expands radially by ~5% as the VP4 proteins, pocket factors, and N-termini of VP1 proteins exit the capsid at the site of receptor engagement. This work shows the power of DED technology and suggests that it can be used for studies of numerous protein-protein interactions in the future. Despite the severity of disease caused by deformed wing virus infections in honey bees also infected with the ectoparasitic mite Varroa destructor the molecular biology literature detailing the virus is limited. The first structures of DWV were solved by cryo-EM and represent two distinct empty capsid forms. One capsid form is decorated at the 5-fold vertices with open finger-like projections, similar to the related triatoma virus (TrV). The second empty capsid form is similary decorated, however the density protrustions form a closed ring. Both capsids are held together by pentamer-pentamer contacts at the viral 2-fold axes. These connections resemble the strand swapping mechanism used by TrV and other related viruses. We propose that DWV capsids also participate in strand swapping. The work presented here opens many doors for future investigations. The EV71 A-particle formed after receptor stimulation needs to be studied to draw parallels between specific and non-specific triggers. The function of other picornavirus procapsids should be investigated to understand the evolutionary conservation of the production of this noninfectious capsid form. The combination of nanodisc technology with DED data collection opens the door for the study of many protein-protein interactions, including interactions between pleomorphic viruses and their receptors. The DWV infectious capsid needs to be isolated so that its structure can be determined. This will help determine the function of each empty capsid form. These studies will add to the growing body of structural virology work that helps to define the function of proteins in large complexes.