Cryoelectron Microscopy (CryoEM)-Based Supramolecular Structural Study of Virus-Host Interactions

2002 Nobel prize of chemistry is awarded to three scientists who develop the NMR techniques to solve protein 3-D structure indicating the importance of knowing the 3-D structure of proteins in living organisms. Indeed, interactions between proteins constitute fundamental mechanisms for the homeostasis of biological organisms. Dysregulation of such interactions can be regarded as a molecular basis for diseases. Structural modifications of proteins are known to be essential to biological functions, such as membrane channel opening, signal transduction, and gene expression. X-ray crystallography and nuclear magnetic resonance (NMR) have proven to be useful in solving protein structures. However, crystallography is limited by the difficulty in crystal formation. Moreover, proteins in crystalloid states are by no means “natural.” Recently, advancements in cyroelectron microscopy (cryoEM) and computation power of computers have brought much hope to the resolution of “supramolecular structures,” i.e., extra-large protein complexes with multiple forming units or components. Using liquid nitrogen to rapidly freeze of supramolecular protein samples in solutions that have been rendered a thin-film on electron microscope grids, cryoEM is capable of acquiring frozen molecular images at various angles under defocusing conditions without staining. The images are then digitized and subjected to analysis in the Fourier space. Finally, the images are reconstructed in three-dimensional display. With the theoretical resolution at atomic level, cryoEM has become a major tool of structural analysis in the post-genomic era. We propose to establish a cryoEM-based structural research modality, using human hepatitis B virus (HBV) core particle as a model system since we have constructed plasmids carrying three mutant forms of HBV core particles that can be expressed in E. coli and readily form particles. Structural analysis of these mutant particles may provide information on mechanisms of viral particle formation. To understand the mechanisms of host-virus interaction, we also propose to establish transgenic mice expressing HBV particles in the liver. CryoEM has been demonstrated to be able to view budding virus. Lastly, due to the shortage of experts in cryoEM which will be highly demanded in studying high molecule weight proteins or complex in the next few years, we propose to emphasize educational programs that will support international collaboration, graduate student exchange, and international technical counseling. Yang-Ming University has accumulated a critical mass for the pursuit of cryoEM-based structural analysis. This includes research groups of protein biochemistry and virology, long tradition of EM service (with two full-time highly experienced EM operators), as well as Graduate Programs of Structural Biology and Bioinformatics. If this project is funded in conjunction with the cryoEM core, our endeavor can be readily extended to service of supramolecular structural analysis that may be incorporated by researchers in the Genomic Medicine Program.

Project ID:PA9308-2266
External Project ID:NSC93-3112-B182-003