The goal of the proposed research is to determine mechanisms of nonenveloped virus internalization, disassembly, and membrane penetration using mammalian reovirus as an experimental system. Given its capacity to target multiple tissue types and organs in mice, reovirus provides an attractive experimental platform to determine the role of viral entry steps in disease pathogenesis. Reovirus entry requires ?1 integrin-mediated internalization, proteolytic removal of outer-capsid protein ?3, and penetration of endosomal membranes by outer-capsid protein ?1. As with many other pathogenic viruses, the biochemical processes that regulate these progressive events in reovirus entry are not understood, nor is it known how the entry steps influence the course of reovirus disease. These gaps in knowledge have hindered efforts to develop antivirals that act at the earliest stages of infection and highly selective viral vectors for gene-delivery applications.
Three specific aims are proposed to elucidate mechanisms of reovirus entry into cells.
In Specific Aim 1, the means by which ?1 integrin promotes reovirus internalization and endocytic transport will be determined. Mutant viruses with alterations in integrin-binding motifs (IBMs) will be engineered using a newly developed plasmid-based reverse genetics system and tested for the capacity to bind recombinant ?1 integrin and infect cells. Mechanisms of ?1 integrin-mediated virus uptake and endocytic transport will be defined using pathway-specific inhibitors, infectivity assays, and cell imaging. The role of ?1 integrin in reovirus pathogenesis will be determined using mice expressing mutant ?1 integrin and IBM mutant viruses.
In Specific Aim 2, viral determinants of reovirus disassembly and membrane penetration will be identified. Mutant viruses with structure- guided alterations in ?3 and ?1 will be generated by reverse genetics and assessed for disassembly, membrane penetration, and infection. The role of endocytic proteases cathepsins B, L, and S in reovirus pathogenesis will be defined using cathepsin-deficient mice and cathepsin inhibitors.
In Specific Aim 3, the structural basis for reovirus disassembly and membrane penetration will be elucidated. Structures of sequential reovirus disassembly intermediates will be determined at subnanometer resolution using cryo-EM and 3D image reconstruction. Structures of a protease-hypersensitive ?3 mutant and the ?1 ? domain, which is required for membrane penetration, will be determined by X-ray crystallography. This research will reveal how viral and cellular proteins cooperate in a highly ordered biochemical pathway that culminates in cell entry by a nonenveloped virus. These studies also will illuminate new targets for therapy against pathogens that use the endocytic pathway and cathepsin proteases to parasitize host cells.
Virus entry into host cells initiates the process of infection and plays a decisive role in cell and tissue tropism and disease. The proposed research uses reovirus, a powerful experimental model for studies of virus cell entry and pathogenesis, to elucidate mechanisms of nonenveloped virus internalization, disassembly, and membrane penetration. This work will enhance a basic understanding of endocytic uptake and processing of intracellular pathogens and identify the individual contributions of these events to disease. This research also will reveal new therapeutic targets for interference with pathogen entry into cells and accelerate development of reovirus as a vector for vaccine delivery and oncolysis.
|Eaton, Heather E; Kobayashi, Takeshi; Dermody, Terence S et al. (2017) African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions. J Virol 91:|
|Cox, Reagan G; Mainou, Bernardo A; Johnson, Monika et al. (2015) Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes. PLoS Pathog 11:e1005303|
|Mainou, Bernardo A; Ashbrook, Alison W; Smith, Everett Clinton et al. (2015) Serotonin Receptor Agonist 5-Nonyloxytryptamine Alters the Kinetics of Reovirus Cell Entry. J Virol 89:8701-12|
|Doyle, Joshua D; Stencel-Baerenwald, Jennifer E; Copeland, Courtney A et al. (2015) Diminished reovirus capsid stability alters disease pathogenesis and littermate transmission. PLoS Pathog 11:e1004693|
|Fernández de Castro, Isabel; Zamora, Paula F; Ooms, Laura et al. (2014) Reovirus forms neo-organelles for progeny particle assembly within reorganized cell membranes. MBio 5:|
|Trask, Shane D; Boehme, Karl W; Dermody, Terence S et al. (2013) Comparative analysis of Reoviridae reverse genetics methods. Methods 59:199-206|
|Danthi, Pranav; Holm, Geoffrey H; Stehle, Thilo et al. (2013) Reovirus receptors, cell entry, and proapoptotic signaling. Adv Exp Med Biol 790:42-71|
|Trask, Shane D; Wetzel, J Denise; Dermody, Terence S et al. (2013) Mutations in the rotavirus spike protein VP4 reduce trypsin sensitivity but not viral spread. J Gen Virol 94:1296-300|
|Mainou, Bernardo A; Zamora, Paula F; Ashbrook, Alison W et al. (2013) Reovirus cell entry requires functional microtubules. MBio 4:|
|Mainou, Bernardo A; Dermody, Terence S (2012) Transport to late endosomes is required for efficient reovirus infection. J Virol 86:8346-58|
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