The primary objective of the proposed research is to determine how the balance between the protective and genome delivery functions of the viral capsid influences viral pathogenesis. The goal will be attained using the tractable mammalian reovirus system as an experimental model. In its native state, the ?1 protein of the reovirus outer capsid confers stability to the viral particle. During entry into cells, ?1 is exposed and cleaved to generate ?1N, ?, and ? fragments. These ?1-derived peptides are buried and remain associated with the virus particle. Upon interaction with host membranes, the ?1 protein undergoes a dramatic conformational rearrangement to expose and release ?1N and ?. The released peptides form pores in target membranes that allow delivery of the ~70 nm viral inner capsid (core) across membranes. Three integrated aims are proposed to define how the mutually antagonistic, protective and genome delivery functions of ?1 are balanced, and how this balance modulates viral disease.
In Aim 1, how the reovirus capsid is destabilized to promote conformational changes required for cell entry will be determined. Viral determinants that modulate capsid stability and conformational flexibility will be identified. The mechanisms by which host proteases promote conformational changes in the viral capsid will be defined. A subnanometer structure of the conformationally-altered reovirus particle will be solved by cryo-electron microscopy (cryo-EM).
In Aim 2, how membranes are breached by ?1 will be elucidated. The minimal number of pore-forming peptides needed to initiate infection will be determined. Features within ?1N and ? that allow these peptides to form pores will be defined. Determinants within the capsid that control the shape and size of the pore formed by reovirus during cell entry will be identified. Structures of viral entry intermediates in association with model membranes will be determined by cryo-EM.
In Aim 3, how functions of ?1 in maintaining capsid stability and allowing genome delivery influence viral pathogenesis will be determined. The capacity of ?1 mutant viruses with altered stability, conformational flexibility, or genome delivery efficiency to replicate at initial sites of infection, disseminate to secondary sites of infection, and replicate at secondary sites will be evaluated in a newborn mouse model. The virulence of ?1 mutant viruses will be compared. The effect of capsid stability on transmission between animals will also be determined. Successful completion of these goals will define how different functions of the viral capsid are regulated, provide unprecedented snapshots of changes occurring in the viral capsid during its transit across the membrane, and identify a relationship between viral capsid properties, tissue tropism, and viral disease. This work will help identify critical control points in the conserved cell entry pathway of nonenveloped viruses that could serve as potential targets for antiviral therapeutics. In addition, results of these studies could foster the development of a more efficacious reovirus oncolytic.

Public Health Relevance

The proposed research uses a highly tractable model to define how viral and host factors maintain a balance between the genome protection and genome delivery function of viral capsids. The studies will reveal how altering this balance influences viral pathogenesis and transmission. This work will also highlight critical steps in the conserved pathway for cell entry utilized by a variety of non-enveloped viruses that can serve as potential targets for antiviral therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI110637-04
Application #
9315588
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Park, Eun-Chung
Project Start
2014-08-01
Project End
2019-07-31
Budget Start
2017-08-01
Budget End
2019-07-31
Support Year
4
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Indiana University Bloomington
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47401
Snyder, Anthony J; Danthi, Pranav (2018) Infectious Subviral Particle-induced Hemolysis Assay for Mammalian Orthoreovirus. Bio Protoc 8:
Thete, Deepti; Danthi, Pranav (2018) Protein Mismatches Caused by Reassortment Influence Functions of the Reovirus Capsid. J Virol 92:
Snyder, Anthony J; Danthi, Pranav (2018) Cleavage of the C-Terminal Fragment of Reovirus ?1 Is Required for Optimal Infectivity. J Virol 92:
Snyder, Anthony J; Danthi, Pranav (2018) Infectious Subviral Particle to Membrane Penetration Active Particle (ISVP-to-ISVP*) Conversion Assay for Mammalian Orthoreovirus. Bio Protoc 8:
Berger, Angela K; Hiller, Bradley E; Thete, Deepti et al. (2017) Viral RNA at Two Stages of Reovirus Infection Is Required for the Induction of Necroptosis. J Virol 91:
Snyder, Anthony J; Danthi, Pranav (2017) The reovirus ?1 340-343 loop controls entry related conformational changes. J Virol :
Morais, Marc C (2016) Breaking the symmetry of a viral capsid. Proc Natl Acad Sci U S A 113:11390-11392
Snyder, Anthony J; Danthi, Pranav (2016) Lipids Cooperate with the Reovirus Membrane Penetration Peptide to Facilitate Particle Uncoating. J Biol Chem 291:26773-26785
Thete, Deepti; Snyder, Anthony J; Mainou, Bernardo A et al. (2016) Reovirus ?1 Protein Affects Infectivity by Altering Virus-Receptor Interactions. J Virol 90:10951-10962
Snyder, Anthony J; Danthi, Pranav (2016) Lipid Membranes Facilitate Conformational Changes Required for Reovirus Cell Entry. J Virol 90:2628-38

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