Symmetrical and asymmetrical structural studies of icosahedral virus interactions with host receptors, antibodies, and antiviral drug
Open Access
- Author:
- Lee, Hyunwook
- Graduate Program:
- Microbiology and Immunology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 16, 2016
- Committee Members:
- Susan Hafenstein, Dissertation Advisor/Co-Advisor
Susan Hafenstein, Committee Chair/Co-Chair
Jianming Hu, Committee Member
Neil D. Christensen, Committee Member
B. Tracy Nixon, Outside Member
David Spector, Committee Member - Keywords:
- Cryo-EM
Single Particle Anaysis
3D-Reconstruction
HPV16
EV71
CVB3
Icosahedral Symmetry - Abstract:
- Cryo-electron microscopy (cryo-EM) is a type of transmission electron microscopy where the sample is studied at cryogenic temperature, below -180 °C in general. A vitrified biological specimen can be examined in its hydrated native state using cryo-EM and the three-dimensional (3D) molecular structure can be reconstructed by single-particle image processing. Recent technological advances in the field have significantly improved the quality of the outputs so that cryo-EM 3D maps are now reaching atomic resolutions. Icosahedral viruses have been advantageous objects for single particle cryo-EM analysis because of their large molecular sizes and the high degree of symmetry, which facilitate the alignment of noisy particle images. Innumerous 3D reconstructions have been generated with icosahedral averaging for various viruses of different forms, revealing many molecular mechanisms that the virus capsids undergo during their lifecycles. The symmetry averaging, however, can obscure asymmetric structures on the virus capsids, which are related with biological functions of the capsids. As many biological reactions occur asymmetrically, especially virus-host interactions, sometimes it is necessary to examine the structures asymmetrically. The main purpose of this thesis is to explore the asymmetric features of icosahedral viruses by developing and exploiting the single particle image processing techniques. Human papillomaviruses and enteroviruses are icosahedral viruses that infect millions of people around the world and cause serious diseases, including various cancers and neurological diseases. Using single particle cryo-EM methods, the work presented here on these clinically important viruses has focused on the virus interactions with cellular components, including neutralizing antibodies and virus receptors. In reality, conformational changes on the virus capsids and molecular interactions between viral and cellular parts do not happen symmetrically as the environment surrounding the virus is not symmetric. Thus, in order to investigate physiologically accurate 3D structures, the icosahedral symmetry imposed during the image processing should be abandoned to recover the asymmetry of viruses interacting with host proteins. Different approaches were applied to solve these issues in each of the presented studies. Cryo-EM 3D reconstructions of human papillomavirus 16 (HPV16) in complex with Fab molecules were generated for the clinically important HPV16-specific neutralizing antibody, H16.V5. During the analysis of the 3D maps new conformational epitopes of H16.V5 as well as its mechanism of neutralization were elucidated. In another experiment, conformational changes of the virus capsids were observed under a highly-concentrated condition and after binding of the antibody. The arrangement of the pentavalent and hexavalent capsomers caused steric hindrance between two Fab molecules binding on each capsomer and resulted in breaking the icosahedral symmetry. Binding dynamics of the two competing Fabs confirmed a hyperstabilization model of neutralization by H16.V5. Enterovirus 71 (EV71) was incubated with a neutralizing antibody (MA28-7), a receptor (PSGL-1), or a drug (NF449) to study the binding modes of the target molecules. The cryo-EM analyses revealed the binding sites of the molecules, a mechanism of neutralization, and a mechanism of strain-specificities of the antibody and of the receptor. Specifically, the results elucidated how a single amino acid residue, VP1-145, determines the MA28-7 and PSGL-1 binding phenotypes of the virus. The three molecules bound at the positively charged surface on the icosahedral 5-fold vertex where symmetry averaging can result in the most extreme build-up of noisy artifact. Because of the steric hindrance, only one molecule, instead of five, could bind on each vertex, breaking the icosahedral symmetry. Moreover, partial occupancies of the bound molecules weakened the cryo-EM densities and made the structural analyses difficult; therefore, different methods were applied to investigate the symmetry-mismatched components, depending on their molecular sizes. In addition, an atomic model of EV71 strain 1095 was built de novo into the cryo-EM reconstruction, completing the atomic structures of the three most prevalent EV71 groups for the residue VP1-145. Two studies were conducted in different time frames with traditional and innovative cryo-EM technologies. The results of each study demonstrate the recent transformative improvements of cryo-EM hardware and software. The coxsackievirus B3 (CVB3) was locally stimulated by incubating with full-length receptors embedded in lipid bilayer discs, called nanodiscs. This novel approach resulted in the first high-resolution asymmetric 3D structure of a picornavirus entry-intermediate, also called an “Altered particle” (A-particle). The resulting structure was significantly different from previously characterized A-particles since the asymmetric binding of the receptor induced global capsid expansion as well as local conformational changes at the site of interaction, including multiple channels and extrusions of the viral proteins. Interpretation of the results opened new gates for studies of picornavirus entry mechanisms and led to a new model that proposed global and local capsid stimulations resulted in different A-particle structures. Remarkably, by imposing icosahedral symmetry, an atomic resolution 3D map was generated from the asymmetric particles with severe local conformational changes. This should raise an alert to the researchers in the field as to the risks of applying icosahedral averaging exclusively to study viruses. Studying asymmetric structures in the icosahedral virus capsids required different approaches using various programs and algorithms. Imposing icosahedral symmetry has been simple but not using the symmetry or reversing the imposed symmetry was not an easy task. Compatibility between different programs was also limited. To handle the issue, therefore, we developed a new C++ function for RELION, the most commonly used software for high-resolution single particle analysis, and applied it to solve the asymmetric structure of the CVB3 A-particle.