Paramyxoviruses are among the most common organisms in acute respiratory tract infections, and these ubiquitous pathogens are responsible for a high degree of morbidity and mortality worldwide. These RNA viruses have evolved mechanisms that control both the level and the particular type of viral RNA synthesized during an infection. cis-acting sequences contained within the paramyxovirus genome are a major control point for regulating RNA synthesis. The proposed work will test several new concepts that have emerged concerning features of the viral nucleocapsid template which regulate genome replication and gene expression for the prototypic paramyxovirus SV5.
The first aim i s to determine the role in viral RNA replication of three factors: (i) sequence within two essential and discontinuous promoter elements, (ii) the spacing of these elements along one face of a helix and (iii) the hexameric phase in the promoter region. A reverse genetics system based on model RNA minigenome analogs will be used along with site specific mutagenesis and an in vivo selection method to determine the relative contribution of each of these factors to promoter activity. Chimeric RNA genomes containing exchanges between the genomic and antigenomic promoters will be used along with polymerase binding assays to determine the basis for differential synthesis of viral RNA from these two promoters. For some paramyxoviruses, there is a very high level of transcriptional readthrough at the junction between the viral M and F genes. In the second aim, a transcription-competent model dicistronic RNA genome will be used to determine the molecular basis for transcriptional readthrough at the M-F junction. In the third aim, recombinant viruses containing altered M-F intergenic regions will be isolated from a full length cDNA. These mutant viruses will be employed to test the two hypotheses that during the course of an infection, M-F readthrough transcription serves to (i) downregulate the level of fusion-competent F protein or (ii) increase the number of polymerase molecules that can access the 5' end of the viral genome. These experiments will test new concepts on how the paramyxovirus nucleocapsid-associated RNA template can modulate the activities of the multifunctional viral polymerase. Results from the proposed work will form a critical base for a rational reverse-genetics approach to parainfluenza vaccines, since it may be possible to design ideal attenuated recombinant viruses which have altered promoter or intergenic transcription activities.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Experimental Virology Study Section (EVR)
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Rubin, Fran A
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Wake Forest University Health Sciences
Schools of Medicine
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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; 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 (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; 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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