Receptor recognition is the first step in viral infection and plays an essential role in target-cell selection in the infected host. Many viruses use cell-adhesion molecules or cell-surface carbohydrates as receptors. However, general rules governing receptor recognition at an atomic level have not been established, and contributions of multiple receptors to viral attachment and cell entry are poorly understood. The proposed research uses reovirus, a highly tractable experimental model that shows promise for oncolytic and vaccine applications, to define the structural basis of virus-receptor interactions at atomic resolution. Following primary infection in the murine intestine, reovirus disseminates to the central nervous system (CNS), where it exhibits serotype-specific differences in tropism and pathogenesis attributable to viral attachment protein ?1. The ?1 protein is a filamentous trimer consisting of an N-terminal tail and a C-terminal head. The ?1 tail of strain T3D reovirus binds sialic acid (SA), and the ?1 head of all three reovirus serotypes binds immunoglobulin superfamily receptor junctional adhesion molecule-A (JAM-A). Three integrated specific aims are proposed to define the structural and functional basis of ?1 interactions with its receptors.
In Specific Aim 1, structures of the three serotypes of ?1 in complex with JAM-A will be determined using X-ray crystallography. Residues in each serotype required for JAM-A binding will be identified by structure-guided mutagenesis of intact virus using a newly developed plasmid-based reverse genetics system. The role of JAM-A binding in reovirus tropism in the murine CNS will be defined using mutants altered in JAM-A utilization and primary cultures of ependymal cells and neurons.
In Specific Aim 2, the structure of T3D ?1 in complex with SA will be determined using X-ray crystallography. Carbohydrate ligands of the three serotypes will be identified using glycan array screening and functional assays. Minimum sequence units required for carbohydrate binding in strains T1L and T3D ?1 will be defined using chimeric viruses and assays of viral binding and infectivity.
In Specific Aim 3, functional relationships between the ?1 receptor-binding domains in reovirus attachment and cell entry will be elucidated using mutant viruses with alterations in ?1 flexibility and length. Adhesive properties of the SA-binding region and its interaction with JAM-A binding will be determined by engineering additional SA-binding sites into the ?1 tail. The ?1 head will be replaced with the adenovirus fiber knob to define the function of receptor specificity in reovirus binding, internalization, and disassembly. These studies will enhance a basic understanding of mechanisms by which pathogenic viruses engage cellular receptors and accelerate the rational design of viral vectors for therapeutic purposes.

Public Health Relevance

Virus-receptor interactions serve a pivotal function in viral disease. The proposed research uses reovirus, a powerful experimental system for studies of viral attachment and pathogenesis, to define general mechanisms by which viruses bind to cellular receptors. This work will contribute important new information about how viruses select cellular targets and aid in the development of new antiviral vaccines and therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI076983-06
Application #
7759118
Study Section
Virology - A Study Section (VIRA)
Program Officer
Cassetti, Cristina
Project Start
2009-01-16
Project End
2013-12-31
Budget Start
2010-01-01
Budget End
2010-12-31
Support Year
6
Fiscal Year
2010
Total Cost
$319,162
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Pediatrics
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Stettner, Eva; Dietrich, Melanie H; Reiss, Kerstin et al. (2015) Structure of Serotype 1 Reovirus Attachment Protein ?1 in Complex with Junctional Adhesion Molecule A Reveals a Conserved Serotype-Independent Binding Epitope. J Virol 89:6136-40
Stencel-Baerenwald, Jennifer; Reiss, Kerstin; Blaum, Bärbel S et al. (2015) Glycan engagement dictates hydrocephalus induction by serotype 1 reovirus. MBio 6:e02356
Monteiro, Ana C; Luissint, Anny-Claude; Sumagin, Ronen et al. (2014) Trans-dimerization of JAM-A regulates Rap2 and is mediated by a domain that is distinct from the cis-dimerization interface. Mol Biol Cell 25:1574-85
Stencel-Baerenwald, Jennifer E; Reiss, Kerstin; Reiter, Dirk M et al. (2014) The sweet spot: defining virus-sialic acid interactions. Nat Rev Microbiol 12:739-49
Katen, Sarah P; Dermody, Terence S (2014) Repurposing staples for viruses: applying peptide design to RSV prophylaxis. J Clin Invest 124:1889-91
Danthi, Pranav; Holm, Geoffrey H; Stehle, Thilo et al. (2013) Reovirus receptors, cell entry, and proapoptotic signaling. Adv Exp Med Biol 790:42-71
Monteiro, Ana C; Sumagin, Ronen; Rankin, Carl R et al. (2013) JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function. Mol Biol Cell 24:2849-60
Reiss, Kerstin; Stencel, Jennifer E; Liu, Yan et al. (2012) The GM2 glycan serves as a functional coreceptor for serotype 1 reovirus. PLoS Pathog 8:e1003078
Frierson, Johnna M; Pruijssers, Andrea J; Konopka, Jennifer L et al. (2012) Utilization of sialylated glycans as coreceptors enhances the neurovirulence of serotype 3 reovirus. J Virol 86:13164-73
Bokiej, Magdalena; Ogden, Kristen M; Ikizler, Mine et al. (2012) Optimum length and flexibility of reovirus attachment protein ?1 are required for efficient viral infection. J Virol 86:10270-80

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