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Principal Investigator: Dr. John Parker
Contact Information: E-mail: jsp7@cornell.edu - Phone: 607-256-5626
Sponsor: NIH-NIAID
Grant Number: 1 R01 AI063036-01A1
Title: Reovirus Factories: Structure, Function, and Dynamics
Annual Direct Cost: $100,000
Project Period: 07/15/05-12/31/10
Studies of reoviruses have defined many basic mechanisms of viral replication and pathogenesis. Reoviruses are being developed as therapeutic agents for human cancers. Reoviruses replicate and assemble within cytoplasmic structures called viral factories (VFs). The long-term goals 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 outer capsid 1: 3 heterohexamer assembly and in regulating heterohexamer 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 double-layered particle. The effect of proteasomal inhibition and inhibition of chaperone function on the movement of the VF matrix and the molecular dynamics of the matrix protein will be assessed by time-lapse IF microscopy and fluorescence-based experiments to assess the diffusional mobilities of the VF matrix protein.
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