Alphaviruses are a group of arthropod-transmitted, enveloped, positive-strand RNA viruses classified in the Togaviridae family. The 30 plus members of the alphavirus genus differ significantly in the severity of disease induced in humans and other animals, from sub-clinical infections with Sindbis virus to fatal encephalitis that can be seen with eastern and Venezuelan equine encephalitis viruses. They have a lipid bilayer envelope and an icosahedral structure, and they are important human and animal pathogens. Our studies investigating the assembly pathway of alphaviruses have proven extremely fruitful in understanding the morphogenesis pathway and structure of this group of simple, yet important, enveloped viruses. Therefore, we propose to study alphavirus assembly using our previously successful approach, encompassing molecular genetics, protein and nucleic acid biochemistry, and structural biology. Our studies will build on very recent advances made in collaboration with our long-time colleague Michael Rossmann that have finally revealed the structure of the Sindbis virus E1-E2 spike using X-ray crystallography. The alphaviruses represent an excellent model system to understand how enveloped icosahedral viruses organize themselves into ordered arrays of proteins. The expertise of my colleagues at Purdue in protein and virus structure will complement our expertise in viral molecular genetics and biochemistry and maintain the dynamic environment in which these studies have, and will continue, to flourish. High resolution atomic structures of the alphavirus nucleocapsid protein and the envelope glycoproteins will be used in conjunction with pseudo-atomic resolution cryo-electron microscopy image reconstructions as a guide for the molecular genetic analysis of the nucleocapsid core and envelope proteins. We will rely heavily on the use of the large collection of biological reagents that we have produced and the expertise in genetics and structural biology accumulated during the previous period of this grant. In addition, we will use a variety of biophysical techniques that include X-ray crystallography, cryo-electron microscopy and electron tomography to probe the structure and assembly of both wild type and mutant viruses. The proposed experiments will focus on the spatial and temporal aspects of alphavirus assembly that guide oligomerization of the envelope glycoproteins into a spike arrangement, the association of the spikes with the pre-formed nucleocapsid core, and the incorporation of 6K protein and formation of the virus particle during budding. Our approach will be to incorporate recent and developing structural information together with genetic and cell biology approaches to develop a comprehensive understanding of virus assembly and egress. This insight will enable us, and others, to pursue novel strategies for virus and disease intervention.

Public Health Relevance

The alphaviruses are mosquito-borne enveloped RNA viruses that cause severe and debilitating illness in humans. In this project, our laboratory will employ state of the art approaches in genetics, cell biology, and structural biology to interrogate the process of virus assembly and egress from the infected cell. We intend to understand this vital aspect of the virus life cycle so that novel intervention strategies could be used in future.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056279-14
Application #
8333966
Study Section
Virology - A Study Section (VIRA)
Program Officer
Sakalian, Michael
Project Start
1997-09-01
Project End
2015-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
14
Fiscal Year
2012
Total Cost
$298,288
Indirect Cost
$98,288
Name
Purdue University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Apte-Sengupta, Swapna; Sirohi, Devika; Kuhn, Richard J (2014) Coupling of replication and assembly in flaviviruses. Curr Opin Virol 9:134-42
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
Aggarwal, Megha; Dhindwal, Sonali; Kumar, Pravindra et al. (2014) trans-Protease activity and structural insights into the active form of the alphavirus capsid protease. J Virol 88:12242-53
Snyder, Jonathan E; Kulcsar, Kirsten A; Schultz, Kimberly L W et al. (2013) Functional characterization of the alphavirus TF protein. J Virol 87:8511-23
Dai, Hong-Sheng; Liu, Zheng; Jiang, Wen et al. (2013) Directed evolution of a virus exclusively utilizing human epidermal growth factor receptor as the entry receptor. J Virol 87:11231-43
Jose, Joyce; Przybyla, Laralynne; Edwards, Thomas J et al. (2012) Interactions of the cytoplasmic domain of Sindbis virus E2 with nucleocapsid cores promote alphavirus budding. J Virol 86:2585-99
Snyder, Jonathan E; Azizgolshani, Odisse; Wu, Bingbing et al. (2011) Rescue of infectious particles from preassembled alphavirus nucleocapsid cores. J Virol 85:5773-81
Tang, Jinghua; Jose, Joyce; Chipman, Paul et al. (2011) Molecular links between the E2 envelope glycoprotein and nucleocapsid core in Sindbis virus. J Mol Biol 414:442-59
Gualtieri, E J; Guo, F; Kissick, D J et al. (2011) Detection of membrane protein two-dimensional crystals in living cells. Biophys J 100:207-14
Jose, Joyce; Snyder, Jonathan E; Kuhn, Richard J (2009) A structural and functional perspective of alphavirus replication and assembly. Future Microbiol 4:837-56

Showing the most recent 10 out of 30 publications