Using structural approaches (cryo-electron microscopy and x-ray crystallography), we will investigate the mechanisms used by coxsackievirus B3 (CVB3) to enter host cells by engaging two receptors (decay accelerating factor and coxsackievirus and adenovirus receptor). CVB3 is a human pathogen that causes myocarditis, pancreatitis, and has been implicated in the onset of juvenile diabetes mellitus. Our proposed studies of CVB3 entry are a continuation of a productive collaboration with Dr. Jeffery Bergelson, Division of Infectious Diseases at Children's Hospital at Philadelphia. A combination of traditional and innovative methods will be used to visualize both receptor binding and the resulting conformational changes that lead to entry and uncoating. Of particular interest is a novel entry intermediate that has never been observed (Aim 1). This particle has been isolated with fingerlike density projections extruding from the capsid, and these densities are hypothesized to be peptides used to anchor the particle to the host membrane. We will investigate the capability of this new particle (and other entry intermediates) to bind receptors and infect the host. In addition to conventional structural studies made with purified virus and recombinant receptor proteins in solution (Aim 2), the full-length receptor will be anchored onto a lipid bilayer disc to form virus-receptor complexes for an in situ asymmetric study (Aim 3). This approach simulates the physiological binding event, in which the virus interacts with receptors at a single, focused region on its capsid. Preliminary results with this asymmetric approach suggest the virus behaves differently when interacting with a few membrane-bound receptors compared to many soluble receptors. Specifically our in situ A-particle retains an intact genome (as the A-particle does in an infection) and undergoes different protein rearrangements compared to particles that are treated with soluble receptors or heat. The proposed study is directed at understanding the structural alterations that an enterovirus capsid must undergo to uncoat after successfully finding and attaching to the receptor in situ.

Public Health Relevance

Enteroviruses cause a wide spectrum of acute disease, ranging from mild upper respiratory illness to aseptic meningitis, encephalitis and acute flaccid paralysis. In addition to acute infections, enteroviuses, especially coxsackieviruses of the B group, have also been associated with severe myocarditis and type 1 diabetes mellitus. This study will investigate the mechanisms of entry beginning with receptor recognition by coxsackievirus B3. Binding of receptor is a critical first step as it sets in motion events that promote host entry as well as initiating conformational changes to the virus capsid that are necessary for release of the viral genome.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI107121-04
Application #
9378063
Study Section
Virology - A Study Section (VIRA)
Program Officer
Park, Eun-Chung
Project Start
2014-11-01
Project End
2019-10-31
Budget Start
2017-11-01
Budget End
2018-10-31
Support Year
4
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033
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Subramanian, Suriyasri; Organtini, Lindsey J; Grossman, Alec et al. (2017) Cryo-EM maps reveal five-fold channel structures and their modification by gatekeeper mutations in the parvovirus minute virus of mice (MVM) capsid. Virology 510:216-223
Organtini, Lindsey J; Shingler, Kristin L; Ashley, Robert E et al. (2017) Honey Bee Deformed Wing Virus Structures Reveal that Conformational Changes Accompany Genome Release. J Virol 91:
Organtini, Lindsey J; Lee, Hyunwook; Iketani, Sho et al. (2016) Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus. J Virol 90:9733-9742
Lee, Hyunwook; Shingler, Kristin L; Organtini, Lindsey J et al. (2016) The novel asymmetric entry intermediate of a picornavirus captured with nanodiscs. Sci Adv 2:e1501929
Guan, Jian; Bywaters, Stephanie M; Brendle, Sarah A et al. (2015) Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization. Virology 483:253-63
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Organtini, Lindsey J; Makhov, Alexander M; Conway, James F et al. (2014) Kinetic and structural analysis of coxsackievirus B3 receptor interactions and formation of the A-particle. J Virol 88:5755-65