The proposed research uses mammalian reovirus (MRV) to define how a single capsid protein, ?1, influences distinct functions of the viral capsid. MRV particles are comprised of two concentric protein shells, the outer capsid and the inner core. The ?1 protein is a major component of the outer capsid. Published evidence and our preliminary data indicate that ?1 performs at least three functions. First, even though the two proteins are not in physical contact, properties of ?1 influence virus assembly in a way that alters the presentation of the sigma1 attachment protein on the virion. Second, two ?1 peptides, generated during virus disassembly, cooperate with host lipid membranes to facilitate further uncoating of the virus and permeabilize host membranes. Third, ?1 influences inter-particle interactions to form multivirion infectious units. With the aid of the following three Aims, the proposed research seeks to provide insight into the functions of ?1.
In Aim 1, the role of ?1 in maintaining assembly fidelity will be determined. The position of sigma1 on the particle will be determined by biochemical studies and cryoelectron microscopy. The contribution of altering the strength of interaction between ?1 and adjacent capsid proteins on the presentation of sigma1 will be evaluated using genetic analysis. How changes to sigma1 encapsidation and sigma1 conformation influence virus replication in vivo will be determined using a mouse model of viral disease.
In Aim 2, the function of ?1 in delivering core particles into the host cytoplasm will be defined. How ?1 peptides recruit entry intermediates to membranes will be determined by biochemical and genetic studies. The minimal number of ?1 peptides needed for successful recruitment of a virus entry intermediate, for pore formation and for successful infection will be quantified. The structure of the virus entry intermediate associated with the membrane will be determined by cryoelectron microscopy. Host proteins that associate with capsids following disassembly and influence the efficiency of infection will be identified by affinity purification and mass spectrometry.
In Aim 3, the contribution of ?1 to MVIU formation will be identified. Regions important for inter-particle interactions will be determined by limited proteolysis and mass spectrometry. The relationship between MVIU formation, coinfection efficiency, and reassortment frequency will be determined. Whether MVIU formation also determines reassortment in vivo and if the determinants of MVIU formation and those that influence the recovery of reassortant progeny correlate, will be determined. Completion of this work will provide comprehensive insight into how ?1 completes each of these functions and define the properties of ?1 that influence the capacity of MRV to replicate in cell culture, produce reassortant progeny, and elicit disease.

Public Health Relevance

The proposed research is focused on delineating how functions of the reovirus capsid protein, 1, impact viral infection. The work will define the role of 1 in promoting entry of virus into host cells. It will also demonstrate the mechanism by which 1 controls the assembly fidelity of reovirus capsids and influences interaction between virus particles to form multivirion infectious units.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Virology - A Study Section (VIRA)
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Park, Eun-Chung
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Indiana University Bloomington
Schools of Arts and Sciences
United States
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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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