Crystallography was the primary structural tool that first opened up structural investigation of viruses at near-atomic resolution. However, larger viruses with lipid envelopes are too variable in their structures and have too small a proportion of their surface involved in lattice contacts to make stable crystals. Cryo-electron microscopy later emerged as the leading tool for advancing Structural Biology to give the "big picture", with crystallography playing a supporting role in the determination of the crystallizable viral components. Single particle averaging of cryo-electron microscopic images is now approaching almost atomic resolution in favorable cases but, once again, the limits of this technology are becoming apparent in the investigations of progressively more complex pleomorphic assemblies. Cryo-electron tomography has now emerged as an invaluable tool for the determination of the structure of individual viral particles without reliance on averaging between symmetrically equivalent components or on the availability of identical homogeneous particles. However, the resolution of a tomographic reconstruction is insufficient to determine structure in atomic detail. Thus, a part of this grant application is dedicated towards extracting more information from tomograms, especially as applied to the slightly pleomorphic, structurally largely unknown, Rubella virus. Alphaviruses and Rubella virus constitute the Togavirus family because of their similarity in gene order and morphology. Among alphaviruses, Chikungunya virus is a major world health concern because of its recent re-emergence in Asia and Africa. We plan to extend our previous studies of the icosahedral, lipid containing alphaviruses, and tackle the more difficult structural analysis of pleomorphic Rubella virus. Specifically, we are planning to extend the resolution of Chikungunya virus-like particles to near-atomic resolution using cryo-electron microscopy in part as an aid to the design of a vaccine against Chikungunya virus with Gary Nabel at the NIH Vaccine Research Center. We also plan to determine the structure of Rubella virus by analyses of electron tomograms. We plan to use antibodies and peptide inhibitors to block maturation, attachment and fusion with host cells in order to study the structural intermediates encountered in the viral life cycle. We are planning to determine the remaining unknown structures of the Rubella virus components and use both crystallography and electron microscopy to study their interactions with each other and with antibodies.
Alphaviruses can be lethal human pathogens and the recent emergence of Chikungunya virus (an alphavirus) in India and Southeast Asia is a major concern to world health. Thus, our studies with neutralizing antibodies and peptides that inhibit infection are relevant to major current health concerns. Furthermore, our studies depend on extending current structural techniques to pleomorphic viruses, thus making possible structural studies of numerous, lipid membrane containing enveloped viruses (such as Rubella virus, an alphavirus homologue) that were previously out of range.
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