Reoviruses are model agents for studying basic viral pathobiology and are developing into exciting new agents for human cancer therapy. Reoviruses replicate and assemble within cytoplasmic structures called viral factories (VFs). The long-term goals of this proposal are to understand how cellular and viral factors interact within VFs to regulate viral assembly and replication. Based on our preliminary findings, we hypothesize that assembly of reovirus virions within VFs requires cellular proteins and is regulated by remodeling of the VF matrix.
The specific aims are: 1. To identify cellular proteins associated with VFs and their function(s) in viral assembly and/or replication. Cellular proteins associated with VFs, will be purified and analyzed by 2-dimensional electrophoresis and mass spectrometry. Association of candidate proteins with VFs in infected cells will be confirmed by immunofluorescence (IF) microscopy. Selected proteins will then be functionally analyzed for their requirement during viral replication using RNA interference, 2. To identify the role(s) of HSP70 chaperones in assembling outer capsid mul:sigma3 heterohexamers and in regulating their recruitment to VFs. The functional role of chaperones in assembly of heterohexamers and their recruitment to VFs will be tested using dominant interfering hsc70 mutants and microinjection experiments. 3. To determine the role that remodeling of the VF matrix plays in assembly of the reovirus virion. The functional effects of proteasomal inhibitors on VF matrix remodeling and assembly of virions will be addressed. The effects of inhibiting proteasomal degradation and chaperone action on the movement of VFs and the molecular dynamics of ?NS in living cells will be assessed and correlated with virion assembly using time-lapse IF microscopy and fluorescence-based experiments to assess the diffusional mobilities of the VF matrix protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI063036-02
Application #
7093542
Study Section
Virology - B Study Section (VIRB)
Program Officer
Cassetti, Cristina
Project Start
2005-07-15
Project End
2009-12-31
Budget Start
2006-01-01
Budget End
2006-12-31
Support Year
2
Fiscal Year
2006
Total Cost
$308,574
Indirect Cost
Name
Cornell University
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Kaufer, Susanne; Coffey, Caroline M; Parker, John S L (2012) The cellular chaperone hsc70 is specifically recruited to reovirus viral factories independently of its chaperone function. J Virol 86:1079-89
Wisniewski, Meagan L; Werner, Brenda G; Hom, Louis G et al. (2011) Reovirus infection or ectopic expression of outer capsid protein micro1 induces apoptosis independently of the cellular proapoptotic proteins Bax and Bak. J Virol 85:296-304
Kim, Jae-Won; Lyi, Sangbom M; Parrish, Colin R et al. (2011) A proapoptotic peptide derived from reovirus outer capsid protein {micro}1 has membrane-destabilizing activity. J Virol 85:1507-16
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
Coffey, Caroline M; Sheh, Alexander; Kim, Irene S et al. (2006) Reovirus outer capsid protein micro1 induces apoptosis and associates with lipid droplets, endoplasmic reticulum, and mitochondria. J Virol 80:8422-38