Crystallography was the primary structural tool that first opened up structural investigation of viruses at near-atomic resolution. For instance, the first near-atomic resolution structure of an animal virus, namely a common cold virus, was determined by us in the mid-1980s using crystallographic methods (Nature 317:145-153, 1985). 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 well-ordered crystals. Cryo-electron microscopy has now emerged as the leading tool for advancing structural biology. Single particle averaging of cryo-electron microscopic images is now frequently approaching atomic resolution with the use of direct electron detection devices and technology to compensate for blurring of images due to random motion during data acquisition. However, 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 the availability of identical homogeneous particles. However, the resolution of tomographic reconstructions is usually insufficient to determine structures in atomic detail. Thus, a small part of this grant application is dedicated towards extending the resolution of tomographic maps. Alphaviruses and rubella virus constitute the Togavirus family because of their similarity in gene order. Among alphaviruses, Chikungunya virus is a major world health concern because of its 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 the pleomorphic rubella virus. Specifically, we are planning to obtain the structures of various alphaviruses when complexed with host receptor molecules and with antibodies and small molecule inhibitors that block maturation, attachment and fusion with host cells in order to study the structural intermediates encountered in the viral life cycle. This information will provide viral targets that can be exploited for the design of specific antiviral therapeutics. We are also developing new technology aimed at stretching and pulling individual cryoEM images to make them more similar and homogenous to improve the resolution of tomographic images of pleomorphic biological assemblies such as rubella virus.
Alphaviruses can be lethal human pathogens and the emergence of Chikungunya epidemics in India and Southeast Asia is a major concern to world health. Thus, our studies with neutralizing antibodies and small molecule compounds 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 pleomorphic viruses that were previously out of range.
|Hasan, S Saif; Sun, Chengqun; Kim, Arthur S et al. (2018) Cryo-EM Structures of Eastern Equine Encephalitis Virus Reveal Mechanisms of Virus Disassembly and Antibody Neutralization. Cell Rep 25:3136-3147.e5|
|Urakami, Akane; Sakurai, Atsuko; Ishikawa, Momoko et al. (2017) Development of a Novel Virus-Like Particle Vaccine Platform That Mimics the Immature Form of Alphavirus. Clin Vaccine Immunol 24:|
|Yap, Moh Lan; Klose, Thomas; Urakami, Akane et al. (2017) Structural studies of Chikungunya virus maturation. Proc Natl Acad Sci U S A 114:13703-13707|
|Porta, Jason; Mangala Prasad, Vidya; Wang, Cheng-I et al. (2016) Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission. J Virol 90:1169-77|
|Long, Feng; Fong, Rachel H; Austin, Stephen K et al. (2015) Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity. Proc Natl Acad Sci U S A 112:13898-903|
|Fox, Julie M; Long, Feng; Edeling, Melissa A et al. (2015) Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress. Cell 163:1095-1107|
|Yap, Moh Lan; Klose, Thomas; Plevka, Pavel et al. (2014) Structure of the 3.3MDa, in vitro assembled, hubless bacteriophage T4 baseplate. J Struct Biol 187:95-102|
|Porta, Jason; Jose, Joyce; Roehrig, John T et al. (2014) Locking and blocking the viral landscape of an alphavirus with neutralizing antibodies. J Virol 88:9616-23|
|Sun, Siyang; Xiang, Ye; Akahata, Wataru et al. (2013) Structural analyses at pseudo atomic resolution of Chikungunya virus and antibodies show mechanisms of neutralization. Elife 2:e00435|
|Mangala Prasad, Vidya; Willows, Steven D; Fokine, Andrei et al. (2013) Rubella virus capsid protein structure and its role in virus assembly and infection. Proc Natl Acad Sci U S A 110:20105-10|
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