The negative-stranded RNA viruses comprise some of the most serious human pathogens in terms of both mortality and morbidity. The purpose of the research described here is to study the mechanism of replication of the prototypical negative-stranded rhabdovirus, vesicular stomatitis virus (VSV) and the unusual human paramyxovirus respiratory syncytial (RS) virus. This research is designed to understand at the molecular level the factors relevant in controlling expression of the information on the genome of these viruses. We will investigate the relative roles of two factors: (i) individual viral proteins and (ii) primary RNA sequence, in controlling the decision to express the genome by transcription to yield discrete mRNAs or by replication to provide a complete copy of the genome. The requirement for individual VSV specific proteins in replication is being dissected using an in vitro replication system in which replication is a function of ongoing protein synthesis programmed by addition of individual mRNAs. A method to generate specific mutations in RNA genomes at the cDNA level and to recover precise RNA transcripts of that altered cDNA is proposed. This method will be used to evaluate the role of sequence in controlling RNA synthetic events and to investigate other structure-function relationships.
The specific aims of this grant are as follows: 1) Determine whether proteins other than N are involved in regulation the transition from transcription to replication. 2) Develop a system to create defined mutations in negative-strand RNA viruses. 3) Determine the N protein encapsidation site using deletion and site-specific mutagenesis. 4) Analyze the role of sequence at the leader:N and intercistronic junctions in controlling the decision to transcribe or replicate. 5) Analyze RS virus RNA transcription and the role of RS virus specific nonstructural proteins in RNA synthesis. 6) Develop an in vitro RNA synthesis system for respiration syncytial virus transcription and replication.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Method to Extend Research in Time (MERIT) Award (R37)
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Virology Study Section (VR)
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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Mondal, Arindam; Victor, Ken G; Pudupakam, R S et al. (2014) Newly identified phosphorylation site in the vesicular stomatitis virus P protein is required for viral RNA synthesis. J Virol 88:1461-72
Harouaka, Djamila; Wertz, Gail W (2012) Second-site mutations selected in transcriptional regulatory sequences compensate for engineered mutations in the vesicular stomatitis virus nucleocapsid protein. J Virol 86:11266-75
Rainsford, Edward W; Harouaka, Djamila; Wertz, Gail W (2010) Importance of hydrogen bond contacts between the N protein and RNA genome of vesicular stomatitis virus in encapsidation and RNA synthesis. J Virol 84:1741-51
Harouaka, Djamila; Wertz, Gail W (2009) Mutations in the C-terminal loop of the nucleocapsid protein affect vesicular stomatitis virus RNA replication and transcription differentially. J Virol 83:11429-39
Galloway, Summer E; Wertz, Gail W (2009) A temperature sensitive VSV identifies L protein residues that affect transcription but not replication. Virology 388:286-93
Hinzman, Edward E; Barr, John N; Wertz, Gail W (2008) Selection for gene junction sequences important for VSV transcription. Virology 380:379-87
Galloway, Summer E; Wertz, Gail W (2008) S-adenosyl homocysteine-induced hyperpolyadenylation of vesicular stomatitis virus mRNA requires the methyltransferase activity of L protein. J Virol 82:12280-90
Barr, John N (2007) Bunyavirus mRNA synthesis is coupled to translation to prevent premature transcription termination. RNA 13:731-6
Green, Todd J; Zhang, Xin; Wertz, Gail W et al. (2006) Structure of the vesicular stomatitis virus nucleoprotein-RNA complex. Science 313:357-60
Martinez, Isidoro; Wertz, Gail W (2005) Biological differences between vesicular stomatitis virus Indiana and New Jersey serotype glycoproteins: identification of amino acid residues modulating pH-dependent infectivity. J Virol 79:3578-85

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