Mammalian reoviruses are highly virulent in newborn mice, producing injury to several organ systems, including the central nervous system and heart. Apoptotic cell death plays a major role in the pathogenesis of reovirus disease. Reovirus-induced apoptosis requires viral disassembly in cellular endosomes, intracellular signal transduction, and changes in the expression of cellular genes under the control of transcription factor NF-icB. Following reovirus infection, mice deficient in the p50 subunit of NF-icB display diminished apoptosis in the brain, yet enhanced apoptosis in the heart, as compared to wild-type animals. These features provide a tractable experimental system to investigate tissue-specific mechanismsof reovirus-induced apoptosis and pathogenesis. The central hypothesis of this proposal is that viral disassembly in the endocytic compartment stimulates the NF-icB signal transduction pathway, reprograms cellular gene expression, and either activates or suppresses the apoptotic machinery in a cell-type specific manner. Three integrated specific aims are proposed to test this hypothesis.
In Specific Aim 1, mechanisms by which viral disassembly induces NF-icB activation and leads to apoptosis will be determined. These experiments focus on the membrane-penetration protein, ja1, which functions following disassembly to deliver the viral core into the cytoplasm and is genetically linked to strain-specific differences in apoptosis induction. Functional domains in ^.1that mediate apoptosis will be identified using mutant viruses and viral cores recoated with mutant forms of ^1. Apoptosis-defective j^1 mutants will be tested for the capacity to disrupt endosomes, injure mitochondria, and activate NF-icB.
In Specific Aim 2, ja1-directed mechanisms of reovirus-induced NF-KB activation and apoptosis will be defined. Signaling intermediates that link |a1 with NF-KB during reovirus infection will be identified using genetically engineered cell lines and RNA interference. A novel pathway of NF-KB activation dependent on the IKKct catalytic and IKKy regulatory subunits of the multicomponent ItcB kinase will be investigated.
In Specific Aim 3, the role of tissue-specific NF-KB signaling pathways in reovirus pathogenesis will be elucidated. Mice lacking IKKa, IKK(3, or NF-KB p50 in either the brain or heart will be infected with reovirus and assessed for apoptosis and disease. Tissue- specific p50-null mice will be used for microarray experiments to identify host genes under NF-KB control that are activated in response to reovirus infection in the brain and heart. Results of these experiments will provide insight into mechanisms by which viruses perturb cellular signaling pathways to cause cell death and disease. Information gained from these studies should foster development of new antiviral strategies designed to modulate apoptosis that may be applicable to a wide array of microbial pathogens.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI050080-10
Application #
7992396
Study Section
Virology - B Study Section (VIRB)
Program Officer
Cassetti, Cristina
Project Start
2001-09-01
Project End
2013-11-30
Budget Start
2010-12-01
Budget End
2013-11-30
Support Year
10
Fiscal Year
2011
Total Cost
$332,071
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
Stebbing, Rachael E; Irvin, Susan C; Rivera-Serrano, Efrain E et al. (2014) An ITAM in a nonenveloped virus regulates activation of NF-*B, induction of beta interferon, and viral spread. J Virol 88:2572-83
Danthi, Pranav; Holm, Geoffrey H; Stehle, Thilo et al. (2013) Reovirus receptors, cell entry, and proapoptotic signaling. Adv Exp Med Biol 790:42-71
Knowlton, Jonathan J; Dermody, Terence S; Holm, Geoffrey H (2012) Apoptosis induced by mammalian reovirus is beta interferon (IFN) independent and enhanced by IFN regulatory factor 3- and NF-ýýB-dependent expression of Noxa. J Virol 86:1650-60
Boehme, Karl W; Ikizler, Mine; Kobayashi, Takeshi et al. (2011) Reverse genetics for mammalian reovirus. Methods 55:109-13
Sen, Adrish; Pruijssers, Andrea J; Dermody, Terence S et al. (2011) The early interferon response to rotavirus is regulated by PKR and depends on MAVS/IPS-1, RIG-I, MDA-5, and IRF3. J Virol 85:3717-32
Holm, Geoffrey H; Pruijssers, Andrea J; Li, Lianna et al. (2010) Interferon regulatory factor 3 attenuates reovirus myocarditis and contributes to viral clearance. J Virol 84:6900-8
Danthi, Pranav; Pruijssers, Andrea J; Berger, Angela K et al. (2010) Bid regulates the pathogenesis of neurotropic reovirus. PLoS Pathog 6:e1000980
Danthi, Pranav; Guglielmi, Kristen M; Kirchner, Eva et al. (2010) From touchdown to transcription: the reovirus cell entry pathway. Curr Top Microbiol Immunol 343:91-119
Zurney, Jennifer; Kobayashi, Takeshi; Holm, Geoffrey H et al. (2009) Reovirus mu2 protein inhibits interferon signaling through a novel mechanism involving nuclear accumulation of interferon regulatory factor 9. J Virol 83:2178-87
Danthi, Pranav; Coffey, Caroline M; Parker, John S L et al. (2008) Independent regulation of reovirus membrane penetration and apoptosis by the mu1 phi domain. PLoS Pathog 4:e1000248

Showing the most recent 10 out of 25 publications