Our recent work has led to the overall hypothesis that following infection, paramyxoviruses delay abundant RNA synthesis to provide time for virus-mediated suppression of host anti-virus responses. This hypothesis emerged from our results with variants of the prototype paramyxovirus Simian Virus 5 (SV5) that have either accelerated or delayed kinetics of RNA synthesis. A remarkable result from this work was our finding that mutations in the P/V gene that accelerate viral RNA synthesis also convert SV5 from a noncytopathic virus into a virus that activates type I interferon (IFN) and kills cells by apoptosis. Thus, the overall goal of our work is to understand mechanisms that can accelerate or delay paramyxovirus RNA synthesis and how changes in the timing of viral gene expression affect key virus:host cell interactions. ? We have isolated a naturally-occurring SV5 variant (WF-CPIV) that shows a remarkable delay in viral gene expression compared to WT SV5.
In Aim 1, we will determine the basis for the delay in WF-CPIV gene expression. Real-time PCR assays will be used to test the hypotheses that delayed WF-CPIV gene expression is due to a defect in either primary or secondary transcription. Recombinant SV5 viruses containing exchanges of polymerase-associate genes will identify WF-CPIV component(s) responsible for delayed gene expression.
Aim 2 addresses our finding that an rSV5 with substitutions in the P/V gene expresses viral mRNA and proteins earlier and to higher levels than WT rSV5. Novel rSV5 viruses will be generated that express WT or mutant P and V proteins from separate transcription units. Biochemical and minigenome replication assays will be used to test the hypotheses that P/V substitutions either decrease the ability of V to inhibit SV5 genome replication or confer higher RNA synthesis activity on the P protein.
In Aim 3, we will test the hypothesis that the cytopathic rSV5 variant containing P/V substitutions induces interferon (IFN) synthesis and apoptosis due to premature expression of viral gene products. Cell lines with inducible expression of WT and mutant P and V proteins, and new rSV5 P/V mutants will be used to test a two-step model for the role of accelerated and delayed SV5 RNA synthesis in the induction of IFN and apoptosis. ? We hypothesize that paramyxoviruses have evolved to optimize the timing of the onset of viral gene expression to avoid host antiviral responses, and changes that accelerate or significantly delay RNA synthesis can activate IFN and/or apoptotic pathways. Our work will increase our understanding of important virus-host cell interactions, as well as provide new approaches to improve the safety and potency of vaccine vectors. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI042023-07
Application #
6830742
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Kim, Sonnie
Project Start
1998-07-01
Project End
2008-11-30
Budget Start
2004-12-01
Budget End
2005-11-30
Support Year
7
Fiscal Year
2005
Total Cost
$251,125
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
937727907
City
Winston-Salem
State
NC
Country
United States
Zip Code
27157
Johnson, John B; Schmitt, Anthony P; Parks, Griffith D (2013) Point mutations in the paramyxovirus F protein that enhance fusion activity shift the mechanism of complement-mediated virus neutralization. J Virol 87:9250-9
Briggs, Caitlin M; Holder, Robert C; Reid, Sean D et al. (2011) Activation of human macrophages by bacterial components relieves the restriction on replication of an interferon-inducing parainfluenza virus 5 (PIV5) P/V mutant. Microbes Infect 13:359-68
Manuse, Mary J; Parks, Griffith D (2010) TLR3-dependent upregulation of RIG-I leads to enhanced cytokine production from cells infected with the parainfluenza virus SV5. Virology 397:231-41
Manuse, Mary J; Briggs, Caitlin M; Parks, Griffith D (2010) Replication-independent activation of human plasmacytoid dendritic cells by the paramyxovirus SV5 Requires TLR7 and autophagy pathways. Virology 405:383-9
Manuse, Mary J; Parks, Griffith D (2009) Role for the paramyxovirus genomic promoter in limiting host cell antiviral responses and cell killing. J Virol 83:9057-67
Gainey, Maria D; Manuse, Mary J; Parks, Griffith D (2008) A hyperfusogenic F protein enhances the oncolytic potency of a paramyxovirus simian virus 5 P/V mutant without compromising sensitivity to type I interferon. J Virol 82:9369-80
Gainey, Maria D; Dillon, Patrick J; Clark, Kimberly M et al. (2008) Paramyxovirus-induced shutoff of host and viral protein synthesis: role of the P and V proteins in limiting PKR activation. J Virol 82:828-39
Arimilli, Subhashini; Johnson, John B; Alexander-Miller, Martha A et al. (2007) TLR-4 and -6 agonists reverse apoptosis and promote maturation of simian virus 5-infected human dendritic cells through NFkB-dependent pathways. Virology 365:144-56
Dillon, Patrick J; Parks, Griffith D (2007) Role for the phosphoprotein P subunit of the paramyxovirus polymerase in limiting induction of host cell antiviral responses. J Virol 81:11116-27
Connor, John H; McKenzie, Margie O; Parks, Griffith D et al. (2007) Antiviral activity and RNA polymerase degradation following Hsp90 inhibition in a range of negative strand viruses. Virology 362:109-19

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