Icosahedral reconstruction of immature Zika Virus particles using single particle Cryo-electron Microscopy
Open Access
- Author:
- Das, Sayan
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- October 27, 2023
- Committee Members:
- Susan Hafenstein, Thesis Advisor/Co-Advisor
B Tracy Nixon, Committee Member
Santhosh Girirajan, Program Head/Chair
Joyce Jose, Thesis Advisor/Co-Advisor - Keywords:
- Flavivirus
CryoEM
structural biology
Zika - Abstract:
- Cryogenic-sample electron microscopy (CryoEM) is a popular technique in structural biology that can help us advance our understanding of the structure of proteins and protein-complexes at near atomic resolution. This in turn helps us decipher their biological significance with more confidence. The work presented here illustrates the functionality of cryoEM with the reconstruction of membranous small, icosahedrally symmetric viruses. Flaviviruses are small (~50nm) viruses that belong to the family Flaviviridae. These viruses are transmitted through vectors like mosquitoes and ticks, and they encompass a positive sense single stranded RNA genome which codes for all the proteins required by the virion. These virions are composed of 3 structural proteins: the envelope (E) protein, the membrane (M) protein and the capsid (C) proteins. A major structural interplay among these 3 proteins happens during the maturation process of the virion, which ultimately results in the formation of mature, infectious virions. Structural studies of flavivirus have been a challenge because the virion is inherently flexible due to the presence of the lipid membrane that confers fluidity to the virion. Because of this fluidity, the virion does not obey perfect icosahedral symmetry. Currently, the best-resolved structure of an immature Zika Virus (ZIKV) is reported to be at 9Å. These data were collected using FEI Titan Krios electron microscope equipped with a Gatan K2 Summit detector. The magnification for the data collection was 22500x (Super-resolution mode) at a pixel size of 0.65Å. To improve upon this result, we collected a small dataset at a magnification of 59000x, having a pixel size of 1.1Å. The data were collected on Titan Krios with a Falcon 4 detector using counting mode. The resulting 7.6 Å map is a better-resolved structure of the immature ZIKV where we can observe the structural proteins E and M rearranging to give it a “spiky” appearance. A high-resolution structure of immature virus could be revolutionary for the field since it could provide us with more information about critical residues that maintain interactions during maturation. It can also provide us with a clearer idea of the interaction between the capsid protein and other structural proteins. These residues could be targeted for vaccine development as well as structure-guided anti-viral therapy. Moreover, a high-resolution structure of the immature flavivirus can complement the available structures of mature flaviviruses and help us bridge the gap in understanding the mechanism of the maturation process in flavivirus.