HIGH RESOLUTION 3-D RECONSTRUCTION OF VIRUSES USING SINGLE PARTICLE ELECTRON CR

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Viral infections have been and remain one of the major threats to human health. Viruses are large assemblies of proteins and nucleic acids that rely on infection of hosts to complete their life cycle and sustain their propagation. Many viruses consist of a highly symmetric icosahedral protein shell that encapsulates the genetic materials. Multiple processes, including capsid assembly, genome packaging, maturation and virus-host interaction, must occur for a virus to successfully complete its life cycle and cause severe, often fatal, damage to the host. High resolution 3-D structure of the virus particles, if available, will provide important insights to understanding of these processes and the development of effective prevention and treatment strategies. Cryo-electron microscopy (cryo-EM) is an emerging technique for structural determination of large macromolecular complexes and viruses. In recent years, there are significant progresses in cryo-EM and it is now becoming routine to solve the structure of a virus to sub-nanometer resolutions (6-10 ), and more recently to near atomic resolution (~4 ). However, the image processing and 3-D reconstruction of cryo-EM images are computational resource demanding. In this proposal, we apply for the computational resources to solve the structures of bacterial virus T7/T3 at their many stages of assembly and DNA packaging process, which is an active NIH-funded R01 project. T7 and T3, which are close homologues, are dsDNA phages in the Podoviridae family that have a short tail and infect E. coli.. In this project, we aim to characterize the mechanisms of T7/T3 capsid assembly, maturation and genome packaging using cryo-EM and 3-D reconstruction techniques. For each of the four major states of the phage life cycle, procapsid, capsid II, dsDNA partially packaged particles (ipDNA-capsid), and the final infectious particles, we will purify the particles, image the particles using cryo-EM, and solve the high resolution structure by image processing and 3-D reconstruction.